Characterization And Culture Of Microspheres Isolated Directly From Tumor Tissues Of Breast Cancer Patients Received Neoadjuvant Chemotherapy

In recent years, there is a growing body of evidence that cells with stem cell characteristics, termed cancer stem cells (CSCs) or tumor-initiating cells (TICs), are the driving force of tumor formation in many cancers including cancers of human acute myeloid and lymphoid leukemia, brain, prostate, liver, pancreas, colon, and breast. Cancer stem cells, which are self-renewal, driving tumorigenicity, recurrence, and metastasis, and they can differentiate into multiple lineages, giving rise to a heterogeneous population of cancer cells which constitute the main body of the tumor. Because the differentiated cells are lack of self-renewal capacity and with limited proliferation capacity, they have no tumorigenicity.Breast cancer stem cells (BrCSCs) bearing the surface makers CD44⁺CD24^-Lin- were originally identified from primary tumor specimen and cell lines by Al-Hajj and his colleagues. Subsequently, Ponti et al reported that almost all the cells of mammospheres derived from two primary and one recurrent breast cancers were CD44⁺CD24^-/low, and established a culture system of BrCSCs in vitro successfully. In a subsequent study, it was found that BrCSCs isolated from breast tumors had high ALDH activity, and the cells bearing both ALDH1 and CD44⁺CD24^- phenotype had high tumorigenicity and could generate a tumor from as few as 20 cells. In addition, BrCSCs can be enriched by sorting for side-population (SP) cells that efflux Hoechst 33342 or rhodamine, or Brdu label retention assays. However, BrCSCs isolated from breast cancer cells are only a subpopulation of tumorigenic cells that are composed of BrCSCs, progenitor cells and their differentiation of cells. The ture BrCSCs are still hiding among them.The CSCs theory holds that CSCs are likely share some properties of nomal setm cells, including relative quiescence, expression of high levels of ABC drug transporters and resisitance to apoptosis, so that tumors may have a small group of chemotherapeutics-resistance cells that can survive chemotherapy. The BrCSCs were also shown to be resistant to chemotherapy drugs, such as epirubicin. Utilizing the drug resistance characteristics of BrCSCs, they can be enriched in vivo and in vitro. Yu et al verified that primary breast cancers from patients received neoadjuvant chemotherapy were enriched for BrCSCs compared to untreated patients. Li et al developed a system that BrCSCs could be enriched by suspension culture combined with anti-cancer regimens. However, the form of enriched BrCSCs in breast cancer after chemotherapy remains unknown.In the present study, we isolated micirospheres, similar to the mammospheres Ponti et al reported, directly from tumor tissues of breast cancer patients received neoadjuvant chemotherapy. In order to facilitate presentation and distinguish with mammospheres previously reported, we name it chemotherapy microspheres (CMSs). By using the serum-free culture, induction of differentiation, cell phenotype analysis and stem cell marker detection, we showed that chemotherapy microsphere-derived cells (CMSDCs) had stem/progenitor cell features and were able to form tumors when as few as 103 cells were injected into the mammary fat pad of NOD/SCID mice. Accordingly, we speculated that chemotherapy pressure could lead to the amplification of BrCSC s and formation of sphere-like CMSs.Partâ… Isolation, Propagation and Phenotypic Charaterization of Chemotherapy Microsphere-derived CellsObjective: To explore the self-renewal and multilineage differentiation capacity of CMSDCs derived from three specimens, observe the morphological differences between CMSs and MSs, charaterize the cell phenotypes of CMSDCs and their differentiated cells and identify whether CMSDCs have cancer stem cells features.Methods: Fresh specimens of breast tumors were collected in DMEM/F12 containing penicilin and streptomycin before surgery, received in the Laboratory within 30 minutes and dissociated mechanically with scissors into samll pieces. Erythrocyte lysis buffer was used to remove red blood cells and trypsin digestion was not