Oligo-Fucoidan Prevents M2 Macrophage Differentiation and HCT116 Tumor Progression

Reactive oxygen species (ROS) produced during intracellular metabolism or triggered by extrinsic factors can promote neoplastic transformation and malignant microenvironment that mediate tumor development. Oligo-Fucoidan is a sulfated polysaccharide isolated from the brown seaweed. Using human THP-1 monocytes and murine Raw264.7 macrophages as well as human HCT116 colorectal cancer cells, primary C6P2-L1 colorectal cancer cells and human MDA-MB231 breast cancer cells, we investigated the effect of Oligo-Fucoidan on inhibiting M2 macrophage differentiation and its therapeutic potential as a supplement in chemotherapy and tumor prevention. We now demonstrate that Oligo-Fucoidan is an antioxidant that suppresses intracellular ROS and mitochondrial superoxide levels in monocytes/macrophages and in aggressive cancer cells. Comparable to ROS inhibitors (DPI and NAC), Oligo-Fucoidan directly induced monocyte polarization toward M1-like macrophages and repolarized M2 macrophages into M1 phenotypes. DPI and Oligo-Fucoidan also cooperatively prevented M2 macrophage invasiveness. Indirectly, M1 polarity was advanced particularly when DPI suppressed ROS generation and supplemented with Oligo-Fucoidan in the cancer cells. Moreover, cisplatin chemoagent polarized monocytes and M0 macrophages toward M2-like phenotypes and Oligo-Fucoidan supplementation reduced these side effects. Furthermore, Oligo-Fucoidan promoted cytotoxicity of cisplatin and antagonized cisplatin effect on cancer cells to prevent M2 macrophage differentiation. More importantly, Oligo-Fucoidan inhibited tumor progression and M2 macrophage infiltration in tumor microenvironment, thus increasing of anti-tumor immunity.

By evaluating M2 macrophages' response, we also found that the expression levels of M1 marker CD68 and CD86 were much induced upon DPI and Oligo-Fucoidan treatment of the differentiated THP-1 M2 macrophages than NAC ( Figure 1H) and the percentages of CD163(+)/CD206(+) M2 macrophages were significantly reduced ( Figure 1I), as compared with untreated group (MOCK) and isotope IgG control (Supplementary Figure S2A); revealing that antioxidants repolarized M2 macrophages.
These results indicate that antioxidants and Oligo-Fucoidan together advance monocyte polarization toward M1 polarity and further repolarize M2 macrophages into M1 plasticity.

ROS Produced in Cancer Cells Influence Macrophage Differentiation
To inspect whether ROS produced in cancer cells can impact macrophage plasticity, two isogenic HCT116 colon cancer cell lines without (p53 −/− ) or with (p53 +/+ ) wild-type p53 were treated with DPI. When DPI suppressed intracellular ROS ( Figure 2A) and mitochondrial superoxide levels ( Figure 2B), the phosphorylation of p47 phox (Ser345) was significantly decreased by DPI independent of the p53 status ( Figure 2C) and DPI dramatically induced the active phosphorylation of p53 (Ser15) and the accumulation of p53 in the p53 +/+ cancer cells.
Next, HCT116 cells were treated with DPI (1 M) and/or Oligo-Fucoidan (400 g/mL) and the preconditioned cancer cells were placed in the upper compartment of a Boyden chamber transwell and cocultured with THP-1 monocytes placed in the bottom chamber for 48 h. The results showed that the DPI-pretreated cancer cells stimulated monocyte polarization toward the phenotypes of F4/80 (high) M0 ( Figure 2D) and CD80 (high) M1 ( Figure 2E) macrophages. Interestingly, the Oligo-Fucoidan-treated cancer cells also upregulated F4/80 and CD80 mRNA levels ( Figure 2D,E) and these effects were additively enhanced by DPI cotreatment. Thus, ROS inhibition in the cancer cells can indirectly promote M0 and M1 differentiation.
Although M2 macrophages were highly invasive ( Figure 2F), combined DPI and Oligo-Fucoidan treatment significantly suppressed their invasive abilities than the individual treatments. Reciprocally, the invasiveness of HCT116 cancer cells was markedly inhibited when they encountered the M2 macrophages those were pretreated by DPI and/or Oligo-Fucoidan ( Figure 2G). Accordingly, DPI and Oligo-Fucoidan treatment can suppress M2 macrophage invasion and renovate M0 and M1 plasticity which prevent cancer cell invasion. Although M2 macrophages were highly invasive ( Figure 2F), combined DPI and Oligo-Fucoidan treatment significantly suppressed their invasive abilities than the individual treatments. Reciprocally, the invasiveness of HCT116 cancer cells was markedly inhibited when they encountered the M2 macrophages those were pretreated by DPI and/or Oligo-Fucoidan ( Figure 2G). Accordingly, DPI and Oligo-Fucoidan treatment can suppress M2 macrophage invasion and renovate M0 and M1 plasticity which prevent cancer cell invasion. Mitochondrial superoxide levels in HCT116 cells were measured after treatment with DPI (1 μM) for 1 h (B) NADPH subunit p-p47 phox (Ser345), p47 phox , p-p53 (Ser15) and total p53 amounts were compared after treatment of HCT116 with DPI (0-3 M) for 48 h (C) Protein level was normalized to the β-actin protein level and then compared with the level of MOCK control. HCT116 cells were pretreated with DPI (1 μM) and/or Oligo-Fucoidan (400 g/mL) for 48 h, followed by incubation with THP-1 monocytes for 48 h in a Boyden chamber transwell. The surface markers of M0 (F4/80) (D) and M1 (CD80) (E) macrophages were assessed by quantitative RT-PCR. The M2 macrophage invasiveness was examined upon DPI and/or Oligo-Fucoidan treatment (F) The invasion ability of HCT116 cells was examined after coculture with the M2 macrophages those were pre-treated with DPI and/or Oligo-Fucoidan in the invasion transwell (G) Crystal violet staining of the invasive M2 macrophages and HCT116 cancer cells (F,G) The results are expressed as the mean ± SD (n = 3). Student's t test determined the statistical significance of pairwise comparisons (* p < 0.05; ** p < 0.01; and *** p < 0.001).

