Heterogeneity Of Primary Tumor Research Articles

Abstract 4129: Chloroquine and romidepsin: combination therapy for treatment of breast cancer metastases

Abstract Cancer metastasis is favored through dynamic regulation of gene expression and comprehensive cellular processes including epigenetic regulation. Aberrant gene expression by altered epigenetic regulation plays a fundamental role in the maintenance of breast cancer stem cell (BCSC) and drug resistance. Additionally, intrinsic tumor heterogeneity in primary tumors and the presence of the breast cancer stem cells complicates treatment options even further. Thus, it is hypothesized that the breast cancer metastasis can be cured by an effective treatment, targeting both the bulk and the BCSCs at the same time. Previously, chloroquine (CQ), inhibited spontaneous breast cancer metastasis to lungs very effectively through inhibition of autophagy as well as DNA hypomethylation. Romidepsin (RO), a highly potent and FDA-approved histone deacetylases inhibitor, is known to exert outstanding anti-cancer effects on numerous cancer types. Thus, we propose a combination therapy of chloroquine and romidepsin to target the breast cancer metastasis. The goal of this study is to test if the combined therapy will effectively inhibit breast cancer metastasis by targeting both bulk tumors and BCSCs compared to each single agent treatment. We evaluated in vitro anti-tumor effects of the combination therapy of chloroquine and romidepsin by measuring mammosphere forming efficiency (MSFE) and by analyzing apoptosis through Annexin V staining in paclitaxel sensitive and resistant Hs578t cells. Secondly, we investigated anti-tumor effects of vehicle (Con), CQ, RO, or their combination (RO-CQ) using a cell line and a patient-derived orthotopic xenograft models. Thirdly, we assessed BCSC populations and tumor initiation ability by FACS analysis, MSFE measurement, and limiting dilution assays (LDA). Finally, we evaluated the metastatic potential of tumors after the each treatment using an in vivo MDA-MB-231 lung metastasis model. We observed that the RO-CQ treatment resulted in the most significant reduction of MSFE and induction of apoptosis in BCSCs and bulk tumors compared to other treatment groups in both paclitaxel sensitive and resistant Hs578t cells. Secondly, only the RO-CQ treatment caused significant inhibition of tumor growth in vivo with substantial decrease in the CD44+/CD24- or Aldefluor+ BCSCs. The reduction of BCSCs by the RO-CQ treatment was well correlated with the severely reduced primary and the secondary MSFE. Also, the reduction of BCSCs was well reflected in the LDA, in which the RO-CQ treatment impaired tumor initiating ability. Finally, we confirmed in vivo that CQ alone and the RO-CQ treatments substantially abolished the metastatic potential of highly metastatic MDA-MB-231 cells. Our data suggest that the two FDA-approved drugs on breast cancer metastases will facilitate the rapid translation of basic discoveries into clinical approaches which can lead to improved survival for breast cancer patients with refractory metastases. Citation Format: Dong Soon Choi, Jenny C. Chang. Chloroquine and romidepsin: combination therapy for treatment of breast cancer metastases. [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 4129. doi:10.1158/1538-7445.AM2015-4129

New and emerging factors in tumorigenesis: an overview.

This article provides an overview of the genes and cellular processes that have emerged recently as new key factors in tumorigenesis. We review these in the context of three broad categories. First, genome-scale sequencing studies have revealed a set of frequently mutated genes in cancer. Genes that are mutated in >5% of all cancers across tissue types are discussed, with a highlighted focus on the two most frequently mutated genes, TP53 and PIK3CA. Second, the mechanisms of resistance to targeted therapy are reviewed. These include acquired resistance under targeted therapy selection owing to mutations and amplification of genes in the same or parallel signaling pathways. Importantly, sequencing of primary tumors has revealed that therapy-resistant clones already exist prior to targeted therapy, demonstrating that tumor heterogeneity in primary tumors confers a mechanism for inherent therapy resistance. Third, “metastasis-specific genes”, or rather lack thereof, are discussed. While many genes have been shown to be capable of promoting metastasis in experimental systems, no common genetic alterations have been identified specific to metastatic lesions. Rather, the same gene mutations frequently found in primary tumors are also found prevalent in metastases, suggesting that the genes that drive tumorigenesis may also drive metastasis. In this light, an emerging view of metastatic progression is discussed. Collectively, these recent advances in cancer research have refined our knowledge on cancer etiology and progression but also present challenges that will require innovative new approaches to treat and manage cancer.

Molecular Heterogeneity in Primary Breast Carcinomas and Axillary Lymph Node Metastases Assessed by Genomic Fingerprinting Analysis.

