The Effects of Natural Epigenetic Therapies in 3D Ovarian Cancer and Patient-Derived Tumor Explants: New Avenues in Regulating the Cancer Secretome

High mortality rates in ovarian cancer have been linked to recurrence, metastasis, and chemoresistant disease, which are known to involve not only genetic changes but also epigenetic aberrations. In ovarian cancer, adipose-derived stem cells from the omentum (O-ASCs) play a crucial role in supporting the tumor and its tumorigenic microenvironment, further propagating epigenetic abnormalities and dissemination of the disease. Epigallocatechin gallate (EGCG), a DNA methyltransferase inhibitor derived from green tea, and Indole-3-carbinol (I3C), a histone deacetylase inhibitor from cruciferous vegetables, carry promising effects in reprograming aberrant epigenetic modifications in cancer. Therefore, we demonstrate the action of these diet-derived compounds in suppressing the growth of 3D ovarian cancer spheroids or organoids as well as post-treatment cancer recovery through proliferation, migration, invasion, and colony formation assays when compared to the synthetic epigenetic compound Panobinostat with or without standard chemotherapy. Finally, given the regulatory role of the secretome in growth, metastasis, chemoresistance, and relapse of disease, we demonstrate that natural epigenetic compounds can regulate the secretion of protumorigenic growth factors, cytokines, extracellular matrix components, and immunoregulatory markers in human ovarian cancer specimens. While further studies are needed, our results suggest that these treatments could be considered in the future as adjuncts to standard chemotherapy, improving efficiency and patient outcomes.


Introduction
Despite countless advancements in care and treatment, ovarian cancer continues to be a challenging and deadly disease. In the United States alone, 19,880 new cases of ovarian cancer were estimated to be diagnosed in 2022. Overall, ovarian cancer ranks fifth among causes of death in women, leading to more deaths than any other gynecologic cancer [1][2][3]. The current standard treatment comprises a combination of cytoreductive surgery, with or without platinum-based chemotherapy [3][4][5]. While initial management aims to provide a cure, diagnosis at advanced stages, alongside resistance and recurrence of disease, have posed challenges to the optimal management of ovarian cancer [6,7]. Therefore, there is a pressing need for new and effective treatment avenues in the field of oncology, targeting modalities such as epigenetics and immunotherapies.
Given that mortality rates in ovarian cancer have been linked primarily to chemoresistance and metastasis of occult microdisease [8], epigenetic therapies offer a promising solution to this treatment predicament [9][10][11]. Recent studies have shown that both cancer

Migration and Invasion
Additionally, pretreated 3D spheroids and organoid-derived cells were tested for their ability to migrate and invade for 24-48 h using the CytoSelectTM 24-Well Cell Migration and Invasion Assay according to the manufacturer's instructions (8 µm, Colorimetric Format), Cat# CBA-100C, Cell Biolabs, San Diego, CA, USA). After microscopic images for migration and invasion were obtained with an inverted microscope (Leica Microsystems), labeled cells from membranes were extracted and tested for optical density (OD) at 560 nm at a plate reader.

Observation of Colony Formation Following Treatment in 3D Culture
5690 cells from pre-treated spheroids and organoids were transferred to 100 mm plates (100 cells/mm 2 ) and incubated for 14 days in cell-appropriate media as previously published in our lab [29]. After that, plates were washed with PBS and incubated with 0.05% Crystal Violet staining solution (Sigma-Aldrich) for 10 min. Plates were again washed Figure 1. Effects of dose concentration on spheroid growth. Spheroids were treated with different concentrations of EGCG (10 µM, 50 µM) and I3C (50 µM, 200 µM) in a single dosage and allowed to grow for 7 days. Percent growth was obtained as a measure of outcome over time, with robust suppression of growth noted in a dose-dependent manner by EGCG 50 µM (p < 0.05) and I3C 200 µM (p < 0.05) when compared to lower doses of the same compounds. Significant differences were also noted between EGCG 50 µM (downregulation of growth of 90%, * p < 0.05) and I3C 200 µM (downregulation of growth of 91%, p < 0.05) relative to control. Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05.
Once the dose efficacy of our natural epigenetic compounds was tested, 3D ovarian cancer spheroids were exposed to monotherapy treatments of EGCG 50 µM, I3C 200 µM, Pano 10 nM, and standard chemotherapy (CT) at concentrations previously demonstrated in our lab [22,29] on days 0 and 3, with percent growth obtained in an extended kinetic assay (days 0, 5, 10, 14, and 17, pictured below) and compared to the control (or drug-free medium) group. As demonstrated below, downregulation of ovarian cancer tumor growth is noted both through microscopy ( Figure 2) and graphical representation of the data, including statistical analysis ( Figure 3). to grow for 7 days. Percent growth was obtained as a measure of outcome over time, with robust suppression of growth noted in a dose-dependent manner by EGCG 50 µM (p < 0.05) and I3C 200 µM (p < 0.05) when compared to lower doses of the same compounds. Significant differences were also noted between EGCG 50 µM (downregulation of growth of 90%, * p < 0.05) and I3C 200 µM (downregulation of growth of 91%, p < 0.05) relative to control. Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05.
Once the dose efficacy of our natural epigenetic compounds was tested, 3D ovarian cancer spheroids were exposed to monotherapy treatments of EGCG 50 µM, I3C 200 µM, Pano 10 nM, and standard chemotherapy (CT) at concentrations previously demonstrated in our lab [22,29] on days 0 and 3, with percent growth obtained in an extended kinetic assay (days 0, 5, 10, 14, and 17, pictured below) and compared to the control (or drug-free medium) group. As demonstrated below, downregulation of ovarian cancer tumor growth is noted both through microscopy ( Figure 2) and graphical representation of the data, including statistical analysis ( Figure 3).
While the control group demonstrates exponential growth, our natural epigenetic monotherapies ascertain a more efficacious response in suppressing cancer growth when compared both to the control and standard chemotherapy-treated groups. The same was observed in the Pano-treated group. Furthermore, when 3D spheroids were treated with EGCG, I3C, and Pano, an overall reversal of growth, or shrinkage, occurred in relation to the control group in an extended kinetic assay. The same effect, however, was not observed when treatment with standard chemotherapy alone took place. As validated by our statistical analysis, on day 10 ( Figure 3, left upper panel) of our experiment, EGCG (96% inhibition of growth, p < 0.05), I3C, and Pano (both led to 100% inhibition of growth with further shrinkage or negative downregulation of growth, p < 0.05) significantly suppressed 3D ovarian cancer growth as compared to control. On day 14 of the experiment (Figure 3, right upper panel), EGCG, I3C, and Pano more drastically suppressed 3D ovarian cancer growth as compared to control (100% inhibition of growth was noted in all three epigenetic treatment groups, with further shrinkage or negative downregulation of growth noted, p < 0.05). Details regarding shrinkage or negative downregulation of growth are stated in "Material and Methods". The effects of our epigenetic treatments were also noted to be robust relative to the chemotherapy-treated groups both on day 10 ( Figure 3, left bottom panel) and day 14 (Figure 3, right bottom panel). While chemotherapy alone has an overall effect in suppressing ovarian cancer growth in relation to the control treated groups (67% on day 10, 33% on day 14), it did not suppress 3D spheroid ovarian cancer growth as efficaciously as compared to our epigenetic treatments (Figure 3, bottom row). As observed in the projection boxes in Figure 3 (top row), no statistically significant difference in efficiency was found between natural and synthetic epigenetic compounds. Microscopic representation of 3D ovarian cancer growth after epigenetic monotherapy treatments. The assay includes treatments with EGCG, I3C, and Pano which effectively suppress spheroid 3D growth over time when compared to control conditions and standard chemotherapy (CT). Additionally, these epigenetic compounds are noted to provide overall uniform shrinkage of spheroids in the absence of the spread of peripheral tumor microdisease. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.  Microscopic representation of 3D ovarian cancer growth after epigenetic monotherapy treatments. The assay includes treatments with EGCG, I3C, and Pano which effectively suppress spheroid 3D growth over time when compared to control conditions and standard chemotherapy (CT). Additionally, these epigenetic compounds are noted to provide overall uniform shrinkage of spheroids in the absence of the spread of peripheral tumor microdisease. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.