Cisplatin and Oligo-Fucoidan Cooperatively Promote the Polarization of M1 Macrophages
To further analyze the chemotherapeutic effect on macrophage plasticity, THP-1 monocytes were treated with cisplatin (15 μM) and/or Oligo-Fucoidan (400 μg/mL). As detected by WST-1 cell viability assay, THP-1 cell viability was significantly reduced (41%) by cisplatin but less affected by Mitochondrial superoxide levels in HCT116 cells were measured after treatment with DPI (1 µM) for 1 h (B) NADPH subunit p-p47 phox (Ser345), p47 phox , p-p53 (Ser15) and total p53 amounts were compared after treatment of HCT116 with DPI (0-3 M) for 48 h (C) Protein level was normalized to the β-actin protein level and then compared with the level of MOCK control. HCT116 cells were pretreated with DPI (1 µM) and/or Oligo-Fucoidan (400 g/mL) for 48 h, followed by incubation with THP-1 monocytes for 48 h in a Boyden chamber transwell. The surface markers of M0 (F4/80) (D) and M1 (CD80) (E) macrophages were assessed by quantitative RT-PCR. The M2 macrophage invasiveness was examined upon DPI and/or Oligo-Fucoidan treatment (F) The invasion ability of HCT116 cells was examined after coculture with the M2 macrophages those were pre-treated with DPI and/or Oligo-Fucoidan in the invasion transwell (G) Crystal violet staining of the invasive M2 macrophages and HCT116 cancer cells (F,G) The results are expressed as the mean ± SD (n = 3). Student's t test determined the statistical significance of pairwise comparisons (* p < 0.05; ** p < 0.01; and *** p < 0.001).
Manganese superoxide dismutase (MnSOD) [33], an O 2− scavenger in mitochondria, is overexpressed in carcinogenesis. Glutathione peroxidase (GPX) diminishes ROS buildup and reduces oxidative damage [34]. Under the oncogenic stress [35], YY1 induction and EGFR activation are stimulated that lead to MnSOD expression and ROS generation. Similarly, cisplatin and Oligo-Fucoidan collectively amplified the ROS signaling pathway in HCT116 (p53 −/− and p53 +/+ ) cells (Supplementary Figure S7A), which showed increase of YY1, EGFR, EGFR phosphorylation (p-EGFR) (Tyr1068), MnSOD and γ-H2AX but decrease of GPX. To confirm whether combined treatment indeed amplifies the oxidative damaging cascade, ROS inhibitors (NAC, MitoQ and DPI) were treated HCT116 cancer cells followed by administration with cisplatin and Oligo-Fucoidan (Supplementary Figure S7B). The results showed that YY1, p-EGFR (Tyr1068), ROS scavenger (MnSOD and catalase) and the phosphorylation (Ser15) and accumulation of p53 were particularly reduced by MitoQ and DPI, representing that combined treatment indeed promoted the ROS signaling.  Figure S7A), which showed increase of YY1, EGFR, EGFR phosphorylation (p-EGFR) (Tyr1068), MnSOD and γ-H2AX but decrease of GPX. To confirm whether combined treatment indeed amplifies the oxidative damaging cascade, ROS inhibitors (NAC, MitoQ and DPI) were treated HCT116 cancer cells followed by administration with cisplatin and Oligo-Fucoidan (Supplementary Figure S7B). The results showed that YY1, p-EGFR (Tyr1068), ROS scavenger (MnSOD and catalase) and the phosphorylation (Ser15) and accumulation of p53 were particularly reduced by MitoQ and DPI, representing that combined treatment indeed promoted the ROS signaling. Histograms show the relative cell apoptotic and necrotic effects. The results are expressed as the mean ± SD (n = 3). Student's t test determined the statistical significance of the indicated comparisons (* p < 0.05; ** p < 0.01; and *** p < 0.001).
Excessive mitochondrial ROS levels cause adenosine triphosphate (ATP) exhaustion and cell death [36]. An examination of cancer cell death in response to cisplatin and/or Oligo-Fucoidan confirmed that combined treatment further promoted the cleaved PARP (Asp214) and active caspase 3 levels compared with monotherapies ( Figure 4C, lane eight vs. lanes six and seven), particularly in Excessive mitochondrial ROS levels cause adenosine triphosphate (ATP) exhaustion and cell death [36]. An examination of cancer cell death in response to cisplatin and/or Oligo-Fucoidan confirmed that combined treatment further promoted the cleaved PARP (Asp214) and active caspase 3 levels compared with monotherapies ( Figure 4C, lane eight vs. lanes six and seven), particularly in Cancers 2020, 12, 421 9 of 22 the p53 +/+ context. Moreover, FITC-Annexin V and PI staining, followed by flow cytometry analysis revealed that cisplatin induced more necrotic and late apoptotic events in p53 +/+ cells (13% and 66.4%, respectively) ( Figure 4E) than in p53 −/− cells (3.1% and 20%, respectively) ( Figure 4D). Combined treatment advanced the outcomes of p53 −/− cell apoptosis (34.3%) ( Figure 4D) and p53 +/+ cell necrosis (39.1%) ( Figure 4E), thus exacerbating the cancer cell death. Histograms indicate the necrotic, early and late apoptotic results in individual treatments of the p53 −/− cells and the p53 +/+ cells.
In study of the primary C6P2-L1 cell lines derived from the colorectal cancer patients ( Figure 5), we also identified that Oligo-Fucoidan supplementation advanced cisplatin effect on inducing the PARP (Asp214) cleavage and caspase 3 activation ( Figure 5A), thus evolving the apoptotic events more than the individual treatments ( Figure 5B). Clearly, although Oligo-Fucoidan has antioxidative ability, it would not defeat cytotoxic effect of cisplatin in treatment of cancer cells. the p53 +/+ context. Moreover, FITC-Annexin V and PI staining, followed by flow cytometry analysis revealed that cisplatin induced more necrotic and late apoptotic events in p53 +/+ cells (13% and 66.4%, respectively) ( Figure 4E) than in p53 −/− cells (3.1% and 20%, respectively) ( Figure 4D). Combined treatment advanced the outcomes of p53 −/− cell apoptosis (34.3%) ( Figure 4D) and p53 +/+ cell necrosis (39.1%) ( Figure 4E), thus exacerbating the cancer cell death. Histograms indicate the necrotic, early and late apoptotic results in individual treatments of the p53 −/− cells and the p53 +/+ cells.
In study of the primary C6P2-L1 cell lines derived from the colorectal cancer patients ( Figure 5), we also identified that Oligo-Fucoidan supplementation advanced cisplatin effect on inducing the PARP (Asp214) cleavage and caspase 3 activation ( Figure 5A), thus evolving the apoptotic events more than the individual treatments ( Figure 5B). Clearly, although Oligo-Fucoidan has antioxidative ability, it would not defeat cytotoxic effect of cisplatin in treatment of cancer cells. To inspect whether the cisplatin and/or Oligo-Fucoidan-treated cancer cells can influence macrophage plasticity, THP-1 monocytes were incubated with the pre-conditioned HCT116 cells in a Boyden chamber transwell. The results indicated the THP-1 cells increased F4/80 (M0 marker) mRNA levels after incubation with the p53 −/− cells (3.28-fold) or the p53 +/+ cells (2.24-fold) those were experienced combination treatment more than monotherapies ( Figure 6A). In addition, the enriched CD86 mRNA levels were detected when THP-1 monocytes encountered the p53 −/− cells or the p53 +/+ cells pre-treated with cisplatin or Oligo-Fucoidan ( Figure 6B); in particular, M1 polarity was advanced by the p53 −/− cells (2.35-fold) or the p53 +/+ cells (2.11-fold) those were cotreated by both agents. A flow cytometry study also confirmed that CD80(+) M1 populations were expanded when monocytes encountered the p53 −/− cells (Supplementary Figure S8A) or the p53 +/+ cells (Supplementary Figure S8B) experienced combined treatment.
Unexpectedly, the cisplatin-treated p53 −/− cancer cells also activated monocyte polarization into the M2 phenotype with CD206 increase (1.16-fold) while the Oligo-Fucoidan-treated p53 −/− cells decreased the CD206 level (0.63-fold) compared to MOCK control ( Figure 6C). However, Oligo-Fucoidan supplementation antagonized the negative effect of cisplatin on the p53 −/− cells ( Figure 6C), suppressing the CD206 level to 0.65-fold in the polarized macrophages. Similar results were identified in the p53 +/+ cancer cells experienced cisplatin and/or Oligo-Fucoidan treatment ( Figure 6C). Consistently, the polarized THP-1 cells revealed the induced amounts of M1 marker (iNOS and CD80) and the reduced amounts of M2 marker (CD163 and Arginase-1) while reacting with the cancer cells those were pretreated with Oligo-Fucoidan or in combination with cisplatin ( Figure 6D). Also, THP-1 cells encountered breast cancer MDA-MB 231 cells those were pretreated with cisplatin and/or Oligo-Fucoidan expressed the higher mRNA levels of M0 (F4/80) (Supplementary Figure S9A)   To analyze M2 macrophage invasiveness in the treatment, THP-1 monocytes were stimulated with PMA and then IL-4, the M2 macrophage invasion ability was substantially suppressed upon cisplatin and/or Oligo-Fucoidan treatment ( Figure 6E). The M2 macrophage polarity also significantly shifted toward the M0 and M1 phenotypes in combined treatment, as indicated by increasing of F4/80 ( Figure 6F) and CD80 ( Figure 6G) but decreasing of CD163 ( Figure 6H) mRNA levels. To analyze M2 macrophage invasiveness in the treatment, THP-1 monocytes were stimulated with PMA and then IL-4, the M2 macrophage invasion ability was substantially suppressed upon cisplatin and/or Oligo-Fucoidan treatment ( Figure 6E). The M2 macrophage polarity also significantly shifted toward the M0 and M1 phenotypes in combined treatment, as indicated by increasing of F4/80 ( Figure 6F) and CD80 ( Figure 6G) but decreasing of CD163 ( Figure 6H) mRNA levels.
Therefore, Oligo-Fucoidan and/or cisplatin can directly impede M2 macrophage invasion and repolarize the M2 phenotype to the M0 and M1 phenotypes. Further, the cancer cells pre-treated with Oligo-Fucoidan alone or in combination with cisplatin can indirectly promote M0 and M1 plasticity and suppress M2 polarity.

