Distinct Cancer Cell Populations Research Articles

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p < 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p < 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR < 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p < 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p < 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p < 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-09.

Abstract 6836: Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape

Abstract Myxoid/Round Cell Liposarcoma (MRCL) is an understudied soft tissue sarcoma that typically affects young adults and accounts for 30% of all liposarcomas. MRCL is characterized by oncogenic DDIT3 fusions, with FUS::DDIT3 being present in over 90% of cases. However, the cellular origins and oncogenic mechanisms of MRCL remain incompletely defined. Here we report oncogene transcription, and tumor-immune interactions in a cohort of 5 patient tumors with matched normal tissue for which we sequenced the transcriptomes of approximately 45,000 single cells using 10X Genomics, and full-length transcripts in approximately 20,000 single cells via Oxford Nanopore in 2 of these patients. Our results reveal two distinct cancer cell populations: Early Cancer Cells (ECC) and Advanced Cancer Cells (ACC). These cells appear to derive from adipose stem progenitor cells (ASPC) and fall along an ASPC→ECC→ACC pseudo-time axis. Along this axis, a distinct pattern emerges where oncogenes tend to be upregulated and tumor suppressor genes (TSG) tend to be downregulated and these appear to be mutually exclusive. Comparing ECC to ASPC we see increased expression of HIPPO and PI3K pathway oncogenes, FGFR2 and IGF1R RTKs, and downregulation of the TSG CDKN2A and CDKN1A. ACC show upregulation of hedgehog, ErbB, WNT, HIPPO, PI3K, and MAPK signaling pathways compared to ECC. Full-length transcript sequencing at the single-cell level shows a consistent expression of the FUS::DDIT3 fusion transcript in both ECC and ACC populations, confirming that these populations are cancer cells. Furthermore, MRCL displays several immune escape mechanisms, including poor immune infiltration and a marked decline of HLA Class I expression along the pseudo-time axis from ECC to ACC, suggesting a complex interplay between oncogenic signaling and immune evasion. These results suggest that MRCL likely originates from ASPC and expression of FUS:DDIT3 oncoprotein induces early HIPPO, PI3K, FGFR2, and insulin signaling pathway expression in ECC with further hedgehog, ErbB, MAPK, and WNT pathway activation in ACC. This progressive activation in oncogenic signaling is associated with immune evasion. This is the first single-cell RNA seq analysis to detect the presence of the FUS::DDIT3 fusion in ECC and ACC significantly advancing our understanding of the mechanisms of MRCL oncogenesis, immune escape, and identifies potential novel therapeutic targets. Citation Format: Rodrigo Gularte-Mérida, Evan Seffar, George Li, Young-Mi Kim, Narasimhan P. Agaram, Nicholas Socci, Francisco Sanchez-Vega, Nikolaus Schultz, Samuel Singer. Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6836.

Inverse agonists of retinoic acid receptor/retinoid X receptor signaling as lineage-specific antitumor agents against human adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the coexistence within tumor tissues of 2 distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these 2 cell types, and their differential vulnerability to antitumor treatments, remains unknown. Using single-cell RNA sequencing, we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDXs) of human ACCs. Using prospective xenotransplantation experiments, we compared the tumor-initiating capacity of the 2 cell types and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the 2 cell types and tested their role as lineage-specific therapeutic targets. Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (all-trans retinoic acid, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells and in vivo antitumor activity against PDX models of human ACC. In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

Cell-of-Origin and Genetic, Epigenetic, and Microenvironmental Factors Contribute to the Intra-Tumoral Heterogeneity of Pediatric Intracranial Ependymoma.

