Cancer Stem Cells and Somatic Stem Cells as Potential New Drug Targets, Prognosis Markers, and Therapy Efficacy Predictors in Breast Cancer Treatment

New drug targets, markers of disease prognosis, and more efficient treatment options are an unmet clinical need in breast cancer (BC). We have conducted a pilot study including patients with luminal B stage breast cancer IIA–IIIB. The presence and frequency of various populations of cancer stem cells (CSC) and somatic stem cells were assessed in the blood, breast tumor tissue, and normal breast tissue. Our results suggest that patients with BC can be divided into two distinct groups based on the frequency of aldehyde dehydrogenase positive cells (ALDH1+ cells) in the blood (ALDH1hi and ALDH1low). In the ALDH1hi cells group, the tumor is dominated by epithelial tumor cells CD44+CD24low, CD326+CD44+CD24−, and CD326−CD49f+, while in the ALDH1low cells group, CSCs of mesenchymal origin and epithelial tumor cells (CD227+CD44+CD24− and CD44+CD24−CD49f+) are predominant. In vitro CSCs of the ALDH1low cells group expressing CD326 showed high resistance to cytostatics, CD227+ CSCs of the ALDH1hi cells group are sensitive to cytostatics. Epithelial precursors of a healthy mammary gland were revealed in normal breast tissue of patients with BC from both groups. The cells were associated with a positive effect of chemotherapy and remission in BC patients. Thus, dynamic control of their presence in blood and assessment of the sensitivity of CSCs to cytostatics in vitro can improve the effectiveness of chemotherapy in BC.


Introduction
Despite advances in diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer death in women [1]. More than 1 million new cases of the disease are registered annually [2]. After the diagnosis of BC, prognosis of complications and the choice of optimal drug therapy are crucial [3]. Traditional prognostic measures are the definition of metastases in the lymph nodes, the size of the tumor, and the type of differentiation of tumor cells [1,3]. When searching for tumor markers, much attention is paid to the tumor subtype, which is determined by the presence (or absence) of the estrogen receptor (ER), progesterone receptor (PR), and the protein associated with the

Patients
The study included 12 patients with IIA-IIIB (T1-4N0-3M0) breast cancer of the luminal B and triple-negative molecular subtypes aged from 38 to 66 years (average age of 50.6 ± 3.02 years) who received treatment at the Cancer Research Institute of Tomsk NRMC (Tomsk, Russia) from 2017-2018. Imaging of the primary breast lesion was performed by mammography and ultrasonography. An immunohistochemical study was conducted to determine the molecular subtype of the tumor before treatment. The luminal B subtype of breast cancer was defined as ER+, PR+ or −, Ki67 > 30%, and all patients with the luminal B subtype were HER2-negative. Some patients showed no expression of ER, PR, and HER2, and they were classified as a triple-negative subtype. Histological diagnosis was confirmed for all samples. Table S1 presents the clinical indicators of tumors in all 12 patients. Blood samples from 10 healthy women of similar age were used as control.
This was a pilot investigation. Informed consent was obtained from all individual participants included in the study. All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Design of Investigation
Blood samples were obtained from patients one day before surgery (Figure 1). Breast tumor and normal breast tissue were obtained from patients on the day of surgery. and/or national research committee and with the 1964 Helsinki declaration and its amendments or comparable ethical standards.

Design of Investigation
Blood samples were obtained from patients one day before surgery (Figure 1). B tumor and normal breast tissue were obtained from patients on the day of surgery.

Isolation of Blood Mononuclear Cells
Lympholyte-H (CEDARLANE, Netherlands, Cedarlane Laboratories, Cat#CL protocol was used for the elimination of erythrocytes and dead cells from human b and receiving mononuclear cells.

Flow Cytometry
Mononuclear cells from blood, human mammary tissue, and breast cancer t were isolated as described above and the expression of surface markers on mononu cells was analyzed using flow cytometry. Fc-receptors were blocked by pre-incubati the cells with unconjugated anti-CD16/CD32 antibodies for 10 min (eBioscience Diego, CA, USA, Cat# 464219, 1/100 dilution) in 50 μL of 0.1% saponin (Sigma-Aldric Louis, MO, USA, Cat# S4521) and 1% BSA (Sigma-Aldrich, St. Louis, MO, USA, A3059-100G) in phosphate buffered saline (PBS) per tube. After the pre-incubation, suspensions were stained with fluorophore-conjugated monoclonal antibodies.