Oligo-Fucoidan Inhibits Tumor Progression and M2 Macrophage Infiltration
To investigate the therapeutic effect of Oligo-Fucoidan and/or cisplatin, HCT116 cells (2 × 10 6 ) were subcutaneously injected into BALB/c nude mice. Two weeks after inoculation, the xenograft mice were treated with cisplatin for 2 weeks and/or supplemented with Oligo-Fucoidan for 5 weeks ( Figure 7A). Control mice were processed with phosphate-buffered saline (PBS) alone. Tumor growth rates were suppressed dramatically in the p53 +/+ cancer cell-bearing mice upon Oligo-Fucoidan (F) or in combination with cisplatin treatment (F + C) compared with cisplatin (C) or PBS treatment ( Figure 7B). However, neither the monotherapy (C or F) nor combined therapy (C + F) significantly affected p53 −/− tumor development ( Figure 7C). At week 7, the p53 −/− tumor burdens were more progressive than the p53 +/+ tumor burdens ( Figure 7D). Although cisplatin alone (C) did not effectively decrease p53 −/− tumor progression, Oligo-Fucoidan alone (F) and in combination with cisplatin (C + F) improved the therapeutic efficacy. Importantly, p53 +/+ tumor regression was achieved in mice upon Oligo-Fucoidan monotherapy (F) or combination therapy (C + F) ( Figure 7E), which also inhibited the tumor necrosis (denoted by arrowheads). As indicated by immunohistochemistry (IHC) staining of vascular endothelial growth factor receptor 2 (VEGFR2) ( Figure 7F), Oligo-Fucoidan (F) alone or combined treatment (C + F) also repressed the angiogenesis effect in the p53 +/+ and p53 −/− tumors, while cisplatin only well inhibited the p53 +/+ tumor angiogenesis. Hence, Oligo-Fucoidan alone or combined with cisplatin substantially impedes tumor progression and angiogenesis, especially in presence of p53. Therefore, Oligo-Fucoidan and/or cisplatin can directly impede M2 macrophage invasion and repolarize the M2 phenotype to the M0 and M1 phenotypes. Further, the cancer cells pre-treated with Oligo-Fucoidan alone or in combination with cisplatin can indirectly promote M0 and M1 plasticity and suppress M2 polarity.