Molecular heterogeneity within primary breast carcinomas and among axillary lymph node (LN) metastases may impact diagnosis and confound treatment. In this study, we used short tandem repeated sequences to assess genomic heterogeneity and to determine hereditary relationships among primary tumor areas and regional metastases from 30 breast cancer patients. We found that primary carcinomas were genetically heterogeneous and sampling multiple areas was necessary to adequately assess genomic variability. LN metastases appeared to originate at different time periods during disease progression from different sites of the primary tumor and the extent of genomic divergence among regional metastases was associated with a less favorable patient outcome (P = 0.009). In conclusion, metastasis is a complex process influenced by primary tumor heterogeneity and variability in the timing of dissemination. Genomic variation in primary breast tumors and regional metastases may negatively impact clinical diagnostics and contribute to therapeutic resistance.

P38MAPK activation mediates tumor necrosis factor-α-induced apoptosis in glioma cells

Gliomas are a type of heterogeneous primary central nervous system tumor, which arise from the glial cells; these types of tumor generally respond poorly to surgery, radiation and conventional chemotherapy. Tumor necrosis factor‑α (TNF‑α) has been suggested to produce an antitumor effect by binding to specific receptors on the tumor cell membrane to induce apoptosis. TNF‑α is known to activate a number of signaling pathways, including extracellular signal‑regulated protein kinase, c‑Jun N‑terminal kinase (JNK), p38 mitogen‑activated protein kinase (p38MAPK), nuclear factor‑κB and caspase cascades, depending on the cell type. However, the involvement of p38MAPK signaling in TNF‑α‑induced apoptosis in glioma cells remains unclear. In the current study, the role of p38MAPK in TNF‑α‑induced apoptosis in rat glioma C6 cells was investigated. TNF‑α was observed to induce cell apoptosis and the phosphorylation of p38MAPK in C6 cells. In addition, the inhibition of p38MAPK markedly reduced TNF‑α‑induced apoptosis, while JNK inhibition did not affect apoptosis. Furthermore, p38MAPK transfection altered the cell cycle of glioma cells and increased the rate of apoptosis. It also led to an increase in the level of soluble TNF‑α in the culture supernatant and membrane TNF receptor I levels in tumor cells. In conclusion, the results of the current study demonstrated that the activation of p38MAPK mediates TNF‑α‑induced apoptosis in glioma C6 cells, suggesting p38MAPK as a potential target for glioma therapy.

Abstract 5170: Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in colorectal cancer

Abstract Complex sets of driver mutations critically control clinical course and treatment response in colorectal cancer (CRC). It is expected that multiple mutations in different signaling pathways interact to establish an individual cancer cell phenotype. Furthermore, mutational patterns can evolve under selective pressure, for instance during tumor metastasis or targeted therapy. Yet only few mutations, such as these in KRAS, NRAS and BRAF, are routinely assessed in the clinic today. With the introduction of next generation sequencing (NGS) many more genes can be simultaneously sequenced, allowing detailed analyses of mutational patterns. We have assembled the CRC5.2 NGS panel for application with Ion Torrent PGM covering 100 frequently mutated genes with 784 amplicons (covering 21,000 COSMIC mutations), going well beyond the scope of commercially available cancer panels. In particular, exon coverage was optimized to embrace all genomic information related to drug sensitivity, using several drug and signaling network databases. The German Consortium for Translational Cancer Research (DKTK) comprises the top cancer centers in Germany. As a part of this group, we employ our custom CRC5.2 panel for the analysis of mutational patterns in CRC. We address several key questions relevant for current diagnostics and treatment in colorectal cancer. In retrospective and prospective clinical studies, we assess mutational heterogeneity in primary tumors, synchronous and metachronous metastases of individual patients. In-depth NGS mutational analyses, transcriptome analysis and methylation studies will identify (epi-)genetic alterations occurring as a consequence of selective pressure during tumor evolution e.g. during metastasis, chemotherapy or targeted tumor therapy. Thus, we will uncover new links between cancer-specific mutations, clinical course and treatment response as well as novel mechanisms of secondary resistance. In addition we aim to identify novel predictive and prognostic mutational markers for the benefit of future CRC patients Citation Format: Soulafa Mamlouk, Liam Childs, Thorben Redmer, Pawel Durek, Dirk Schumacher, Hendrik Bläker, Daniela Aust, Moritz von Winterfeld, Volker Heinemann, Dominik Modest, Kerstin Möhr, Markus Morkel, Reinhold Schäfer, Christine Sers. Linking tumor evolution and therapy response using diagnostic targeted next generation sequencing in colorectal cancer. [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 5170. doi:10.1158/1538-7445.AM2014-5170

Abstract 3020: Patient-derived xenograft (PDX) models to study the role of breast cancer stem cells in metastasis formation

