Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

G Protein-Coupled Receptor 87 (GPR87) Promotes the Growth and Metastasis of CD133+ Cancer Stem-Like Cells in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a prevalent disease worldwide, and the majority of HCC-related deaths occur due to local invasion and distant metastasis. Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have been hypothesized to be responsible for metastatic disease. Recently, we and others have identified a CSC population from human HCC cell lines and xenograft tumors characterized by their expression of CD133. However, the precise molecular mechanisms by which CD133⁺ cancer stem-like cells mediate HCC metastasis have not been sufficiently analyzed. Here, we have sorted HCC cells using CD133 as a cancer stem cell (CSC) marker by magnetic-activated cell sorting (MACS) and demonstrated that the CD133⁺ HCC cells not only possess greater migratory and invasive capacity in vitro but are also endowed with enhanced metastatic capacity in vivo and in human HCC specimens when compared to CD133⁻ HCC cells. Gene expression analysis of the CD133⁺ and CD133⁻ cells of the HCC line SMMC-7721 revealed that G protein-coupled receptor 87 (GPR87) is highly expressed in CD133⁺ HCC cells. In this study, we explored the role of GPR87 in the regulation of CD133 expression. We demonstrated that the overexpression of GPR87 up-regulated CD133 expression, promoted CSC-associated migratory and invasive properties in vitro, and increased tumor initiation in vivo. Conversely, silencing of GPR87 expression reduced the levels of CD133 expression. Conclusion: GPR87 promotes the growth and metastasis of CD133⁺ cancer stem-like cells, and our findings may reveal new targets for HCC prevention or therapy.     

Response to Stress in Early Tumor Colonization Modulates Switching of CD133-Positive and CD133-Negative Subpopulations in a Human Metastatic Colon Cancer Cell Line,SW620

According to the cancer stem cell (CSC) model, higher CD133 expression in tumor tissue is associated with metastasis and poor prognosis in colon cancer. As such, the CD133-positive (CD133⁺) subpopulation of cancer cells is believed to play a central role in tumor development and metastatic progression. Although CD133⁺ cells are believed to display more CSC-like behavior and be solely responsible for tumor colonization, recent research indicates that CD133⁻ cells from metastatic colon tumors not only also possess colonization capacity but also promote the growth of larger tumors in a mouse model than CD133⁺ cells, suggesting that an alternative mechanism of metastasis exists. This study investigated this possibility by examining the cell viability, tumorigenicity, and proliferation and growth capacity of the CD133⁺ and CD133⁻ subpopulations of the SW620 cell line, a human metastatic colon cancer cell line, in both an in vitro cell model and an in vivo mouse model. While both SW620 ^CD133− and SW620^CD133+ cells were found to engage in bidirectional cell-type switching in reaction to exposure to environmental stressors, including hypoxia, a cell adhesion-free environment, and extracellular matrix stimulation, both in vitro and in vivo, CD133⁻ cells were found to have a growth advantage during early colonization due to their greater resistance to proliferation inhibition. Based on these findings, a hypothetical model in which colon cancer cells engage in cell-type switching in reaction to exposure to environmental stressors is proposed. Such switching may provide a survival advantage during early colonization, as well as that explain previous conflicting observations.     

