Tumor-initiating Cells Activity Research Articles

Hypoxia Controls the Glycome Signature and Galectin-8–Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that β1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low β1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed β1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity invivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Abstract 2852: A gene activating in vivo screen identifies EBAG9 as novel metastasis regulator in human colorectal cancer

Abstract As colorectal cancer (CRC) mortality remains high due to metastatic spread in patients with advanced disease stages, we here aimed at identifying novel metastasis regulators in CRC and at understanding their mechanism of action. In CRC, tumor progression and metastasis formation are driven by cells that possess tumor-initiating cell (TIC) activity. We therefore employed a collection of primary patient-derived CRC tumor spheroid cultures (TSC) that are enriched for cells with TIC activity to perform a gene activating in vivo screen, intending to identify genes that enhance metastasis formation. To this end, TSC (n=4) were transduced with a lentiviral trapping vector that integrates into the genome and thereby drives the overexpression of genes located in the vicinity of the integration site (IS). Transduced TSC were then serially transplanted under the kidney capsule of immunodeficient mice (NSG) and monitored for the induction of metastasis formation. Analysis of lentiviral IS by linear amplification-mediated (LAM)-PCR revealed an enrichment of an IS within the gene locus of Estrogen Receptor Binding Site Associated Antigen 9 (EBAG9) in metastases. RNA sequencing confirmed the trapping vector mediated upregulation of a truncated version of the EBAG9 gene (exon 5-7), which strongly suggested a contribution of EBAG9 to metastatic spread. To validate these findings, both the truncated (TR) as well as the physiologically occurring full length (FL) EBAG9 gene were overexpressed in TSC (n=4) and serially transplanted into NSG mice. In addition to an augmentation in metastasis upon EBAG9 overexpression compared to controls (FL: 1.4-fold, TR: 2.4-fold), we also detected the occurrence of lymphogenous metastases when EBAG9 levels were increased, indicating that EBAG9 not only contributes to metastatic spread but also modulates tropism. To further characterize the underlying processes, global gene and protein expression analyses were performed in primary TSC as well as in the CRC cell line DLD1. Compared to control transduced cells, EBAG9 overexpression induced expression changes in cell adhesion and microtubule molecules, which led us to investigate cell migration and adhesion as potential contributing mechanisms to metastasis formation. We here observed an increase both in cell migration as well as in adhesion to the extracellular matrix protein collagen I upon EBAG9 overexpression in DLD1. Interestingly, DLD1 adhesion to lymphatic endothelial cells was significantly higher in EBAG9 FL (p=0.008) and EBAG9 TR (p=0.016) overexpressing cells, underscoring the role of EBAG9 in tropism modulation. In summary, we here identified EBAG9 as novel metastasis regulator in CRC which will not only contribute to a better mechanistic understanding of metastatic spread but also to the development of novel future strategies for preventing and targeting advanced disease stages. Citation Format: Karin Laaber, Taronish D. Dubash, Anna Maria Melzer, Tania Christiansen, Nati Ha, Benedikt Brors, Martin Schneider, Friederike Herbst, Hanno Glimm, Claudia R. Ball. A gene activating in vivo screen identifies EBAG9 as novel metastasis regulator in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2852.