Abstract Although advances have been made in the treatment of localized breast cancer, there has been less progress in the treatment of advanced metastatic disease. This disease is an oncoming therapeutic challenge for breast cancer since it is always fatal. Conventional therapies may fail to target cancer stem cells (CSC). Metastatic breast cancer may thus benefit from current intensive research in the CSC field. Several studies suggest that CSCs are the only cancer cells responsible for metastasis formation. CSCs may have the ability to disseminate in blood circulation as circulating tumor cells (CTCs) and to colonize new sites. To better understand the biological processes that link CSCs and metastasis, we have generated a xenobank of 50 patient-derived xenografts (PDXs) of primary tumors grafted in the humanized mammary fat pad of immunodeficient mice. These models reflect the heterogeneity of primary tumors both at molecular and cellular level. Using a lentiviral vector, we infected our PDXs to obtain GFP-luciferase-positive models designed to monitor metastasis development from the orthotopic site. To mimick the patient therapeutic management, metastasis formation is monitored during tumor growth but also after tumor surgical removal. Metastases are collected and analyzed for histology and phenotype. The different metastatic patterns are correlated with the mutational profile of PDX. CTCs are also monitored at different time of the metastatic process using a specific filter (ISET) for cell characterization and phenotype. PDXs can be classified in tumor groups with similar metastatic tropisms. To depict the role of CSC from primary tumors in the tropism of their metastases, we collected and genomic profiled the ALDEFLUOR-positive CSCs in orthotopic tumors from different models with distinct metastatic pattern. These different PDX models lead to a better understanding of the role of breast CSCs in metastasis formation from CTCs to well-constituted metastasis and their study may identify targets able to design future therapeutic strategies. Citation Format: Christophe Ginestier, Danae Lartigue, Olivier Cabaud, Aurelie Malzac, Julien Wicinski, Emmanuelle Josselin, Pascal Finetti, Jose Adelaide, Francois Bertucci, Max Chaffanet, Daniel Birnbaum, Emmanuelle Charafe-Jauffret. Patient-derived xenograft (PDX) models to study the role of breast cancer stem cells in metastasis formation. [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 3020. doi:10.1158/1538-7445.AM2014-3020

Abstract 1205: In vivo animal models with patient-derived tumor xenografts for better clinical prediction

Abstract More predictive in vivo animal tumor models are urgently needed to test efficacy of newly developed cancer therapies and predict clinical response. At SRI we have developed highly innovative and predictive models initiated with single tumor cells isolated from cancer patient specimens, including colon, lung, and breast tumor xenografts. These tumors propagated in immunocompromised mice recapitulate primary tumor heterogeneity as shown by histology. In our models, the tumor cells are implanted subcutaneously to evaluate inhibition of primary tumor, or at orthotopic sites to recapitulate advanced metastatic disease and analyze circulating tumor cells (CTCs) or disseminated tumor cells (DTCs). Tumor cells are also implanted in organs, e.g. kidney capsule, to mimic the vascularized tumor microenvironment or in the human skin graft to analyze the tumor interactions with human stroma and vasculature, and the effect of treatment on angiogenesis. The cellular suspension method offers several advantages over the widely employed tumor fragments, including the ability to assess the phenotype of tumor cell populations, tumorigenicity, and tumor initiating cells. Cells isolated from tumors of mice in control and treatment groups are mouse-lineage depleted prior to evaluation in a number of in vitro or ex vivo assays, including migration and invasion through extracellular matrix proteins. To determine whether treatment affects frequency of tumor initiating cells (TICs) and metastasis initiating cells (MICs), we will perform in vivo limiting dilution assay (LDA). Moreover, to evaluate the ability of our tumor models to induce angiogenesis we utilized the chicken embryo chorioallantoic membrane (CAM) assay and scored the vascularity of mouse lineage-depleted tumor cells using an established CAM scoring guide with 5 as the highest score and 0 as the lowest score. One patient derived ER- invasive ductal carcinoma breast cancer (SRI-B1) with high growth rate demonstrated strong pro-angiogenic activity, inducing blood vessels in the CAM assay with a score of 4 compared to 5 for a VEGF positive control and 0 of negative control. The high vascular density induced by SRI-B1 tumor cells correlated with in vivo metastatic dissemination to distant organs upon orthotopic implantation in NOD/SCID mice, suggesting that tumor models can be screened in the CAM assay to evaluate their metastatic potential. We plan to further use these models to test the anticancer activity of experimental therapies that target metastasis. Citation Format: Jun Li, Jessica Kalra, Dominic Dinh, Wan-ru Chao, Xiaohe Liu, Lidia Sambucetti, Lucia Beviglia. In vivo animal models with patient-derived tumor xenografts for better clinical prediction. [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 1205. doi:10.1158/1538-7445.AM2014-1205

A novel genome-wide in vivo screen for metastatic suppressors in human colon cancer identifies the positive WNT-TCF pathway modulators TMED3 and SOX12.