Apoptosis and Necrosis Assays in Pre-Treated Spheroids
Given that a reversal of growth, or shrinkage, was noted following treatment with the above epigenetic compounds, we sought to investigate the extent of apoptosis and necrosis resulting from our treatments. This assay additionally allows for direct visualization regarding the retention or loss of overall 3D cancer morphology. As demonstrated by our data in Figure 3, downregulation of growth efficiently took place when ovarian cancer cells were exposed to our various epigenetic monotherapies when compared to CT and control groups. In correlation, EGCG, I3C, and Pano treatments induced programmed cell death (apoptosis/necrosis) and significant loss of overall 3D morphology after 17 days of treatments ( Figure 4). Following CT treatments, significant downregulation in 3D morphology is noted when compared to control, with high induction of apoptosis/necrosis being observed. Therefore, our epigenetic compounds efficiently induce programmed cell death in 3D ovarian cancer ( Figure 4). Ovarian cancer tumor growth is efficaciously suppressed by treatments with EGCG, I3C, and Pano in relation to the control (top row) treated groups on days 10 (96% by EGCG, 100% with further negative downregulation of growth by I3C and Pano, p < 0.05) and day 14 (100% inhibition of growth was noted in all three epigenetic treatment groups with further shrinkage or negative downregulation of growth noted, p < 0.05). CT suppressed ovarian cancer growth in relation to the control-treated groups (67% on day 10, 33% on day 14); however, not as efficiently when compared to our epigenetic treatments (bottom row). On the top row, a projection box allows for visualization of the effects of our epigenetic treatments. No significant differences were noted between natural compounds and Pano on days 10 and 14, suggesting similar efficiency of action between them (p > 0.05). Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. The asterisk (*) represents p < 0.05.

Lateral Spread of Microdisease
In addition to the above results, which indicate that epigenetic therapies efficiently induce downregulation of tumor growth, loss of 3D spheroid morphology, and apoptosis/necrosis of cells, we further examined the role of epigenetic therapies in suppressing the lateral spread of disease. As demonstrated by the appearance of 3D spheroids at day 5 ( Figure 5), epigenetic therapies have properties that prevent the lateral dispersion of single cells or tumor particles. The same effect is not visualized in the standard chemotherapytreated group, with lateral spread of particles or a thin monolayer of disease being noted as early as day 5 after treatments.
This phenomenon is also observed across days of treatments in an extended kinetic assay, with epigenetic therapies progressively inducing symmetric downregulation of tumor growth and lateral spread of disease ( Figure 2). As days of treatment progressed, however, a thin monolayer of disease surrounding a central 3D core was noted in spheroids treated with a combination of cisplatin and taxol (CT), suggesting that standard chemotherapy alone may not fully halt microspread of disease in ovarian cancer patients. Moreover, single cells noted to shed from 3D structures in the CT-treated group were noted to lead to de novo formation of spheroids, perhaps demonstrating how microdisease can lead to recurrence and spread of ovarian cancer.

Lateral Spread of Microdisease
In addition to the above results, which indicate that epigenetic therapies efficiently induce downregulation of tumor growth, loss of 3D spheroid morphology, and apoptosis/necrosis of cells, we further examined the role of epigenetic therapies in suppressing the lateral spread of disease. As demonstrated by the appearance of 3D spheroids at day 5 ( Figure 5), epigenetic therapies have properties that prevent the lateral dispersion of single cells or tumor particles. The same effect is not visualized in the standard chemotherapy-treated group, with lateral spread of particles or a thin monolayer of disease being noted as early as day 5 after treatments. Figure 5. Representation of the lateral spread of microdisease. In 3D culture, both natural (EGCG 50 µM and I3C 200 µM) and synthetic (Pano 10 nM) epigenetic compounds suppress the lateral dispersion of tumor cells or particles. In contrast, the same response is not noted in spheroids exposed to standard chemotherapy (CT), as visualized here on day 5 of experiments. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. This phenomenon is also observed across days of treatments in an extended kinetic assay, with epigenetic therapies progressively inducing symmetric downregulation of tumor growth and lateral spread of disease ( Figure 2). As days of treatment progressed,

Lateral Spread of Microdisease
In addition to the above results, which indicate that epigenetic therapies efficiently induce downregulation of tumor growth, loss of 3D spheroid morphology, and apoptosis/necrosis of cells, we further examined the role of epigenetic therapies in suppressing the lateral spread of disease. As demonstrated by the appearance of 3D spheroids at day 5 ( Figure 5), epigenetic therapies have properties that prevent the lateral dispersion of single cells or tumor particles. The same effect is not visualized in the standard chemotherapy-treated group, with lateral spread of particles or a thin monolayer of disease being noted as early as day 5 after treatments. Figure 5. Representation of the lateral spread of microdisease. In 3D culture, both natural (EGCG 50 µM and I3C 200 µM) and synthetic (Pano 10 nM) epigenetic compounds suppress the lateral dispersion of tumor cells or particles. In contrast, the same response is not noted in spheroids exposed to standard chemotherapy (CT), as visualized here on day 5 of experiments. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. This phenomenon is also observed across days of treatments in an extended kinetic assay, with epigenetic therapies progressively inducing symmetric downregulation of tumor growth and lateral spread of disease ( Figure 2). As days of treatment progressed, Figure 5. Representation of the lateral spread of microdisease. In 3D culture, both natural (EGCG 50 µM and I3C 200 µM) and synthetic (Pano 10 nM) epigenetic compounds suppress the lateral dispersion of tumor cells or particles. In contrast, the same response is not noted in spheroids exposed to standard chemotherapy (CT), as visualized here on day 5 of experiments. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.

Can Epigenetic Therapies Affect Standard Chemotherapy Efficiency in the Treatment of 3D Ovarian Cancer?
As demonstrated by our prior results, EGCG, I3C, and Pano monotherapies efficiently downregulate 3D spheroid ovarian cancer growth while suppressing lateral dispersion of single cells or tumor particles when compared to standard chemotherapy alone (CT) (Figures 2 and 5). Considering such findings, we further tested the efficiency of our epigenetic treatments when combined with CT in order to assess whether combination treatments can augment or optimize the efficiency of standard chemotherapy in the treatment of 3D ovarian cancer ( Figure 6). ciently downregulate 3D spheroid ovarian cancer growth while suppressing lateral dispersion of single cells or tumor particles when compared to standard chemotherapy alone (CT) (Figures 2 and 5). Considering such findings, we further tested the efficiency of our epigenetic treatments when combined with CT in order to assess whether combination treatments can augment or optimize the efficiency of standard chemotherapy in the treatment of 3D ovarian cancer ( Figure 6). Figure 6. Microscopic representation of 3D ovarian cancer spheroid growth after combo therapy with epigenetic treatments and standard chemotherapy. In 3D culture, combinations of EGCG/CT, I3C/CT, and Pano/CT further suppress tumor growth in comparison to standard chemotherapy (CT) alone. EGCG/CT and Pano/CT have a more robust effect on volume inhibition, while Pano/CT promotes loss of 3D morphology. Lateral dispersion of disease can be visually observed in the CTtreated group. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.
When EGCG, I3C, and Pano are combined with chemotherapy, overall suppression of tumor growth is noted in a more efficacious manner than in the CT-treated group. More specifically, the EGCG/CT and Pano/CT combos promulgate a more robust and symmetric inhibition of tumor growth across days of treatments, indicating a downregulatory effect of these combination treatments on 3D spheroid growth. Regarding overall morphology, all three combinations suppress dispersion of the lateral monolayer of tumor particles in relation to the chemotherapy-treated group, as observed above. However, despite the efficiency of Pano/CT in downregulating 3D spheroid growth, shedding of single cells can be noted ( Figure 6) to a greater extent in the EGCG/CT and I3C/CT treatment groups. Figure 6. Microscopic representation of 3D ovarian cancer spheroid growth after combo therapy with epigenetic treatments and standard chemotherapy. In 3D culture, combinations of EGCG/CT, I3C/CT, and Pano/CT further suppress tumor growth in comparison to standard chemotherapy (CT) alone. EGCG/CT and Pano/CT have a more robust effect on volume inhibition, while Pano/CT promotes loss of 3D morphology. Lateral dispersion of disease can be visually observed in the CT-treated group. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.
When EGCG, I3C, and Pano are combined with chemotherapy, overall suppression of tumor growth is noted in a more efficacious manner than in the CT-treated group. More specifically, the EGCG/CT and Pano/CT combos promulgate a more robust and symmetric inhibition of tumor growth across days of treatments, indicating a downregulatory effect of these combination treatments on 3D spheroid growth. Regarding overall morphology, all three combinations suppress dispersion of the lateral monolayer of tumor particles in relation to the chemotherapy-treated group, as observed above. However, despite the efficiency of Pano/CT in downregulating 3D spheroid growth, shedding of single cells can be noted ( Figure 6) to a greater extent in the EGCG/CT and I3C/CT treatment groups. Additionally, Pano/CT appears to promote loss of the overall 3D spheroid morphology when compared to EGCG/CT and I3C/CT treatments ( Figure 6, day 17 of treatment). Overall, when our epigenetic treatments are combined with standard chemotherapy, a more robust downregulation of growth is noted when compared to monotherapy treatments.