Oligo-Fucoidan Inhibits Tumor Progression and M2 Macrophage Infiltration
To investigate the therapeutic effect of Oligo-Fucoidan and/or cisplatin, HCT116 cells (2 × 10 6 ) were subcutaneously injected into BALB/c nude mice. Two weeks after inoculation, the xenograft mice were treated with cisplatin for 2 weeks and/or supplemented with Oligo-Fucoidan for 5 weeks ( Figure 7A). Control mice were processed with phosphate-buffered saline (PBS) alone. Tumor growth rates were suppressed dramatically in the p53 +/+ cancer cell-bearing mice upon Oligo-Fucoidan (F) or in combination with cisplatin treatment (F + C) compared with cisplatin (C) or PBS treatment ( Figure  7B). However, neither the monotherapy (C or F) nor combined therapy (C + F) significantly affected p53 −/− tumor development ( Figure 7C). At week 7, the p53 −/− tumor burdens were more progressive than the p53 +/+ tumor burdens ( Figure 7D). Although cisplatin alone (C) did not effectively decrease p53 −/− tumor progression, Oligo-Fucoidan alone (F) and in combination with cisplatin (C + F) improved the therapeutic efficacy. Importantly, p53 +/+ tumor regression was achieved in mice upon Oligo-Fucoidan monotherapy (F) or combination therapy (C + F) ( Figure 7E), which also inhibited the tumor necrosis (denoted by arrowheads). As indicated by immunohistochemistry (IHC) staining of vascular endothelial growth factor receptor 2 (VEGFR2) ( Figure 7F), Oligo-Fucoidan (F) alone or combined treatment (C + F) also repressed the angiogenesis effect in the p53 +/+ and p53 −/− tumors, while cisplatin only well inhibited the p53 +/+ tumor angiogenesis. Hence, Oligo-Fucoidan alone or combined with cisplatin substantially impedes tumor progression and angiogenesis, especially in presence of p53.  Afterward, TAMs were assessed by IHC analysis, showing that more CD163(+) M2 macrophages (indicated by asterisks) infiltrated into the PBS-treated p53 −/− tumors than the Oligo-Fucoidan-or cisplatin-treated p53 −/− tumors ( Figure 8A). Also, more M2 macrophages were accumulated in the stromal region of the PBS-treated p53 −/− tumors than that of the Oligo-Fucoidan-or cisplatin-treated p53 −/− tumors ( Figure 8B) and the retention of M2 macrophages in tumoral and stromal regions were greatly inhibited by combined therapy (C + F) ( Figure 8A,B).
Afterward, TAMs were assessed by IHC analysis, showing that more CD163(+) M2 macrophages (indicated by asterisks) infiltrated into the PBS-treated p53 −/− tumors than the Oligo-Fucoidan-or cisplatin-treated p53 −/− tumors ( Figure 8A). Also, more M2 macrophages were accumulated in the stromal region of the PBS-treated p53 −/− tumors than that of the Oligo-Fucoidan-or cisplatin-treated p53 −/− tumors ( Figure 8B) and the retention of M2 macrophages in tumoral and stromal regions were greatly inhibited by combined therapy (C + F) ( Figure 8A,B). Also, M2 macrophage infiltration was more abundant in the PBS-treated p53 +/+ tumors and they were noticeably reduced by cisplatin and/or Oligo-Fucoidan treatment ( Figure 8C). As compared with the p53 −/− tumors ( Figure 8B), the p53 +/+ tumors had less stromal M2 macrophage accumulation ( Figure 8D), which were also reduced by each monotherapy or eliminated by combo therapy. IHC images (2× magnification) of the tumor and stroma regions in the indicated treatment were shown in Also, M2 macrophage infiltration was more abundant in the PBS-treated p53 +/+ tumors and they were noticeably reduced by cisplatin and/or Oligo-Fucoidan treatment ( Figure 8C). As compared with the p53 −/− tumors ( Figure 8B), the p53 +/+ tumors had less stromal M2 macrophage accumulation ( Figure 8D), which were also reduced by each monotherapy or eliminated by combo therapy. IHC images (2× magnification) of the tumor and stroma regions in the indicated treatment were shown in Supplementary Figure S10. Taking together, Oligo-Fucoidan alone or in combination with cisplatin capably prevents M2 macrophage infiltration that renovates the tumor suppressive microenvironment.

