Cancer Stem Cell Hierarchy Research Articles

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-μFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-μFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-μFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-μFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-μFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

SYST-26 INVESTIGATING THE FUNCTIONAL ROLE OF GPNMB IN GLIOBLASTOMA AND THE TUMOR IMMUNE MICROENVIRONMENT AND ITS TARGETED ELIMINATION USING CAR-T CELLS

Abstract INTRODUCTION Glycoprotein nonmetastatic melanoma protein B (GPNMB) is active in the extracellular matrix of glioblastoma and presents a promising immunotherapy target for both tumor cells and immunosuppressive macrophages. METHODS Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue samples of 16 patient-matched primary/recurrent GBMs and 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. CRISPR/Cas9 was used to eliminate expression in GBM lines and proliferation and mouse survival times measured. GPNMB knockout clones generated in GL261 were engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNA sequencing at endpoint. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated using in vitro assays and GBM PDX models. RESULTS GPNMB was absent in most normal tissues but detected in residual PDX tumors treated orthotopically with CD133 CAR-Ts. Tissue microarrays and whole cell proteomics showed GPNMB upregulation in recurrent GBMs compared to primary (p=0.0349 vs. p=0.0033) while absent in normal tissues. Single cell sequencing data of patient GBMs revealed GPNMB was also highly expressed in tumor-associated macrophages. Eliminating GPNMB in GBM cell lines decreased proliferation (P<0.001) and prolonged survival times in all mouse models (P<0.01). GPNMB knockout clones displayed downregulation of hallmark signaling pathways of GBM such as PDGFR, TGF-beta, Integrins and Stats, and decreased innate/adaptive immune activation. CAR-T cytotoxicity and activation was observed in vitro and in vivo resulting in decreased tumor burden (P<0.001) and increased survival times (P<0.001). CONCLUSIONS We show GPNMB influences tumor-intrinsic biology of GBM and is also active in macrophages in the recurrent GBM immune microenvironment. By targeting GPNMB alongside CD133, combinatorial therapeutic regimens could target both the cancer stem cell hierarchy and its supportive niche. Administration of both CAR-T cell therapies to humanized mice engrafted with patient-derived GBMs is underway.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

Proteomic profiling and ROC analysis identify CD151 and ELAVL1 as potential therapy response markers for the antiviral drug in resistant TNBC.

Triple-negative breast cancer is high heterogeneous, aggressive, and metastatic with poor prognosis. Despite of advances in targeted therapies, TNBC has been reported to cause high morbidity and mortality. A rare subpopulation within the tumor microenvironment organized into a hierarchy of cancer stem cells is responsible for therapy resistance and tumor recurrence. Repurposing of antiviral drugs for cancer treatment is gaining momentum due to reduced cost, labour, and research time, but limited due to lack of prognostic, and predictive markers. The present study investigates proteomic profiling and ROC analysis to identify CD151 and ELAVL1 as potential therapy response markers for the antiviral drug 2-thio-6-azauridine (TAU) in resistant TNBC. The stemness of MDA-MB 231 and MDA-MD 468 adherent cells was enriched by culturing them under non-adherent and non-differentiation conditions. Then, CD151+ subpopulation was isolated and characterized for the enrichment of stemness. This study found that CD151 has overexpressed in stemness enriched subpopulations, and also showed CD44 high and CD24 low expression along with stem cell-related transcription factors octamer-binding transcription factor 4 (OCT4) and Sex determining Y-box 2 (SOX2). This study also found that TAU induced significant cytotoxicity and genotoxicity in the CD151+TNBC subpopulation and inhibited their proliferation by inducing DNA damage, cell cycle arrest at the G2M phase, and apoptosis. Further, a proteomic profiling study showed that the expression of CD151 along with ELAVL1, an RNA-binding protein, was significantly reduced with TAU treatment. KM plotter showed correlation of CD151 and ELAVL1 gene expression with a poor prognosis of TNBC. ROC analysis predicted and validated CD151 and ELAVL1 as best therapy response marker for TAU in TNBC. These findings provide new insight into repurposing antiviral drug TAU for treatment of metastatic and drug resistant TNBC.

Extirpating the cancer stem cell hydra: Differentiation therapy and Hyperthermia therapy for targeting the cancer stem cell hierarchy.