Measurement of ALDH Activity
An aldehyde dehydrogenase-based cell detection kit (StemCell Technolo Vancouver, BC, Canada, Cat#01700) was used to determine ALDH1 enzymatic activ the blood, breast tumor, and normal breast tissue. Cells were suspended in aldefluor a buffer and incubated with the ALDH enzyme substrate, BODIPY-aminoacetalde (BAAA), for 40 min at 37 °C. As a control, cells were also treated diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH enzyme activity. Fluoresc

Isolation of Blood Mononuclear Cells
Lympholyte-H (CEDARLANE, Netherlands, Cedarlane Laboratories, Cat#CL5015) protocol was used for the elimination of erythrocytes and dead cells from human blood and receiving mononuclear cells.

Flow Cytometry
Mononuclear cells from blood, human mammary tissue, and breast cancer tissue were isolated as described above and the expression of surface markers on mononuclear cells was analyzed using flow cytometry. Fc-receptors were blocked by pre-incubation of the cells with unconjugated anti-CD16/CD32 antibodies for 10 min (eBioscience, San Diego, CA, USA, Cat# 464219, 1/100 dilution) in 50 µL of 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA, Cat# S4521) and 1% BSA (Sigma-Aldrich, St. Louis, MO, USA, Cat# A3059-100G) in phosphate buffered saline (PBS) per tube. After the pre-incubation, cells suspensions were stained with fluorophore-conjugated monoclonal antibodies.

Measurement of ALDH Activity
An aldehyde dehydrogenase-based cell detection kit (StemCell Technologies, Vancouver, BC, Canada, Cat#01700) was used to determine ALDH1 enzymatic activity in the blood, breast tumor, and normal breast tissue. Cells were suspended in aldefluor assay buffer and incubated with the ALDH enzyme substrate, BODIPY-aminoacetaldehyde (BAAA), for 40 min at 37 • C. As a control, cells were also treated with diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH enzyme activity. Fluorescence was determined using a BD FACS Canto II flow cytometer and analyzed using FACSDiva software (BD Biosciences).

Detection of Hemopoietic Stem Cells
A two-color research reagent CD45/CD34 (Becton Dickinson, San Jose, CA, USA, Cat# 341071) was used to determine the presence of HSCs. The reagent contains FITC-labeled CD45, clone 2D1, and PE-labeled CD34, clone 8G12. We determined HSC according to the ISHAGE protocol [18].
All samples were run on a Becton Dickenson FACSCanto II flow cytometer. The instrument was set up and standardized using BD Cytometer Setup and Tracking (CS&T) procedures according to manufacturer specifications. Data were analyzed using FACSDiva 8.0 software.

Isolation of Breast Cancer Cells
The CD326 + and CD227 + cell fractions were isolated using the EasySep™ Human CD326 (StemCell Technologies, Canada, Vancouver, Catalog #18356) or CD227 (StemCell Technologies, Canada, Vancouver, Catalog #18359) positive selection kit according to the technical protocol supplied by StemCell Technologies (StemCell Technologies, Canada, Vancouver). For EasySep™ cell separation, the labeled cell suspension was placed in the EasySep™ magnet for 5 min, and the cells that were not magnetically labeled were discarded. Labeled cells were resuspended, and the separation was repeated a total of 6 times. The EasySep™ cell separation was evaluated by flow cytometry. A two-color research reagent CD45/CD34 (Becton Dickinson, San Jose, CA, USA, Cat# 341071) was used to determine the presence of HSCs. The reagent contains FITClabeled CD45, clone 2D1, and PE-labeled CD34, clone 8G12. We determined HSC according to the ISHAGE protocol [18].
All samples were run on a Becton Dickenson FACSCanto II flow cytometer. The instrument was set up and standardized using BD Cytometer Setup and Tracking (CS&T) procedures according to manufacturer specifications. Data were analyzed using FACSDiva 8.0 software.