Discussion
The oxidative microenvironment fuels the function and recruitment of M2 macrophages and TAMs [25] and the high mitochondrial ROS can induce cell malignancy and tumor initiation [37]. Overexpression of Cu-SOD and Zn-SOD catalyze the generation of H 2 O 2 which can serve as the redox switch to induce polarization of M2 macrophages [11], enhancing Arginase-1 and urea levels but reducing the iNOS levels and NO synthesis required for M1 differentiation. ROS accumulation in cancerous or stromal cells may activate inflammatory factors [38][39][40], which promote cell transformation and tumor initiation, progression and metastasis.
H 2 O 2 oxidant also modulates the activity of YY1 [41] and EGFR [42] by oxidative modification of cysteine residues. Paradoxically, the oxidative stress-provoked YY1 expression potentiates antioxidant machinery in irradiation-induced neuronal damage via amplification of NRF2-mediated transcriptional activation of antioxidant responsive elements [43], thereby protecting neuronal cells against oxidative damage. Conversely, NRF2 promotes EGFR expression to fuel mRNA translation in maintenance of pancreatic tumor [44]. Furthermore, K-Ras(G12D) [45], B-Raf(V619E) and Myc oncogenes induce NRF2 antioxidant program, by which enhance ROS detoxification and tumorigenesis. Besides, the activation of MCT-1 oncogene capably induces YY1-EGFR signaling axis that promotes MnSOD expression [35], mitochondrial ROS generation, lung cancer cell invasion and tumor progression. Here, we identify that Oligo-Fucoidan combined with cisplatin treatment induces ROS signaling in colorectal cancer cells which can be suppressed by antioxidants (NAC, MitoQ and DPI) (Supplementary Figure  S7B), demonstrating that Oligo-Fucoidan and cisplatin together induce the oxidative signaling axis of YY1/EGFR/MnSOD (Supplementary Figure S7). How this antioxidant mechanism in cancer cells reprogram the macrophage polarity remained unclear.
We found that Oligo-Fucoidan is an idea antioxidant that decreases mitochondrial superoxide and intracellular ROS in monocytes (Figures 1D and 3A) and cancer cells ( Figure 4A,B). Importantly, M1-like polarity was promoted after inhibiting intracellular ROS generation in monocytes ( Figure 1A-E), supporting that an antioxidative mechanism can directly activate tumor suppressive function of macrophages ( Figure 9). Furthermore, DPI and Oligo-Fucoidan cotreatment directly activated polarization of M0 and M1 from monocytes (Supplementary Figure S4A When M2 macrophages were treated with DPI and/or Oligo-Fucoidan ( Figure 2F), the invasiveness of M2 macrophages was effectively inhibited. Mutually, the cancer cell invasion ability enhanced by the M2 macrophages was repressed while pre-treating the M2 macrophages with DPI and/or Oligo-Fucoidan ( Figure 2G).
As cisplatin chemotherapy produced high levels of mitochondrial superoxide in monocytes ( Figure 3A), monocytes were differentiated into not only M0 ( Figure 3D) and M1 ( Figure 3B,E) but also M2 ( Figure 3C,F) phenotypes. Similarly, cisplatin activated the M0 macrophage polarization into not only M1 ( Figure 3G) but also M2 ( Figure 3H) types, whereas Oligo-Fucoidan supplementation blunted cisplatin effect on promoting M2 polarity. Thus, Oligo-Fucoidan supplement may capably relief the problem of M2 promotion after chemotherapy. ROS inhibitors such as DPI, NAC, MitoQ and Oligo-Fucoidan may proficiently prevent M2 macrophage differentiation and attenuate the chemotherapeutic side effect and renovate a healthy microenvironment. It will be important to inspect whether antioxidant(s) can reduce M2 macrophage infiltration in tumors.
Fucoidan also regulates immune responses that affect the production of chemokines and proinflammatory cytokines as well as the number and activity of immune cells [46][47][48][49]. For example, the expression of the M2-type chemokine CCL22 through the NF-κB pathway in M2 macrophages is downregulated by Fucoidan [50], which may inhibit tumor cell migration and regulatory T cell recruitment. These suggest that Fucoidan supplementation may alter cancer immunity that helps disease treatment. We have demonstrated that Oligo-Fucoidan reduces IL-6 and MCP-1/CCL2 expression and secretion in the ETO-treated cancer cells [13]; thus, Oligo-Fucoidan may control autocrine loops in cancer cells and paracrine pathways in the innate and/or adaptive immune systems. By changing the oxidative metabolism and cytokine/chemokine profile of the immune cells and chemotherapeutic cells, different types of Fucoidan may improve therapeutics and disease-free survival and attenuate chemotherapy-related illnesses, distress and/or inflammation in patients.
14 downregulated by Fucoidan [50], which may inhibit tumor cell migration and regulatory T cell recruitment. These suggest that Fucoidan supplementation may alter cancer immunity that helps disease treatment. We have demonstrated that Oligo-Fucoidan reduces IL-6 and MCP-1/CCL2 expression and secretion in the ETO-treated cancer cells [13]; thus, Oligo-Fucoidan may control autocrine loops in cancer cells and paracrine pathways in the innate and/or adaptive immune systems. By changing the oxidative metabolism and cytokine/chemokine profile of the immune cells and chemotherapeutic cells, different types of Fucoidan may improve therapeutics and disease-free survival and attenuate chemotherapy-related illnesses, distress and/or inflammation in patients. Figure 9. Schematic summary of Oligo-Fucoidan, ROS inhibitor and cisplatin effect on macrophage polarization and cancer treatment. Cisplatin induces ROS production but ROS inhibitor and Oligo-Fucoidan suppress ROS generation in aggressive cancer cells, which indirectly change macrophage polarity. Direct inhibition of ROS generation in monocytes or M2 macrophages by ROS inhibitor and Oligo-Fucoidan could synergistically promote M1 phenotype and prevent M2 polarity. Thereby, an antioxidant mechanism potentially establishes the tumor suppressive microenvironment(s) that prevent tumor progression.