Does the Tumorigenic Microenvironment Affect the Efficiency of Epigenetic Therapies?
In a clinical scenario, the tumorigenic microenvironment carries the ability to impair the efficiency of standard chemotherapy while promoting cancer growth, chemoresistance, and metastasis of disease. This effect is highly driven by omentum stem cell populations, as previously shown in the literature [18][19][20][21]. Given that stem cells create a supportive environment for cancer, we further investigated the effects of our treatments in 3D ovarian cancer organoids, comprising both ovarian cancer tumor cells and omentum derived stem cells (O-ASCs). Therefore, after combining these two cell populations (see details in material and methods), organoids were yielded through the hanging drop method and initially exposed to monotherapy treatments of EGCG, I3C, Pano, and CT on days 0 and 3 of experiments. Percent growth was obtained in an extended kinetic assay (days 0, 5, 7, 10, 14, and 17) and compared to the control (or drug-free medium) group, following the same model previously applied to the spheroid population. As observed in Figure 7 (days 5, 10, and 17 pictured), although organoids contain two different cell populations, their 3D morphology is similar in appearance and structure to that of spheroids. On day 5 following treatments, our epigenetic treatments and standard chemotherapy (CT) are noted to suppress growth and the lateral spread of disease. However, as experimental days progressed, lateral spread of disease and suboptimal downregulation of percent growth were progressively noted in the CT and control groups on days 10 and 17 of experiments when compared to the monotherapy epigenetic groups. In the Pano group, loss of overall 3D morphology also took place, as noted on day 17 of treatment ( Figure 7). supportive environment for cancer, we further investigated the effects of our treatments in 3D ovarian cancer organoids, comprising both ovarian cancer tumor cells and omentum derived stem cells (O-ASCs). Therefore, after combining these two cell populations (see details in material and methods), organoids were yielded through the hanging drop method and initially exposed to monotherapy treatments of EGCG, I3C, Pano, and CT on days 0 and 3 of experiments. Percent growth was obtained in an extended kinetic assay (days 0, 5, 7, 10, 14, and 17) and compared to the control (or drug-free medium) group, following the same model previously applied to the spheroid population. As observed in Figure 7 (days 5, 10, and 17 pictured), although organoids contain two different cell populations, their 3D morphology is similar in appearance and structure to that of spheroids. On day 5 following treatments, our epigenetic treatments and standard chemotherapy (CT) are noted to suppress growth and the lateral spread of disease. However, as experimental days progressed, lateral spread of disease and suboptimal downregulation of percent growth were progressively noted in the CT and control groups on days 10 and 17 of experiments when compared to the monotherapy epigenetic groups. In the Pano group, loss of overall 3D morphology also took place, as noted on day 17 of treatment ( Figure 7).

Figure 7.
Morphology of organoids in 3D culture following epigenetic or CT treatment. Treatments with natural (EGCG, I3C) and synthetic (Pano) epigenetic compounds were performed on days 0 and 3, with response to therapy recorded in percent growth on days 0, 5, 7, 10, 14, and 17. As illustrated, as the days of the experiment progressed, both the CT and control groups provided suboptimal downregulation of percent growth and lateral spread of disease when compared to the monotherapy epigenetic treatment groups. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. Morphology of organoids in 3D culture following epigenetic or CT treatment. Treatments with natural (EGCG, I3C) and synthetic (Pano) epigenetic compounds were performed on days 0 and 3, with response to therapy recorded in percent growth on days 0, 5, 7, 10, 14, and 17. As illustrated, as the days of the experiment progressed, both the CT and control groups provided suboptimal downregulation of percent growth and lateral spread of disease when compared to the monotherapy epigenetic treatment groups. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group.
From the perspective of an extended kinetic assay, when organoids were treated with our epigenetic compounds, significant downregulation of growth was induced relative to the control group on day 10 ( Figure 8, left panel) by EGCG (98% inhibition of growth, p < 0.01), I3C (97% inhibition of growth, p < 0.01) and Pano (100% inhibition of growth with further shrinkage or negative downregulation of growth noted, p < 0.01). Similar effects were also observed on day 17 of experiments ( Figure 8, right panel) following treatment with EGCG (98.2% inhibition of growth, p < 0.01), I3C (98.1% inhibition of growth, p < 0.01) and Pano (100% inhibition of growth with further shrinkage or negative downregulation of growth noted, p < 0.01). While it has been established in the literature that chemotherapy alone efficiently suppresses 3D organoid ovarian cancer growth, 75% inhibition of growth was noted in the CT group on day 10 ( Figure 8, left bottom panel), with only 23.2% inhibition of growth noted on day 17 (Figure 8, right bottom panel) of experiments relative to the control group. All our epigenetic treatments also efficiently downregulated tumor growth in relation to the chemotherapy-treated group in our experiments ( Figure 8, bottom row). Additionally, live/dead immunostaining was performed on organoid cultures following 17 days of our various treatments, showing the same trend and supporting the results shown in Figure 8 (80% live cells were noted in the control group, approximately 15% live cells were noted in the EGCG and I3C groups, and 3% live cells were noted in the Pano group, data not shown). Therefore, our results suggest that epigenetic therapies can efficiently suppress tumor growth even in the presence of tumorigenic omentum-derived stem cells, known to be crucial in providing support for tumor propagation and chemoresistance of disease. Figure 8. Effects of epigenetic monotherapies and standard chemotherapy (CT) on organoid growth in a prolonged kinetic assay on days 10 (left panel) and 17 (right panel). Ovarian cancer organoid tumor growth is efficaciously suppressed by treatments with EGCG, I3C, and Pano in relation to the control (top row) treated groups on days 10 (98% by EGCG, 97% by I3C, 100% by Pano with further negative downregulation of growth, p < 0.01) and day 17 (98.2% by EGCG, 98.1% by I3C, 100% by Pano with further negative downregulation of growth, p < 0.01). CT suppressed ovarian cancer growth in relation to the control-treated groups (75% on day 10, 23% on day 14, p < 0.05), but not as efficiently when compared to our epigenetic treatments (bottom row). On the top row, a projection box allows for visualization of the effects of our epigenetic treatments. No significant differences were noted between natural compounds and Pano on days 10 and 17, suggesting similar efficiency of action between them (p > 0.05). Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. The asterisk (*) represents p < 0.05 and (**) represents p < 0.01.
Once we established the efficacy of our epigenetic monotherapies in suppressing tumor propagation, we further tested their efficacy when combined with chemotherapy. While both epigenetic monotherapy treatments and combo treatments efficiently downregulated 3D ovarian cancer growth in relation to control ( Figure 9, top panel, p < 0.05 for all treatment groups), when ovarian cancer 3D organoids were exposed to combo treatments of EGCG/CT, I3C/CT, and Pano/CT, a more robust effect on suppression of tumor growth was noted in relation to monotherapy treatments, suggesting additive effects between chemotherapy and epigenetic regimens ( Figure 9, bottom panel). As illustrated in Figure 9 (bottom panel), significant downregulation took place when organoids were treated with EGCG/CT (82%, p < 0.05) and I3C/CT (90%, p < 0.05) in relation to EGCG and I3C monotherapies. Contrarily, no significant differences were noted in the downregulation of tumor growth between the Pano/CT and Pano treatment groups (p > 0.05). Overall, our results indicate that a combination of natural epigenetic treatments and chemotherapy efficiently halts cancer growth in a 3D organoid ovarian cancer model. ments of EGCG/CT, I3C/CT, and Pano/CT, a more robust effect on suppression of tumor growth was noted in relation to monotherapy treatments, suggesting additive effects between chemotherapy and epigenetic regimens ( Figure 9, bottom panel). As illustrated in Figure 9 (bottom panel), significant downregulation took place when organoids were treated with EGCG/CT (82%, p < 0.05) and I3C/CT (90%, p < 0.05) in relation to EGCG and I3C monotherapies. Contrarily, no significant differences were noted in the downregulation of tumor growth between the Pano/CT and Pano treatment groups (p > 0.05). Overall, our results indicate that a combination of natural epigenetic treatments and chemotherapy efficiently halts cancer growth in a 3D organoid ovarian cancer model. Figure 9. Effects of epigenetic treatments alone or in combination with chemotherapy in 3D ovarian cancer organoids. As indicated in the top panel, organoid's percent growth was significantly inhibited by treatment with our natural epigenetic treatments when used alone or in combination with chemotherapy relative to the control group (p < 0.05 for all treatment groups). Higher efficacy is noted both in the EGCG/CT (82%, p < 0.05) and I3C/CT (90%, p < 0.05) groups when compared to epigenetic monotherapy treatments on day 10 of experiments. No significant differences were noted when Pano was administered alone or in combination with chemotherapy. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. The asterisk (*) represents p < 0.05.