Abstract 1491: Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer

Abstract Tumor progression in colorectal cancer (CRC) is driven by a subpopulation of cells with tumor-initiating cell (TIC) activity. In the past, bulk experiments provided strong evidence for functional heterogeneity of the CRC TIC compartment with distinct types of TICs driving CRC progression. However, in these retrospective experiments direct assignment of functional states to individual functionally relevant cell types in CRC has not been possible. We here asked whether functional states of individual cells can be assigned to specific CRC cell subpopulations. Therefore, 4,663 single-cell profiles were generated by single-cell RNA-sequencing (scRNA-seq) of patient-derived CRC spheroid cultures (n=12). Non-negative matrix factorization identified 13 gene expression modules linked to cell states or cell types that resemble differentiation states of normal intestinal epithelial cells. To evaluate their presence in CRC patient tumors, the identified transcriptional programs were applied to publicly available expression data (TCGA cohort; n=459). Clustering of samples based on the contribution of individual expression signatures revealed six patient clusters with significantly different progression-free survival (p=0.043) and a trend towards different overall survival (p=0.12), indicating a relevance for patient outcome. Comparing cell state meta-signature expression in cell type-associated subpopulations revealed differences in functional states, i.e. proliferative and metabolic activity. Interestingly, stem-like cells showed significantly increased oxidative phosphorylation (OXPHOS) and decreased glycolysis scores, suggesting a link between metabolic states and cell differentiation. scRNA-seq of patient-derived organoid and xenograft models as well as primary tumors revealed similar trends. To assess whether metabolic states are linked to TIC activity, CRC spheroid cells were sorted according to mitochondrial membrane potential (MMP), and sphere forming cell (SFC) as well as TIC frequency were assessed by limiting dilutions. Strikingly, MMPhigh cells exhibited a pronounced increase of SFC frequencies in vitro compared to MMPlow cells in 80% of patients (n=5). In addition, TIC frequencies were strongly increased in the MMPhigh compared to the MMPlow subpopulation in vivo (n=2; 1/46,535 vs. 1/211,305 and 1/249 vs. 1/2,089), demonstrating that TIC activity relies on OXPHOS. In line with this, pretreatment with the OXPHOS inhibitor CCCP decreased SFC frequencies (n=3), indicating targetable metabolic vulnerability of CRC spheroid cells. In summary, we here show that transcriptional heterogeneity identifies functional states during CRC TIC differentiation. Targeting context-dependent regulation of tumor cell differentiation might unravel novel vulnerabilities for therapeutic intervention in human CRC. Citation Format: Martina K. Zowada, Stephan M. Tirier, Sebastian M. Dieter, Teresa Krieger, Ava Oberlack, Robert L. Chua, Mario Huerta, Foo Wei Ten, Karin Laaber, Jeongbin Park, Katharina Jechow, Torsten Müller, Mathias Kalxdorf, Mark Kriegsmann, Katharina Kriegsmann, Friederike Herbst, Jeroen Krijgsveld, Martin Schneider, Roland Eils, Hanno Glimm, Christian Conrad, Claudia R. Ball. Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1491.

CtBP\u2014a targetable dependency for tumor-initiating cell activity and metastasis in pancreatic adenocarcinoma

Ctbp2 is a uniquely targetable oncogenic transcriptional coregulator, exhibiting overexpression in most common solid tumors, and critical to the tumor-initiating cell (TIC) transcriptional program. In the “CKP” mouse pancreatic ductal adenocarcinoma (PDAC) model driven by mutant K-Ras, Ctbp2 haploinsufficiency prolonged survival, abrogated peritoneal metastasis, and caused dramatic downregulation of c-Myc, a known critical dependency for TIC activity and tumor progression in PDAC. A small-molecule inhibitor of CtBP2, 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) phenocopied Ctbp2 deletion, decreasing tumor burden similarly to gemcitabine, and the combination of 4-Cl-HIPP and gemcitabine further synergistically suppressed tumor growth. Pharmacodynamic monitoring revealed that the 4-Cl-HIPP/gemcitabine combination induced robust and synergistic tumor apoptosis and marked downregulation of the TIC marker CD133 in CKP PDAC tumors. Collectively, our data demonstrate that targeting CtBP represents a fruitful avenue for development of highly active agents in PDAC that cooperate with standard therapy to limit both primary and metastatic tumor burden.

Tumor suppressor miR-215 counteracts hypoxia-induced colon cancer stem cell activity

Cancer stem cells, also known as tumor-initiating cells (TICs), are a population of aggressive and self-renewing cells that are responsible for the initiation and progression of many cancers, including colorectal carcinoma. Intratumoral hypoxia, i.e. reduced oxygen supply following uncontrolled proliferation of cancer cells, is thought to support TIC activity by inducing specific hypoxia-responsive mechanisms that are not yet entirely understood. Using previously established and fully characterized patient-derived TIC cultures, we could observe increased sphere and colony formation under hypoxic conditions. Mechanistically, microRNA (miRNA)-profiling experiments allowed us to identify miR-215 as one of the main hypoxia-induced miRNAs in primary colon TICs. Through stable overexpression of miR-215, followed by a set of functional in vitro and in vivo investigations, miR-215 was pinpointed as a negative feedback regulator, working against the TIC-promoting effects of hypoxia. Furthermore, we could single out LGR5, a bona fide marker of non-neoplastic intestinal stem cells, as a downstream target of hypoxia/miR-215 signaling. The strong tumor- and TIC-suppressor potential of miR-215 and the regulatory role of the hypoxia/miR-215/LGR5 axis may thus represent interesting points of attack for the development of innovative anti-CSC therapy approaches.

The effects of adipose-derived stem cells on CD133-expressing bladder cancer cells.

