Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment

Many types of tumors are organized in a hierarchy of heterogeneous cell populations. The cancer stem-like cells (CSCs) hypothesis suggests that tumor development and metastasis are driven by a minority population of cells, which are responsible for tumor initiation, growth and recurrences. The inability to efficiently eliminate CSCs during chemotherapy, together with CSCs being highly tumorigenic and invasive, may result in treatment failure due to cancer relapse and metastases. CSCs are emerging as a promising target for the development of translational cancer therapies. Ideal panacea for cancer would kill all malignant cells, including CSCs and bulk tumor cells. Since both chemotherapy and CSCs-specific therapy are insufficient to cure cancer, we propose combination therapy with CSCs-targeted agents and chemotherapeutics for improved breast cancer treatment. We generated in vitro mammosphere of 2 breast cancer cell lines, and demonstrated ability of mammospheres to grow and enrich cancer cells with stem-like properties, including self-renewal, multilineage differentiation and enrichment of cells expressing breast cancer stem-like cell biomarkers CD44⁺/CD24^?/low. The formation of mammospheres was significantly inhibited by salinomycin, validating its pharmacological role against the cancer stem-like cells. In contrast, paclitaxel showed a minimal effect on the proliferation and growth of breast cancer stem-like cells. While combination therapies of salinomycin with conventional chemotherapy (paclitaxel or lipodox) showed a potential to improve tumor cell killing, different subtypes of breast cancer cells showed different patterns in response to the combination therapies. While optimization of combination therapy is warranted, the design of combination therapy should consider phenotypic attributes of breast cancer types.     

Testing the cancer stem cell hypothesis in melanoma: The clinics will tell

Whether tumorigenic cancer stem cells (CSCs) exist in melanoma has been the focus of much controversy in recent years. A number of studies have pointed to the existence of melanoma cell sub-populations that act as CSCs and can be distinguished from other tumor cells based on specific surface marker expression or specific properties such as the capacity for extensive self-renewal. Other studies failed to identify melanoma stem cells and proposed that the potential to initiate tumors is a wide spread feature in melanoma inherent to most if not all cells of the tumor mass. As with normal stem cells, the term CSC is based on an operational definition, indicating not just a tumor-initiating cell, but also a cell with the capacity to sustain long-term tumor propagation. Therefore, the experimental set-up chosen to identify putative CSCs in melanoma is crucial: Both the method of tumor cell preparation and the procedure used to assess CSC properties in vivo influence the experimental outcome and hence its interpretation. In this review, we summarize our current knowledge on CSCs and the role of stem cell properties in melanoma and discuss recent findings with respect to their clinical relevance.     

Cancer stem cells: distinct entities or dynamically regulated phenotypes?

The origins of tumor-propagating neoplastic stem-like cells [cancer stem cells (CSC)] and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features (i.e., transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of therapeutic strategies to deplete tumors of their tumor-propagating and treatment-resistant cell subpopulations.     

Cancer stem-like cells contribute to cisplatin resistance and progression in bladder cancer

A variety of cancer stem-like cells (CSCs) have been shown to be responsible for cancer tumorigenicity, relapse and metastasis. Despite several reports demonstrating the presence of CSCs in human bladder cancer, their identities are still under debate, and few studies have examined their roles in cisplatin resistance and related tumor progression. In this study, a subpopulation of CSCs was enriched following cisplatin selection from the bladder cell line T24. The cisplatin-resistant T24 cells displayed a greater self-renewal capacity as demonstrated by higher levels of sphere formation and stem cell marker expression, contained a larger proportion of side population cells and exhibited higher tumorigenicity. They also possessed epithelial-mesenchymal transition characteristics. Furthermore, a strong correlation between the levels of Bmi1 and Nanog expression and the degree of malignancy of urothelial cell carcinomas tissues was observed. We provide the first direct evidence that CSC-like cells exist in the population of cisplatin-resistant bladder cancer cells and may play a role in the progression and drug resistance of bladder cancer.     