Do epigenetic Therapies Suppress Post-Treatment Regrowth, Metastasis, and Migration of 3D Derived Ovarian Cancer Cells?
3.6.1. Post-Treatment Ability of 3D-Derived Spheroid and Organoid Cells to Regrow and Proliferate Figure 9. Effects of epigenetic treatments alone or in combination with chemotherapy in 3D ovarian cancer organoids. As indicated in the top panel, organoid's percent growth was significantly inhibited by treatment with our natural epigenetic treatments when used alone or in combination with chemotherapy relative to the control group (p < 0.05 for all treatment groups). Higher efficacy is noted both in the EGCG/CT (82%, p < 0.05) and I3C/CT (90%, p < 0.05) groups when compared to epigenetic monotherapy treatments on day 10 of experiments. No significant differences were noted when Pano was administered alone or in combination with chemotherapy. Two independent experiments were performed, including triplicates or tetraplicates for each treatment group. The asterisk (*) represents p < 0.05. So far, our epigenetic treatments have efficiently suppressed 3D ovarian cancer growth both in a spheroid and organoid model. Considering that ovarian cancer patients treated with chemotherapy often experience recurrence of disease, we sought to investigate whether epigenetic treatments affect the ability of pre-treated 3D-derived cancer cells from spheroids and organoids to regrow, metastasize, invade, and produce colony forming units (CFU). In order to evaluate the post-treatment potential for regrowth and proliferation, an MTT assay was performed on 3D-derived ovarian cancer cells from pre-treated spheroids and organoids. As illustrated in Figure 10 (left panel), pre-treatment of spheroids with EGCG, I3C, and Pano suppressed the proliferation of 3D-derived cancer cells by approximately 45%, 66%, and 89%, respectively (p < 0.05) relative to the control or drug-free group. Similar effects were observed in organoid-derived cells ( Figure 10, right panel), in which the ability of cells to proliferate after our epigenetic treatments was significantly suppressed by approximately 59%, 70%, and 76% in the EGCG, I3C, and Pano groups, respectively (p < 0.05). In contrast, pre-treatment with standard chemotherapy did not significantly affect the ability of cancer cells to regrow following treatment, both in the spheroid and organoid models.
ing units (CFU). In order to evaluate the post-treatment potential for regrowth and proliferation, an MTT assay was performed on 3D-derived ovarian cancer cells from pre-treated spheroids and organoids. As illustrated in Figure 10 (left panel), pre-treatment of spheroids with EGCG, I3C, and Pano suppressed the proliferation of 3D-derived cancer cells by approximately 45%, 66%, and 89%, respectively (p < 0.05) relative to the control or drugfree group. Similar effects were observed in organoid-derived cells ( Figure 10, right panel), in which the ability of cells to proliferate after our epigenetic treatments was significantly suppressed by approximately 59%, 70%, and 76% in the EGCG, I3C, and Pano groups, respectively (p < 0.05). In contrast, pre-treatment with standard chemotherapy did not significantly affect the ability of cancer cells to regrow following treatment, both in the spheroid and organoid models. Figure 10. Regrowth ability of cells following epigenetic and chemotherapy treatments in spheroids (left panel) and organoids (right panel). An MTT assay was utilized to quantify the regrowth ability of 3D-derived cancer cells following treatment with EGCG, I3C, Pano, and CT. Pre-treatment of spheroids and organoids with EGCG, I3C, and Pano significantly suppressed the proliferation of cancer cells following treatments (p < 0.05 for all epigenetic groups). Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05.
Results obtained through MTT assays were further confirmed by microscopic contrast images. Regrowth of 3D-derived spheroid ( Figure 11, top rows) and organoid ( Figure  11, bottom rows) cells demonstrates a high regrowth ability in the control or drug-free group. In the CT-pretreated group, regrowth ability is mildly decreased when compared to the control group, both in spheroid and organoid-derived cells. Comparatively, pretreatment with EGCG and I3C monotherapies moderately suppressed recovery of cells, while pre-treatment with Pano robustly suppressed regrowth following treatment in both models, as correlated with MTT data ( Figure 10). In order to correlate with the clinical scenario in which epigenetic therapies may hold the potential to augment the effects of standard chemotherapy, we additionally tested the regrowth ability following treatment with a combination of chemotherapy and epigenetic compounds. Combinations of CT with EGCG, I3C, and Pano robustly inhibited the ability of cancer cells to regrow when compared to epigenetic treatments alone in both models ( Figure 11). An MTT assay was utilized to quantify the regrowth ability of 3D-derived cancer cells following treatment with EGCG, I3C, Pano, and CT. Pre-treatment of spheroids and organoids with EGCG, I3C, and Pano significantly suppressed the proliferation of cancer cells following treatments (p < 0.05 for all epigenetic groups). Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05.
Results obtained through MTT assays were further confirmed by microscopic contrast images. Regrowth of 3D-derived spheroid ( Figure 11, top rows) and organoid ( Figure 11, bottom rows) cells demonstrates a high regrowth ability in the control or drug-free group. In the CT-pretreated group, regrowth ability is mildly decreased when compared to the control group, both in spheroid and organoid-derived cells. Comparatively, pre-treatment with EGCG and I3C monotherapies moderately suppressed recovery of cells, while pretreatment with Pano robustly suppressed regrowth following treatment in both models, as correlated with MTT data ( Figure 10). In order to correlate with the clinical scenario in which epigenetic therapies may hold the potential to augment the effects of standard chemotherapy, we additionally tested the regrowth ability following treatment with a combination of chemotherapy and epigenetic compounds. Combinations of CT with EGCG, I3C, and Pano robustly inhibited the ability of cancer cells to regrow when compared to epigenetic treatments alone in both models ( Figure 11).