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. They are also considered as a preferred cell source for urinary tract reconstruction. However, as MSCs exhibit affinity to tumor microenvironment, possible activation of tumor-initiating cells remains a major concern in the application of stem cell-based therapies for patients with a bladder cancer history. To analyze the influence of adipose-derived stem cells (ASCs) on bladder cancer cells with stem cell-like properties, we isolated CD133-positive bladder cancer cells and cultured them in conditioned medium from ASCs (ASC-CM). Our results showed that parental 5637 and HB-CLS-1 cells showed induced clonogenic potential when cultured in ASC-CM. Soluble mediators secreted by ASCs increased proliferation and viability of unsorted cells as well as CD133+ and CD133- subpopulations. Furthermore, incubation with ASC-CM modulated activation of intracellular signaling pathways. Soluble mediators secreted by ASCs increased phosphorylation of AKT1/2/3 (1.4-fold, P < 0.05), ERK1/2 (1.6-fold, P < 0.02), and p70 S6K (1.4-fold) in CD133+ cells isolated from 5637 cell line. In turn, decreased phosphorylation of those three proteins involved in PI3K/Akt and MAPK signaling was observed in CD133+ cells isolated from HB-CLS-1 cell line. Our results revealed that bladder cancer stem-like cells are responsive to signals from ASCs. Paracrine factors secreted by locally-delivered ASCs may, therefore, contribute to the modulation of signaling pathways involved in cancer progression, metastasis, and drug resistance.

Abstract 2893: IGF2 is essential for tumor initiating cell activity in human colorectal cancer

Abstract A small fraction of all cells within individual tumours from colorectal cancer (CRC) patients drives long term tumor growth and metastases in immune-compromised mice. Targeting these tumor-initiating cells (TIC) may improve the long-term outcome in advanced CRC. To identify candidate genes which drive proliferation and survival of TIC, we have performed a large scale high throughput loss of function shRNA screen in three-dimensional TIC enriched patient spheroids. Spheroids were transduced with the barcoded Cellecta decipher library comprising 27,500 shRNAs targeting 5043 genes associated with cell signaling pathways (Module 1). Two weeks later, cells were harvested for DNA isolation, barcode amplification and high-throughput barcode sequencing. Amongst others, we found 5/6 shRNAs targeting Insulin growth factor 2 (IGF2) scoring within a 20% depletion threshold, presenting depletion levels very similar to positive control shRNAs. mRNA expression profiling and qPCR analyses demonstrated low to moderate expression of the IGF2 gene product in the majority of patient spheroid cultures analyzed (n=17). In contrast, two out of 15 patient derived spheroid cultures analyzed demonstrated very pronounced IGF2 overexpression (&amp;gt;250 fold). Integrative SCNA profiling, expression and TAD profiling using the CESAM algorithm, followed by 4C Seq, demonstrated a tandem duplication of the IGF2 locus in these two patients which interrupts a IGF2 adjacent TAD boundary and results in de novo contact domain formation between the IGF2 promoter and a normally hidden distant super-enhancer. A dual luciferase reporter assay revealed that the hijacked enhancer is functionally active in human CRC cells and thereby may drive unphysiological IGF2 expression following enhancer hijacking. To assess the functional relevance of IGF2 tandem duplications, spheroids were transduced with RFP expressing lentiviral vectors encoding for 3 shRNAs targeting IGF2 as well as control shRNAs targeting EIF3A or scrambled shRNA. Strikingly, IGF2 knockdown led to a marked reduction of RFP+ cells in competitive proliferation assays and markedly reduced viability assessed by ATPlight assay. Moreover, IGF2 knockdown strongly reduced tumor formation following xenotransplantation into immune deficient NSG mice. These data demonstrate that IGF2 is required for survival, proliferation and tumor-initiation of primary human TIC enriched spheroids. Notably, oncogenic miRNA-483 is encoded within intron 8 of IGF2, however, its function in IGF2 locus tandem duplicated cells remains elusive. Understanding the mechanisms of IGF2 dependency and the role of miRNA-483 in this context will be essential for the future development of therapeutic approaches targeting IGF2 expression in this patient subset harbouring tandem-duplications of the IGF2 locus. Citation Format: Taronish D. Dubash, Christine Siegl, Sebastian M. Dieter, Joachim Weischenfeldt, Alexandros P. Drainas, Laura Schwarzmueller, Malgorzata Oles, Balca Mardin, Mikolaj Slabicki, Huber Wolfgang, Martin Schneider, Jan Korbel, Hanno Glimm, Claudia R. Ball. IGF2 is essential for tumor initiating cell activity in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2017-2893

Succession of transiently active tumor-initiating cell clones in human pancreatic cancer xenografts.