The progression of tumors to the metastatic state involves the loss of metastatic suppressor functions. Finding these, however, is difficult as in vitro assays do not fully predict metastatic behavior, and the majority of studies have used cloned cell lines, which do not reflect primary tumor heterogeneity. Here, we have designed a novel genome-wide screen to identify metastatic suppressors using primary human tumor cells in mice, which allows saturation screens. Using this unbiased approach, we have tested the hypothesis that endogenous colon cancer metastatic suppressors affect WNT-TCF signaling. Our screen has identified two novel metastatic suppressors: TMED3 and SOX12, the knockdown of which increases metastatic growth after direct seeding. Moreover, both modify the type of self-renewing spheroids, but only knockdown of TMED3 also induces spheroid cell spreading and lung metastases from a subcutaneous xenograft. Importantly, whereas TMED3 and SOX12 belong to different families involved in protein secretion and transcriptional regulation, both promote endogenous WNT-TCF activity. Treatments for advanced or metastatic colon cancer may thus not benefit from WNT blockers, and these may promote a worse outcome.

Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits

Ample evidence supports genetic and functional heterogeneity in primary tumors, but it remains unclear whether circulating tumor cells (CTCs) also exhibit the same hierarchical organization. We examined the functional diversity of viable, single CTCs using an array of subnanoliter wells (nanowells). The compartmentalization of single cells by nanowells allowed clonal comparison and mapping of heterogeneity of single cells or preformed clusters of cells. By measuring the short-term viability, invasiveness and secretory profiles of individual CTCs, it was evident that only a rare subset of CTCs possessed malignant traits indicative of metastatic potential in late-stage, progressing metastatic castration-resistant prostate cancer (mCRPC) patients. These CTCs were resistant to anoikis after being in the circulation, were invasive in their epithelial state, or secreted proteases capable of cleaving peptide substrates. Every CTC observed, however, did not exhibit such metastatic potential, suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients.

Assessment of changes in tumor heterogeneity following neoadjuvant chemotherapy in primary esophageal cancer

To assess the changes in computed tomography (CT) tumor heterogeneity following neoadjuvant chemotherapy in esophageal cancer. Thirty-one consecutive patients who received neoadjuvant chemotherapy for esophageal cancer were identified. Analysis of primary tumor heterogeneity (texture) was performed on staging and post-chemotherapy CT scans. Image texture parameters (mean grey-level intensity, entropy, uniformity, kurtosis, skewness, standard deviation of histogram) were derived for different levels of image filtration (0-2.5). Proportional changes in each parameter following treatment were obtained. Comparison between pathological tumor response and texture parameters was analyzed using Mann-Whitney U-test. The relationship between CT texture and overall survival) was estimated using the Kaplan-Meier method. Tumor texture became more homogeneous after treatment with a significant decrease in entropy and increase in uniformity (filter 1.0 and 2.5). Pretreatment (filter 1.5, P = 0.006) and posttreatment standard deviation of histogram (filter 1.0, P = 0.009) showed a borderline association with pathological tumor response. A proportional change in skewness <0.39 (filter 1.0) was associated with improved survival (median overall survival 36.1 vs. 11.1 months; P < 0.001). CT tumor heterogeneity decreased following neoadjuvant chemotherapy and has the potential to provide additional information in primary esophageal cancer.

The safety and efficacy of magnetic nano-iron hyperthermia therapy on rat brain glioma

Gliomas are a group of heterogeneous primary central nervous system tumors arising from glial cells. These tumors are associated with high morbidity and mortality. New opportunities for the development of effective therapies for malignant gliomas are urgently needed. Magnetic nano-particles can heat up tumor tissues and induce the killing of cancer cells. However, the in vivo action of magnetic nano-iron hyperthermia on brain gliomas has not been widely investigated. The safety, efficacy, and suitable dose of hyperthermia therapy remain unknown. We successfully established a rat model of brain glioma by injecting C6 glioma cells into the right caudate nuclei of rats. Fixed doses (2.5, 5, or 10mg) of magnetic nano-iron were then injected into the tumors of tumor-bearing rats. The survival time of tumor-bearing rats was subsequently observed, and imaging studies were conducted on the brain tumors. Of the 80 rats that underwent C6 glioma cell implantation, 70 exhibited decreased mobility and appetite, and wasting. Establishment of this brain glioma model was confirmed to be successful by magnetic resonance imaging. After injection of different doses of magnetic nano-iron, the survival times of the different dose groups of tumor-bearing rats were not significantly different. However, the tumor size exhibited a significant decrease with magnetic nano-iron hyperthermia therapy. Injection of various doses of magnetic nano-iron was safe in tumor-bearing rats. The effective doses were 2.5 and 5mg. Magnetic nano-iron hyperthermia significantly shrank the brain gliomas in tumor-bearing rats.