Simple SummaryIntra-cranial ependymoma (EPN) accounts for approximately 10% of pediatric brain tumors. The current therapeutic strategies have not significantly improved prognosis, which is still dismal in nearly 40% of patients. Major challenges for treatment are chemorefractoriness of EPN, tendency to recur, and high intra-tumoral heterogeneity (ITH). It is increasingly emerging that stalled neurodevelopmental programs driven by cancer stem cells (CSCs)/progenitor cells are at the root of oncogenesis and ITH of pediatric brain tumors, including EPN. This is the first review that examines how genetic and heritable epigenetic alterations and environmental selection forces drive ITH of pediatric intra-cranial EPN in the perspective of the CSC model. This review also summarizes how improvement in the single-cell technology has deepened the comprehension of the complexity, cell-of-origin, and developmental trajectories of EPN, paving the way for novel therapeutic options.Intra-tumoral heterogeneity (ITH) is a complex multifaceted phenomenon that posits major challenges for the clinical management of cancer patients. Genetic, epigenetic, and microenvironmental factors are concurrent drivers of diversity among the distinct populations of cancer cells. ITH may also be installed by cancer stem cells (CSCs), that foster unidirectional hierarchy of cellular phenotypes or, alternatively, shift dynamically between distinct cellular states. Ependymoma (EPN), a molecularly heterogeneous group of tumors, shows a specific spatiotemporal distribution that suggests a link between ependymomagenesis and alterations of the biological processes involved in embryonic brain development. In children, EPN most often arises intra-cranially and is associated with an adverse outcome. Emerging evidence shows that EPN displays large intra-patient heterogeneity. In this review, after touching on EPN inter-tumoral heterogeneity, we focus on the sources of ITH in pediatric intra-cranial EPN in the framework of the CSC paradigm. We also examine how single-cell technology has shed new light on the complexity and developmental origins of EPN and the potential impact that this understanding may have on the therapeutic strategies against this deadly pediatric malignancy.

Single-Cell Transcriptomics Reveals the Expression of Aging- and Senescence-Associated Genes in Distinct Cancer Cell Populations.

The human aging process is associated with molecular changes and cellular degeneration, resulting in a significant increase in cancer incidence with age. Despite their potential correlation, the relationship between cancer- and ageing-related transcriptional changes is largely unknown. In this study, we aimed to analyze aging-associated transcriptional patterns in publicly available bulk mRNA-seq and single-cell RNA-seq (scRNA-seq) datasets for chronic myelogenous leukemia (CML), colorectal cancer (CRC), hepatocellular carcinoma (HCC), lung cancer (LC), and pancreatic ductal adenocarcinoma (PDAC). Indeed, we detected that various aging/senescence-induced genes (ASIGs) were upregulated in malignant diseases compared to healthy control samples. To elucidate the importance of ASIGs during cell development, pseudotime analyses were performed, which revealed a late enrichment of distinct cancer-specific ASIG signatures. Notably, we were able to demonstrate that all cancer entities analyzed in this study comprised cell populations expressing ASIGs. While only minor correlations were detected between ASIGs and transcriptome-wide changes in PDAC, a high proportion of ASIGs was induced in CML, CRC, HCC, and LC samples. These unique cellular subpopulations could serve as a basis for future studies on the role of aging and senescence in human malignancies.

A statistical approach for tracking clonal dynamics in cancer using longitudinal next-generation sequencing data.

MotivationTumours are composed of distinct cancer cell populations (clones), which continuously adapt to their local micro-environment. Standard methods for clonal deconvolution seek to identify groups of mutations and estimate the prevalence of each group in the tumour, while considering its purity and copy number profile. These methods have been applied on cross-sectional data and on longitudinal data after discarding information on the timing of sample collection. Two key questions are how can we incorporate such information in our analyses and is there any benefit in doing so?ResultsWe developed a clonal deconvolution method, which incorporates explicitly the temporal spacing of longitudinally sampled tumours. By merging a Dirichlet Process Mixture Model with Gaussian Process priors and using as input a sequence of several sparsely collected samples, our method can reconstruct the temporal profile of the abundance of any mutation cluster supported by the data as a continuous function of time. We benchmarked our method on whole genome, whole exome and targeted sequencing data from patients with chronic lymphocytic leukaemia, on liquid biopsy data from a patient with melanoma and on synthetic data and we found that incorporating information on the timing of tissue collection improves model performance, as long as data of sufficient volume and complexity are available for estimating free model parameters. Thus, our approach is particularly useful when collecting a relatively long sequence of tumour samples is feasible, as in liquid cancers (e.g. leukaemia) and liquid biopsies.Availability and implementationThe statistical methodology presented in this paper is freely available at github.com/dvav/clonosGP.Supplementary information Supplementary data are available at Bioinformatics online.