Post-Treatment Invasion and Migration Ability of 3D-Derived Spheroid and Organoid Cells Following Epigenetic Treatments
The ability of ovarian cancer cells to metastasize is an ongoing and concerning problem. The tumorigenic microenvironment provides a supportive environment for proliferation, migration, and invasion of cancer cells, preventing optimal treatment and cure. Considering that, cell migration and invasion assays (see material and methods) were performed on cells derived from both models. In Figure 12 (left panel, monotherapy), our results demonstrate that the ability of spheroid-derived ovarian cancer cells to invade was only significantly impaired by EGCG (13%, p < 0.05) and Pano treatments (20%, p < 0.01). Upon addition of standard chemotherapy to these epigenetic treatments, invasion ability was significantly inhibited across all combo treatments when compared to the standard chemotherapytreated group ( Figure 12, left panel, combo therapy). In the organoid derived model, which represents cancer in the presence of a tumorigenic microenvironment, EGCG (p < 0.01), I3C (p < 0.01), and Pano (p < 0.001) significantly downregulated the post-treatment invasion ability of cancer cells by approximately 57%, 21%, and 73%, respectively ( Figure 12, right panel, monotherapy). When epigenetic treatments were combined with chemotherapy, the invasion ability of pre-treated 3D organoid-derived cells was significantly downregulated by EGCG/CT and Pano/CT by 70% (p < 0.001) and 41% by I3C/CT relative to the control group (p < 0.01) ( Figure 12, right panel, combo therapy).
The above results are further supported by representative images from the invasion assay ( Figure 13).
Next, we tested the effect of our epigenetic treatments on migration capacity in organoid-derived cells. Given the significance of the organoid model to translational studies, we concentrated our migration assay on this model only. As shown in Figure 14 (left panel), EGCG, I3C, and Pano progressively suppressed the ability of cancer cells to migrate relative to the control group by approximately 48.7%, 65%, and 82.6%, respectively (p < 0.001). When CT monotherapy alone was used, migration capacity was suppressed by only 27% relative to the control group (p < 0.01), as observed both in the left and right panels. In a similar manner, when epigenetic treatments were combined with chemotherapy ( Figure 14, right panel), significant suppression of migration capacity was noted following combo treatments with EGCG/CT, I3C/CT, and Pano/CT relative to the CT monotherapy treated group of approximately 45% (p < 0.01), 62% (p < 0.01), and 81% (p < 0.001), respectively. The ability of ovarian cancer cells to metastasize is an ongoing and concerning problem. The tumorigenic microenvironment provides a supportive environment for proliferation, migration, and invasion of cancer cells, preventing optimal treatment and cure. Considering that, cell migration and invasion assays (see material and methods) were performed on cells derived from both models. In Figure 12 ( Figure 11. Microscopic representation of the regrowth of a spheroid (top rows) derived cells or organoid (bottom rows) derived cells of epigenetic versus combo treatments with chemotherapy. 3D-derived cells were plated following 17 days of our various treatments and allowed to regrow in normal conditions for an additional 7 days. Cells in the control group showed high regrowth abilities. In cells pre-treated with epigenetic monotherapies, EGCG and I3C moderately suppressed the recovery of cells in comparison to the control group, while Pano more robustly suppressed growth in both models. The combination of epigenetic treatments with chemotherapy further suppresses regrowth ability when compared to monotherapy treatments. Two independent experiments were performed, including triplicates for each treatment group.

Figure 12.
Ability of 3D-derived spheroid (left panel) and organoid (right panel) cells to invade following epigenetic monotherapy or combo treatments. An invasion assay was used to evaluate the invasion capacity of spheroids and organoids following pre-treatment with various therapies. The ability of 3D-derived organoid cells pre-treated with monotherapy epigenetic treatments was significantly affected by EGCG (57%, p < 0.01), I3C (21%, p < 0.01), and Pano (73%, p < 0.001) relative to control. When combo treatments of EGCG/CT and Pano/CT were used, the post-treatment invasion ability of organoid-derived cells was downregulated relative to standard chemotherapy alone by approximately 70% (p < 0.001), with 41% inhibition induced by treatment with I3C/CT (p < 0.01). Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05, (**) represents p < 0.01, and (***) represents p < 0.001.
The above results are further supported by representative images from the invasion assay ( Figure 13).  Figure 12. Ability of 3D-derived spheroid (left panel) and organoid (right panel) cells to invade following epigenetic monotherapy or combo treatments. An invasion assay was used to evaluate the invasion capacity of spheroids and organoids following pre-treatment with various therapies. The ability of 3D-derived organoid cells pre-treated with monotherapy epigenetic treatments was significantly affected by EGCG (57%, p < 0.01), I3C (21%, p < 0.01), and Pano (73%, p < 0.001) relative to control. When combo treatments of EGCG/CT and Pano/CT were used, the post-treatment invasion ability of organoid-derived cells was downregulated relative to standard chemotherapy alone by approximately 70% (p < 0.001), with 41% inhibition induced by treatment with I3C/CT (p < 0.01). Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05, (**) represents p < 0.01, and (***) represents p < 0.001. following epigenetic monotherapy or combo treatments. An invasion assay was used to evaluate the invasion capacity of spheroids and organoids following pre-treatment with various therapies. The ability of 3D-derived organoid cells pre-treated with monotherapy epigenetic treatments was significantly affected by EGCG (57%, p < 0.01), I3C (21%, p < 0.01), and Pano (73%, p < 0.001) relative to control. When combo treatments of EGCG/CT and Pano/CT were used, the post-treatment invasion ability of organoid-derived cells was downregulated relative to standard chemotherapy alone by approximately 70% (p < 0.001), with 41% inhibition induced by treatment with I3C/CT (p < 0.01). Two independent experiments were performed, including triplicates for each treatment group. The asterisk (*) represents p < 0.05, (**) represents p < 0.01, and (***) represents p < 0.001.
The above results are further supported by representative images from the invasion assay ( Figure 13).  Figure 13. Microscopic representation of invasion assay from post treated 3D derived organoid cells following epigenetic monotherapy (top row) or combination (bottom row) treatments. In organoids, the ability to invade is significantly affected by pre-treatment with EGCG and Pano, both in monotherapy and groups. The dark color represents cell invasion; see Materials and Methods. Two independent experiments were performed, including triplicates for each treatment group.
grate relative to the control group by approximately 48.7%, 65%, and 82.6%, respectively (p < 0.001). When CT monotherapy alone was used, migration capacity was suppressed by only 27% relative to the control group (p < 0.01), as observed both in the left and right panels. In a similar manner, when epigenetic treatments were combined with chemotherapy ( Figure 14, right panel), significant suppression of migration capacity was noted following combo treatments with EGCG/CT, I3C/CT, and Pano/CT relative to the CT monotherapy treated group of approximately 45% (p < 0.01), 62% (p < 0.01), and 81% (p < 0.001), respectively. Figure 14. Migration ability of 3D organoid-derived cells following epigenetic monotherapy (left panel) or combination treatments with chemotherapy (right panel). A migration assay was used to evaluate the capacity of cancer cells to migrate following pretreatment with our various therapies. As observed, epigenetic monotherapies halt the migration of cancer cells. Two independent experiments were performed, including triplicates for each treatment group. The asterisk (**) represents p < 0.01 and (***) represents p < 0.001.

Post-Treatment Ability of 3D-Derived Cells to Form Colony Forming Units (CFU)
So far, our experiments have assessed the capacity of 3D ovarian cancer cells to regrow, migrate, and invade following treatment with epigenetic monotherapies and chemotherapy alone, as well as following combination treatments. Next, we tested the ability of pre-treated organoid-derived cells to form CFU. As observed below ( Figure 15, top row), pre-treatment with epigenetic monotherapies overall suppresses CFU formation capacity when compared to the control group, with robust effects observed in the I3C and Pano-treated groups. Comparatively, when epigenetic treatments are combined with chemotherapy, a robust effect is noted across combination treatments ( Figure 15, bottom row). Efficacious suppression of CFU formation is equally observed in the EGCG/CT, I3C/CT, and Pano/CT combo groups in relation to the control and CT groups. Figure 14. Migration ability of 3D organoid-derived cells following epigenetic monotherapy (left panel) or combination treatments with chemotherapy (right panel). A migration assay was used to evaluate the capacity of cancer cells to migrate following pretreatment with our various therapies. As observed, epigenetic monotherapies halt the migration of cancer cells. Two independent experiments were performed, including triplicates for each treatment group. The asterisk (**) represents p < 0.01 and (***) represents p < 0.001.