Isolation of Breast Cancer Cells
The CD326 + and CD227 + cell fractions were isolated using the EasySep™ Human CD326 (StemCell Technologies, Canada, Vancouver, Catalog #18356) or CD227 (StemCell Technologies, Canada, Vancouver, Catalog #18359) positive selection kit according to the technical protocol supplied by StemCell Technologies (StemCell Technologies, Canada, Vancouver). For EasySep™ cell separation, the labeled cell suspension was placed in the EasySep™ magnet for 5 min, and the cells that were not magnetically labeled were discarded. Labeled cells were resuspended, and the separation was repeated a total of 6 times. The EasySep™ cell separation was evaluated by flow cytometry.  Separation-sorted human breast cancer cell concentrations were determined using a cell counter and then seeded at densities of 5 × 10 5 cells/mL in low adherence 6-well plates. The cultures were maintained in Human EpiCult-C (StemCell Technologies, Vancouver, Canada), supplemented with 5% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) for 24 h, and then the medium was replaced with serum-free medium and Separation-sorted human breast cancer cell concentrations were determined using a cell counter and then seeded at densities of 5 × 10 5 cells/mL in low adherence 6-well plates.

In Vitro Tumor Study
The cultures were maintained in Human EpiCult-C (StemCell Technologies, Vancouver, Canada), supplemented with 5% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) for 24 h, and then the medium was replaced with serum-free medium and maintained for an additional 10 days. At the end of the assays, the colonies were counted under a microscope. After 10 days, the medium was removed and the plates gently rinsed with PBS. The cultured cells were then counted. The procedure was repeated three times. Sorted cells were seeded and cultivated in MammoCult TM (StemCell Technologies, Vancouver, Canada, Cat#05620) supplemented with 0.48 µg/mL freshly dissolved hydrocortisone (StemCell Technologies, Vancouver, Canada, Cat#07904) and 4 µg/mL heparin (StemCell Technologies, Vancouver, Canada, Cat#07980) and 10 ng/mL IL-6 (Sigma-Aldrich, St. Louis, MO, USA, Cat# SRP3096) to induce greater numbers of mammospheres and tumorspheres, and the cultures were maintained for an additional 7 to 10 days. At the end of the assay, cells were assessed by flow cytometry and image processing of each well with Cytation™ 3.
CD227 + -and CD326 + -sorted cell populations were cultivated in the presence of 10 ng/mL cytostatics (docetaxel (Taxotere (T)) + adriamycin (A) + cyclophosphamide (C), TAC). Levels of apoptosis were evaluated after 2 h culture using flow cytometry and image processing of each well with Cytation™ 3.
In the next stage, we chose a patient with a high grade of malignancy and a high level of ALDH1 + cells circulating in the blood. The patient received cycles of TAC-based chemotherapy. We studied the dynamics of the count of tumor cells in the blood before and after surgery, after courses of chemotherapy. Moreover, we studied in vitro culture tumor cells isolated from breast tumor tissue of this patient with and without cytostatics. For the in vitro study, cytostatics which the patient received were used. Figure 3 shows the experimental design of these investigations in vitro. maintained for an additional 10 days. At the end of the assays, the colonies were counted under a microscope. After 10 days, the medium was removed and the plates gently rinsed with PBS. The cultured cells were then counted. The procedure was repeated three times. Sorted cells were seeded and cultivated in MammoCult TM (StemCell Technologies, Vancouver, Canada, Cat#05620) supplemented with 0.48 μg/mL freshly dissolved hydrocortisone (StemCell Technologies, Vancouver, Canada, Cat#07904) and 4 μg/mL heparin (StemCell Technologies, Vancouver, Canada, Cat#07980) and 10 ng/mL IL-6 (Sigma-Aldrich, St. Louis, MO, USA, Cat# SRP3096) to induce greater numbers of mammospheres and tumorspheres, and the cultures were maintained for an additional 7 to 10 days. At the end of the assay, cells were assessed by flow cytometry and image processing of each well with Cytation™ 3. CD227 + -and CD326 + -sorted cell populations were cultivated in the presence of 10 ng/mL cytostatics (docetaxel (Taxotere (T)) + adriamycin (A) + cyclophosphamide (C), TAC). Levels of apoptosis were evaluated after 2 h culture using flow cytometry and image processing of each well with Cytation™ 3.
In the next stage, we chose a patient with a high grade of malignancy and a high level of ALDH1 + cells circulating in the blood. The patient received cycles of TAC-based chemotherapy. We studied the dynamics of the count of tumor cells in the blood before and after surgery, after courses of chemotherapy. Moreover, we studied in vitro culture tumor cells isolated from breast tumor tissue of this patient with and without cytostatics. For the in vitro study, cytostatics which the patient received were used. Figure 3 shows the experimental design of these investigations in vitro.