Although tumor‐initiating cell (TIC) self‐renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long‐term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self‐renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.

Methylparaben stimulates tumor initiating cells in ER+ breast cancer models.

A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF-7 breast cancer cells and a patient-derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 μg per day) led to increased tumor size of MCF-7 xenografts and ER+ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF-7 cell proliferation or induction of canonical estrogen-responsive genes (pS2 and progesterone receptor), in contrast to 17β-estradiol (E2) treatment. MCF-7 and PDX-derived mammospheres exhibited increased size and up-regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA-MB-231 (ER- ) mammospheres. As tumor-initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti-estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non-classic estrogen response or an ER-independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract 3316: The effects of dopamine receptor 2 on breast cancer tumor initiating cells

Abstract Despite advances in early detection and treatment of breast cancers, metastatic disease remains a significant problem and a large cause of mortality in the US. Although tumors are characterized as a mass of uncontrolled cell division, it is thought that only small populations of cells, known as tumor initiating cells (TICs), are primarily responsible for metastasis. TICs are less differentiated than most tumor cells and many TICs show an epithelial-to-mesenchymal transition (EMT). Recently, the FDA-approved drug thioridazine, a dopamine receptor 2 (DRD2) antagonist, has been shown to inhibit pluripotency gene expression only in transformed stem cells. To test whether DRD2 can regulate TICs in breast cancer cells, two main assays were performed. The tumorsphere assay is used to measure TIC activity within a population of cells in vitro. In this assay, single cells are cultured in suspension, in the absence of serum. Cells must escape anoikis and grow without cell-cell contacts or the assortment of factors contained in serum. However, the gold standard in vivo assay to measure tumor initiation is the transplantation assay at limiting dilutions. Cells are injected into immunocompromised mice in decreasing numbers and the number of tumors each population of cells is capable of forming is measured. In this study the tumorsphere assay and transplantation assay both show that DRD2 supports TICs in breast cancers. Interestingly, our data suggest that DRD2 regulates TICs and cellular differentiation through the activity of the STAT3 transcription factor. Activation of STAT3 has been shown to induce gene expression changes that support TICs and upregulate the pluripotency factor OCT4. STAT3 activity can also induce EMT and resistance to some therapeutics. By inducing the expression of its own activator, IL-6, STAT3 can initiate a feed forward loop to maintain continual expression to promote TICs, EMT, and drug resistance. Halting the continual activation of STAT3 in these cells should be an important objective. Our data supports the concept that DRD2 may represent a new, and druggable target for therapies to decrease metastasis and/or recurrence by interfering with STAT3 and inhibiting TIC-like activities. Citation Format: Matthew Tegowski, Charlene Santos, Albert Baldwin. The effects of dopamine receptor 2 on breast cancer tumor initiating cells. [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 3316.

ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models.

BackgroundHypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion.MethodsWe used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f + cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49fneg or low cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients.ResultsWe report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes.ConclusionsOur data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f + cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0510-x) contains supplementary material, which is available to authorized users.

Phenotypic differentiation does not affect tumorigenicity of primary human colon cancer initiating cells

Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions.Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.

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.

Abstract LB-145: Pten deletion in SOX9+ cells synergizes with hepatic injury to drive tumor development in mouse liver

Abstract Liver cancer is an extremely deadly disease ranked as the third most common cancer and the second leading cause of male cancer-related death worldwide. Among primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the two most frequent subtypes, accounting for 85-95% of the total liver cancer cases, and both types of liver cancer have been found to originate from the expansion and differentiation of tumor initiating cells (TICs). Thus, it is critically important to understand mechanisms that regulate liver TICs activation. By utilizing a Pten(loxp/loxp); Albumin-Cre+ mouse liver cancer model (L-PKO), our previous studies have confirmed the activation of TICs during liver tumorigenesis. We also found that SOX9 was expressed in both HCC and CC, suggesting that SOX9 might serve as a liver TICs marker. We thus used the Pten(loxp/loxp); SOX9-CreER+ mouse model (TIC-PKO) to study the role of PTEN in TICs during liver tumorigenesis. Liver tumor development was observed in TIC-PKO mice at the age of 12 months old, and immunohistochemical analysis revealed that the tumors were heterogeneous, indicating TICs were involved in the tumorigenesis process. In addition, treatment of DDC, which is a liver toxin and thus causes hepatocyte death, dramatically increased the incidence and led to the early onset of liver tumorigenesis in the mutant but not the control mice, suggesting that liver injury served as an essential microenvironmental niche in promoting liver tumor development. Together, our observations suggested that the PTEN signaling is critical for TICs regulation and Pten deletion synergizes with liver injury to drive both TICs activation and liver tumorigenesis. Citation Format: Ni Zeng, Anketse Kassa, Janel Kopp, Lina He, Maike Sander, Bangyan Stiles. Pten deletion in SOX9+ cells synergizes with hepatic injury to drive tumor development in mouse liver. [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 LB-145. doi:10.1158/1538-7445.AM2015-LB-145