Breast cancer stem cells as last soldiers eluding therapeutic burn: A hard nut to crack

Cancer stem cells (CSCs) are found in many cancer types, including breast carcinoma. Breast cancer stem cells (BCSCs) are considered as seed of cancer formation and they are associated with metastasis and genotoxic drug resistance. Several studies highlighted the presence of BCSCs in tumor microenvironment and they are accentuated with several carcinoma events including metastasis and resistance to genotoxic drugs and they also rebound after genotoxic burn. Stemness properties of a small population of cells in carcinoma have provided clues regarding the role of tumor microenvironment in tumor pathophysiology. Hence, insights in cancer stem cell biology with respect to molecular signaling, genetics and epigenetic behavior of CSCs have been used to modulate tumor drug resistance due to genotoxic drugs and signaling protein inhibitors. This review summarizes major scientific breakthroughs in understanding the contribution of BCSCs towards tumor's capability to endure destruction inflicted by molecular as well as genotoxic drugs.     

Cancer stem cells and metastasis

Cancer stem cells (CSCs) represent a subpopulation of tumour cells endowed with self-renewal and multi-lineage differentiation capacity but also with an innate resistance to cytotoxic agents, a feature likely to pose major clinical challenges towards the complete eradication of minimal residual disease in cancer patients. Operationally, CSCs are defined by their tumour-propagating ability when serially transplanted into immune-compromised mice and by their capacity to fully recapitulate the original heterogeneity of cell types observed in the primary lesions they are derived from. CSCs were first identified in haematopoietic malignancies and later in a broad spectrum of solid tumours including those of the breast, colon and brain. Notably, several CSC characteristics are relevant to metastasis, such as motility, invasiveness and, as mentioned above, resistance to DNA damage-induced apoptosis. Here, we have reviewed the current literature on the relation between CSCs and metastasis formation. Preliminary studies on cancer cell lines and patient-derived material suggest a rate-limiting role for stem-like cells in the processes of tumour cell dissemination and metastasis formation. However, additional studies are needed to deliver formal proof of their identity as the cell of origin of recurrences at distant organ sites. Nevertheless, several studies have already provided pre-clinical evidence of the efficacy of novel therapies directed against disseminated CSCs.     

Tumor stem cells

Stem cells possess two basic characteristics: they are able to renew themselves and to develop into different cell types. The link between normal stem cells and tumor cells could be examined in three aspects: what are the differences and similarities in the control of self-renewal capacity between stem cells and tumor cells; whether tumor cells arise from stem cells; do tumorous stem cells exist? Since tumor cells also exhibit self-renewal capacity, it seems plausible that their regulation is similar to that of the stem cells. The infinite self-renewal ability (immortalization) is assured by several, so far only partly known, mechanisms. One of these is telomerase activity, another important regulatory step for survival is the inhibition of apoptosis. Other signal transduction pathways in stem cell regulation may also play certain roles in carcinogenesis: e.g. Notch, Sonic hedgehog (SHH), and Wnt signals. Existence of tumor stem cells was suggested since it is simpler to retain the self-renewal capacity than to reactivate the immortality program in an already differentiated cell. Moreover, stem cells live much longer than the differentiated ones, and so they are exposed for a long period of time to impairments, collecting gene errors leading to the breakdown of the regulation. However, it is still an open question whether all cells in the tumor possess the capacity that produces this tissue or not, that is: are there tumor stem cells or there are not. If tumor stem cells exist, they would be the main target for therapy: only these must be killed since the other tumor cells possess limited proliferative capacity, therefore limited life span. The only problem is that during tumor progression stem-like cells can develop continuously and the identification but mainly the prevention of their formation is still a great challenge.(Pathology Oncology Research Vol 10, No 2, 69–73)     

Complexity of cancer stem cells

Heterogeneity of tumor tissue has been accounted for in recent years by a hierarchy‐based model in which cancer stem cells (CSCs) have the ability both to self‐renew and to give rise to differentiated tumor cells and are responsible for the overall organization of a tumor. Research into CSCs has progressed rapidly and concomitantly with recent advances in the biology of normal tissue stem cells, resulting in the identification of CSCs in a wide range of human tumors. Studies of mouse models of human cancer have provided further insight into the characteristics of CSCs as well as a basis for the development of novel therapies targeted to these cells. However, recent studies have revealed complexities, such as plasticity of stem cell properties and clonal diversity of CSCs, in certain tumor types that have led to revision of the original CSC model. In this review, we summarize the history of the discovery and characterization of CSCs, as well as address recent advances that have revealed the complexity of these cells and their therapeutic implications.     