Imaging
Images of CD227 + and CD326 + cells were obtained using a Cytation 3 cell imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, USA) tuned to DAPI, GFP, and Texas Red light cubes.
At the end of the incubation period, CD227 + and CD326 + were counter stained with Hoechst 33342 (blue) for cell number measurements, or Annexin V-iFluor™ 350 Conjugate

Imaging
Images of CD227 + and CD326 + cells were obtained using a Cytation 3 cell imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, USA) tuned to DAPI, GFP, and Texas Red light cubes.
At the end of the incubation period, CD227 + and CD326 + were counter stained with Hoechst 33342 (blue) for cell number measurements, or Annexin V-iFluor™ 350 Conjugate and 7-AAD fluorescent probes for apoptosis/ necrosis assessments. Then, a Cytation 3 (magnification of 4× or 20×) was imaged, followed by cell analysis using Gen5™ data analysis software (Bad Friedrichshall, Germany).
All collected images were pre-processed to align the background before applying analytical methods. Cell analysis was performed on a blue channel to determine cell count based on the number of Hoechst-stained nuclei. The default settings resulted in adequately calculated data for further analysis.

Statistical Analysis
All statistical analyses were carried out by using SPSS statistical software (version 15.0, SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using the Mann-Whitney U test. Additionally, p values < 0.05 were considered to indicate statistically significant differences. All quantitative data presented are the mean value and standard error.

