Stromal Response Research Articles

The type I insulin-like growth factor regulates the liver stromal response to metastatic colon carcinoma cells.

Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.

SPARC is a possible predictive marker for albumin-bound paclitaxel in non-small-cell lung cancer.

ObjectivesNanoparticle albumin-bound paclitaxel (nab-paclitaxel) produced good tumor response in cases with lung squamous cell carcinoma, one of the most difficult cancers to treat. Secreted protein acidic and rich in cysteine (SPARC) binds to albumin, suggesting that SPARC plays an important role in tumor uptake of nab-paclitaxel. There is as yet no predictive marker for cytotoxic agents against non-small-cell lung cancer (NSCLC), and hence we believed that SPARC expression might be associated with tumor response to nab-paclitaxel.Patients and methodsWe studied stromal SPARC reactivity and its association with clinicopathological characteristics in 200 cases of NSCLC using a custom tissue microarray fabricated in our laboratory by immunohistochemical staining. We also investigated the relationship between stromal SPARC reactivity and tumor response to nab-paclitaxel using biopsy or surgical specimens obtained from advanced or recurrent lung cancer patients.ResultsHigh SPARC stromal reactivity (>50% of optical fields examined) was detected in 16.5% of cases and intermediate SPARC reactivity (10%–50%) in 56% of cases. High expression in cancer cells was rare (five cases). Stromal SPARC level was correlated with smoking index, squamous cell carcinoma, and vessel invasion. Furthermore, patients with high stromal SPARC reactivity in biopsy specimens such as transbronchial lung biopsy or surgical specimens tended to respond better to nab-paclitaxel.ConclusionStromal SPARC was detected by immunohistochemical staining in ∼70% of NSCLC cases, and good tumor response to nab-paclitaxel was correlated with high stromal SPARC reactivity. SPARC may be a useful predictive marker for selecting patients likely to respond favorably to nab-paclitaxel treatment.

Roles of Nanofiber Scaffold Structure and Chemistry in Directing Human Bone Marrow Stromal Cell Response

Nanofiber technology has emerged as a promising tool to recapitulate native extracellular matrix structure; however the properties of nanofibers governing cell-material interactions are still largely undetermined. We have investigated the role of both nanofiber structure and chemistry in directing the response of human bone marrow stromal cells (hBMSCs). We developed a poly (e-caprolactone) (PCL) nanofiber material system where scaffold structure is maintained while surface chemistry is modified via mild sodium hydroxide (NaOH) hydrolysis to introduce surface carboxyl groups. Surface carboxyl groups was verified via x-ray photoelectron spectroscopy (XPS), water contact angle (WCA), and toluidine blue-O biochemical assay and scaffold structure was verified with scanning electron microscopy (SEM). hBMSC response was investigated for proliferation, differentiation (alkaline phosphatase activity and mineralization) , cell morphology, and microarray gene expression profiles. We found that both nanofiber structure and chemistry play a role in hBMSC osteogenic differentiation where PCL nanofiber scaffolds were able to elicit an osteogenic phenotype, while chemically modified PCL nanofiber scaffolds did not. Scaffold structure was found to have a more dominant effect on cell morphology and extracellular matrix gene expression while nanofiber chemistry had a larger effect on genes related to metabolism and cytoskeletal rearrangement. The nanofiber scaffold material system developed here can serve as a platform to further investigate the role of micro environmental cues on osteogenic differentiation. The NaOH modified NF scaffolds are particularly useful as a negative control for osteogenic differentiation in NF scaffolds.

The Role of Stromal Components in Pancreatic Cancer Progression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, and is known for its extremely poor prognosis. Because of the location and composition of the organ, early symptoms cannot be visualized as easily as in other solid tumors. In the past decades, researchers have been mostly working on the genetic and epigenetic alterations of the cancer cells themselves, and therapies on pancreatic cancer cells alone have failed to significantly improve patient outcome. With the identification of abundant tumor stromal responses, the focus of pancreatic cancer research has begun to change. Increasing evidence has proved that the tumor stroma, especially the cell components (such as pancreatic stellate cells, tumor-associated macrophages, mast cells etc.) plays a key role in the development of PDAC. In this review, we discuss the interactions between cancer cells and several important cell components of the tumor stroma, as well their role in tumor growth, migration, invasion, angiogenesis and immune recognition.