Dependence of Tumor Cell Lines and Patient-Derived Tumors on the NAD Salvage Pathway Renders Them Sensitive to NAMPT Inhibition with GNE-618

Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity.

Abstract A58: An ovarian patient-derived xenograft model to identify the chemoresistant population

Abstract Introduction: A cornerstone of preclinical ovarian cancer research over the last 30 years has been the use of cell lines both in vitro and in vivo. This resource has underperformed in its ability to identify effective novel therapeutics and evaluate the heterogeneity of an ovarian neoplasm. Developing a patient-derived xenograft (PDX) allows for a comprehensive study of the heterogeneous tumor and potentially the identification of cellular populations responsible for chemoresistance and recurrence, but has not been fully characterized for ovarian cancer. We have developed an ovarian PDX model to demonstrate many important similarities and differences between these and primary patient tumors, and begun to explore its utility in identifying mediators of chemoresistance and personalizing therapy. Methods: Tumors removed from patients undergoing primary tumor reductive surgery were implanted into the subcutaneous flanks of SCID mice. Once a tumor developed, it was expanded into additional mice to propagate a PDX line. Tumors were analyzed for heterogeneity by examination of the tumor initiating cell populations, its stromal content with HLA immunohistochemistry, and proliferation with Ki-67. Cohorts of PDX lines were treated with maximum-tolerated dose of combined carboplatin and paclitaxel or vehicle and response was compared between the PDX and the patient. Chemoresistant PDX lines were generated by treatment with MTD carboplatin and paclitaxel until no evidence of disease (NED), observation until recurrence, and retreatment until fully resistant. RNAseq was conducted comparing treated PDX lines to the untreated PDX lines (n=6 pair) to identify important mediators of chemotherapy resistance. Finally, PDX lines were characterized for defects in homologous recombination (HR) repair with an ex vivo assay, correlating HR status with response to PARP inhibition in vivo. Results: Refinement of xenografting procedures yields an 85% success rate in establishing a PDX. PDX tumors maintain the patient's histology, but the stromal component is replaced by murine cells in the first generation. The xenografts retain primary tumor heterogeneity, at least in expression of putative tumor initiating cells (TIC) (ALDH: 17% vs 19%, CD44: 2.4% vs 5%, CD133 10% vs 3%). However, treatment with chemotherapy significantly increases these populations (ALDH1 increased to 36.1% from 16.2% (p=0.002); CD133 increased to 33.8% from 16.2% (p=0.026), suggesting a role in chemotherapy resistance. The treated PDX tumors showed a decrease in Ki67 staining (from 64% to 34%, p=0.001), suggesting dormancy is induced in the surviving population. The PDX tumors show similar response to chemotherapy as patient tumors, in that PDX tumors from patients with a partial response respond more slowly (if at all) than those from patients achieving a complete response (mean change in tumor volume -5.11% vs -63.73%, p=0.029). RNAseq comparing treated and untreated PDX lines indicate only 54 common genes with a significant change in mRNA (p&amp;lt;0.05), suggesting great heterogeneity among patients not only in primary tumor content, but also mediators of chemoresistance. Finally, patient tumors predicted to respond to PARP inhibition by an ex vivo assay show regression of tumor or stable disease with single-agent PARP inhibitor therapy. Conclusions: The ovarian PDX model recapitulates patient tumor heterogeneity and mirrors response to chemotherapy. This model may prove superior in predicting response to therapy in patients, and allows a better model to study complex tumor biology that is impacted by tumor heterogeneity, such as survival of small populations with chemotherapy. Citation Format: Zachary C. Dobbin, Ashwini K. Katre, Monjri M. Shah, Britt K. Erickson, Hauping Chen, Ronald D. Alvarez, Michael G. Conner, Dongquan Chen, Charles N. Landen. An ovarian patient-derived xenograft model to identify the chemoresistant population. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A58.

Differential drug responses of circulating tumor cells within patient blood

Personalized medicine holds great promise for cancer treatment, with the potential to address challenges associated with drug sensitivity and interpatient variability. Circulating tumor cells (CTC) can be useful for screening cancer drugs as they may reflect the severity and heterogeneity of primary tumors. Here we present a platform for rapidly evaluating individualized drug susceptibility. Treatment efficacy is evaluated directly in blood, employing a relevant environment for drug administration, and assessed by comparison of CTC counts in treated and control samples. Multiple drugs at varying concentrations are evaluated simultaneously to predict an appropriate therapy for individual patients.