Results and Discussion
The ideal way to identify and track disease recurrence and/or progression in cancer patients is through surrogate marker approaches that are minimally invasive, reliable, and allow for longitudinal testing of accessible sample types, such as blood. At present, no single molecular or cellular blood marker has been proven to have such properties in BC. The most commonly used molecular markers are carcinoembryonic antigen or products of the MUC-1 gene expressed by tumor cells [19]. The high heterogeneity of the CSC phenotype in cancer was the reason for the study of various oncoantigens in our study. When studying blood samples from patients with BC, we found an increase in the number of tumor cells with overexpression of CD227 and CD326, as well as CSCs of mesenchymal origin (CD44 + CD24 − ) compared to the levels in healthy volunteers ( Figure 4). Moreover, we observed an increase in the HSCs and CD309 + endothelial cells in the blood relative to healthy volunteers. In addition, in BC an increase in the number of ALDH1 + cells circulating in the blood was observed.
Our results are consistent with the literature, which indicates a correlation between the tumor progression and the number of HSCs and endothelial cells in the blood [15]. Therefore, it is important to investigate the potential presence of HSC and endothelial cells in the peripheral blood of patients with BC, in addition to the already known tumor markers.
The tumor fraction ALDH1 is widely used as a biomarker of metastatic activity and a determinant of the clinical outcome of breast cancer [20]. The collection of material for biopsy is a rather painful intervention. Notably, prognostic potential of ALDH1 + CTC in patients with BC was suggested in the literature [20][21][22]. ALDH expression by circulating tumor cells correlates with poor clinical outcome, metastatic progression, and the response to therapy in patients with metastatic breast cancer [22]. Another study suggested that ALDH1 expression in primary breast tumors correlates with the presence of CTCs and clinical outcome in patients with non-metastatic disease [22]. Objectively, ALDH1 + cells circulating in the blood can be used as diagnostic markers. In our study, we found that patients with BC differed in the content of ALDH1 + cells in the blood. We identified a group of patients with a significant number of ALDH1 + cells (≥0.9% of all isolated mononuclear cells, group ALDH1 hi cells) and a group of patients with a small number of ALDH1 + cells (≤0.9% of all isolated mononuclear cells, group ALDH1 low cells) ( Figure 5). It is important to note that the level of ALDH1 + cells in the tumor of patients of ALDH1 hi cells group was also higher than that of patients of the ALDH1 low cells group. cines 2021, 9, x FOR PEER REVIEW 7 of 14  found that patients with BC differed in the content of ALDH1 cells in the blood. We identified a group of patients with a significant number of ALDH1 + cells (≥0.9% of all isolated mononuclear cells, group ALDH1 hi cells) and a group of patients with a small number of ALDH1 + cells (≤0.9% of all isolated mononuclear cells, group ALDH1 low cells) ( Figure 5). It is important to note that the level of ALDH1 + cells in the tumor of patients of ALDH1 hi cells group was also higher than that of patients of the ALDH1 low cells group. In BC, metastases in their development go through the mesenchymal and epithelial phases of the metastatic cascade [22]. ALDH1 is involved in the epithelial and more proliferative phase of metastatic tissue colonization. In this regard, the use of ALDH1 as a key biomarker of the metastases risk may be more useful in the diagnosis of advanced breast cancer (epithelial phase) [22]. In patients with an unexpanded disease, which is characterized by an intravascular and more mesenchymal phase of the metastatic cascade, the level of ALDH1 expression is significantly reduced. We found confirmation of this in the present study. According to our data, a patient of the ALDH1 hi cells group had metastases in the lungs.
When determining other antigens, we found intergroup differences in other CSCs. The ALDH1 hi cells group showed an increased number of epithelial tumor cells CD44 + CD24 low , CD326 + CD44 + CD24 -, and CD326 -CD49f + (Figure 6). At the same time, in the ALDH1 low cells group, the content of CSCs of mesenchymal origin (CD44 + CD24 -), In BC, metastases in their development go through the mesenchymal and epithelial phases of the metastatic cascade [22]. ALDH1 is involved in the epithelial and more proliferative phase of metastatic tissue colonization. In this regard, the use of ALDH1 as a key biomarker of the metastases risk may be more useful in the diagnosis of advanced breast cancer (epithelial phase) [22]. In patients with an unexpanded disease, which is characterized by an intravascular and more mesenchymal phase of the metastatic cascade, the level of ALDH1 expression is significantly reduced. We found confirmation of this in the present study. According to our data, a patient of the ALDH1 hi cells group had metastases in the lungs.
Despite the increased level of ALDH1 in the patients with BC, we did not find an increase in the numbers of HSC and CD309 + endothelial cell levels in the blood or tumor tissue compared to patients with ALDH1 low . It is known that endothelial cells play a role in tumor progression by promoting the progression of avascular micrometastasis to vascularized macrometastases [23]. HSCs play an important role in the development of inflammation and tumor progression [14].