Cancer Cell Invasion of Mammary Organoids with Basal-In Phenotype.

This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal-in phenotype is the use of a minimal gel scaffold that the epithelial cells self-assemble around and encapsulate. When MDA-MB-231 breast cancer cells are co-cultured with epithelial cells from day 0 under these conditions, cells self-organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA-MB-231 invasion in a tumor progression-relevant direction relative to epithelial cell-basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.

Stromal fibroblasts induce metastatic tumor cell clusters via epithelial-mesenchymal plasticity.

Emerging evidence supports the hypothesis that multicellular tumor clusters invade and seed metastasis. However, whether tumor-associated stroma induces epithelial-mesenchymal plasticity in tumor cell clusters, to promote invasion and metastasis, remains unknown. We demonstrate herein that carcinoma-associated fibroblasts (CAFs) frequently present in tumor stroma drive the formation of tumor cell clusters composed of two distinct cancer cell populations, one in a highly epithelial (E-cadherinhiZEB1lo/neg: Ehi) state and another in a hybrid epithelial/mesenchymal (E-cadherinloZEB1hi: E/M) state. The Ehi cells highly express oncogenic cell-cell adhesion molecules, such as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 that associate with E-cadherin, resulting in increased tumor cell cluster formation and metastatic seeding. The E/M cells also retain associations with Ehi cells, which follow the E/M cells leading to collective invasion. CAF-produced stromal cell-derived factor 1 and transforming growth factor-β confer the Ehi and E/M states as well as invasive and metastatic traits via Src activation in apposed human breast tumor cells. Taken together, these findings indicate that invasive and metastatic tumor cell clusters are induced by CAFs via epithelial-mesenchymal plasticity.

Metformin Results in Diametrically Opposed Effects by Targeting Non-Stem Cancer Cells but Protecting Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been shown as a distinct population of cancer cells strongly implicated with resistance to conventional chemotherapy. Metformin, the most widely prescribed drug for diabetes, was reported to target cancer stem cells in various cancers. In this study, we sought to determine the effects of metformin on head and neck squamous cell carcinoma (HNSCC). CSCs and non-stem HNSCC cells were treated with metformin and cisplatin alone, and in combination, and cell proliferation levels were measured through MTS assays. Next, potential targets of metformin were explored through computational small molecule binding analysis. In contrast to the reported effects of metformin on CSCs in other cancers, our data suggests that metformin protects HNSCC CSCs against cisplatin in vitro. Treatment with metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, OCT-4, and NANOG. On the other hand, we observed that metformin successfully decreased the proliferation of non-stem HNSCC cells. Computational drug–protein interaction analysis revealed mitochondrial complex III to be a likely target of metformin. Based on our results, we present the novel hypothesis that metformin targets complex III to reduce reactive oxygen species (ROS) levels, leading to the differential effects observed on non-stem cancer cells and CSCs.