Abstract B13: Human pancreatic cancer organoids for functional dependency profiling

Abstract Pancreatic ductal adenocarcinoma is a deadly disease with few effective targeted therapies. As such, there is great interest in the development of in vitro and in vivo models from patient-derived samples to better understand disease drivers and to identify potential therapeutic targets. In collaboration with the Division of Gastrointestinal Oncology at the Dana-Farber Cancer Institute and the Cancer Cell Line Factory at the Broad Institute we have developed a pipeline to culture primary pancreatic tumor specimens as patient-derived xenografts (PDXs), organoids, and cell lines. We have established a cohort of 18 PDXs from primary pancreatic tumors and 3 from metastatic specimens. In addition, we have initiated a panel of 10 3D organoid cultures, including from core biopsies and fine needle aspirates, and have also attempted 2D culture of 17 tumor specimens using the conditionally reprogrammed cell (CRC) culture protocol, with successful generation of 5 tumor cell lines. In cases where a 2D cell line could not be established from the primary specimen, outgrowth of fibroblasts associated with the tumor was a common mode of failure. Consequently, we now enrich for the epithelial tumor population through several passages in 3D organoid culture before adapting to 2D surfaces to enable more efficient cell line generation. We are characterizing organoids and cell lines by targeted amplicon and/or whole exome sequencing for comparison to the primary specimen. Pancreatic tumors are notable for having a pronounced stromal response, but the impact of extracellular matrix (ECM)-mediated signaling on tumor cell behavior remains unclear. As such, one goal of our efforts is to characterize and understand how the ECM environment influences tumor cell signaling and drug responses. To this end, we have performed compound screening of a 3D organoid line in parallel with a matched 2D cell line to identify novel therapeutic targets and to begin to dissect therapeutic sensitivities that may be specific to the culture environment. Our preliminary results suggest that while there is significant concordance in drug sensitivity of cells cultured in 2D and 3D contexts, there are also several dependencies that are unique to the specific environmental context. We are performing RNA sequencing and additional molecular analyses of cells in these various adhesive conditions to identify key molecules or pathways that mediate these differential responses. The combination of compound and genetic screening in a variety of environmental contexts using these novel patient-derived culture models will improve our understanding of pancreatic cancer biology and facilitate more comprehensive pancreatic cancer dependency profiling. Citation Format: Srivatsan Raghavan, Shubhroz Gill, Andrew J. Aguirre, Yuen-Yi M. Tseng, Anson Peng, Paula Keskula, Xiaoyun Wu, Coyin Oh, Jamie Cheah, Gregory Kryukov, Ewa Sicinska, Brian M. Wolpin, Matthew H. Kulke, Charles S. Fuchs, Paul Clemons, Todd R. Golub, Stuart Schreiber, Jesse S. Boehm, William C. Hahn. Human pancreatic cancer organoids for functional dependency profiling. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B13.

Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour’s dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.

TSLP Down-Regulates S100A7 and ß-Defensin 2 Via the JAK2/STAT3-Dependent Mechanism

Elevated T-helper type 2 cytokines in atopic skin, such as IL-4 and IL-13, were thought to be responsible for an impaired expression of antimicrobial proteins, which may contribute to the increased susceptibility to skin infections in patients with atopic dermatitis. In this study, the relationship between thymic stromal lymphopoietin and antimicrobial proteins and the involved molecular pathway was defined in normal human epidermal keratinocytes and human skin equivalent model. Stimulation of normal human epidermal keratinocytes with thymic stromal lymphopoietin decreased both mRNA and levels of S100A7 and human β-defensin 2 in a dose-dependent manner, and the regulation was JAK2/STAT3-dependent. Thymic stromal lymphopoietin decreased the antimicrobial protein expression, even in the presence of IL-17, which is their strong inducer. STAT3 directly regulated the S100A7 and human β-defensin 2 promoters in normal human epidermal keratinocytes. Immunohistochemically, lesional atopic skin stained more intensely with phospho-STAT3 compared with healthy control. Our results show that up-regulated thymic stromal lymphopoietin may contribute to the deficiency of antimicrobial proteins in atopic dermatitis, including S100A7 and human β-defensin 2, by a JAK2/STAT3-dependent mechanism and that STAT3/Sin3a might directly control the transcriptional activity of the antimicrobial protein promoters in normal human epidermal keratinocytes. Taken together, a key role of the JAK2/STAT3/Sin3a signaling pathway in thymic stromal lymphopoietin-mediated immune response in normal human epidermal keratinocytes might give us clues to understanding the pathological signal transductions in atopic dermatitis.