used to avoid the damage of the spheres in the process. After filtration through a 74μm pore sieve, CMSs were seeded in serum free DMEM/F12 media containing 10 ng/ml bFGF, 20 ng/ml hEGF, B27, in a 37℃incubator containing 5% CO₂. The sphere shape and refraction changes were observed every day. The CMSs were passaged by enzymatical dissociation to single cells after 3 days in culture and reseeded in the media described above at 1×103 cells/ml. The newly formed CMSs after 5 days were collected by centrifugation at 1000 rpm. For serial passaging to determine self-renewal ability of CMSDCs, total number of CMSs obtained at each passage was analyzed micoroscopically for single cells and reseeded in culture bottles. Conversely, the differentiation of CMSDCs was induced by culturing them in DMEM/F12 supplemented with 5% fetal bovine serum without growth factors. The medium was changed about 3 days and follow-up experiments were conducted when the cultures reached 90% of confluence. The expression of epithelial specific antigen (ESA) and Nestin on CMSDCs was detected by immunofluorescence under confocal laser scanning microscopy, in addition, the expression cytokeratin 14, 18 and 19 on CMSDCs and their differentiated cells was detected using the same method as above. The ratio of ALDH1⁺ cells of CMSDCs and their differentiated cells was evaluated by flow cytometry.Results: The cell clusters of CMSs had discrepancy in size, weak light refraction. After 24 hours, a small number of single cells shed from the surface of microspheres, which had strong light refraction and were similar to MSs previously reported in the literature. A layer of necrotic cells could be seen after 3 days of culture. The CMSs were dissociated into single cells via trypsin at each passage and further inoculated into similar medium in the presence of growth factors for serial mammosphere assays. after 2 days, it can be seen that the new CMSs formed, gradually increased. Five days later, the CMSs with strong light refraction were no longer increasing. The necrotic cells did not exist on the outer layer of the newly formed CMSs. The time interval between each passaging was 5 days. The CMSs could be expanded as floating spheres at least up to 15 generations in vitro. When CMSDCs were cultured under differentiating conditions (i.e., after withdrawal of growth factors and addition of 5% FBS) for 3 days, floating cells could adhere and differentiate, acquiring an epithelial morphology. The CMSDCs could form new CMSs with strong refraction by serial sphere formation assay. The CMSDCs expressed the breast stem cell maker cytokeratin 19, but not cytokeratin 14 and 18, which mark the myoepithelial and luminal cells, respectively, but the differentiated cells expressed cytokeratin 14 and 18, but not cytokeratin 19. In three specimens, the CMSDCs harbor highly ALDH1-positive cells which accounted for only 0.50%-1.28% of the total cells in their differentiated cells. Only the CMSDCs of one specimen contained CD44+CD24- phenotype cells.Conclusion: The CMSDCs isolated from breast cancer tissues have self-renewal capacity, multiple differentiation potential and contain a high proportion of of ALDH1+ phenotype cells. CMSs may be the microspheres enriched for BrCSCs which rapidly undergo amplification under chemotherapy pressure.Partâ…¡The Drug Resistance of CMSDCs and Expression of Oct-4 and ABCG2 in the CMSDCsObjective: To further identify the stem cell-like properties of CMSDCs derived from the other three specimens by detecting drug resistance of CMSDCs and their differentiated progeny, the expression of stem cell markers Oct-4 and the multiple drug-resistant marker ABCG2 in the CMSDCs and their differentiated cells.Methods: The growth inhibition of CMSDCs and their differentiated counterparts was determined by MTT assay. The expression of Oct-4 gene in the CMSDCs and their differentiated cells was analyzed by RT-PCR; The expression level of ABCG2 protein in the CMSDCs and their differentiated cells was analyzed by Western blot.Results: The CMSDCs were resistant to docetaxel and epirubicin, but their differentiated counterparts were sensitive to these drugs. High levels of Oct-4 and ABCG2 expression were observed