Abstract 42: MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells

Abstract Leukemia is hierarchically organized into two distinct population of cancer cells, cancer stem cells (CSCs) and non-stem cancer cells (NSCC). Numerous studies suggest that CSCs may express embryonic stem (ES) cells related stemness genes including Nanog, Sox-2, and Oct-4. However, potential cell surface markers that could specifically enrich the leukemia stem cell (LSC) subfraction based on the expression of these stemness genes have not yet been identified. Previously, we reported that ABCG2, a drug efflux pump, could enrich a subpopulation of cells exhibiting very high level of Nanog, Sox-2, and Oct-4 in ES cells. Additionally, these ABCG2+ ES cells also exhibited high HIF-2α, a transcription factor, which in cooperation with MYC-regulated both Nanog and Sox-2. In addition to these features, ABCG2+ cells demonstrated highly cytoprotective altruistic behavior by secreting high levels of glutathione. In the present study, we evaluated whether ABCG2+ subfraction of human LSCs exhibits high level of Nanog, Sox2, and Oct4. We also investigated the altruistic behavior of the LSCs, a potential novel mechanism of drug resistance and disease relapse. Methods: ABCG2+ cells from cervical lymph node and peripheral blood of T- cell acute lymphoblastic lymphoma/leukemia (T-ALL) and chronic myeloid leukemia (CML) patients (n=12) were enriched using immunomagnetic sorting. These cells were then expanded and used for different experiments including flow cytometry, ChiP on ChiP assay, in vivo transplantation assay, and siRNA inhibition treatment, to demonstrate the role of HIF-2α and MYC in regulating the altruistic stemness program in human LSCs. Results: We were able to enrich ABCG2+ cell subfraction from human T-ALL (n=5) and CML (n=7) patients exhibiting high levels of stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2α. Higher engraftment potential was observed in ABCG2+ cells in comparison to ABCG2- cells as indicated by in vivo transplantation assay in NOD/SCID mice. The inhibition of stemness genes Nanog and Sox2 by siRNA gene silencing led to the loss of expression of MYC and HIF-2α, indicating that these genes are regulated MYC and HIF-2α. We then investigated the altruistic stemness phenotype of the ABCG2+ cells by studying the distinct molecular signature of MYC binding to HIF-2α in these cells. siRNA HIF-2α treatment led to ABCG2+ cell loss of proliferative capacity and reduced GSH levels, suggesting reduction of cytoprotective altruistic behavior in vitro. Additionally, siRNA HIF-2α treated ABCG2+ cells, when injected in mice, exhibited increased survival. Importantly, treatment of ABCG2+ cell harboring mice with FM19G11, a HIF inhibitor, led to marked loss in the secondary engraftment in NOD/SCID mice, thus indicating loss of ABCG2+ cell self-renewal capacity. Then we found that siRNA MYC inhibition led to loss of HIF-2α expression, suggesting that MYC might be regulating HIF-2α mediated altruistic stemness program in the ABCG2+ cells. Conclusion: To summarize, we found that ABCG2 is an excellent marker to enrich the LSC subfraction having high expression of stemness genes. The ABCG2+ LSCs exhibit altruistic stemness phenotype that includes high secretion of GSH and the distinct binding of MYC to HIF-2a, Sox2, and Nanog. Thus, we now report that a subfraction of LSC could also be enriched such that it exhibits altruistic stemness phenotype which was previously reported by us in ES cells. Our findings may open up new understanding of the LSC-mediated cancer relapse and drug resistance. Citation Format: Bidisha Pal, Anupam Sarma, Joyeeta Talukdar, Seema Bhuyan, Sora Sandhya, Sukanya Gayan, Gayatri Gogoi, Debabrat Baishya, Amal Chandra Kataki, Dean W. Felsher, Bikul Das. MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 42.

Implications of cancer evolution for drug development.

Tumour evolution, which results in the existence of multiple distinct populations of cancer cells within the same tumour and the same patient, is increasingly appreciated to have a key role in drug resistance. In this article, we discuss the implications for drug development, including approaches to reduce the likelihood of the emergence of drug resistance.