Abstract IA04: Stromal regulation of pancreatic cancer epigenome and metabolome

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the dramatic accumulation of stromal components implicated in tumor growth, metastasis, and resistance to therapy. This creates a living chimera in which stroma-derived growth factors, matrix proteins and inflammatory cytokines are thought to nurture the nascent tumor. This study reveals that the stromal secretome exerts rapid epigenomic changes in the cancer cells that effectively marshals up metabolic and genomic networks needed to power cell growth. To tackle the complexity of the tumor microenvironment (TME), we devised a three-dimensional culture system to dissect the adaptive impact of the stromal secretome on the cancer cell. This integrative approach identified the rapid mobilization of more than a dozen gene networks (cell cycle, steroid biosynthesis, metabolic pathways, DNA replication, etc.), as well as a commensurate adaptive metabolome. A major unanswered question relates to the sequence of events that result in the formation and ultimate function of these networks, in part generated by rapid changes in acetylation of histone H3 at lysines 9 and 27 (H3K9ac and H3K27ac). Our work suggests the existence of a molecular and-gate such that tumor activation is the consequence of mutant Ras and the tumor micro-environment. To mediate this molecular hand-shake, we find that BRD2 acts in the epithelial compartment to transduce stromal cues at the genomic level. Based on the above, and consistent with recent Mazur PK et al Nat Med (2015), we show the BET bromodomain inhibitor JQ1 targets a key synergy point and reduces pancreatic tumor growth in vivo. Collectively, this work describes a novel connection between the stromal response and a driver oncogene and may offer new insights into the role of the TME in the treatment of ras-driven tumor. Citation Format: Mara H. Sherman, Ronald Evans. Stromal regulation of pancreatic cancer epigenome and metabolome. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA04.

Abrogation of prostaglandin E-EP4 signaling in osteoblasts prevents the bone destruction induced by human prostate cancer metastases

The metastasis of tumors to bone is known to be promoted by prostaglandin E2 (PGE2) produced by the tumor host stromal tissue. Although bone metastases frequently occur in prostate cancer patients, the significance of PGE2 in stromal responses to the tumor is not known. In this study, we report that PGE2 and its receptor EP4 play a pivotal role in bone destruction and metastasis in an experimental metastasis model of prostate cancer in nude mice. Using human prostate cancer PC-3 cells that are stably transfected with luciferase, we showed that the development of bone metastasis was accompanied by increased osteoclastic bone resorption in the bone metastasis microenvironment, and could be abrogated by an EP4 receptor antagonist. The growth of PC-3 cells in vitro was not influenced by PGE2 or by the EP4 receptor. However, cell-cell interactions between fixed PC-3 cells and host osteoblasts induced PGE2 production and RANKL expression in the osteoblasts. Addition of an EP4 antagonist suppressed both PGE2 and RANKL expression induced by the PC3-osteoblast interaction, which would have consequent effects on osteoclast activation and osteolysis. These results indicate that the blockage of PGE2-EP4 signaling prevents the bone destruction required for prostate cancer metastases, and that this is, in part due to the abrogation of bone cell responses. The study provides further evidence that an EP4 antagonist is a candidate for the treatment of prostate cancer in the blockade of bone metastasis.