Abstract 1417: Clonal succession in pancreatic cancer progression is not driven by genetic instability

Abstract Our group has recently shown that human pancreatic cancer (PDAC) progression is driven by a succession of transiently active tumor initiating cell (TIC) clones during serial xenotransplantation. Genetic labeling demonstrated that serial PDAC xenograft tumors and even tumors of parallel mice transplanted with cells from the same donor xenografts harbored very little to no overlap of active TIC clones, indicating substantial changes in the proliferative activity of individual TIC predominantly producing progeny without detectable tumor-initiating activity. We now asked whether observed clonal activation and inactivation is caused by acquisition of de novo mutations during evolution of genetic subclones or by functional plasticity of genetically stable TIC clones. Therefore, we monitored somatic non-synonymous mutations in culture and during PDAC progression in genetically marked serial xenografts of two patients. DNA was isolated from xenografts, primary TIC cultures and corresponding normal pancreas or primary tumor tissue. Following paired-end exome sequencing, reads were aligned to a concatenated hs37d5 human and mm10 mouse genome assembly and human specific single nucleotide variants (SNVs) and small insertions/deletions (indels) were identified. We found a total of 45 altered gene coding genomic loci (P1 = 10; P2 = 35) not present in control tissue. Strikingly, most SNVs detected were present in all samples, only very few SNV were acquired during serial transplantation. In P1, 4 novel SNVs not present in the original patient tumor sample were detected within coding regions of TTC13, OR4K15, SSPO and TPGS1. Allele frequencies ranged from 2-27% in serial xenografts. In xenografts of P2 we detected 35 SNVs not present in healthy tissue. Of these, one mutation in the gene C10orf12 aroused after serial transplantation with a maximum altered allele frequency of 17%. None of these mutations is a known cancer driver or was found as recurrent in large scale cancer sequencing approaches. To evaluate whether the clonal TIC dynamics within established tumors are recapitulated in vitro, we analysed individual TIC clone kinetics in serially passaged cultures and in cultures derived from transduced xenografts. Strikingly, the kinetics in vitro were similar to those observed within serially transplanted xenografts. Every culture passage was formed by a distinct set of actively proliferating cell clones without significant overlap between individual serial passages indicating that clonal succession of TIC activity in PDAC is not dependent on the cellular context in tumors in vivo. The remarkable genetic stability of xenografts during serial transplantation strongly indicates that changes in the functional state of PDAC cells and not genetic instability drive clonal succession of TIC activity in PDAC. Citation Format: Karl Roland Ehrenberg, Claudia R. Ball, Felix Oppel, Naveed Ishaque, Taronish D. Dubash, Sebastian M. Dieter, Christopher M. Hoffmann, Ulrich Abel, Moritz Koch, Jens Werner, Frank Bergmann, Manfred Schmidt, Christof von Kalle, Wilko Weichert, Jürgen Weitz, Benedikt Brors, Hanno Glimm. Clonal succession in pancreatic cancer progression is not driven by genetic instability. [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 1417. doi:10.1158/1538-7445.AM2015-1417