Constitutive expression and activation of stress response genes in cancer stem-like cells/tumour initiating cells: Potent targets for cancer stem cell therapy

Abstract

Cancer stem-like cells (CSCs)/tumour-initiating cells (TICs) are defined as the small population of cancer cells that have stem cell-like phenotypes and high capacity for tumour initiation. These cells may have a huge impact in the field of cancer therapy since they are extremely resistant to standard chemoradiotherapy and thus are likely to be responsible for disease recurrence after therapy. Therefore, extensive efforts are being made to elucidate the pathological and molecular properties of CSCs/TICs and, with this information, to establish efficient anti-CSC/TIC targeting therapies. This review considers recent findings on stress response genes that are preferentially expressed in CSCs/TICs and their roles in tumour-promoting properties. Implications for a novel therapeutic strategy targeting CSCs/TICs are also discussed.     

Targeting the K-Ras - JNK axis eliminates cancer stem-like cells and prevents pancreatic tumor formation

Cancer cells with self-renewal and tumor-initiating capacity, either quiescent (cancer stem cells, CSCs) or proliferating (cancer stem-like cells, CSLCs), are now deemed responsible for the pervasive therapy resistance of pancreatic cancer, one of the deadliest human cancers characterized by high prevalence of K-Ras mutation. However, to date, much remains unknown how pancreatic CSCs/CSLCs are regulated. Here we show that the K-Ras – JNK axis plays a pivotal role in the maintenance of pancreatic CSCs/CSLCs. In vitro inhibition of JNK, either pharmacological or genetic, caused loss of the self-renewal and tumor-initiating capacity of pancreatic CSLCs. Importantly, JNK inhibition in vivo via systemic JNK inhibitor administration, which had no discernible effect on the general health status of mice, efficiently depleted the CSC/CSLC population within pre-established pancreatic tumor xenografts. Furthermore, knockdown of K-Ras in pancreatic CSLCs with K-Ras mutation led to downregulation of the JNK pathway as well as in loss of self-renewal and tumor-initiating capacity. Together, our findings suggest that pancreatic CSCs/CSLCs are dependent on K-Ras activation of JNK and also suggest that the K-Ras – JNK axis could be a potential target in CSC/CSLC-directed therapies against pancreatic cancer.     

Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic

Recently, several human cancers including leukemia and breast and brain tumors were found to contain stem-like cancer cells called cancer stem cells (CSC). Most of these CSCs were identified using markers that identify putative normal stem cells. In some cases, stem-like cancer cells were identified using the flow cytometry-based side population technique. In this study, we first show that approximately 30% of cultured human cancer cells and xenograft tumors examined ( approximately 30 in total) possess a detectable side population. Purified side population cells from two cell lines (U373 glioma and MCF7 breast cancer) and a xenograft prostate tumor (LAPC-9) are more tumorigenic than the corresponding non-side population cells. These side population cells also possess some intrinsic stem cell properties as they generate non-side population cells in vivo, can be further transplanted, and preferentially express some "stemness" genes, including Notch-1 and beta-catenin. Because the side population phenotype is mainly mediated by ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, we subsequently studied ABCG2+ and ABCG2- cancer cells with respect to their tumorigenicity in vivo. Although side population cells show increased ABCG2 mRNA expression relative to the non-side population cells and all cancer cells and xenograft tumors examined express ABCG2 in a small fraction (0.5-3%) of the cells, highly purified ABCG2+ cancer cells, surprisingly, have very similar tumorigenicity to the ABCG2- cancer cells. Mechanistic studies indicate that ABCG2 expression is associated with proliferation and ABCG2+ cancer cells can generate ABCG2- cells. However, ABCG2- cancer cells can also generate ABCG2+ cells. Furthermore, the ABCG2- cancer cells form more and larger clones in the long-term clonal analyses and the ABCG2- population preferentially expresses several "stemness" genes. Taken together, our results suggest that (a) the side population is enriched with tumorigenic stem-like cancer cells, (b) ABCG2 expression identifies mainly fast-cycling tumor progenitors, and (c) the ABCG2- population contains primitive stem-like cancer cells.     