Abstract 4145: Stromal heterogeneity and immune response in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer (HGSOC). Despite the increasing evidence on stromal involvement in tumor progression, the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) in ovarian cancer is not fully understood. How tumor heterogeneity contributes to a different in immune response remains a challenge question that needs to be addressed. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors. By laser microdissection of tumor tissue samples from HGSOC patients, we generated cell type specific expression profiles and identified a CAF-specific gene signature for ovarian cancer. Genes expressed exclusively by CAFs that are associated with differential immune response were identified by comparing CAF expression profiles from HGSOC patients with high and low tumor-infiltrated CD8+ T cell densities. Among the differentially expressed genes identified, immunostaining results showed a significant inverse correlation between stromal MFAP5 expression and intratumoral CD8+ T cell density in HGSOC tissue samples (p = 0.006). The results were further confirmed by correlating stromal MFAP5 protein expression and intratumoral CD8+ T cell density. Our recent studies showed that increased stromal MFAP5 expression is associated with poorer survival in HGSOC patients and MFAP5 modulates ovarian caner invasion and motility potential. Together with our preliminary data showing that MFAP5 modulates the expression of immune-related genes, we hypothesize that stromal MFAP5 may generate an immunosuppressive microenvironment through suppressing CD8+ T cell activation and trafficking in the ovarian tumor tissue. Cell culture experiment showed that recombinant MFAP5 protein treatment induced expression of CD47, an immune checkpoint protein, in cancer cells and downregulated CXCL13, a chemokine essential for immune cell adhesion, in endothelial cells. Using animal models, these findings were further validated. Based on our data, animal studies will be performed to further evaluate the efficacy of immune activation by targeting stromal MFAP5 in ovarian cancer treatment. Delineating the molecular mechanism by which MFAP5 modulates the immune responses in ovarian cancer will help to design novel treatment modalities based on stromal MFAP5 blockade, which will promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Stromal heterogeneity and immune response in ovarian cancer. [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 4145.

Abstract 4143: Characterizing the immunogenicity of gastric cancer by transcriptomic expression based immune phenotyping

Abstract One of the main challenges in immuno-oncology is to quantify different types of immune cells in the tumor microenvironment, as such data can greatly facilitate our understanding of the mechanism of action of, and response to cancer immunotherapies. Traditionally immune profiling is performed by immunohistochemistry or flow cytometry experiments using surface markers specific to each immune cell type. However, both approaches suffer from practical limitations such as reagent availability and real world specimen conditions. Recently several computational deconvolution approaches using expression profiles of the bulk tumor tissue samples have been developed. This alternative approach utilizes existing database of purified immune cell gene signatures and relies on a rigorous mathematical framework of signal deconvolution. One of such algorithms, called CIBERSORT (Newman et al. 2015), has been demonstrated to perform robustly in deconvoluting the relative fractions of 22 human leukocyte subsets in solid tumor tissue samples, and benchmarked favorably against FACS analysis. In this work, we performed immune phenotyping by the CIBERSORT-based expression deconvolution on a cohort of 100 gastric cancer (GC) patients. We found that the fractions of activated CD4 memory T cells, rather than CD8+ T cell, are most significantly correlated with tumor neo-antigen load, whereas the latter is most significantly associated with the expression of a “Cytolytic Activity” metagene and an interferon gamma signature. There is stronger pre-existing host immune response against the MSI and EBV subtypes of GCs mediated by either cytotoxic T cells or Natural Killer cells. Lastly, we found a high M2/M1 tumor associated Macrophages (TAMs) ratio is strongly associated with poor GC prognosis, corroborating recent reports on the role of a TAM and stromal response in gastric cancer angiogenesis. Taken together, we demonstrated in this work that critical information about the tumor infiltrating immune cells can be gleaned from the computational deconvolution of bulk tumor gene expression profiling data. The comprehensiveness and cost-effectiveness of this approach can complement other immune profiling techniques in characterizing a wide variety of tumor specimens under various treatment conditions. We foresee its application in characterizing pharmacodynamics, understanding immunological mechanism of action and monitoring treatment response and disease progression for cancer immunotherapies and their combinations in both pre-clinical and clinical settings. Citation Format: Kai Wang, Hoicheong Siu, Shibing Deng, Jadwiga R. Bienkowska, Suet Yi Leung. Characterizing the immunogenicity of gastric cancer by transcriptomic expression based immune phenotyping. [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 4143.