Post-Treatment Ability of 3D-Derived Cells to Form Colony Forming Units (CFU)
So far, our experiments have assessed the capacity of 3D ovarian cancer cells to regrow, migrate, and invade following treatment with epigenetic monotherapies and chemotherapy alone, as well as following combination treatments. Next, we tested the ability of pretreated organoid-derived cells to form CFU. As observed below ( Figure 15, top row), pretreatment with epigenetic monotherapies overall suppresses CFU formation capacity when compared to the control group, with robust effects observed in the I3C and Pano-treated groups. Comparatively, when epigenetic treatments are combined with chemotherapy, a robust effect is noted across combination treatments ( Figure 15, bottom row). Efficacious suppression of CFU formation is equally observed in the EGCG/CT, I3C/CT, and Pano/CT combo groups in relation to the control and CT groups.  Figure 15. Ability of 3D-derived organoid cells to form CFU following epigenetic monotherapies (top row) and combo treatments with CT (bottom row). CFU formation assay was performed as described in Materials and Methods. Overall, suppression of the post-treatment capacity of CFU to establish themselves is noted after treatment with epigenetic monotherapies in relation to the control group, as well as in combination treatments in relation to CT alone. Two independent experiments were performed.

Can Epigenetic Treatments Alter the Expression of Protumorigenic Markers in Ovarian
Cancer Human Specimens?

Effects on the Secretion of Protumorigenic Factors by ELISA
The secretome has been established as the major regulator of tumors and their microenvironments [54,55]. The secretion of cytokines, growth factors, and components of the extracellular matrix promotes proliferation, metastasis, and invasion and is also involved in the process of chemoresistance to standard chemotherapy [56,57]. Therefore, we aimed to further investigate how our natural and synthetic epigenetic therapies regulate the se- Figure 15. Ability of 3D-derived organoid cells to form CFU following epigenetic monotherapies (top row) and combo treatments with CT (bottom row). CFU formation assay was performed as described in Materials and Methods. Overall, suppression of the post-treatment capacity of CFU to establish themselves is noted after treatment with epigenetic monotherapies in relation to the control group, as well as in combination treatments in relation to CT alone. Two independent experiments were performed.

Can Epigenetic Treatments Alter the Expression of Protumorigenic Markers in Ovarian Cancer
Human Specimens?

Effects on the Secretion of Protumorigenic Factors by ELISA
The secretome has been established as the major regulator of tumors and their microenvironments [54,55]. The secretion of cytokines, growth factors, and components of the extracellular matrix promotes proliferation, metastasis, and invasion and is also involved in the process of chemoresistance to standard chemotherapy [56,57]. Therefore, we aimed to further investigate how our natural and synthetic epigenetic therapies regulate the secretion of growth factors and cytokines involved in tumorigenesis progression in human ovarian cancer specimens in the presence of a tumorigenic omental micro-environment. As described in material and methods, cancer tissue and omentum obtained from a patient with ovarian cancer were co-cultured in the presence of our epigenetic therapies for 4 days. Conditioned medium from co-cultured specimens was collected and tested for secretion of the following human cytokines: VEGF, IL-6, IL-10, and TNF-α. As demonstrated in Figure 16, secretion of the angiogenic factor VEGF was significantly downregulated by EGCG and I3C (17% and 26%, respectively, p < 0.05), with no effects resulting from treatment with Pano or CT. The secretion of IL-10, associated with the induction of proliferation and metastasis, was efficiently suppressed by EGCG (90.1%, p < 0.05), I3C (96.7%, p < 0.05), Pano (86.3%, p < 0.05), and CT (89.1%, p < 0.05). Secretion of TNF-α, a proinflammatory marker, was significantly downregulated by all of our treatments, with an overall less robust effect observed following treatment with I3C (26.4%, p < 0.05) when compared to the remaining treatments (EGCG 79%, Pano 88.6%, CT 91.3% downregulation, p < 0.05; calculations are relative to control). The secretion of IL-6, involved in the induction of proliferation in ovarian cancer, was significantly downregulated by EGCG and I3C (80% and 32.5%, respectively, p < 0.05). Given that our epigenetic treatments seem to regulate the secretory machinery, we expanded our investigation to a larger scale, evaluating their effects on additional cytokines, growth factors, and extracellular matrix components through protein arrays. ELISA assays were performed as described in material and methods. Overall, secretion levels of growth factors and cytokines were determined by a standard curve with appropriate recombinant growth factors. Significant differences are indicated with an asterix (*) in relation to the control or drug-free treatment with p < 0.05. Two independent experiments were performed, including triplicates for each treatment group.

Effects on the Secretion of Cytokines, Growth Factors, MMPs, and TIMPs by Protein Array
Subsequently, the conditioned medium of co-culture specimens was collected and tested for secretion of human cytokines and growth factors relevant to cancer propagation and chemoresistance through protein arrays ( Figure 17).

IL-10
TNF-⍺ IL-6 Figure 16. Effects of epigenetic treatments on cytokine secretion in ovarian cancer tissue by ELISA. ELISA assays were performed as described in material and methods. Overall, secretion levels of growth factors and cytokines were determined by a standard curve with appropriate recombinant growth factors. Significant differences are indicated with an asterix (*) in relation to the control or drug-free treatment with p < 0.05. Two independent experiments were performed, including triplicates for each treatment group.

Effects on the Secretion of Cytokines, Growth Factors, MMPs, and TIMPs by Protein Array
Subsequently, the conditioned medium of co-culture specimens was collected and tested for secretion of human cytokines and growth factors relevant to cancer propagation and chemoresistance through protein arrays ( Figure 17). Figure 16. Effects of epigenetic treatments on cytokine secretion in ovarian cancer tissue by ELISA. ELISA assays were performed as described in material and methods. Overall, secretion levels of growth factors and cytokines were determined by a standard curve with appropriate recombinant growth factors. Significant differences are indicated with an asterix (*) in relation to the control or drug-free treatment with p < 0.05. Two independent experiments were performed, including triplicates for each treatment group.

Effects on the Secretion of Cytokines, Growth Factors, MMPs, and TIMPs by Protein Array
Subsequently, the conditioned medium of co-culture specimens was collected and tested for secretion of human cytokines and growth factors relevant to cancer propagation and chemoresistance through protein arrays ( Figure 17). Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively.
Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be i in cancer progression and metastasis, such as matrix metalloproteases (MMPs) a inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein indicated in Figure 18.

IL-8
Stimulates ovarian cancer growth and proliferation Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be i in cancer progression and metastasis, such as matrix metalloproteases (MMPs) a inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein indicated in Figure 18.

IL-11
Promotes chemoresistance, proliferation and survival of metastatic ovarian cancer cells 36 ± 3.6 18 ± 2.7 50% collected from ovarian cancer human specimens pretreated with EGCG, I3C, and Pano was tested by protein array for cytokines and growth factors expression. See Materials and Methods.
Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
collected from ovarian cancer human specimens pretreated with EGCG, I3C, and Pano was tested by protein array for cytokines and growth factors expression. See Materials and Methods.
Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18.
collected from ovarian cancer human specimens pretreated with EGCG, I3C, and Pano w by protein array for cytokines and growth factors expression. See Materials and Methods.
Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be i in cancer progression and metastasis, such as matrix metalloproteases (MMPs) a inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein indicated in Figure 18. array data were analyzed using optical density comparisons between control ious treatments, with a summary of pertinent optical density changes being ed in Table 1. Our natural epigenetic treatments downregulated the secretion sociated with induction of proliferation, migration, and immunosuppression cancer [58,59], by more than 70%, an effect comparable to that obtained by ith the synthetic compound Pano. In contrast, IL-8 secretion, also associated n cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, cor-chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. 34 Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Word 16.73 was used to produce this table.

IL-6R
Next, the secretion of components of the extracellular matrix known to be i in cancer progression and metastasis, such as matrix metalloproteases (MMPs) a inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein indicated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be i in cancer progression and metastasis, such as matrix metalloproteases (MMPs) a inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein indicated in Figure 18.