in the CMSDCs, but the expression of Oct-4 and ABCG2 was reduced or absent in the differentiated cells.Conclusion: Our results demonstrate that CMSDCs express the stem cell maker Oct-4 and the multiple drug-resistant marker ABCG2. CMSDCs show characteristics consistent with those of cancer stem cells.Partâ…¢Tumorigenicity of CMSDCs in the Mammary Fat Pad of NOD/SCID MiceObjective: To assess the tumorigenic potential of the CMSDCs derived from the three specimens mentioned in the first part of this dissertation in NOD/SCID mice, investigate the biological characteristics of mouse tumor cells and further identify whether the CMSDCs have the characteristics of BrCSCs.Methods: Tumorigenicity of CMSDCs in vivo was evaluated by implanting them into NOD/SCID mice to constitute animal model. The ratio of ALDH1⁺ cells of mouse tumor cells was evaluated by flow cytometry. The changes in cell morphology were observed when the mouse tumor cells were plated in serum-free medium containing growth factors. Differentiation was induced by culturing spheroid cells in medium supplemented with serum without growth factors.Results: New tumor could form when 10^\3 CMSDCs were injected into the mammary fat pad of NOD/SCID mice, but no tumor generated when the same number of differentiated cells were injected. The mouse tumor cells contained a small amount of ALDH1⁺ phenotype cells. MSs could be formed when the mouse tumor cells were cultured in serum-free medium. The MSDCs displayed stem-like properties for they could maintain in continuous passage in serum-free medium and differentiate to monolayer of spindle-shaped cells in the media supplemented with serum.Conclusion: The mouse tumor cells have the same properties as the parental cell population. CMSDCs is highly tumorigenic, with stem cell characteristics, can be used for breast cancer stem/progenitor cell research.Partâ…£The Influence of Different Cycles of Chemotherapy on the Formation of CMSDCsObjective: To isolate and culture of BrCSCs from 29 breast cancer patients received different cycles of chemotherapy, investigate their proliferation and differentiation biological characteristics in vitro and the impact of neoadjuvant chemotherapy on the isolation and culture of BrCSCs.Methods: Freshly isolated cells from tumor tissues of breast cancer patients received neoadjuvant chemotherapy were cultured in serum-free media supplemented with growth factors. It was observed whether there was sphere-like cell clusters formation. Proliferative capacity of CMSDCs in serum free medium was assessed by determination of growth curve. The self-renewal capacity of CMSDCs was evaluated by colony formation assay. Conversely, the differentiation of CMSDCs was induced by culturing them in media supplemented with 5% fetal bovine serum without growth factors. Differentiation potential of CMSDCs was also assessed by determination of growth curve. The ratio of ALDH1⁺ cells of CMSDCs was evaluated by flow cytometry.Results: The 7 specimens with one cycle of chemotherapy cultured in serum free medium were no CMSs formation, but the 3 of 6 specimens with two cycle of chemotherapy could form CMSs in the medium described above. Interestingly, the 11 of 16 specimens with more than three cycle of chemotherapy could be directly isolated MSs, but the remaining 5 cases obtaind only a small amount of necrotic cells and tissue fragments. CMSDCs could adhere and differentiate in media under differentiating conditions. The CMSDCs received more than 3 cycles of chemotherapy had greater proliferation capacity, self-renewal capacity, differentiation capacity in vitro and a higher proportion of ALDH1⁺ phenotype cells compared to that of CMSDCs received 2 cycles of chemotherapy. CMSs were more common among basal-like and Her2 subtypes tumors than other subtypes. Conclusion: Isolation of BrCSCs from breast cancer tissues after adjuvant chemotherapy should select samples after three cycles of chemotherapy; BrCSCs can be enriched by new adjuvant chemotherapy; The phenotype of ALDH1⁺ BrCSCs distributed more widely in breast cancer tissues than that of the CD44⁺CD24^- ones. The formation of CMSs was associated with the basal-like and Her2 subtypes of breast carcinoma.