Abstract 3183: Deep-sequencing reveals distinct clonal evolution patterns of DLBCL relapse and identifies mutant MLL2 as potential early driver.

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is an aggressive lymphoma with 40% of the patients relapse to chemoresistant disease. Our current understanding of the molecular mechanisms associated with relapse is poor. Mainly, how clonal heterogeneity in primary tumors contributes to relapse is unclear. Therefore, we performed high-throughput sequencing of rearranged VDJ junctions in 20 pairs of matched diagnosis-relapse tumors. In DLBCL, VDJ junctions effectively tag clonal tumor populations. Moreover, somatic hypermutations (SHM) within rearranged VDJ provide additional information to study heterogeneity and track evolution. To achieve high coverage, we sequenced (2X150bp) across the 300-350bp VDJ sequences, generating 0.25-0.5 million VDJ junctions per tumor. We developed a custom bioinformatics pipeline to identify the VDJ and SHM patterns of each sequence. In most pairs (n=19), diagnosis and relapse are clonally related, harboring the same major VDJ rearrangement. Phylogenetic analysis on the SHM profiles of the major VDJ arrangement uncovered two distinct scenarios of DLBCL relapse. In scenario I (n=17), the major diagnosis and relapse clones clustered together, indicating a linear progression. We performed exome sequencing on 6 such pairs. As expected, diagnosis and relapse tumors harbored many common mutations, including mutations in CARD11, NOTCH1, and TP53. Furthermore, we observed that relapse tumors had acquired additional 70±22 mutations, among which 14±5 are in genes previously found mutated in DLBCLs, including EP300, EZH2, and MYC. Taken together, we hypothesize that in this scenario, a portion of the diagnosis tumor cells survived chemotherapy, acquired additional “facilitator” mutations, and developed into relapse tumors. In scenario II (n=2), the major relapse clone clustered together with a highly divergent minor diagnosis clone forming a separate branch from the rest of the diagnosis clones on the phylogenetic tree. This particular pattern indicates that in this scenario although the relapse and diagnosis tumors came from the same B cell, they diverged early, acquired different mutations, and rose at different times. Indeed, exome sequencing on these pairs revealed that both diagnosis and relapse tumors had their distinctive DLBCL related mutations. Furthermore, there were several mutations common between diagnosis and relapse tumors, indicating that these mutations were acquired very early in the history of the tumors. Interestingly, MLL2 mutations were found in the common set of mutations in both pairs, strongly suggesting that MLL2 mutation could act as a “driver” or “facilitator” mutation in the earliest steps of DLBCL lymphomagenesis. Taken together, we discovered two modes of DLBCL relapse by coupling deep-sequencing of the VDJ rearrangements and exomes, providing new knowledge about molecular pathogenesis of DLBCL relapse. Citation Format: Yanwen Jiang, David Redmond, Kui Nie, Peter Martin, Ari Melnick, Wayne Tam, Olivier Elemento. Deep-sequencing reveals distinct clonal evolution patterns of DLBCL relapse and identifies mutant MLL2 as potential early driver. [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 3183. doi:10.1158/1538-7445.AM2013-3183

Abstract C73: Src-beta catenin-SNAI1 axis is critical for the stemness and bone metastasis of E-cadherin-lacking prostate cancer cells