We suggest that patients with different levels of ALDH1 in the blood can suffer from tumors at different stages. The populations of CSC, HSCs, and endothelial cells isolated in this study can be used as markers for personalized tumor therapy, and disease prognosis.
Since we did observe group differences in the content of tumor CD326 + CD44 + CD24 − cells and CD227 + CD44 + CD24 − cells from the tumor of patient A of the ALDH1 hi cells group and patient B of the ALDH low cells group, we isolated CD326 + and CD227 + cells, obtained primary cultures, and characterized them. At the same time, we evaluated the effects of IL-6 (a factor that stimulates CSC [24]) and cytostatics proposed for chemotherapy (CT) in vitro. endothelial cells in the tumor prevailed.
Despite the increased level of ALDH1 in the patients with BC, we did not find an increase in the numbers of HSC and CD309 + endothelial cell levels in the blood or tumor tissue compared to patients with ALDH1 low . It is known that endothelial cells play a role in tumor progression by promoting the progression of avascular micrometastasis to vascularized macrometastases [23]. HSCs play an important role in the development of inflammation and tumor progression [14]. We suggest that patients with different levels of ALDH1 in the blood can suffer from tumors at different stages. The populations of CSC, HSCs, and endothelial cells isolated in this study can be used as markers for personalized tumor therapy, and disease prognosis.
Since we did observe group differences in the content of tumor CD326 + CD44 + CD24cells and CD227 + CD44 + CD24 -cells from the tumor of patient A of the ALDH1 hi cells group and patient B of the ALDH low cells group, we isolated CD326 + and CD227 + cells, obtained primary cultures, and characterized them. At the same time, we evaluated the effects of IL-6 (a factor that stimulates CSC [24]) and cytostatics proposed for chemotherapy (CT) in vitro.
CD227 + cells of patient B and CD326 + cells of patient A were investigated (Figure 7a). We found that in patient B, the culture of CD227 + cells was characterized by the absence of cells in apoptosis, insignificant clonal activity (mammosphere) and an increase in cell mass during the cultivation cycle (Figure 7d-f). In culture of CD227 + cells of patient B, IL- CD227 + cells of patient B and CD326 + cells of patient A were investigated (Figure 7a). We found that in patient B, the culture of CD227 + cells was characterized by the absence of cells in apoptosis, insignificant clonal activity (mammosphere) and an increase in cell mass during the cultivation cycle (Figure 7d-f). In culture of CD227 + cells of patient B, IL-6 increased the frequency of mammosphere formation. After co-cultivation with cytostatics, the proportion of apoptotic CD227 + cells was 70% (Figure 7b,c).
The culture of CD326 + cells of patient A withstood three passages and by the end of the cultivation cycle its cell mass significantly increased. Moreover, the frequency of mammosphere formation increased in the presence of IL-6 ( Figure 7a). The activity of the cells of patient A in culture was superior to those of patient B. On the other hand, patient A's CD326 + cells were resistant to cytostatics: the number of cells in apoptosis was 14% versus 27% in patient B.
These data made it possible to suggest that when diagnosing breast cancer, it is desirable to determine the level of ALDH1 + cells circulating in the blood and the ratio of CD326 + and CD227 + tumor cells in the tumor. These cells can act as tumor progression factors and inducers of metastases in all groups, regardless of ALDH1 expression. To increase the effectiveness of chemotherapy, we suggest in vitro selection of cytostatics. This approach can enhance effective patient care.
In the next stage, we studied the count of tumor cells in the blood before and after surgery, after courses of chemotherapy of the patient received cycles of TAC-based chemotherapy (Figure 8a). We observed a decrease in the content of CD227 + CD44+CD24 − , CD227 + , and CD44 + CD24 low cells in the blood after surgery. However, we observed an increase in the content of these cell populations after courses of chemotherapy. We suggest that changes in blood can be associated with the resistance of cancer cells to chemotherapy. We studied in vitro cultured CSCs and tumor cells isolated from breast tumor tissue of this patient with and without cytostatics (Figure 8b-d). We found no changes in the count of dead tumor cells and apoptotic tumor cells. Moreover, no differences were observed in the count of tumor cells (CD326 − CD49 + , CD326 + CD227 + , CD326 + CD44 + CD24 − , CD227 + , CD227 + CD44 + CD24 − ) after a cycle of cultivation with cytostatics (TAC) treatment.
Thus, dynamic control of circulating CSCs of patients with BC makes it possible to detect cells with the potential for tumor progression and metastasis in the blood. In addition, in vitro assessment of the sensitivity of the CSC to the chemotherapy may allow a quick correction of the patient's treatment.
