Kien Pham

Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of basal-like breast cancer cells through novel mechanisms

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

Expression and Functional Heterogeneity of Chemokine Receptors CXCR4 and CXCR7 in Primary Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of these tumors indicates a need to identify new therapeutic targets. Therapy resistance of GBMs is attributed to heterogeneity of the glioblastoma due to genetic alterations and functional subpopulations. Chemokine receptors CXCR4 and CXCR7 play important roles in progression of various cancers although the specific functions of the CXCL12−CXCR4−CXCR7 axis in GBM are less characterized. In this study we examined the expression and function of CXCR4 and CXCR7 in four primary patient-derived GBM cell lines of the proliferative subclass, investigating their roles in in vitro growth, migration, sphere and tube formation. CXCR4 and CXCR7 cell surface expression was heterogeneous both between and within each cell line examined, which was not reflected by RT-PCR analysis. Variable percentages of CXCR4+CXCR7− (CXCR4 single positive), CXCR4−CXCR7+ (CXCR7 single positive), CXCR4+CXCR7+ (double positive), and CXCR4−CXCR7− (double negative) subpopulations were evident across the lines examined. A subpopulation of slow cell cycling cells was enriched in CXCR4 and CXCR7. CXCR4+, CXCR7+, and CXCR4+/CXCR7+ subpopulations were able to initiate intracranial tumors in vivo. CXCL12 stimulated in vitro cell growth, migration, sphere formation and tube formation in some lines and, depending on the response, the effects were mediated by either CXCR4 or CXCR7. Collectively, our results indicate a high level of heterogeneity in both the surface expression and functions of CXCR4 and CXCR7 in primary human GBM cells of the proliferative subclass. Should targeting of CXCR4 and CXCR7 provide clinical benefits to GBM patients, a personalized treatment approach should be considered given the differential expression and functions of these receptors in GBM.

Patient-Derived Xenograft Models for Pancreatic Adenocarcinoma Demonstrate Retention of Tumor Morphology through Incorporation of Murine Stromal Elements

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma. Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived pancreatic adenocarcinoma xenografts, establishing the integration of murine cells into networks of invading cancer cells. In addition, we provide methods and observations in the establishment and maintenance of a patient-derived pancreatic adenocarcinoma xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Patient demographics, staging, pathological analysis, and outcomes were analyzed. After successful engraftment of tumors, histological and immunofluorescence analyses were performed on explanted tumors. Pancreatic adenocarcinoma specimens were successfully engrafted in 15 (60%) of 25 attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages, and tumors retain metastatic potential. Interestingly, despite morphological similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions.

An in vivo model of epithelial to mesenchymal transition reveals a mitogenic switch

The epithelial to mesenchymal transition (EMT) is a process by which differentiated epithelial cells transition to a mesenchymal phenotype. EMT enables the escape of epithelial cells from the rigid structural constraints of the tissue architecture to a phenotype more amenable to cell migration and, therefore, invasion and metastasis. We characterized an in vivo model of EMT and discovered that marked changes in mitogenic signaling occurred during this process. DNA microarray analysis revealed that the expression of a number of genes varied significantly between post-EMT and pre-EMT breast cancer cells. Post-EMT cancer cells upregulated mRNA encoding c-Met and the PDGF and LPA receptors, and acquired increased responsiveness to HGF, PDGF, and LPA. This rendered the post-EMT cells responsive to the growth inhibitory effects of HGF, PDGF, and LPA receptor inhibitors/antagonists. Furthermore, post-EMT cells exhibited decreased basal Raf and Erk phosphorylation, and in comparison to pre-EMT cells, their proliferation was poorly inhibited by a MEK inhibitor. These studies suggest that therapies need to be designed to target both pre-EMT and post-EMT cancer cells and that signaling changes in post-EMT cells may allow them to take advantage of paracrine signaling from the stroma in vivo.

CCL5, CCR1 and CCR5 in murine glioblastoma: immune cell infiltration and survival rates are not dependent on individual expression of either CCR1 or CCR5.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Microglia/macrophages are found within human GBM where they likely promote tumor progression. We report that CCL5, CCR1, and CCR5 are expressed in glioblastoma. Individual deletion of CCR1 or CCR5 had little to no effect on survival of tumor bearing mice, or numbers of glioblastoma-infiltrated microglia/macrophages or lymphocytes. CCL5 promoted in vitro migration of wild type, CCR1- or CCR5-deficient microglia/macrophages that was blocked by the dual CCR1/CCR5 antagonist, Met-CCL5. These data suggest that CCL5 functions within the glioblastoma microenvironment through CCR1 and CCR5 in a redundant manner.