Abstract Bone metastasis of prostate cancer (PCA) cells is the main cause of high mortality and morbidity in patients. Recent studies have suggested that cancer stem cells not only sustain the growth and heterogeneity of primary tumors but also play a role in metastasis. Therefore, a better molecular understanding of metastatic cancer stem cells could be exploited towards targeting bone metastasis. Here, we analyzed the role of E-cadherin and Src-beta catenin-SNAI1 axis in regulating the stemness and bone metastasis of PCA cells. Human PCA PC3 cells were transfected with shRNA specific for E-cadherin or random shRNA, and selected for stable knock-down of E-cadherin (ShEC-PC3) or respective control cells (Sh-PC3). MTT, clonogenic and xenograft assays were performed to compare the in vitro and in vivo proliferation potential of Sh-PC3 and ShEC-PC3 cells. Stem cell population was characterized by FACS and prostasphere formation. Orthotopic xenograft model and optical imaging tools were employed to study metastasis. Immunoblotting and immunohistochemistry (IHC) methods were used to analyze proliferation, stemness, EMT, and osteomimicry biomarkers. Our results showed that ShEC-PC3 cells possessed mesenchymal phenotype, enhanced invasiveness and clonogenicity as well as higher growth rate both in cell culture and xenografts. Importantly, compared to Sh-PC3 cells, ShEC-PC3 cells formed significantly higher number and bigger sized prostaspheres suggesting an increase in the stem cell population with E-cadherin knock-down. Also, the ShEC-PC3 prostaspheres disintegration in the presence of serum generated bigger mass of attached proliferating cells compared to Sh-PC3. Cell sorting for established PCA stem cell phenotypic markers (CD44highCD24low) confirmed a two-fold increase in stem cell population along with a nine fold higher CD44high/CD44low population in ShEC-PC3 cells compared to Sh-PC3 cells. Immunoblotting and IHC analyses showed that E-cadherin knock-down increased the expression of stemness markers (CD44, Notch1 and Egr-1) as well as EMT and invasivness markers (Vimentin, pSrc-tyr416, beta catenin, NF-kappaB, integrin alpha 5 and beta 3) in both cell culture and xenograft tissues. Next, to define the role of E-cadherin in PCA metastasis, we orthotopically injected Sh-PC3 or ShEC-PC3 cells in the prostate of nude mice and after 4 weeks, imaged the mice using IR-dye (EGF optical probe). Mice injected with ShEC-PC3 cells showed remarkable metastasis to distant organs mainly to the bones, while mice injected with Sh-PC3 cells showed only prostate localized growth. We also observed increased expression of several osteomimicry molecules namely CXCR4, uPA, RANKL and Runx2 in ShEC-PC3 cells in cell culture as well as xenograft tissues. Importantly, we observed a remarkable increase in SNAI1 expression in both cytoplasmic and nuclear fractions from prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, the SNAI1 knock-down by specific siRNA completely inhibited the prostasphere formation, clonogenicity and invasiveness of ShEC-PC3 cells. The SNAI1 inhibition also decreased the level of pSrc-tyr416 and beta catenin in ShEC-PC3 cells suggesting SNAI1 role in regulating the expression of these molecules. Interestingly, inhibition of Src (by dasatinib) or beta catenin (by FH535) also decreased the SNAI1 expression. These results clearly showed that E-cadherin knock-down in PCA cells promotes the formation of Src-beta catenin-SNAI1 axis which then controls the stemness, invasiveness, and metastasis of PCA cells. Therefore, E-cadherin activators and/or Src-beta catenin-SNAI1 repressors could be useful in targeting the PCA stemness and bone metastasis, and lowering the PCA burden. Citation Format: Gagan Deep, Anil K. Jain, Samiha Mateen, Kavitha C. Vijendra, Subhash C. Gangar, Chapla Agarwal, Rajesh Agarwal. Src-beta catenin-SNAI1 axis is critical for the stemness and bone metastasis of E-cadherin-lacking prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C73.

Effect of homotypic and heterotypic interaction in 3D on the E-selectin mediated adhesive properties of breast cancer cell lines

Hematogenous metastasis involves a glycoprotein mediated adhesion cascade of tumor cells with E-selectin on the endothelial layer of the blood vessels. Cell–cell interactions play a major role in cancer metastasis and invasiveness. Intercellular communication between two cancer cells or between a cancer cell with a stromal cell in the microenvironment such as fibroblasts or inflammatory cells play an important role in metastatic invasion. Culturing tumor cells as 3D spheroids can recapitulate these physiologically relevant cell–cell interactions. The heterogeneity in primary tumors is attributed to cell subpopulations with varying degree of invasiveness. Co-culturing cancer cells with different phenotypes as 3D spheroids can mimic this heterogeneity. Here we report the effect of homotypic and heterotypic interactions in breast cancer cells cultured as 3D spheroids on polydimethylsiloxane (PDMS) on the adhesion phenotype to E-selectin. We show that breast cancer cell lines (BT20 and MCF7) propagating as 3D spheroids on PDMS exhibit a stronger interaction with human recombinant E-selectin when compared to their respective monolayer grown counterparts on tissue culture plate (TCP). Matrigel invasion assay also indicated that BT20 and MCF7 spheroids were more invasive than BT20 and MCF7 cells grown as monolayers. To mimic tumor heterogeneity in vitro, a co-culture model included tumorigenic cell lines BT20, MCF7 and a non-tumorigenic mammary epithelial cell line MCF10A. These cell lines were cultured together in equal seeding ratio on PDMS to generate co-culture spheroids. The heterotypic interactions in the co-culture model resulted in enhancement of the adhesion of the most invasive BT20 cell line to E-selectin. BT20 cells in co-culture bound to the greatest degree to soluble E-selectin compared to MCF7 and MCF10A cells in co-culture. Co-invasion assay with co-culture spheroids indicated that BT20 cells in co-culture were more invasive than MCF7 and MCF10A cells. The results presented here indicate that homotypic and heterotypic interaction of cancer cells favor adhesion to E-selectin thus representing a complexity beyond planar cell culture. Also, when cells of different phenotypes are mixed, the heterogeneity enhances the adhesive phenotype and invasiveness of the most invasive cell population. The results challenge the classic use of planar cell culture for evaluating the adhesion of cancer cells to E-selectin and establish our co-culture technique as a model that can help investigative studies in metastasis and invasiveness of breast and other types of cancers.