Abstract 5124: Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice

Abstract Breast cancer recurrence remains high in triple-negative cases despite aggressive surgical, radiological, and chemotherapeutic intervention. Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence. However, the tumor microenvironment's role in recurrence is not well understood. We hypothesize that the irradiated tumor stroma and microenvironment may influence tumor cell migration. In this study, we characterize the effects of normal tissue irradiation on tumor and immune cell migration to evaluate how tumor-stromal interactions modulate recurrence after therapy. This work represents the first step toward elucidating how stromal tissue radiation response contributes to tumor and immune cell recruitment. Mouse embryonic fibroblasts (MEF) were irradiated to 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation to be used as a chemoattractant in a transwell assay to investigate the induction of 4T1 murine or MDA-MB-231 human mammary carcinoma cell invasion. An orthotopic breast cancer model was used to evaluate the effect of radiation on tumor cell migration to normal tissues. Nude mice were inoculated with luciferase labeled 4T1 or MDA-MB-231 cells in the mammary fat pad (MFP), and BALB/c mice with depleted CD4 and CD8+ T cells were inoculated with 4T1 MFP tumors. The contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine when tumors were palpable. Cell migration was monitored with bioluminescence imaging (BLI) 10 d after irradiation. Irradiated and control tissues were evaluated using immunohistochemistry (IHC). Tissue sections were stained with F4/80 to determine the extent of macrophage infiltration. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cell populations. Of particular interest were CD11b+F4/80+ macrophages and CD11b+GR1+ myeloid-derived suppressor cells (MDSCs). Radiation enhanced tumor cell migration to normal tissues both in vitro and in vivo. 4T1 and MDA-MB-231 cells exhibited an increase in invasion (p<0.05) in vitro when exposed to irradiated MEF media. Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the MFP and surrounding tissues, including the peritoneum and muscle (p<0.001), in nude and BALB/c mice with depleted CD4 and CD8+ T cells. An increase in macrophage infiltration was found in irradiated tissue sections. Flow cytometry confirmed increases in macrophage and MDSC infiltration in irradiated areas. This study establishes that normal tissue radiation response may play a role in modulating tumor and immune cell migration after radiation. The increase in macrophage and MDSC infiltration after irradiation indicates the immune contribution in tumor cell migration. These results suggest that the tumor stroma may facilitate tumor cell invasion and tumor regrowth following radiotherapy. Citation Format: Marjan Rafat, Marta Vilalta, Todd A. Aguilera, Amato J. Giaccia, Edward E. Graves. Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice. [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 5124.

Abstract 887: N-Myc drives neuroendocrine prostate cancer

Abstract Emerging observations from clinical trials suggest that a subset of castration resistant prostate adenocarcinomas (CRPC) eventually evolve or progress to a predominantly neuroendocrine phenotype (NEPC). This transition is emerging as an important mechanism of treatment resistance. This cell plasticity is characterized by loss of androgen receptor (AR) and prostate specific antigen (PSA), and significant over-expression and gene amplification of MYCN (encoding N-Myc). While N-Myc is a bona fide driver oncogene in several rare tumor types, the molecular mechanisms that underlie N-Myc driven NEPC have yet to be characterized. Integrating a novel genetically engineered mouse (GEM) model of prostate specific N-Myc overexpression, human prostate cancer cell line modeling, and human prostate cancer transcriptome data, we found that N-Myc over-expression leads to the development of poorly differentiated, invasive prostate cancer that is molecularly similar to human NEPC tumors. To determine if N-Myc plays a causal role in driving the NEPC phenotype, we generated GEM lines that carry a CAG-driven lox-stop-lox human MYCN gene integrated into the ROSA26 (LSL-MYCN) locus and either a Tmprss2 driven tamoxifen-activated Cre recombinase (T2-Cre) or probasin (Pb)-Cre. Since PTEN deletion is a frequent alteration in CRPC and PI3K/AKT signaling can enhance N-Myc protein stability we also engineered the mice with a floxed Pten locus. N-Myc over-expression in the context of Ptenf/+ at 3 months post-induction leads to focal mouse high-grade prostatic intraepithelial neoplasia (mHGPIN). T2-Cre; Ptenf/f; LSL-MYCN+/+ mice develop highly proliferative, diffuse mHGPIN which consists of proliferations of cells with nuclear atypia that expand the glands, imparting irregular borders and inducing a mild stromal response, mitotic figures, and incipient necrosis. RNAseq data from N-Myc these mHGPIN lesions show they are molecularly similar to NEPC based on RNAseq data from 203 human CRPC and NEPC samples. At 6 months, Pb-Cre; Ptenf/f; LSL-MYCN+/+ mice develop poorly differentiated, highly proliferative, invasive prostate cancer. Based on the RNAseq data from the N-Myc GEM line, GEM-derived mouse prostate cancer organoid cultures and isogenic cell lines, we found that N-Myc regulates a specific NEPC-associated molecular program that includes a repression of AR signaling, enhanced AKT signaling and repression of Polycomb Repressive Complex 2 target genes. We further showed that N-Myc interacts with AR and this interaction depends on Enhancer of Zeste Homolog 2 (EZH2). Finally, N-Myc expressing cell lines and organoids displayed an enhanced sensitivity to inhibitors targeting the AKT pathway, EZH2 and Aurora-A. Altogether, our data shows that N-Myc drives the neuroendocrine phenotype in prostate cancer and provides rationale for the development of new therapeutic strategies for treating this aggressive subtype of prostate cancer. Citation Format: Etienne Dardenne, Himisha Beltran, Kaitlyn Gayvert, Matteo Benelli, Adeline Berger, Loredana Puca, Joanna Cyrta, Andrea Sboner, Zohal Noorzad, Theresa MacDonald, Cynthia Cheung, Dong Gao, Yu Chen, Martin Eilers, Juan Miguel Mosquera, Brian D. Robinson, Mark A. Rubin, Olivier Elemento, Francesca Demichelis, David S. Rickman. N-Myc drives neuroendocrine prostate cancer. [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 887.