CD133与肿瘤干细胞研究进展

越来越多的证据表明肿瘤中存在肿瘤干细胞(cancer stem cells),并且其与肿瘤的增殖、转移、复发和对放化疗不敏感关系密切.因此,肿瘤治疗应当针对肿瘤干细胞,通过特异表面标记分选肿瘤干细胞是研究其生长特点的前提.近年来,CD133为研究最多的在于细胞(stem cell)和多种组织肿瘤干细胞表面独立表达的特异标记分子.通过CD133可以分选干细胞、前体细胞和肿瘤干细胞.众多研究表明,CD133~+肿瘤细胞与肿瘤的自我更新、分化潜能、信号传导调控、药物耐受、复发和预后等均有相关性.CD133~+细胞有望在干细胞相关疾病的治疗和肿瘤靶向治疗中发挥巨大作用.     

Identification of a cancer stem-like population in the Lewis lung cancer cell line

Objective: Although various human cancer stem cells (CSCs) have been defined, their applications are restricted to immunocompromised models. Developing a novel CSC model which could be used in immunocompetent or transgenic mice is essential for further understanding of the biomolecular characteristics of tumor stem cells. Therefore, in this study, we analyzed murine lung cancer cells for the presence of CSCs. Methods: Side population (SP) cells were isolated by fluorescence activated cell sorting, followed by serum-free medium (SFM) culture, using Lewis lung carcinoma cell (LLC) line. The self-renewal, differentiated progeny, chemosensitivity, and tumorigenic properties in SP and non-SP cells were investigated through in vitro culture and in vivo serial transplantation. Differential expression profiles of stem cell markers were examined by RT-PCR. Results: The SP cell fraction comprised 1.1% of the total LLC population. SP cells were available to grow in SFM, and had significantly enhanced capacity for cell proliferation and colony formation. They were also more resistant to cisplatin in comparison to non-SP cells, and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA expression of Oct-4, ABCG2, and CD44. Conclusion: We identified SP cells from a murine lung carcinoma, which possess well-known characteristics of CSCs. Our study established a useful model that should allow investigation of the biological features and pharmacosensitivity of lung CSCs, both in vitro and in syngeneic immunocompetent or transgenic/knockout mice.     

Cancer stem cell dynamics in tumor progression and metastasis: Is the microenvironment to blame?

Stem cells are defined by their self-renewal capacity and the ability to give rise to all differentiated progeny necessary for one specific organ. These two characteristics are also inherent in cancer stem cells (CSCs), which are thought to be the only subpopulation within a tumor endowed with tumorigenic potential. CSCs combine many features that render cancer one of the leading causes of death in the Western world: metastasis, tumor recurrence, and therapy refractoriness. Strikingly, CSCs are not a fixed entity, but differentiated tumor cells are able to revert to a stem-like state. Thus, CSCs are not only intrinsically programmed to fulfill their detrimental roles, but are orchestrated by stromal cells residing in their vicinity and forming the CSC niche. Yet, this relationship is not a one-way road: CSCs are able to manipulate stromal cells to their needs, not only in the primary tumor, but also in distant organs and thus prime the foreign soil for their arrival by inducing a premetastatic niche. The suggested plasticity between the differentiation states of cancer cells and the regulation by microenvironmental cues provides new starting-points for novel cancer therapies.     

Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation

Introduction  

The irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs.     

Tumor‐derived spheroids: Relevance to cancer stem cells and clinical applications

Recently, many types of in vitro 3‐D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3‐D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2‐D monolayer cultures, and include tumor‐derived organoids, tumor‐derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3‐D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor‐derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell‐related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC‐related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor‐derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor‐derived spheroids may prove to be instrumental for a high‐throughput screening platform or for the cultivation of CSC‐related tumor cells found in the circulation or body fluids.     