Ever since the discovery of cancer stem cells (CSCs), they have progressively attracted more attention as a therapeutic target. Like the mythical hydra, this subpopulation of cells seems to contribute to cancer immortality, spawning more cells each time that some components of the cancer cell hierarchy are destroyed. Traditional modalities focusing on cancer treatment have emphasized apoptosis as a route to eliminate the tumor burden. A major problem is that cancer cells are often in varying degrees of dedifferentiation contributing to what is known as the CSCs hierarchy and cells which are known to be resistant to conventional therapy. Differentiation therapy is an experimental therapeutic modality aimed at the conversion of malignant phenotype to a more benign one. Hyperthermia therapy (HT) is a modality exploiting the changes induced in cells by the application of heat produced to aid in cancer therapy. While differentiation therapy has been successfully employed in the treatment of acute myeloid leukemia, it has not been hugely successful for other cancer types. Mounting evidence suggests that hyperthermia therapy may greatly augment the effects of differentiation therapy while simultaneously overcoming many of the hard-to-treat facets of recurrent tumors. This review summarizes the progress made so far in integrating hyperthermia therapy with existing modules of differentiation therapy. The focus is on studies related to the successful application of both hyperthermia and differentiation therapy when used alone or in conjunction for hard-to-treat cancer cell niche with emphasis on combined approaches to target the CSCs hierarchy.

Abstract 1154: Investigating the functional role of GPNMB in glioblastoma and the tumor immune microenvironment and its targeted elimination using CAR-Ts

Abstract Glycoprotein nonmetastatic melanoma protein B (GPNMB) is known to be active in the extracellular matrix of glioblastoma and has been identified as a promising immunotherapy target for both tumor cells and immunosuppressive macrophages. Methods: Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue samples of 16 patient matched primary/recurrent GBMs as well as 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. Flow cytometry measured surface expression levels of GPNMB to confirm CAR-T accessibility. CRISPR/Cas9 was used to eliminate expression in GBM lines to measure proliferation and mouse survival times. GPNMB knockout clones were generated in GL261 and engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNAseq at endpoint. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated using standard in vitro assays and GBM PDX models. Results: The absence of GPNMB throughout most normal tissues validates the rationale of administering CAR-Ts as a safe modality for patients. GPNMB detected in residual tumors of PDX models treated orthotopically with CD133 CAR-Ts revealed it as a targetable subpopulation of GBM cells and a rational co-target alongside CD133 in the heterogeneous tumor. Tissue microarrays and whole cell proteomics found GPNMB to be upregulated in recurrent GBMs compared to primary (p=0.0349 and p=0.0033 respectively) while being absent in normal tissues. Single cell sequencing data of patient GBMs revealed GPNMB was also highly expressed in tumor-associated macrophages. Eliminating GPNMB in GBM cell lines decreased proliferation (P<0.001) and prolonged survival times in all mouse models (P<0.01) indicating its functional relevance. GPNMB knockout clones displayed downregulation of hallmark signalling pathways of GBM such as PDGFR, TGF-beta, Integrins and Stats, as well as decreased innate/adaptive immune activation. CAR-T cytotoxicity and activation was observed in vitro and in vivo resulting in decreased tumor burden (P<0.001) and increased survival times (P<0.001). Ultimately a CD133+ population was observed in residual tumors of GPNMB CAR-T treated mice at endpoint and surface expression of CD133 and GPNMB revealed co-expression and distinct populations. Conclusions: We show GPNMB influences tumor-intrinsic biology of GBM and is also active in macrophages in the recurrent GBM immune microenvironment. By targeting GPNMB along with CD133, combinatorial therapeutic regimens could target both the cancer stem cell hierarchy and its supportive niche. Administration of both CAR-T cell therapies to humanized mice engrafted with patient-derived GBMs will provide better cytotoxic coverage and potentially provide more durable therapeutic efficacy for GBM patients. Citation Format: Neil Savage, Franz J. Zemp, Nick Mikolajewicz, Hong Han, Chitra Venugopal, Chirayu Chokshi, Nazanin Tatari, Thomas Kislinger, Jason Moffat, Doug Mahoney, Sheila K. Singh. Investigating the functional role of GPNMB in glioblastoma and the tumor immune microenvironment and its targeted elimination using CAR-Ts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1154.