Prominin 1/CD133 Endothelium Sustains Growth of Proneural Glioma

In glioblastoma high expression of the CD133 gene, also called Prominin1, is associated with poor prognosis. The PDGF-driven proneural group represents a subset of glioblastoma in which CD133 is not overexpressed. Interestingly, this particular subset shows a relatively good prognosis. As with many other tumors, gliobastoma is believed to arise and be maintained by a restricted population of stem-like cancer cells that express the CD133 transmembrane protein. The significance of CD133⁺ cells for gliomagenesis is controversial because of conflicting supporting evidence. Contributing to this inconsistency is the fact that the isolation of CD133⁺ cells has largely relied on the use of antibodies against ill-defined glycosylated epitopes of CD133. To overcome this problem, we used a knock-in lacZ reporter mouse, Prom1^lacZ/+, to track Prom1⁺ cells in the brain. We found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that express markers characteristic of the neuronal, glial or vascular lineages. In proneural tumors derived from injection of RCAS-PDGF into the brains of tv-a;Ink4a-Arf^−/− Prom1^lacZ/+ mice, Prom1⁺ cells expressed markers for astrocytes or endothelial cells. Mice co-transplanted with proneural tumor sphere cells and Prom1⁺ endothelium had a significantly increased tumor burden and more vascular proliferation (angiogenesis) than those co-transplanted with Prom1⁻ endothelium. We also identified specific genes in Prom1⁺ endothelium that code for endothelial signaling modulators that were not overexpressed in Prom1⁻ endothelium. These factors may support proneural tumor progression and could be potential targets for anti-angiogenic therapy.     

CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271⁺ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271⁺ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271⁺ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271⁺ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.     

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56^hi/GD2⁺/CD81^hi), primitive neuroectodermal tumors (CD271^hi/CD99⁺), Wilms tumors (>1 cell population), rhabdomyosarcoma (_nuMYOD1⁺/_numyogenin⁺), carcinomas (CD45⁻/EpCAM⁺), germ cell tumors (CD56⁺/CD45⁻/NG2⁺/CD10⁺) and eventually also hemangiopericytomas (CD45⁻/CD34⁺). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

Tumor Cells Positive and Negative for the Common Cancer Stem Cell Markers Are Capable of Initiating Tumor Growth and Generating Both Progenies

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been “identified” in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC⁺ and CSC⁻ cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC⁺ and CSC⁻ cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC⁻ cells exhibit similar tumorigenesis and proliferation as CSC⁺ cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC⁺ and CSC⁻ cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC⁺-derived or CSC⁻-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.     

TGF-β1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line,leading to an increase of migration ability in the CD133+ A549 cell fraction

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-β1 (TGF-β1) is the major inductor of EMT. The aim of this study is to investigate TGF-β1''s effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-β1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133⁻ cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133⁻ cells. On the contrary, wound size reveals that TGF-β1 enhances motility in wild-type A549 as well as CD133⁺ and SP⁺ cells. For CD133⁻ and SP⁻ cells, TGF-β1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133⁺ A549 cells. In particular, SP⁺ and SP⁻ A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133⁻ cells no change in colony number was observable after TGF-β1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.     

Phage display screening identifies a prostate specific antigen (PSA)–/lo prostate cancer cell specific peptide to retard castration resistance of prostate cancer

Patients with prostate cancer (PCa) will eventually progress to castrate-resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) treatment. Prostate-specific antigen (PSA)^−/lo cells which harbor self-renewing long-term tumor-propagating cells that can be enriched using ALDH+CD44+α2β1+ and can initiate tumor development may represent a critical source of CRPC cells. Our purpose was to find a peptide that specifically targets PSA^−/lo PCa cells to retard the development of CRPC. PSA⁺ and PSA^−/lo cells were successfully separated from LNCaP xenograft tumors after prostate- PSAP-GFP vector infection and FACS. A variety of PSA^−/lo cells specifically targeting peptide (named as “TAP1” targeted affinity peptide 1) was identified by using phage display library screening. The highest binding rate in TAP1 binding cell subpopulations are identified to be among ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells. TAP1 significantly inhibited PCa growth both in vitro and in vivo. TAP1 significantly improved the anti-proliferation effect of the anti-androgens (Charcoal dextran-stripped serum (CDSS)+Bicalutamide, Enzalutamide) and chemotherapeutic agents (Abiraterone, Docetaxel, Etoposide) in vitro. TAP1 treatment shortens the length of telomeres in ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells and significantly reduces the expression of Homeobox B9 (HOXB9) and TGF-β2. In conclusion, PSA^−/lo PCa cell-specific targeting peptide (TAP1) that suppressed PCa cell growth both in vitro and in vivo and improved the drug sensitivities of anti-androgens and chemotherapeutic agents at least through shortening the length of telomere and reducing the expression of HOXB9 and TGF-β2. Therapeutic peptides that specifically target prostate cancer stem cell might be a very valuable and promising approach to overcome chemoresistance and prevent recurrence in patients with PCa.     