Abstract 3885: ITGA6 (CD49F) is directly regulated by hypoxia-inducible factors

Abstract Introduction: In response to hypoxic stress, tumors must adapt to the hostile microenvironment by regulating angiogenesis, cellular metabolism, cell survival and motility. The alpha subunits of the Hypoxia-Inducible Factors (HIF-1α and HIF-2α) are oxygen-responsive, master regulators of the hypoxic response. Over-expression of HIF-1α independently correlates with decreased survival of breast cancer patients, and a hypoxic core gene signature is enriched in triple negative breast cancers (TNBC) and HER2+ cancers relative to luminal tumors (ER+). In a variety of solid tumors, HIF-1α or HIF-2α also directly promotes tumor initiating cell (TIC) activity. Methods: Mammary tumor epithelial cells (MTECs) were established from late-stage tumors of MMTV-PyMT+; Hif1α floxed mice (which model ER-negative, luminal-like tumors), and exposed to either Adenovirus-β-gal or -Cre to create wild-type (WT) and knock-out (KO) cells, respectively. MDA-MB-231 cells (a model of TNBC) were generated in which either HIF1A or HIF2A or both genes (DKD) were knocked down (KD) via shRNA. Results: When PyMT+ MTECs were transplanted into female recipients, HIF-1 deletion reduced primary tumor growth by 60%, lung metastasis by 90% and TIC activity by &amp;gt;35-fold (Schwab et al. 2012). The expression of cell surface markers routinely used to enrich for TICs was assayed in PyMT+ and MDA-MB-231 cells. ITGA6 mRNA and CD49f protein expression were significantly decreased in response to HIF deletion at both normoxia and hypoxia. ChIP assays showed that either HIF-1 or HIF-2 bind to at least two HREs identified within the ITGA6 promoter. When WT PyMT tumors were sorted to enrich for CD49fhi cells, limiting dilution transplantation confirmed that TIC activity was increased relative to CD49low cells. Finally, data mining of breast cancer patient repositories showed that ITGA6 mRNA expression is independently and inversely correlated with overall and recurrence free survival. Conclusions: CD49f is a direct transcriptional target of HIF-1/HIF-2. Because CD49f is known to enrich for breast TICs, we suggest that HIF-dependent regulation of ITGA6 expression contributes at least partially to HIFs promotion of TIC activity in ER-negative breast cancers. Citation Format: Danielle L. Peacock, Luciana P. Schwab, Tiffany N. Seagroves. ITGA6 (CD49F) is directly regulated by hypoxia-inducible factors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3885. doi:10.1158/1538-7445.AM2014-3885

Abstract B006: Tumor cell heterogeneity of prolactin-induced ERα+ mammary carcinomas

Abstract Anti-estrogenic therapies have significantly improved the survival of women with estrogen-receptor alpha (ERα) positive breast tumors. Unfortunately 25% of women who suffer from ERα+ cancer experience recurrence of tumors which can exhibit strong therapeutic resistance. The mechanisms underlying the onset of anti-estrogen resistant tumors following treatment are not well understood, but emerging evidence is now implicating tumor-initiating cells (TICs) in tumor growth, treatment resistance, and differentiation. TICs represent a small discrete subpopulation of cells which have the ability to drive the growth of an entire heterogeneous tumor. Elevated circulating prolactin (PRL) levels have been shown to be associated with ERα+ breast tumor incidence, and also correlate with increased metastasis and treatment resistance. To examine the relationship between TICs and treatment resistance in ERα+ mammary carcinomas, we utilized a transgenic mouse model (NRL-PRL), established on a FVB/N background, that mimics the local expression of PRL within the mammary epithelia as observed in women. After a long latency, nulliparous NRL-PRL females develop locally invasive mammary carcinomas, with some observed lung and bone metastases. Importantly, PRL-induced mammary tumors exhibit wide histological diversity and the majority are ERα+ (&amp;gt;75% of tumors). Although development of tumors is accelerated by supplemental estrogen, end stage tumors are insensitive to estrogen, similar to the luminal B subtype. A panel of 20 ERα+ glandular and papillary adenocarcinomas was collected from NRL-PRL females. Their histological appearances and ERα expression were retained after transplantation into mammary glands of wild-type syngeneic recipients. To analyze tumor cell heterogeneity, subpopulations were characterized by subjecting enzymatically dispersed tumor cells to fluorescence-activated cell sorting (FACS). PRL-induced ERα+ tumors consistently displayed high percentages of both luminal (EPCAMhiCD49fmed) and basal (EPCAMloCD49fhi) cells, which is distinct from the luminal but ERα- (MMTV-Her2/neu) and claudin-low (p53-/-) mouse models of breast cancer. The luminal/basal cell ratios for NRL-PRL, MMTV-Her2/neu, and p53-/- derived tumors were 2.4±0.4, 46.0±1.8, and 0.004±0.005, (mean±S.D., N=3-4), respectively. The basal compartment exhibits relatively high TIC enrichment in several well-characterized models of breast cancer. However, recent evidence suggests that the luminal compartment may contain a luminal-progenitor subpopulation which also displays some TIC activity. Interestingly, expression of the surface-marker CD61 in PRL-induced tumors was distributed across both luminal and basal cell compartments. Ongoing limiting-dilution transplantation studies will identify fractions containing TIC activity. Transgenic mammary production of PRL is a useful mouse model to study aggressive ERα+ breast cancer that will provide novel insights into this disease. Supported by R01 CA157675 and T32 ES007015. Citation Format: Michael P. Shea, Kathleen A. O'Leary, Linda A. Schuler. Tumor cell heterogeneity of prolactin-induced ERα+ mammary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B006.