Although Oligo-Fucoidan reduces ROS generation, it would not weaken the oxidative damage and cytotoxicity in the chemotherapeutic cancer cells (Figures 4 and 5). In combination with cisplatin, Oligo-Fucoidan still promoted cancer cell death through increasing p53-p21 signaling (Supplementary Figure S6A), PARP cleavage and caspase-3 activation (Figures 4C and 5A), which may cause the cytotoxicity. Oligo-Fucoidan supplementation not only advanced cisplatin's cytotoxic effects on colon cancer cells ( Figure 4D,E) and breast cancer cells ( Figure 5), Oligo-Fucoidan also promoted TGF-β receptor degradation and Toll-like receptor 4-mediated pathway to induce ER stress [51,52], which triggered lung cancer cell death and executed a tumor-suppressing mechanism [51].
Algae species diversity, growing conditions and purification processes could affect the structure and activity of Fucoidan as well as the therapeutic efficacy. It has been shown that fucoidans mainly act via the PI3K/AKT [53], MAPK and caspase signaling pathways. Although the structure of Oligo-Fucoidan is still unresolved, its functions are similar to that of HMFs in anti-lung cancer [51], genomic protection [13] (Supplementary Figure S6A, lanes 3 and 7) and the M2 inhibition [50]; thus, they may share analogous molecular structures as described previously [54][55][56][57]. Although Oligo-Fucoidan reduces ROS generation, it would not weaken the oxidative damage and cytotoxicity in the chemotherapeutic cancer cells (Figures 4 and 5). In combination with cisplatin, Oligo-Fucoidan still promoted cancer cell death through increasing p53-p21 signaling (Supplementary Figure S6A), PARP cleavage and caspase-3 activation (Figures 4C and 5A), which may cause the cytotoxicity. Oligo-Fucoidan supplementation not only advanced cisplatin's cytotoxic effects on colon cancer cells (Figure 4D,E) and breast cancer cells ( Figure 5), Oligo-Fucoidan also promoted TGF-β receptor degradation and Toll-like receptor 4-mediated pathway to induce ER stress [51,52], which triggered lung cancer cell death and executed a tumor-suppressing mechanism [51].
Algae species diversity, growing conditions and purification processes could affect the structure and activity of Fucoidan as well as the therapeutic efficacy. It has been shown that fucoidans mainly act via the PI3K/AKT [53], MAPK and caspase signaling pathways. Although the structure of Oligo-Fucoidan is still unresolved, its functions are similar to that of HMFs in anti-lung cancer [51], genomic protection [13] (Supplementary Figure S6A, lanes 3 and 7) and the M2 inhibition [50]; thus, they may share analogous molecular structures as described previously [54][55][56][57].
Consistent with cisplatin's direct effect on monocytes and M0 macrophages (Figure 3), the cisplatin-treated cancer cells not only stimulated monocytes differentiating into M0 ( Figure 6A) and M1 ( Figure 6B and Supplementary Figure S8) but also M2 phenotypes ( Figure 6C); however, the M2 polarization promoted by the cisplatin-treated cancer cells was repressed by Oligo-Fucoidan supplement. In particular, while monocytes encountered the p53 +/+ cancer cells pretreated with Oligo-Fucoidan and cisplatin ( Figure 6D), the amounts of M1 marker (iNOS and CD80) were relatively increased but those of M2 marker (CD163 and Arginase-1) were significantly decreased. Combined treatment also directly inhibited the invasiveness of M2 macrophages ( Figure 6E), reduced the M2 phenotype ( Figure 6H) and repolarized the M2 macrophages toward the M0 and M1 phenotypes ( Figure 6F,G). Consequently, the development and progression of p53 +/+ tumors were more inhibited by Oligo-Fucoidan alone and in combination with cisplatin ( Figure 7B,D,E), as compared with the p53 −/− tumors ( Figure 7C-E). Similarly, M2 macrophage recruitment in the p53 −/− tumoral and stromal regions were more abundant ( Figure 8A,B) than in the p53 +/+ microenvironment ( Figure 8C,D). Oligo-Fucoidan monotherapy or combined with cisplatin successfully inhibited M2 macrophage infiltration (Figure 8), signifying that the suppressive TME were renovated. Further inspection of the mechanism(s) of Oligo-Fucoidan and its systematic activity with current drugs or newly developed immunotherapies in anti-tumor immune surveillance will be an important subject.
Colorectal cancer from the patient was minced and dissociated with 200 U/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and passed through a Falcon 40 M cell strainer (Corning, Corning, NY, USA). Red blood cells were lysed with ammonium-chloride-potassium buffer (Thermo Fisher Scientific). The isolated primary cancer cell line (C6P2-L1) was first grown in Dulbecco's Modified Eagle Medium (DMEM) containing 20% FBS, L-glutamine (2 mM), penicillin (100 units/mL) and streptomycin (100 µg/mL) and then subcultured in DMEM supplemented with 10% FBS in a 37°C incubator with 5% CO 2 .