VEGFR inhibitors upregulate CXCR4 in VEGF receptor-expressing glioblastoma in a TGFβR signaling-dependent manner

The failure of standard treatment for patients diagnosed with glioblastoma (GBM) coupled with the highly vascularized nature of this solid tumor has led to the consideration of agents targeting VEGF or VEGFRs, as alternative therapeutic strategies for this disease. Despite modest achievements in survival obtained with such treatments, failure to maintain an enduring survival benefit and more invasive relapsing tumors are evident. Our study suggests a potential mechanism by which anti-VEGF/VEGFR therapies regulate the enhanced invasive phenotype through a pathway that involves TGFβR and CXCR4. VEGFR signaling inhibitors (Cediranib and Vandetanib) elevated the expression of CXCR4 in VEGFR-expressing GBM cell lines and tumors, and enhanced the in vitro migration of these lines toward CXCL12. The combination of VEGFR inhibitor and CXCR4 antagonist provided a greater survival benefit to tumor-bearing animals. The upregulation of CXCR4 by VEGFR inhibitors was dependent on TGFβ/TGFβR, but not HGF/MET, signaling activity, suggesting a mechanism of crosstalk among VEGF/VEGFR, TGFβ/TGFβR, and CXCL12/CXCR4 pathways in the malignant phenotype of recurrent tumors after anti-VEGF/VEGFR therapies. Thus, the combination of VEGFR, CXCR4, and TGFβR inhibitors could provide an alternative strategy to halt GBM progression.

Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

Cancer cells exert mastery over the local tumor-associated stroma (TAS) to configure protective immunity within the tumor microenvironment. The immunomodulatory character of pancreatic lysates of patients with cancer differs from those with pancreatitis. In this study, we evaluated the cross-talk between pancreatic cancer and its TAS in primary human cell culture models. Upon exposure of TAS to pancreatic cancer cell-conditioned media, we documented robust secretion of IL6 and IL8. This TAS response was MyD88-dependent and sufficient to directly suppress both CD4+ and CD8+ T-cell proliferation, inducing Th17 polarization at the expense of Th1. We found that patients possessed a similar shift in circulating effector memory Th17:Th1 ratios compared with healthy controls. The TAS response also directly suppressed CD8+ T-cell-mediated cytotoxicity. Overall, our results demonstrate how TAS contributes to the production of an immunosuppressive tumor microenvironment in pancreatic cancer. Cancer Res; 77(3); 672-83. ©2016 AACR.

Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

Hepatocellular Carcinoma Metastasis and Circulating Tumor Cells

A hallmark of aggressive hepatocellular carcinomas (HCCs) is the ability to metastasize. Metastatic lesions are difficult to manage in clinical practice as the extent of disease typically precludes curative resection and resistance to systemic treatments is common [1, 2]. Metastasis is a multistage process in which cancer cells (1) delaminate from the primary site and locally invade the host stroma (initiation), (2) enter into blood and/or lymphatic vasculature (intravasation), (3) survive and exit the circulation into distant sites (extravasation), and (4) colonize the new microenvironment and proliferate to form a macroscopic secondary tumor (colonization) [3, 4]. This simplified model provides a framework for a sequence of biological properties that must be acquired during cancer dissemination. Different malignancies, however, demonstrate unique regulatory events in this process largely governed by the complex microenvironment in which the metastatic cells originate and that of the distant location(s) where the metastasis is established [5]. Given the anatomic and physiologic complexity of the liver, the HCC microenvironment is one of the most difficult to reproduce experimentally and, consequently, understand in its totality. In the context of this chapter, HCC metastasis is discussed, with an emphasis on the role of the hepatic microenvironment and the role of circulating cancer cells in the metastatic process.

Abstract 1466: Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through the incorporation of murine stromal elements

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma (PC). Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived PC xenografts, establishing the rapidity by which murine cells are integrated into networks of invading cancer cells. In addition, we provide methodology and observations in the establishment and maintenance of a patient-derived PC xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into NOD-SCID mice. Patient demographics, staging, pathologic analysis and outcomes were analyzed. After successful engraftment of tumors, histologic and immunofluorescent analyses were performed on explanted tumors. PC specimens were successfully engrafted in 15 of 25 (60%) of attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages and tumors retain metastatic potential. Interestingly, despite morphologic similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions. Citation Format: Daniel Delitto, Kien Pham, Adrian C. Vlada, George A. Sarosi, Ryan M. Thomas, Kevin E. Behrns, Chen Liu, Steven J. Hughes, Shannon M. Wallet, Jose G. Trevino. Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through the incorporation of murine stromal elements. [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 1466. doi:10.1158/1538-7445.AM2015-1466