9, x FOR PEER REVIEW 10 of 14 6 increased the frequency of mammosphere formation. After co-cultivation with cytostatics, the proportion of apoptotic CD227 + cells was 70% (Figure 7b,c).  CD326 CD44 CD24 , CD227 , CD227 CD44 CD24 ) after a cycle of cultivation with cytostatics (TAC) treatment. Thus, dynamic control of circulating CSCs of patients with BC makes it possible to detect cells with the potential for tumor progression and metastasis in the blood. In addition, in vitro assessment of the sensitivity of the CSC to the chemotherapy may allow a quick correction of the patient's treatment. The content of ALDH1 + , HSC, CD227 + CD44 + CD24 − , CD227 + , and CD44 + CD24 low cells in the blood before and after surgery, after courses of chemotherapy. Cells were analyzed by flow cytometry using antibodies for human CD24, CD44, CD227, CD45, CD34, and ALDH1; (b) the count of dead tumor cells and tumor cells with apoptosis after a cycle of cultivation without cytostatic (tumor) and with cytostatics (docetaxel (taxotere (T)), adriamycin (A) and cyclophosphamide (C)) (tumor + TAC) treatment. Determination of the percentage of dead cells and cells in apoptosis was made by the ratio of cells counted in green and red channel to total cells counted using blue (DAPI) channel; (c) the count of tumor cells (CD326 − CD49 + , CD326 + CD227 + , CD326 + CD44 + CD24 − , CD227 + , CD227 + CD44 + CD24 − ) after a cycle of cultivation without cytostatic (tumor) and with cytostatics (docetaxel (Taxotere (T)), adriamycin (A) and cyclophosphamide (C)) (tumor + TAC). Cells were analyzed by flow cytometry using antibodies for human CD24, CD44, CD227, and CD326; (d) Isotype controls and phenotype establishment and qualitative analysis of CD227 (FITC), CD326 (PerCP-Cy5.5), CD24 (PE-Cy7), CD44 (APC) expression.
In conclusion, it should be noted that in the breast tissue adjacent to the tumor (normal tissue) and tumor tissue of patients with BC, different populations of epithelial progenitor cells of a healthy mammary gland were revealed (Figure 9). We did not find significant differences in their content between the patients of the ALDH1 hi and ALDH1 low groups. In all groups, cells were associated with a positive effect of chemotherapy and remission of patients with BC. Thus, targeting epithelium and endothelium regeneration of the mammary gland might be beneficial and prevent tumors. However, our study has some limitations. Since we performed a short-term single-center study, it has a relatively small sample size, which diminishes the likelihood of generalization. To assess their applicability to a larger population, the results presented here need further validation using multi-center cohorts with large numbers of patients.
In conclusion, it should be noted that in the breast tissue adjacent to the tumor (normal tissue) and tumor tissue of patients with BC, different populations of epithelial progenitor cells of a healthy mammary gland were revealed (Figure 9). We did not find significant differences in their content between the patients of the ALDH1 hi and ALDH1 low groups. In all groups, cells were associated with a positive effect of chemotherapy and remission of patients with BC. Thus, targeting epithelium and endothelium regeneration of the mammary gland might be beneficial and prevent tumors. However, our study has some limitations. Since we performed a short-term single-center study, it has a relatively small sample size, which diminishes the likelihood of generalization. To assess their applicability to a larger population, the results presented here need further validation using multi-center cohorts with large numbers of patients. Figure 9. Characterization of bipotent precursors of breast cells (CD326 low CD49f hi CD227 + ) (а,e), basal tissue-specific stem cells of the mammary gland (CD326 hi CD49f hi ) (b,f), precursors of luminal cells (CD326 low CD49f low CD227 + ) (c,g), and precursors of myoepithelial cells (CD326 low CD49f + ) (d,h) isolated from the breast tissue adjacent to the tumor (normal tissue) (a-d) and tumor tissue (e-h) of patients with BC of the ALDH1 hi and ALDH1 low groups. Cells were analyzed by flow cytometry Figure 9. Characterization of bipotent precursors of breast cells (CD326 low CD49f hi CD227 + ) (a,e), basal tissue-specific stem cells of the mammary gland (CD326 hi CD49f hi ) (b,f), precursors of luminal cells (CD326 low CD49f low CD227 + ) (c,g), and precursors of myoepithelial cells (CD326 low CD49f + ) (d,h) isolated from the breast tissue adjacent to the tumor (normal tissue) (a-d) and tumor tissue (e-h) of patients with BC of the ALDH1 hi and ALDH1 low groups. Cells were analyzed by flow cytometry using antibodies for CD49f, CD227, and CD326. * Differences are significant in comparison with the ALDH1 hi group (p < 0.05).

Conclusions
Patients with IIA-IIIB (T1-4N0-3M0), triple negative BC, and HER+ BC, are divided into a group with a significant number of ALDH1 + cells and a group with a small number of ALDH1 + cells in the blood and tumors. The composition of tumor CSCs and their activity in patients of the ALDH1 hi cells group and the ALDH1 low cell group differ. ALDH1 expression level and ratio of tumor cells CD44 + CD24 low , CD326 + CD44 + CD24 − , CD326 − CD49f + , CSC of mesenchymal origin (CD44 + CD24 − ) and epithelial tumor cells (CD227 + CD44 + CD24 − and CD44 + CD24 − CD49f + ), HSC, and CD309 + endothelial cells in tumors can act as personalized diagnostic markers, predictors of complications and the effectiveness of breast cancer treatment in further research. Moreover, the dynamic control in the blood and assessment of the sensitivity of CSCs to cytostatics in vitro can improve the effectiveness of chemotherapy in BC.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biomedicines9091223/s1, Table S1: Clinical and morphological parameters of the patients included in the study.