Circulating and Tumor-Infiltrating Myeloid-Derived Suppressor Cells in Patients with Colorectal Carcinoma

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of myeloid cells that suppress T cell immunity in tumor-bearing hosts. In patients with colon cancer, MDSCs have recently been described as Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ cells correlating with cancer stage, metastasis and chemotherapy response. To learn in more detail the dynamic change and clinical relevance of circulating and tumor-infiltrating Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSC in colorectal cancer, we harvested the blood from 64 patients with varying stage of colorectal cancer and tumor and matched paraneoplastic tissues from 5 patients with advanced colorectal cancer, subjected them to multicolor flow cytometric analysis of percentage, absolute number and phenotype of MDSC and finally characterized their immunosuppressive functions. Our results demonstrate that peripheral blood from colorectal cancer patients contains markedly increased percentage and absolute number of Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs compared with healthy individuals, and this increase is closely correlated with clinical cancer stage and tumor metastasis but not primary tumor size and serum concentrations of cancer biomarker. A similar increase of MDSCs was also observed in the tumor tissues. Phenotyping MDSCs shows that they express high CD13 and CD39, low CD115, CD117, CD124 and PD-L1, and devoid of CD14, CD15 and CD66b, reminiscent of precursor myeloid cells. MDSCs from cancer patients but not healthy donors have the immunosuppressive activity and were able to inhibit in vitro autologous T-cell proliferation. Collectively, this study substantiates the presence of increased immunosuppressive circulating and tumor-resident Lin^−/lowHLA-DR⁻CD11b⁺CD33⁺ MDSCs in patients with colorectal cancers correlating with cancer stage and metastasis, and suggests that pharmacologic blockade of MDSCs should be considered in future clinical trials.     

Isolation and Characterisation of Mesenchymal Stem/Stromal Cells in the Ovine Endometrium

ObjectiveMesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation.ResultsThere was a small population CD271⁺ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271⁺CD49f^- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271^-CD49f^- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271⁺ cells.ConclusionThis is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.     

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.     

Pancreatic Cancer Stem-like Cells Display Aggressive Behavior Mediated via Activation of FoxQ1

Subpopulations of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors, including pancreatic cancer (PC), and the existence of these cells is clinically relevant. Emerging evidence suggests that CSLCs participate in cell growth/proliferation, migration/invasion, metastasis, and chemo-radiotherapy resistance, ultimately contributing to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that isolated triple-marker-positive (CD44⁺/CD133⁺/EpCAM⁺) cells of human PC MiaPaCa-2 and L3.6pl cells behave as CSLCs. These CSLCs exhibit aggressive behavior, such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (CD44⁺/CD133⁺/EpCAM⁺) exhibit differential expression of more than 1,600 mRNAs, including FoxQ1, compared with the triple-marker-negative (CD44⁻/CD133⁻/EpCAM⁻) cells. The knockdown of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with the inhibition of EpCAM and Snail expression. Mouse xenograft tumor studies showed that CSLCs have a 100-fold higher potential for tumor formation and rapid tumor growth, consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental MiaPaCa-2 cells. The inhibition of FoxQ1 attenuated tumor formation and growth, and expression of CSC markers in the xenograft tumor derived from CSLCs of MiaPaCa-2 cells. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, further suggesting that targeting some of these genes could be important for the development of novel therapies for achieving better treatment outcome of PC.     

Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes

Introduction

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous.

Methods

Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis.

Results

The proportion of cells expressing CD44^highCD24^low/neg, side population (SP) cells, ALDH1⁺, CD49f^high, CD133^high, and CD34^high differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1⁺, CD34^low, and CD49f^high suggested properties of transit amplifying cells. Colony formation was higher from ALDH1⁻ and non-SP cells than ALDH1⁺ and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1⁺ cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined.

Conclusions

These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.