Abstract 4380: Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-sequencing

Abstract Tumors are heterogeneous ecosystems composed of genetically and epigenetically distinct cancer cell populations embedded in an intricate tumor microenvironment. The complexity and cell-to-cell interactions within this system pose a tremendous therapeutic challenge and opportunity. Due to technical constraints, current profiling technologies only provide average signals that do not reflect this intrinsic genetic and phenotypic variability. Here, we applied single-cell RNA-sequencing to examine 4,645 single cells isolated from 19 freshly procured melanomas, profiling malignant, immune and stromal cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, stem-like cells, spatial context, and a drug treatment resistance program. All tumors harbored malignant cells from two distinct transcriptional cell states, such that treatment-sensitive “MITF-high” tumors also contained drug-resistant “AXL-high” tumor cells; similar heterogeneity was present in 18 established melanoma cell lines. The frequency of AXL-high cells increased in post-relapse resistant tumors following treatment with BRAF/MEK inhibitors. Using multiplexed, quantitative single-cell immunofluorescence analysis and FACS, we validated these observations in melanoma cell lines treated with BRAF±MEK inhibitors. Signatures of cell types identified from single-cell analysis revealed distinct patterns of the tumor microenvironment. We inferred cell-to-cell interactions between stromal, immune and malignant cells, and identified factors, including known secreted gene products (e.g. CXCL12) and several complement factors. We validated the association between cancer-associated fibroblast (CAF)-expressed complement factor 3 (C3) and TIL infiltration in an independent set of 308 melanomas. Finally, analysis of TILs revealed T-cell activation dependent and independent exhaustion programs that varied among patients dependent on their exposure to treatment with immune checkpoint-inhibitors. In addition to co-expression of several known co-inhibitory receptors, including PD1, CTLA-4, and TIM-3, we identified common markers associated with cytotoxicity-independent T-cell exhaustion across patients. To identify potential T-cell clones, we classified single T-cells by their isoforms of the V and J segments of the alpha and beta TCR chains, allowing us to identify expanded T-cell clones. We found that clonally expanded T-cells expressed a strong exhaustion program, while non-expanded T-cells lacked this phenotype. This study represents the most comprehensive single-cell genomics analysis in humans to date and begins to unravel the cellular ecosystem of tumors. Single-cell genomics offer new insights with implications for both targeted and immune therapies by simultaneously profiling numerous aspects of a tumor with a single assay. Citation Format: Benjamin Izar, Itay Tirsh, Sanjay Prakadan, Marc Wadsworth, Daniel Treacy, John Trombetta, Asaf Rotem, Christine Lian, George Murphy, Mohammad Fallahi-Sichani, Ken Dutton-Regester, Jia-Ren Lin, Judit Jane-Valbuena, Orit Rozenblatt-Rosen, Charles Yoon, Alex Shalek, Aviv Regev, Levi Garraway. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4380.

Abstract 2025: Inverse relation of Myc and p27 in cribriform pattern prostate cancer

Abstract Background: Prostate cancers composed exclusively of Gleason pattern 3 have an excellent prognosis,while those containing Gleason pattern 4 or higher are more aggressive. Gleason pattern 4 is composed of either fused glands, poorly formed glands or cribriform structures with cribriform structures being most readily recognized and shown to be of independent prognostic significance (PMIDS: 24145642;25189638). However, the biological properties of these cribriform structures have not been studied. There is a spectrum of nuclear features in cribriform glands with cells towards the perimeter containing enlarged nucleoli, and open chromatin as compared to cells towards the center. Since MYC overexpression drives nucleolar enlargement we examined the pattern of expression of MYC protein in these structures. Also, we examined p27 staining since it has been reported to be more intense in the nuclei in the center of these structures (PMID:9731735). Design: Cases with cribriform Gleason score 7 were used for IHC for MYC, p27 and Ki67 as well as double label immunofluorescence staining for p27 and MYC. Slides were scanned and regions were selected containing cribriform glands (N = 18 total regions) and analyzed using image analysis software by Aperio Inc. We quantified the level of nuclear staining for MYC and p27 in relation to the outside/perimeter regions versus the inner aspect of the glands. Chromogenic in-situ hybridization for MYC mRNA was also performed. Results: The increase in MYC staining in the perimeter aspects of the cribriform structures was highly significant as compared to the inner regions (P<0.0001, rank sum). Conversely, p27 staining showed an increase in the central region as compared to the perimeter aspect (P = 0.0024). There was also a marked increase in staining for Ki67 (p<0.0001) as well as MYC mRNA in the perimeter cells. Double label fluorescent staining showed a tight inverse correlation between MYC and nuclear p27 staining. Conclusion: Two distinct populations of cancer cells comprise cribriform carcinomas of the prostate; the central aspect is composed of cells with relatively small nucleoli that are non-proliferating with high levels of nuclear p27, and an outer layer that is composed of metabolically more active cells with increased MYC, nucleolar size and proliferative fraction. These findings provide new biological insights into the unique structure of cribriform prostate cancer lesions and suggest a complex interplay between the two cell types. Future studies are needed to determine how and whether these findings relate to disease aggressiveness. Citation Format: Ibrahim Kulac, Jessica Hicks, Qizhi Zheng, Helen Fedor, Toby C. Cornish, Angelo M. De Marzo. Inverse relation of Myc and p27 in cribriform pattern prostate cancer. [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 2025. doi:10.1158/1538-7445.AM2015-2025

CD24+ Ovarian Cancer Cells Are Enriched for Cancer-Initiating Cells and Dependent on JAK2 Signaling for Growth and Metastasis.