Mitochondrial Superoxide and Intracellular ROS Levels
To exam the ROS signaling cascade, HCT116 cancer cells were pretreated with NAC (1 mM), MitoQ (1 M) and DPI (2 M) for 24 h followed by cotreatment of cisplatin (15 M) and Oligo-Fucoidan (400 g/mL) for 24 h. DPI and NAC were from Sigma-Aldrich. MitoQ was from Cayman Chemical (Ann Arbor, Michigan, USA).

Cytotoxicity Effect and Cell Viability Assay
HCT116 cancer cells and the primary C6P2-L1 colon cancer cells were treated with Oligo-Fucoidan (400 µg/mL) and/or cisplatin (15 µM) for 48 h, washed with ice cold PBS, resuspended in a 1× binding buffer (BD Biosciences, Franklin Lakes, NJ, USA) and then stained with 5 µL of FITC-conjugated Annexin V and 5 µL of PI in a total volume of 100 µL (1 × 10 5 cells) for 15 min in the dark. Afterward, 400 µL of the 1× binding buffer was added to the samples, which were analyzed by a BD FACSCalibur flow cytometer in the FL1 and FL2 emission channels at excitation wavelengths of 488 nm and 543 nm, respectively. Early and late apoptosis events as well as necrosis effect were detected.