Abstract B50: A patient-derived renal cell carcinoma model as a platform for the identification of novel diagnostic markers and therapeutic targets

Abstract Clear-cell renal cell carcinoma (ccRCC) is highly resistant to conventional therapies and to date effective treatment is restricted to surgical resection. Pre-clinical in vitro and in vivo models, which accurately mimic this disease, are critical for the development of novel effective therapies. Previous attempts to culture ccRCC cells have been proven difficult and thus, only few ccRCC cell lines are currently available. In addition long-term culturing often causes loss of primary tumor characteristics including tumor heterogeneity. This might explain why in contrast to other tumor entities, tumor-initiating cell (TIC) subpopulations have so far not been reported for ccRCC. TICs have been associated with tumor progression, metastasis and drug-resistance. Consequently these cells have also been named cancer stem cells (CSCs). Furthermore, distinct combinations of genetic and epigenetic alterations result in profound inter-patient heterogeneity and can in part promote TIC activity. Therefore, the development and characterization of clinically relevant patient derived ccRCC-models would provide an experimental basis to study the complex mechanisms leading to and maintaining ccRCCs. Hence, we established a patient-derived serum-free spheroid culture system that can be used to propagate primary ccRCC cells. These ccRCC cells retain their tumor-initiating potential and mimic the human malignancy upon orthotopic injection into immunodeficient mice. They not only mirror the histological properties but also the molecular features of the parental tumor. Moreover, these cells show the capacity to form de novo metastasis in lungs, which is the most common metastatic site for ccRCC in patients. The established ccRCC cultures were then used as a screening platform for the identification of surface proteins, which are heterogeneously expressed among tumor cells. Functional in vitro and in vivo assays showed that subpopulations defined by expression of these markers also displayed distinct functional characteristics including TIC activity. Gene Set Enrichment Analysis (GSEA) revealed differences in the signaling networks active in these sub-populations. In addition, molecular and cellular assays addressed the significance of these pathways for the maintenance of ccRCC malignancy. Currently the identified surface markers and underlying pathways are validated as potential novel diagnostic and therapeutic targets. Therefore, our novel patient-derived serum-free spheroid culture system serves as a platform to expand patient derived ccRCCs. It further allows the analysis and characterization of different subpopulations, which may ultimately contribute to the understanding of the molecular basis underlying progression, therapy-resistance and metastasis of this disease. Citation Format: Teresa Rigo-Watermeier, Corinna Klein, Vanessa Vogel, Christian Eisen, Thomas Hoefner, Wilko Weichert, Sascha Pahernik, Markus Hohenfellner, Martin R. Sprick, Andreas Trumpp. A patient-derived renal cell carcinoma model as a platform for the identification of novel diagnostic markers and therapeutic targets. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B50.

Abstract 2295: Succession of transiently active TIC clones drives long-term human pancreatic cancer progression .