Ovarian cancer is known to be composed of distinct populations of cancer cells, some of which demonstrate increased capacity for cancer initiation and/or metastasis. The study of human cancer cell populations is difficult due to long requirements for tumor growth, interpatient variability, and the need for tumor growth in immune-deficient mice. We therefore characterized the cancer initiation capacity of distinct cancer cell populations in a transgenic murine model of ovarian cancer. In this model, conditional deletion of Apc, Pten, and Trp53 in the ovarian surface epithelium (OSE) results in the generation of high-grade metastatic ovarian carcinomas. Cell lines derived from these murine tumors express numerous putative stem cell markers, including CD24, CD44, CD90, CD117, CD133, and ALDH. We show that CD24(+) and CD133(+) cells have increased tumor sphere-forming capacity. CD133(+) cells demonstrated a trend for increased tumor initiation while CD24(+) cells versus CD24(-) cells had significantly greater tumor initiation and tumor growth capacity. No preferential tumor-initiating or growth capacity was observed for CD44(+), CD90(+), CD117(+), or ALDH(+) versus their negative counterparts. We have found that CD24(+) cells, compared with CD24(-) cells, have increased phosphorylation of STAT3 and increased expression of STAT3 target Nanog and c-myc. JAK2 inhibition of STAT3 phosphorylation preferentially induced cytotoxicity in CD24(+) cells. In vivo JAK2 inhibitor therapy dramatically reduced tumor metastases, and prolonged overall survival. These findings indicate that CD24(+) cells play a role in tumor migration and metastasis and support JAK2 as a therapeutic target in ovarian cancer.

CD24(+) cells fuel rapid tumor growth and display high metastatic capacity.

IntroductionBreast tumors are comprised of distinct cancer cell populations which differ in their tumorigenic and metastatic capacity. Characterization of cell surface markers enables investigators to distinguish between cancer stem cells and their counterparts. CD24 is a well-known cell surface marker for mammary epithelial cells isolation, recently it was suggested as a potential prognostic marker in a wide variety of malignancies. Here, we demonstrate that CD24+ cells create intra-tumor heterogeneity, and display highly metastatic properties.MethodsThe mammary carcinoma Mvt1 cells were sorted into CD24− and CD24+ cells. Both subsets were morphologically and phenotypically characterized, and tumorigenic capacity was assessed via orthotopic inoculation of each subset into the mammary fat pad of wild-type and MKR mice. The metastatic capacity of each subset was determined with the tail vein metastasis assay. The role of CD24 in tumorigenesis was further examined with shRNA technology. GFP-labeled cells were monitored in vivo for differentiation. The genetic profile of each subset was analyzed using RNA sequencing.ResultsCD24+ cells displayed a more spindle-like cytoplasm. The cells formed mammospheres in high efficiency and CD24+ tumors displayed rapid growth in both WT and MKR mice, and were more metastatic than CD24- cells. Interestingly, CD24-KD in CD24+ cells had no effect both in vitro and in vivo on the various parameters studied. Moreover, CD24+ cells gave rise in vivo to the CD24− that comprised the bulk of the tumor. RNA-seq analysis revealed enrichment of genes and pathways of the extracellular matrix in the CD24+ cells.ConclusionCD24+ cells account for heterogeneity in mammary tumors. CD24 expression at early stages of the cancer process is an indication of a highly invasive tumor. However, CD24 is not a suitable therapeutic target; instead we suggest here new potential targets accounting for early differentiated cancer cells tumorigenic capacity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0589-9) contains supplementary material, which is available to authorized users.