Stem cells, quiescence and rectal carcinoma: an unexplored relationship and potential therapeutic target

Stem cells are responsible for maintaining differentiated cell numbers during normal physiology and at times of tissue stress. They have the unique capabilities of proliferation, self-renewal, clonogenicity and multi-potentiality. It is a widely held belief that stem-like cells, known as cancer stem cells (CSCs), maintain tumours. The majority of currently identified intestinal stem cell populations appear to be rapidly cycling. However, quiescent stem cell populations have been suggested to exist in both normal intestinal crypts and tumours. Quiescent CSCs may have particular significance in the modern management of colorectal cancer making their identification and characterisation a priority. In this review, we discuss the current evidence surrounding the identification and microenvironmental control of stem cell populations in intestinal crypts and tumours as well as exploring the evidence supporting the existence of a quiescent stem and CSC population in the gut and other tissues.     

First among equals: the cancer cell hierarchy

Primary cancer cells exhibit heterogeneity in their proliferative ability. The cancer stem cell (CSC) model accounts for this heterogeneity by proposing that each cancer consists of a small population of CSCs that are capable of unlimited growth and self-renewal and a much larger population of cells, descendants of the CSCs, that have lost self-renewal capacity. The CSC model has important implications for cancer therapy. Eradication of CSCs, the cells responsible for maintenance of the neoplasm, would be necessary and sufficient to achieve cure. By extension, both the frequency of stem cells in a tumor and their propensity to undergo self-renewal (P_sr) would have a direct impact on the curability of that tumor. The P_sr is a critical biological characteristic of CSCs—small differences in P_sr have enormous impact on the probability of success in cancer therapy. Differentiation therapy, defined as treatment that reduces the P_sr of CSCs, is one approach to targeting CSCs.     

High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant

Chemo-resistance is a clinical barrier to more effective anti-cancer therapy. In this context, cancer stem-like cells (CSCs) are thought to be chemo-resistant, resulting in tumor recurrence and distant metastasis. Our hypothesis is that chemo-resistance in CSCs is driven, in part, by enhanced mitochondrial function. Here, we used breast cell lines and metastatic breast cancer patient samples to begin to dissect the role of mitochondrial metabolism in conferring the CSC phenotype. More specifically, we employed fluorescent staining with MitoTracker (MT) to metabolically fractionate these cell lines into mito-high and mito-low sub-populations, by flow-cytometry. Interestingly, cells with high mitochondrial mass (mito-high) were specifically enriched in a number of known CSC markers, such as aldehyde dehydrogenase (ALDH) activity, and they were ESA+/CD24-/low and formed mammospheres with higher efficiency. Large cell size is another independent characteristic of the stem cell phenotype; here, we observed a >2-fold increase in mitochondrial mass in large cells (>12-μm), relative to the smaller cell population (4–8-μm). Moreover, the mito-high cell population showed a 2.4-fold enrichment in tumor-initiating cell activity, based on limiting dilution assays in murine xenografts. Importantly, primary human breast CSCs isolated from patients with metastatic breast cancer or a patient derived xenograft (PDX) also showed the co-enrichment of ALDH activity and mitochondrial mass. Most significantly, our investigations demonstrated that mito-high cells were resistant to paclitaxel, resulting in little or no DNA damage, as measured using the comet assay. In summary, increased mitochondrial mass in a sub-population of breast cancer cells confers a stem-like phenotype and chemo-resistance. As such, our current findings have important clinical implications for over-coming drug resistance, by therapeutically targeting the mito-high CSC population.     

Therapeutic strategies targeting cancer stem cells

Increasing studies have demonstrated a small proportion of cancer stem cells (CSCs) exist in the cancer cell population. CSCs have powerful self-renewal capacity and tumor-initiating ability and are resistant to chemotherapy and radiation. Conventional anticancer therapies kill the rapidly proliferating bulk cancer cells but spare the relatively quiescent CSCs, which cause cancer recurrence. So it is necessary to develop therapeutic strategies acting specifically on CSCs. In recent years, studies have shown that therapeutic agents such as metformin, salinomycin, DECA-14, rapamycin, oncostatin M (OSM), some natural compounds, oncolytic viruses, microRNAs, cell signaling pathway inhibitors, TNF-related apoptosis inducing ligand (TRAIL), interferon (IFN), telomerase inhibitors, all-trans retinoic acid (ATRA) and monoclonal antibodies can suppress the self-renewal of CSCs in vitro and in vivo. A combination of these agents and conventional chemotherapy drugs can significantly inhibit tumor growth, metastasis and recurrence. These strategies targeting CSCs may bring new hopes to cancer therapy.