A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

IntroductionStem-like cancer cells or cancer stem cells (CSCs) may comprise a phenotypically and functionally heterogeneous subset of cells, whereas the molecular markers reflecting this CSC hierarchy remain elusive. The glycolytic enzyme alpha-enolase (ENO1) present on the surface of malignant tumor cells has been identified as a metastasis-promoting factor through its function of activating plasminogen. The expression pattern of surface ENO1 (sENO1) concerning cell-to-cell or CSC heterogeneity and its functional roles await further investigation.MethodsThe cell-to-cell expression heterogeneity of sENO1 was profiled in malignant cells from different types of cancers using flow cytometry. The subcellular localization of sENO1 and its functional roles in the invadopodia formation and cancer cell invasiveness were investigated using a series of imaging, molecular, and in vitro and in vivo functional studies.ResultsWe showed here that ENO1 is specifically localized to the invadopodial surface of a significant subset (11.1%-63.9%) of CSCs in human gastric and prostate adenocarcinomas. sENO1+ CSCs have stronger mesenchymal properties than their sENO1- counterparts. The subsequent functional studies confirmed the remarkable pro-invasive and pro-metastatic capacities of sENO1+ CSCs. Mechanistically, inhibiting the surface localization of ENO1 by downregulating caveolin-1 expression compromised invadopodia biogenesis, proteolysis, and CSC invasiveness.ConclusionsOur study identified the specific expression of ENO1 on the invadopodial surface of a subset of highly invasive and pro-metastatic CSCs. sENO1 may provide a diagnostically and/or therapeutically exploitable target to improve the outcome of patients with aggressive and metastatic cancers.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28 cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10 cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma.

An evolving paradigm of cancer stem cell hierarchies: therapeutic implications.

Over a decade of research has confirmed the critical role of cancer stem-like cells (CSCs) in tumor initiation, chemoresistance, and metastasis. Increasingly, CSC hierarchies have begun to be defined with some recurring themes. This includes evidence that these hierarchies are 'flexible,' with both cell state transitions and dedifferentiation events possible. These findings pose therapeutic hurdles and opportunities. Here, we review cancer stem cell hierarchies and their interactions with the tumor microenvironment. We also discuss the current therapeutic approaches designed to target CSC hierarchies and initial clinical trial results for CSC targeting agents. While cancer stem cell targeted therapies are still in their infancy, we are beginning to see encouraging results that suggest a positive outlook for CSC-targeting approaches.

Cancer stem cells in breast and prostate: Fact or fiction?

Since the introduction of the cancer stem cell (CSC) hypothesis, accumulating evidence shows that most cancers present stem-like niches. However, therapies aimed at targeting this niche have not been as successful as expected. New evidence regarding CSCs hierarchy, similarities with normal tissue stem cells and cell plasticity might be key in understanding their role in cancer biology and how to efficiently eliminate them. In this Chapter, we discuss what is known in breast and prostate CSCs from their initial discoveries to the current therapeutic efforts in the field. Future challenges towards better CSC identification and isolation strategies will be key to shed light into how CSCs could accurately be targeted in combination to traditional therapies to ultimately prolong patient survival.

Personalized regulation of glioblastoma cancer stem cells based on biomedical technologies: From theory to experiment (Review).

Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM represents >50% of primary tumors of the nervous system and ~20% of intracranial neoplasms. Standard treatment involves surgery, radiation and chemotherapy. However, the prognosis of GBM is usually poor, with a median survival of 15 months. Resistance of GBM to treatment can be explained by the presence of cancer stem cells (CSCs) among the GBM cell population. At present, there are no effective therapeutic strategies for the elimination of CSCs. The present review examined the nature of human GBM therapeutic resistance and attempted to systematize and put forward novel approaches for a personalized therapy of GBM that not only destroys tumor tissue, but also regulates cellular signaling and the morphogenetic properties of CSCs. The CSCs are considered to be an informationally accessible living system, and the CSC proteome should be used as a target for therapy directed at suppressing clonal selection mechanisms and CSC generation, destroying CSC hierarchy, and disrupting the interaction of CSCs with their microenvironment and extracellular matrix. These objectives can be achieved through the use of biomedical cellular products.