Embryonic stem cell factors and pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic tumor, is a highly aggressive human cancer with the lowest five-year survival rate of any human maligancy primarily due to its early- metastasis and lack of response to chemotherapy and radiation. Recent research suggests that PDAC cells comprise a hierarchy of tumor cells that develop around a population of cancer stem cells (CSCs), a small and distinct population of cancer cells that mediates tumoregenesis, metastasis and resistance to standard treatments. Thus, CSCs could be a target for more effective treatment options. Interestingly, pancreatic CSCs are subject to regulation by some of key embryonic stem cell (ESC) transctiption factors abberently expressed in PDAC, such as SOX2, OCT4 and NANOG. ESC transcription factors are important DNA-binding proteins present in both embryonic and adult somatic cells. The critical role of these factors in reprogramming processes makes them essential not only for embryonic development but also tumorigenesis. Here we provide an overview of stem cell transcription factors, particularly SOX2, OCT4, and NANOG, on their expression and function in pancreatic cancer. In contrast to embryonic stem cells, in which OCT4 and SOX2 are tightly regulated and physically interact to regulate a wide spectrum of target genes, de novo SOX2 expression alone in pancreatic cancer cells is sufficient to promote self-renewal, de-differentiation and imparting stemness characteristics via impacting specific cell cycle regulatory genes and epithelial-mesnechymal transtion driver genes. Thus, targeting ESC factors, particularly SOX2, could be a worthy strategy for pancreatic cancer therapy.

Radiation resistance of cancer stem cells as an obstacle in cancer therapy

Sh Faisal 14.00 Radiation represents an important therapeutic intervention in many tumor types. Nevertheless, tumor recurrence continues to post major limitation to this type of therapy. Wealth of data suggests that there is a distinct population of cancer cells with exclusive ability to self-renew, termed cancer stem cells (CSCs). This subset of cancer cells has been shown to be resistant to conventional therapy, including chemotherapy and radiation. Failure to eradicate CSCs may result in tumor recurrence. This review article summarized the characteristic and identification of CSCs along with mechanisms of resistance in these cells including increased DNA repair. Understanding the mechanisms underlying radioresistance of CSCs will lead to better combination therapies to overcome resistance of these cells which will ultimately improve outcome for radiation treatment. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

Evidence points to the existence of cancer stem cells

Currently, in solid tumours there are two models for tumour proliferation. First, the classic theory in which all cancer cells have proliferative potential and can replicate and contribute to the expansion of the tumour. Second, the cancer stem cell (CSC) hypothesis suggests that there is a hierarchy of cell proliferation with a distinct population of cancer cells at the top of the pyramid. Now, two studies published in Nature and one in Science have provided evidence for the presence of CSCs in solid tumours for the first time in models of unperturbed tumour growth.

Breast cancer intratumor genetic heterogeneity: causes and implications

There is burgeoning evidence to suggest that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones harbor private genetic aberrations in addition to the founder mutations, and that these distinct populations of cancer cells interact in competitive and mutualistic manners. The combined effect of genetic and epigenetic instability, and differential selective pressures according to the microenvironment and therapeutic interventions, create many different evolutionary routes such that intratumor heterogeneity is inevitable. Numerous cytogenetic, comparative genomic hybridization and, more recently, massively parallel sequencing studies have generated indisputable evidence of this phenomenon. The impact of intratumor heterogeneity on response and resistance to therapy is beginning to be understood; this information may prove crucial for the potentials of personalized medicine to be realized. In this review, the evidence of intratumor heterogeneity in breast cancer, its potential causes and implications for the clinical management of breast cancer patients are discussed.

Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype

Intratumor genetic heterogeneity is a key mechanism underlying tumor progression and therapeutic resistance. The prevailing model for explaining intratumor diversity, the clonal evolution model, has recently been challenged by proponents of the cancer stem cell hypothesis. To investigate this issue, we performed combined analyses of markers associated with cellular differentiation states and genotypic alterations in human breast carcinomas and evaluated diversity with ecological and evolutionary methods. Our analyses showed a high degree of genetic heterogeneity both within and between distinct tumor cell populations that were defined based on markers of cellular phenotypes including stem cell-like characteristics. In several tumors, stem cell-like and more-differentiated cancer cell populations were genetically distinct, leading us to question the validity of a simple differentiation hierarchy-based cancer stem cell model. The degree of diversity correlated with clinically relevant breast tumor subtypes and in some tumors was markedly different between the in situ and invasive cell populations. We also found that diversity measures were associated with clinical variables. Our findings highlight the importance of genetic diversity in intratumor heterogeneity and the value of analyzing tumors as distinct populations of cancer cells to more effectively plan treatments.