RANTES (CCL5)
Alters Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as 38 Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as 31 Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. array data were analyzed using optical density comparisons between control ious treatments, with a summary of pertinent optical density changes being ed in Table 1. Our natural epigenetic treatments downregulated the secretion sociated with induction of proliferation, migration, and immunosuppression cancer [58,59], by more than 70%, an effect comparable to that obtained by ith the synthetic compound Pano. In contrast, IL-8 secretion, also associated n cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, cor-chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, he secretion of components of the extracellular matrix known to be involved ogression and metastasis, such as matrix metalloproteases (MMPs) and their issue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density chang demonstrated in Table 1. Our natural epigenetic treatments downregulated the s of IL-10, associated with induction of proliferation, migration, and immunosup in ovarian cancer [58,59], by more than 70%, an effect comparable to that obta treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also as with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL related with chemoresistance and metastasis of disease [62], was inhibited by 50 and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regar fects on immunoresponse markers, our treatments suppressed levels of RANTES to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fo treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J Mean and SD are represented in OD units, while effect is calculated in percent decrease (do arrow) or increase (upward arrow) in relation to the control or drug free treatment group.
Optical Density (OD) signal was detected and compared between samples using Image J software. Mean and SD are represented in OD units, while effect is calculated in percent decrease (downward arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Microsoft Word 16.73 was used to produce this table.
Protein array data of MMPs and TIMPs were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 2. The secretion of MMP-8, which has been correlated with ovarian cancer's overall prognosis [56,64,65], was downregulated by 30%, 16%, and 7% by EGCG, I3C, and Pano, respectively. Secretion of MMP-13, known to stimulate ovarian cancer metastasis [65], was suppressed by 31%, 36%, and 34% following treatments with EGCG, I3C, and Pano. As expected, the secretion of TIMPs was overall upregulated by our various treatments ( Table 2).
1 Figure 18. Protein array map of MMPs and TIMPs (top row) and experimental images (bottom row) of human specimen pretreated with epigenetic therapies. Conditioned medium collected from ovarian cancer human specimens pretreated with EGCG, I3C, and Pano was tested by protein array for cytokines and growth factors expression. See Materials and Methods. Protein array data were analyzed using optical density comparisons between control our various treatments, with a summary of pertinent optical density changes being onstrated in Table 1. Our natural epigenetic treatments downregulated the secretion -10, associated with induction of proliferation, migration, and immunosuppression arian cancer [58,59], by more than 70%, an effect comparable to that obtained by ment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, cor-ed with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding ef-on immunoresponse markers, our treatments suppressed levels of RANTES, known ter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following ments with EGCG, I3C, and Pano, respectively.

1.
Analysis of cytokines and growth factors' secretion following epigenetic treatments.  al Density (OD) signal was detected and compared between samples using Image J software. and SD are represented in OD units, while effect is calculated in percent decrease (downward ) or increase (upward arrow) in relation to the control or drug free treatment group. Microsoft 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be involved ncer progression and metastasis, such as matrix metalloproteases (MMPs) and their itors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as ated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J software. Mean and SD are represented in OD units, while effect is calculated in percent decrease (downward arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Microsoft Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density changes demonstrated in Table 1. Our natural epigenetic treatments downregulated the se of IL-10, associated with induction of proliferation, migration, and immunosupp in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtai treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also asso with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-1 related with chemoresistance and metastasis of disease [62], was inhibited by 50% and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regard fects on immunoresponse markers, our treatments suppressed levels of RANTES, to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fol treatments with EGCG, I3C, and Pano, respectively. Optical Density (OD) signal was detected and compared between samples using Image J so Mean and SD are represented in OD units, while effect is calculated in percent decrease (dow arrow) or increase (upward arrow) in relation to the control or drug free treatment group. M Word 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be in in cancer progression and metastasis, such as matrix metalloproteases (MMPs) an inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein a indicated in Figure 18.

MMP-3
Remodeling of ECM 10,777 ± 752 10,209 ± 588 5% EVIEW 20 of 27 collected from ovarian cancer human specimens pretreated with EGCG, I3C, and Pano was tested by protein array for cytokines and growth factors expression. See Materials and Methods.
Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density change demonstrated in Table 1. Our natural epigenetic treatments downregulated the se of IL-10, associated with induction of proliferation, migration, and immunosupp in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtai treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also ass with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. ILrelated with chemoresistance and metastasis of disease [62], was inhibited by 50% and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regard fects on immunoresponse markers, our treatments suppressed levels of RANTES, to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fol treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be in in cancer progression and metastasis, such as matrix metalloproteases (MMPs) an inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein a indicated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved in cancer progression and metastasis, such as matrix metalloproteases (MMPs) and their inhibitors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as indicated in Figure 18. 25 Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Protein array data were analyzed using optical density comparisons between control and our various treatments, with a summary of pertinent optical density changes being demonstrated in Table 1. Our natural epigenetic treatments downregulated the secretion of IL-10, associated with induction of proliferation, migration, and immunosuppression in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtained by treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, correlated with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding effects on immunoresponse markers, our treatments suppressed levels of RANTES, known to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following treatments with EGCG, I3C, and Pano, respectively. Protein array data were analyzed using optical density comparisons between and our various treatments, with a summary of pertinent optical density change demonstrated in Table 1. Our natural epigenetic treatments downregulated the se of IL-10, associated with induction of proliferation, migration, and immunosupp in ovarian cancer [58,59], by more than 70%, an effect comparable to that obtai treatment with the synthetic compound Pano. In contrast, IL-8 secretion, also ass with ovarian cancer proliferation [60,61], was downregulated by only 10-20%. ILrelated with chemoresistance and metastasis of disease [62], was inhibited by 50% and 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regard fects on immunoresponse markers, our treatments suppressed levels of RANTES, to alter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% fol treatments with EGCG, I3C, and Pano, respectively. Protein array data were analyzed using optical density comparisons between control our various treatments, with a summary of pertinent optical density changes being onstrated in Table 1. Our natural epigenetic treatments downregulated the secretion -10, associated with induction of proliferation, migration, and immunosuppression arian cancer [58,59], by more than 70%, an effect comparable to that obtained by ment with the synthetic compound Pano. In contrast, IL-8 secretion, also associated ovarian cancer proliferation [60,61], was downregulated by only 10-20%. IL-11, cor-ed with chemoresistance and metastasis of disease [62], was inhibited by 50%, 92%, 97.8% following treatments with EGCG, I3C, and Pano, respectively. Regarding ef-on immunoresponse markers, our treatments suppressed levels of RANTES, known ter T-cells and natural killer cells' function [63], by 96%, 71%, and 76.7% following ments with EGCG, I3C, and Pano, respectively. Next, the secretion of components of the extracellular matrix known to be involved ncer progression and metastasis, such as matrix metalloproteases (MMPs) and their itors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as ated in Figure 18. 32 al Density (OD) signal was detected and compared between samples using Image J software. and SD are represented in OD units, while effect is calculated in percent decrease (downward ) or increase (upward arrow) in relation to the control or drug free treatment group. Microsoft 16.73 was used to produce this table.
Next, the secretion of components of the extracellular matrix known to be involved ncer progression and metastasis, such as matrix metalloproteases (MMPs) and their itors, tissue inhibitors of metalloproteases (TIMPs), was analyzed by protein array as ated in Figure 18. 28 Optical Density (OD) signal was detected and compared between samples using Image J software. Mean and SD are represented in OD units, while effect is calculated in percent decrease (downward arrow) or increase (upward arrow) in relation to the control or drug free treatment group. Microsoft Word 16.73 was used to produce this table.