Hyperthermia Promotes Apoptosis and Suppresses Invasion in C6 Rat Glioma Cells

Gliomas are a group of heterogeneous primary central nervous system tumors. Hyperthermia has proven to be a potential therapeutic tool for cancers in the clinic. However, the molecular mechanisms of hyperthermia remain unclear. The objective of this study was to investigate the effects of hyperthermia on the invasiveness in C6 glioma cells and related molecular pathways. Here our data show hyperthermia stimulated the release of tumor necrosis factor-alpha (TNF-α) and decreased C6 glioma cell migration and invasive capability at 30, 60, 120 and 180 min; with increased spontaneous apoptosis in C6 glioma cells at 120 min. We also found mitogen-activated protein kinase (P38 MAPK) protein expression to be increased and nuclear factor-kappa B (NF-κB) protein expression decreased. Based on the results, we conclude that hyperthermia alone reduced invasion of C6 glioma cells through stimulating TNF-α signaling to activate apoptosis, enhancing P38 MAPK expression and inhibiting the NF-κB pathway, a first report in C6 rat glioma cells.

Differential response to EGFR- and VEGF-targeted therapies in patient-derived tumor tissue xenograft models of colon carcinoma and related metastases

Heterogeneity in primary tumors and related metastases may result in failure of antitumor therapies, particularly in targeted therapies for the treatment of cancer. In this study, patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases were used to evaluate the response to EGFR- and VEGF-targeted therapies. Our results showed that primary colon carcinoma and its corresponding lymphatic and hepatic metastases have a different response rate to anti-EGFR (cetuximab) and anti-VEGF (bevacizumab) therapies. However, the underlying mechanism of these types of phenomenon is still unclear. To investigate whether such phenomena may result from the heterogeneity in primary colon carcinoma and related metastases, we compared the expression levels of cell signaling pathway proteins using immunohistochemical staining and western blotting, and the gene status of KRAS using pyrosequencing in the same primary colon carcinoma and its corresponding lymphatic and hepatic metastatic tissues which were used for establishing the PDTT xenograft models. Our results showed that the expression levels of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR were different in primary colon carcinoma and matched lymphatic and hepatic metastases although the KRAS gene status in all cases was wild-type. Our results indicate that the heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases may result in differences in the response to dual-inhibition of EGFR and VEGF.

Abstract LB-397: Deep sequencing of Xenografts and case-matched blood and primary tumors reveals a 20 folds enrichment of loss of heterozygosity versus somatic mutations suggesting LOH plays an ever important role in tumorigenesis

Abstract The double hits theory suggests a major mechanism of tumorigenesis. However, compared to the somatic mutations, the loss of heterozygosity (LOH) was not frequently identified in many sequencing projects. Unlike the somatic mutations that were detected by the evidence of novel alleles in tumor tissue, the alleles involving in the LOH are present in normal tissues as well. Given the high level of tissue heterogeneity of primary tumors, including both tumorous cells and normal stroma cells, at whole tissue or million cells level LOH can only be detected as a quantitative change between tumor and normal samples, which are not easily distinguished from potential sequencing errors or other artifacts especially with low depth sequencing. By applying whole exome capture deep sequencing on case-matched primary tumor, blood and xenograft samples, we found that xenografts represent a much purer human tumor cell population than primary tumor tissues. These tumor cells growing in mice not only contain the majority of somatic mutations in the primary tumors (&amp;gt;95%), but also reveal a &amp;gt;20 folds enrichment of somatic LOH events, which were not categorized as LOH in primary tumors, versus somatic mutations. All these findings were detected after removing mouse sequence contaminations, which if included will induce about half of the total variations observed in the xenograft samples. We found that these LOH events affect both tumor suppressor genes and oncogenes. Given that driver mutations or other variations that are implicated in oncogenesis are most likely preserved in xenografted tumor cells in immunedeficient mice, we hypothesize that the bigger range of somatic LOH events that we observed in xenograft samples play a potential ever important role in tumorigenesis than what we thought previously. Our finding also sheds a new light on a novel approach for the discovery of potential drug targets by sequencing xenograft samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-397. doi:1538-7445.AM2012-LB-397