CD10\u2212/ALDH\u2212 cells are the sole cisplatin-resistant component of a novel ovarian cancer stem cell hierarchy

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10−/ALDH− CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10−/ALDH− CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10−/ALDH− CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10−/ALDH− CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

Cancer Stem Cells in Oral Cavity Squamous Cell Carcinoma: A Review.

Cancer stem cells (CSCs) have been identified in oral cavity squamous cell carcinoma (OCSCC). CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells and cancer cells that drive tumor growth. Studies of many cancer types including OCSCC have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple CSC subtypes within OCSCC, making investigation reliant on the use of multiple markers. This review examines the current knowledge in CSC markers OCT4, SOX2, NANOG, ALDH1, phosphorylated STAT3, CD44, CD24, CD133, and Musashi-1, specifically focusing on their use and validity in OCSCC CSC research and how they may be organized into the CSC hierarchy. OCSCC CSCs also express components of the renin–angiotensin system (RAS), which suggests CSCs may be novel therapeutic targets by modulation of the RAS using existing medications.

The Cellular Origin and Evolution of Breast Cancer.

In this review, we will discuss how the cell of origin may modulate breast cancer intratumoral heterogeneity (ITH) as well as the role of ITH in the evolution of cancer. The clonal evolution and the cancer stem cell (CSC) models, as well as a model that integrates clonal evolution with a CSC hierarchy, have all been proposed to explain the development of ITH. The extent of ITH correlates with clinical outcome and reflects the cellular complexity and dynamics within a tumor. A unique subtype of breast cancer, the claudin-low subtype that is highly resistant to chemotherapy and most closely resembles mammary epithelial stem cells, will be discussed. Furthermore, we will review how the interactions among various tumor cells, some with distinct mutations, may impact breast cancer treatment. Finally, novel technologies that may help advance our understanding of ITH and lead to improvements in the design of new treatments also will be discussed.

The bad seed gardener: Deubiquitinases in the cancer stem-cell signaling network and therapeutic resistance.

Tumors are comprised of highly heterogeneous populations of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumors in vivo. These rare cancer stem cells (CSCs) have been regarded as the "bad seeds" accounted for tumor initiation, progression, metastasis, relapse and therapeutic resistance. CSC-targeted therapy seems to be a better avenue for radical cure of cancer. Deubiquitinases (DUBs), specifically disassembling ubiquitin chains, have been demonstrated to play an important role in rigidly maintaining the balance between ubiquitination and deubiquitination for protein quality control and homeostasis in normal circumstances. Dysfunction or deregulation of DUBs always leads to a series of disorders, even malignant transformation. Despite the accumulative evidence that DUB inhibitors in cancer remedy mainly target the tumor bulk, side effects like toxicity and resistance are still hard nuts to crack. In this article, we review the concept of ubiquitin proteasome system (UPS) and hallmarks of CSCs related to tumor obstinacy. We primarily summarize the CSC-related factors and signaling pathways and focus on the function of DUBs on biological traits of CSCs. We also illustrate the opportunities and challenges for the application of DUB inhibitors in the CSC-targeted therapy. Finally, we discuss the complexity of cancer stem cell hierarchy complexity and argue that a combination therapy for both CSCs and non-CSCs should be a desirable option.

Abstract 1397: Identification of a hepatocellular carcinoma cell line capable of monitoring the differentiation of CD13(+)CD166(-) cancer stem cells and effects of sorafenib

Abstract Background: Recent cancer stem cell (CSC) research has highlighted the necessity to develop drugs targeting CSCs. However, only few in vitro models are available which show CSC hierarchy and allow monitoring of CSC differentiation and screening of drugs targeting CSC. Aim: In the present study, we aimed to identify such hepatocellular carcinoma (HCC) cell line(s). Methods: Based on a hypothesis that the proportion of CSCs would decrease during culture because of their differentiation into progenitors (population switch), we tested the positivity of several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166) in HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture. Results: Only Li-7 cells showed relevant population switch: the proportion of CD13 decreased, while that of CD166 increased during the culture. High tumorigenicity of Li-7 was lost after the population switch. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, while CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other cell populations. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5FU. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated the hierarchy of Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Interestingly, sorafenib selectively targeted CD166(-) fraction including CD13(+) CSCs, which exhibited higher mRNA expression of FGF3 and FGF4, candidate biomarkers for sorafenib. 5-FU followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than reverse sequence or either alone. Conclusions: We identified an useful cell line Li-7 capable of monitoring the CD13 (+) CSC differentiation and screening the drugs targeting the CSCs of HCC and found that sorafenib preferentially targeted CD13(+) CSCs in vitro. Screening cell lines for “population switch” may be useful to identify novel candidate model systems for screening drugs targeting CSCs. Citation Format: Takeshi Yamada, Masato Abei, Inaho Danjoh, Ryoko Shirota, Taro Yamashita, Shinji Endo, Ichinosuke Hyodo, Yukio Nakamura. Identification of a hepatocellular carcinoma cell line capable of monitoring the differentiation of CD13(+)CD166(-) cancer stem cells and effects of sorafenib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2015-1397