Discussion
Despite advancements in the treatment of ovarian cancer, high mortality rates continue to occur in patients diagnosed with the disease [1][2][3][4]54]. Given challenges with the optimal management of recurrent and metastatic disease, robust efforts from the scientific community have taken place in the past decade, targeting new avenues and treatment options for ovarian cancer patients [7,9,12]. It has been widely established that not only genetic changes, but also epigenetic aberrations play a critical role in cancer propagation, metastasis, and chemoresistance [10,11,16]. Therefore, epigenetic therapies offer an innovative therapeutic approach to such treatment challenges, offering new strategies for augmenting the efficiency of standard chemotherapy, counteracting chemoresistance, and leading to improved clinical outcomes [27][28][29]. Currently, a few synthetic epigenetic compounds have been investigated through clinical trials. However, given toxicity concerns, they have yet to be optimized in order to become widely recommended treatments [30][31][32]. In contrast, natural epigenetic compounds such as EGCG and I3C offer the promise of lesser toxicity and easier patient availability, rising as an option that could be used prior to, in combination with, or following standard chemotherapy treatment [35][36][37][38][39][40]. Regarding their action, natural epigenetic compounds have been previously explored in a multitude of 2D tissue culture cancer models, revealing great potential for suppressing the propagation of cancer [41][42][43][44]. Our study is unique in that it focuses on further exploring the actions of EGCG and I3C versus the synthetic compound Panobinostat (Pano), with or without standard chemotherapy, on 3D ovarian cancer models or human specimens. Our results suggest that natural treatments are efficacious in suppressing ovarian cancer tissue growth alone as well as in the presence of a tumorigenic microenvironment. Taking a step forward, we additionally demonstrate the role of our epigenetic treatments in regulating the secretory machinery that promotes tumor propagation, metastasis, and chemoresistance in 3D models and human tissue specimens.
Regarding cancer progression, our results support that natural epigenetic compounds efficiently inhibit 3D ovarian cancer tumor growth in a dose-dependent manner (Figures 1-3). Interestingly, the efficiency of action of EGCG and I3C was noted not only in spheroids, which represent an ovarian cancer cell population (Figures 1-3), but also in organoids (Figures 7 and 8), in which ovarian cancer cells and tumorigenic omental adipose-derived stem cells are combined, representing the complex interactions between the tumor and its heterogeneous tumorigenic microenvironment (Figures 7 and 8). As the tumorigenic microenvironment supports cancer growth, metastasis of disease, and chemoresistance [18,66,67], our results are promising in evidencing that natural epigenetic therapies can reprogram the tumorigenic microenvironment regarding cancer progression, with comparable effects to those elicited by the synthetic compound Pano (Figures 3 and 8). The advantages of EGCG and I3C when compared to Pano, however, include ease of availability independent of provider and the possibility of incorporation into a patient's diet as a treatment adjunct or even as a prophylactic regimen. In-depth in vivo studies or Phase 1 trials, however, are still needed in order to evaluate the toxicity and safety levels in patients.
Considering that platinum-based chemotherapy in combination with taxol is the firstline treatment recommendation for ovarian cancer patients, it was critical to investigate the efficiency of natural epigenetic therapies in combination with standard chemotherapy. While prior studies have explored the effects of EGCG and I3C in suppressing additional types of cancers in a 2D tissue culture model [39,43,[45][46][47][48]68], there is paucity of data regarding the efficacy of these compounds when combined with chemotherapy. As demonstrated by our results (Figures 6 and 9), combination treatments had higher efficacy in suppressing tumor growth when combined with standard chemotherapy in spheroids ( Figure 6) and organoids ( Figure 9). Contrarily, while Pano is efficacious in suppressing tumor growth as a monotherapy, no significant differences in tumor growth were noted in our study when Pano was combined with standard chemotherapy (Figure 9). Our results therefore suggest a role for natural epigenetic therapies in augmenting or serving as an adjunct to standard of care treatment in ovarian cancer patients.
Given that there is an ongoing challenge in the treatment of patients with recurrent or metastatic ovarian cancer, with rates of recurrence as high as 70% in the first three years following diagnosis [2,5], it was relevant to explore post-treatment outcomes of epigenetic treatments for 3D ovarian cancer. In a clinical scenario, microdiseases can remain in the human body following debulking surgery or chemotherapy treatment, later leading to recur-rence or metastasis [8]. To represent this phenomenon, we additionally showed that natural epigenetic compounds have a lasting effect in suppressing tumor regrowth, migration, invasion, and the capacity to form colonies in 3D-derived ovarian cancer cells previously treated with such compounds. Our study shows that 3D-derived cells from spheroids and organoids previously treated with EGCG and I3C have limited capacity to regrow when compared to the chemotherapy-treated groups ( Figure 10). When chemotherapy is combined with EGCG and I3C, the post-treatment regrowth ability of cells is further suppressed (Figure 11). Similar effects are observed in cells treated with the synthetic epigenetic compound Pano (Figures 10 and 11). Additionally, natural epigenetic compounds suppress the post-treatment ability of 3D organoid-derived cells to invade (Figures 12 and 13), migrate (Figure 14), and form colonies (Figure 15). These results infer that our epigenetic treatments further potentiate post-treatment effects in ovarian cancer, perhaps by altering aberrant gene expression noted in cancer and its tumorigenic environment.
The secretion of growth factors, cytokines, and components of the extracellular matrix (known as the secretome) has been shown to play a crucial role in regulating progression, resistance, and metastasis in various cancers [54][55][56][57]. We therefore tested the role of our epigenetic treatments in influencing the secretome of ovarian cancer tissue in the presence of a tumorigenic microenvironment, given that this model most closely represents the clinical progression of disease (Figures 16-18, Tables 1 and 2). Our results indicate that EGCG, I3C, and Pano significantly downregulate levels of the protumorigenic cytokine IL-11, which is a major factor implicated in the chemoresistance of ovarian cancer cells through the activation of the JAK2-STAT5 pathway [62]. Considering that targeting this pathway can reverse chemoresistance properties and re-sensitize cells to platinum treatment, our natural epigenetic compounds may be promising options for improving the clinical outcomes and prognosis of patients with platinum-resistant ovarian cancer by downregulating levels of IL-11 [62]. Similar to the effects observed following treatment with Pano, EGCG and I3C downregulate the secretion of IL-10, an immunosuppressive factor in ovarian cancer ascites correlated with proliferation and metastasis, as well as maintenance of the tumorigenic microenvironment [58,59]. Therefore, inhibition of IL-10 by our epigenetic treatments can target the growth and metastasis of ovarian cancer cells. IL-6, an inflammatory chemokine associated with tumorigenesis and chemoresistance in disease, was also significantly downregulated by EGCG and I3C [61]. Moreover, critical factors involved in the metastasis of disease and overall prognosis (MMPs) and their inhibitors (TIMPS) were also robustly affected by our treatments [56,64,65]. MMP 8, 10, and 14 were downregulated by EGCG, I3C, and Pano, while levels of TIMPs 1 and 3, as expected, were upregulated (Tables 1 and 2). These results support the idea that natural epigenetic compounds can modulate the secretome of ovarian cancer, serving as new avenues to overcome tumor propagation and metastasis and perhaps aiding in reversing chemoresistance to platinum agents.
Although the focus of this study was not to fully explore the molecular mechanisms of EGCG and I3C, studies available in the literature have demonstrated that suppression of tumor growth can be explained through the induction of apoptosis by upregulation of cell cycle regulatory markers. For example, inhibition of tumorigenic progression by EGCG has been correlated with induction of P53 [45], P16, and P21 [43]. These markers have been widely established as checkpoint inhibitors known to halt cell cycle progression and subsequent tumor growth through apoptosis and cell death. In a similar manner, I3C has been shown to inhibit cancer progression in lung tumors by downregulating levels of cyclin E and its partner CDK2, enhancing apoptotic cell death [47]. Given that EGCG and I3C induced apoptotic cell death in our 3D models (Figure 4), downregulating tumor growth, we hypothesize that the same molecular mechanisms also took place. Further studies uncovering more in-depth molecular mechanisms of EGCG and I3C in ovarian cancer are, however, still needed.
It is also important to highlight that while prior concerns existed regarding hepatocellular toxicity inflicted by EGCG in humans, the pre-FRIEND trial has evaluated the safety of EGCG in premenopausal women, with no drug-induced liver injury demonstrated in any patients [69]. Regarding I3C, most human studies have failed to report overt toxicity [70,71], suggesting that natural epigenetic compounds are plausible treatment options in humans.

Conclusions
To our knowledge, this is the first study to show the high efficiency of natural epigenetic treatments in suppressing 3D ovarian cancer growth and post-treatment recovery, invasion, and migration of disease in the presence of a tumorigenic microenvironment. Additionally, natural epigenetic compounds can regulate the protumorigenic secretome, which is involved in metastasis, chemoresistance, and relapses of disease. While further studies are needed, the effects obtained from natural epigenetic therapies and their limited toxicity when compared to synthetic epigenetic compounds further support their future potential as prophylactic, adjunct, or therapeutic options for improving the clinical outcomes of ovarian cancer patients.