Abstract Despite accumulating evidence indicating a pivotal role of tumor-initiating cells (TIC) in tumor progression and metastases formation of pancreatic cancer (PC), very little is known about the clonal TIC dynamics within PC tumors in vivo. It is unclear whether the high aggressiveness of PC is driven by a homogeneous population of self-renewing TIC or whether distinct classes or activation states of TIC drive long-term disease progression. To address this we tracked the clonal contribution of individual TIC to tumor formation in vivo. Serum-free adherent cultures were established allowing enrichment of PC TIC without restriction to a certain phenotype. These cultures grow as 3-dimensional epithelial colonies with tight cell-to-cell contacts und reliably form subcutaneous tumors in NSG mice. To induce differentiation of TIC, FBS was added and FGF2, FGF10 and Nodal were withdrawn. Under these conditions, irregular shaped cells form a monolayer and down regulate markers previously described for TIC or normal pancreatic progenitors. Strikingly, despite profound changes in morphology and marker expression tumor initiation and self-renewal of TIC in serial transplantation were unchanged. Moreover, sorted CD133+ and CD133− cells equally efficient formed tumors containing similar proportions of CD133+ in vivo. Together these data indicate an unexpected phenotypic plasticity of TIC in PC. To determine the clonal kinetics of individual self-renewing TIC in vivo, early passage serum-free cultures from 3 patients were transduced with a lentiviral vector (LV) and serially transplanted. LV stably integrate into the host cell genome resulting in TIC clone specific fusion sequences which were identified by highly sensitive LAM-PCR and high throughput sequencing. In primary mice, 0.003-0.113% of all transduced cells contributed actively to tumor formation. Unexpectedly, in subsequent generations tumor formation was mainly driven by distinct TIC clones not detectable in earlier but recruited to tumor formation in later generations. Moreover, individual primary xenografts generated pairs of secondary tumors with very little overlap between clonal compositions. Mathematical modeling indicates strong changes in proliferative activity of individual, otherwise homogenous TIC which predominantly produce non-tumorigenic progeny with very limited self-renewal. These data indicate that long-term progression of PC is driven by a succession of transiently active TIC generating tumor cells in temporally restricted bursts. The recruitment of inactive TIC clones to tumor formation after serial transplantation points to a context-dependent regulation of TIC activity within the growing tumor. Further understanding the mechanisms regulating the balance between activated and resting states of TIC may allow developing specific treatment strategies targeting this most relevant cell population in PC. Citation Format: Felix Oppel, Claudia R. Ball, Christopher M. Hoffmann, Sebastian M. Dieter, Taronish D. Dubash, Moritz Koch, Jürgen Weitz, Frank Bergmann, Manfred Schmidt, Ulrich Abel, Christof von Kalle, Hanno Glimm. Succession of transiently active TIC clones drives long-term human pancreatic cancer progression . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2295. doi:10.1158/1538-7445.AM2013-2295

NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro

IntroductionNOTCH activation has been recently implicated in human breast cancers, associated with a poor prognosis, and tumor-initiating cells are hypothesized to mediate resistance to treatment and disease relapse. To address the role of NOTCH1 in mammary gland development, transformation, and mammary tumor-initiating cell activity, we developed a doxycycline-regulated mouse model of NOTCH1-mediated mammary transformation.MethodsMammary gland development was analyzed by using whole-mount analysis and by flow cytometry in nulliparous transgenic mice maintained in the presence/absence of doxycycline (or intracellular NOTCH1). Mammary tumors were examined histologically and immunophenotyped by staining with antibodies followed by flow cytometry. Tumors were transplanted into mammary fat pads under limiting dilution conditions, and tumor-initiating cell frequency was calculated. Mammary tumor cells were also plated in vitro in a tumorsphere assay in the presence/absence of doxycycline. RNA was isolated from mammary tumor cell lines cultured in the presence/absence of doxycycline and used for gene-expression profiling with Affymetrix mouse arrays. NOTCH1-regulated genes were identified and validated by using quantitative real-time polymerase chain reaction (PCR). Mammary tumor-bearing mice were treated with doxycycline to suppress NOTCH1 expression, and disease recurrence was monitored.ResultsSimilar to published studies, we show that constitutive expression of human intracellular NOTCH1 in the developing mouse mammary gland inhibits side branching and promotes luminal cell fate. These mice develop mammary adenocarcinomas that express cytokeratin (CK) 8/18. In vivo limiting-dilution analyses revealed that these mammary tumors exhibit functional heterogeneity and harbor a rare (1/2,978) mammary tumor-initiating cell population. With this dox-regulated NOTCH1 mammary tumor model, we demonstrate that NOTCH1 inhibition results in mammary tumor regression in vivo and prevents disease recurrence in four of six tumors tested. Consistent with the in vivo data, NOTCH1 inhibition reduces mammary tumorsphere activity in vitro. We also identify the embryonic stem cell transcription factor Nanog as a novel NOTCH1-regulated gene in tumorspheres and in mouse and human breast cancer cell lines.ConclusionsThese data indicate that NOTCH1 inhibition results in mammary tumor regression in vivo and interferes with disease recurrence. We demonstrate that NOTCH1-transformed mouse mammary tumors harbor a rare mammary tumor-initiating population and that NOTCH1 contributes to mammary tumor-initiating activity. This work raises the possibility that NOTCH therapeutics may target mammary tumor-initiating cells in certain human breast cancer subtypes.