The Invasion Ability of M2 Macrophages and Cancer Cells
The polarized M2 macrophages (derived from THP-1 monocytes) were resuspended in serum-free medium, plated in the upper chamber of a Matrigel ® invasion transwell (24-well plate, 8-µm pore size) (Corning) and cocultured with DPI and/or Oligo-Fucoidan or cocultured with cisplatin and/or Oligo-Fucoidan in the bottom chamber for 24 h at 37 • C. The noninvasive M2 macrophages remaining on the upper membrane were removed by cotton swabs. The M2 macrophages penetrated through the Matrigel matrix were rinsed twice in PBS, fixed with 3.7% formaldehyde for 10 min, permeabilized with 100% methanol for 20 min, stained with 0.2% crystal violet for 15 min, photographed under an optical microscope and quantified by ImageJ software (version 1.52a, National Institute of Mental Health, Bethesda, Maryland).
HCT116 cancer cells (4 × 10 5 ) suspended in serum-free medium were plated in the upper chamber of a Corning ® BioCoat TM Matrigel ® invasion transwell (24-well plate, 8-µm pore size) (Corning, NY, USA) and cocultured with the DPI and/or Fucoidan-pretreated M2 macrophages in the bottom chamber for 24 h at 37 • C. The noninvasive cancer cells retained on the upper membrane were removed by cotton swabs. The invasive cancer cells through the Matrigel matrix were measured as described above.

Expression Levels of Macrophage Markers Detected by Quantitative RT-PCR
The polarity of THP-1 monocytes and Raw264.7 macrophages as well as the derived M2 macrophages were studied after treatment with ROS inhibitors (NAC, DPI) (Sigma-Aldrich), DPI and/or Oligo-Fucoidan or cisplatin and/or Oligo-Fucoidan for 48 h. Moreover, HCT116 cancer cells (4 × 10 5 ) or MDA-MB231 cancer cells (4 × 10 5 ) were pretreated with cisplatin (15 µM) and/or Oligo-Fucoidan (400 µg/mL) for 48 h and the preconditioned cancer cells were rinsed and seeded in the upper chamber of Corning Falcon ® Cell Culture Inserts (Corning, NY, USA) and incubated with THP-1 monocytes (1 × 10 6 ) placed in the bottom chamber for 48 h. Cellular RNAs of the polarized macrophages were isolated in a TRIzol solution (Thermo Fisher Scientific), treated with DNase I and transcribed into cDNA by SuperScript TM II reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative RT-PCR was conducted using SYBR Green Master Mix, a cDNA template (100 ng) and the primers for F4/80 (forward The quantitative RT-PCR reaction was performed at 95 • C for 15 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. The β-actin expression level was used as an internal control. Relative mRNA levels were calculated by the formula: ∆∆CT = ∆Ct test sample−∆Ct control sample. Fold changes in gene expression were calculated using the 2 −∆∆CT method.

Tumor Growth in Xenograft Mice and a Tumor Immunohistochemistry Study
Six-to eight-week-old BALB/c nude mice (BALB/cAnN.Cg-Foxn1 nu /CrlNarl) were obtained from the National Laboratory Animal Center of Taiwan. The experiments were performed according to the Animal Use Protocol (NHRI-IACUC-105103-A) approved by the National Health Research Institutes (NHRI). Mice were injected subcutaneously with HCT116 cells (2 × 10 6 /100 µL of PBS) and randomly divided into 4 groups (PBS, Oligo-Fucoidan, cisplatin and combined treatment). Two weeks after inoculation, the tumors reached approximately 100 mm 3 in size and the mice were intravenously (i.v.) injected with cisplatin (1 mg/kg) three times per week for 2 weeks and/or orally fed Oligo-Fucoidan (150 mg/kg) two times per week for 5 weeks. Control mice were administered PBS alone. Tumor volumes (Vs) were measured weekly and calculated using the formula: Vs = (length × width 2 )/2.

Disclosure
HLH serves on the Scientific Advisory Board of Hi-Q Marine Biotech International Ltd. The terms of this arrangement have been reviewed and approved by the National Health Research Institutes in accordance with its conflict of interest guidelines.

Statistics
A two-tailed unpaired Student's t test was applied to compare the results of the control and experimental groups. The chi-square test was used to determine the statistical significance of differences in tumor burdens. A p value < 0.05 was considered statistically significant.

Conclusion
We now confirm that Oligo-Fucoidan quenches intracellular ROS and mitochondrial superoxide which can directly or indirectly benefit M1-like macrophage polarization. Importantly, Oligo-Fucoidan suppresses the drawback of chemotherapy on M2 macrophage polarization in vitro and inhibits M2 macrophage infiltration in vivo. Our results first demonstrate that Oligo-Fucoidan supplementation sufficiently enhances chemo-sensitivity in aggressive cancer cells and renovates a healthy microenvironment that prevents tumor progression.