A tumor deconstruction platform identifies definitive end points in the evaluation of drug responses.

Tumor heterogeneity and the presence of drug-sensitive and refractory populations within the same tumor are almost never assessed in the drug discovery pipeline. Such incomplete assessment of drugs arising from spatial and temporal tumor cell heterogeneity reflects on their failure in the clinic and considerable wasted costs in the drug discovery pipeline. Here we report the derivation of a flow cytometry-based tumor deconstruction platform for resolution of at least 18 discrete tumor cell fractions. This is achieved through concurrent identification, quantification and analysis of components of cancer stem cell hierarchies, genetically instable clones and differentially cycling populations within a tumor. We also demonstrate such resolution of the tumor cytotype to be a potential value addition in drug screening through definitive cell target identification. Additionally, this real-time definition of intra-tumor heterogeneity provides a convenient, incisive and analytical tool for predicting drug efficacies through profiling perturbations within discrete tumor cell subsets in response to different drugs and candidates. Consequently, possible applications in informed therapeutic monitoring and drug repositioning in personalized cancer therapy would complement rational design of new candidates besides achieving a re-evaluation of existing drugs to derive non-obvious combinations that hold better chances of achieving remission.

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

BackgroundsCancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC.MethodsBased on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS.ResultsOnly Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(−) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(−), CD13(−)/CD166(−) and CD13(−)/CD166(+) fractions, whereas CD13(−)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(−) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(−) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(−) and CD13(−)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(−) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone.ConclusionsWe identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a “population change” upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of “heterogeneous, unstable” cell line may prove more useful in the CSC era than conventional “homogeneous, stable” cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1297-7) contains supplementary material, which is available to authorized users.

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML 1‐ ETO mouse model

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.

Elimination of both cancer stem cells and progenitor cells as essential for tumor eradication.

e22179 Background: Cancer stem cells (CSC) have been identified in several malignancies. Elimination of CSCs requires precise identification of markers and development of mechanisms to target them specifically. It is possible that in spite of elimination of CSCs, the tumor may recur. Dedifferentiation of progenitor cells may help replenish cancer stem cells. Methods: A mathematical model of a cancer stem cell system with four layers has been constructed to reflect the hierarchy of cancer stem cells, progenitor cells and, transit-amplifying cells and pre-differentiated cells. This model is constructed in the lines of the phase space model described by Kirkland. Dedifferentiation parameters were introduced to allow for dedifferentiation when the stem cell pool receded below 40% of its normal size. The effects of stem cell antagonists (SCA) and differentiating agents (DA) were simulated to study the perturbations caused by them on the growth of the tumor. Results: SCAs when used alone result in the establishment of a new equilibrium across different layers within the tumor by de-differentiation. DAs which act only on progenitor cells cause an increase in the number of cells in the subsequent layers and contribute to an increase in the size of the tumor. DAs which act only on the pre-differentiated layer of cells move cells from this layer into a terminally differentiated layer of cells which results in a new but lower equilibrium. DAs which act on the progenitor, transit amplifying and pre-differentiated cells cause the establishment of a new equilibrium which is lower than that of all agents which act on individual layers of cells. A combination of an SCA and a DA which acts on multiple layers eliminates the re-establishment of a new equilibrium and leads to decline in cell populations in all the layers, thus reducing the possibility of recurrence. Conclusions: SCAs when used individually are not effective in eliminating the tumor. DAs which act only on progenitor cells can fuel tumor growth but DAs which act on multiple layers have a profound effect in slowing the growth of the tumor but do not eliminate the tumor. This model implies that a combination of an SCA and a DA which acts on multiple layers may have a better chance at eliminating the tumor.