Pathologic features of aggressive vulvar carcinoma are associated with epithelial-mesenchymal transition

Factors contributing to aggressive behavior in vulvar squamous cell carcinoma (vSCC) are poorly defined; however, a recent study has shown that vSCCs with an infiltrative pattern of invasion and fibromyxoid stroma are associated with worse outcomes than tumors with a pushing or nested pattern of invasion and lymphoplasmacytic stroma. Epithelial-mesenchymal transition (EMT) has been associated with tumor progression in a number of malignancies, and this study proposes that EMT contributes to tumor aggressiveness in this subset of vSCC. Immunohistochemistry was used to detect nuclear localization of β-catenin, loss of E-cadherin, and presence of vimentin in 58 cases of vSCC. The association of these phenotypic changes with pathologic features and clinical outcomes was tested using Fisher's exact and χ(2) analyses (significance at P≤.05). EMT-associated features were identified in 45 of 58 cases (78%) with 28 cases exhibiting more than one feature. Nuclear β-catenin and presence of vimentin were significantly more likely to occur in tumors with an infiltrative pattern of invasion or a fibromyxoid stromal response. Loss of E-cadherin was significantly associated with an infiltrative pattern, but not a fibromyxoid stroma. Risk for tumor recurrence was significantly increased in tumors with nuclear localization of β-catenin alone or in tumors displaying multiple EMT-associated features. These results suggest that the development of EMT may be a mechanism by which infiltrative vulvar tumors with a fibromyxoid stromal response behave more aggressively and convey worse outcomes than tumors that do not exhibit these pathologic features.

Stromal-Based Signatures for the Classification of Gastric Cancer

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Proteomic Profiles of Vulvar Squamous Cell Carcinoma Variants

Vulvar squamous cell carcinoma (vSCC) is a gynecologic malignancy diagnosed in nearly 4500 women in the US each year. High risk of recurrence and nodal metastasis lead to increased morbidity and mortality in these patients. In previous studies, we identified 2 major variants of vSCC. Some tumors exhibit a pushing invasive pattern and lymphoplasmacytic (LPC) stromal response; others display an infiltrative invasive pattern and a fibromyxoid (FMX) stromal response. We also showed that infiltrative tumors with FMX stroma are more likely to recur and metastasize to the lymph nodes, contain perineural invasion, and undergo an epithelial mesenchymal transition (EMT). Molecular tumor features that contribute to these outcomes have not been well defined in vSCC and may be due to differential expression of key proteins involved in biological processes that aid in tumor growth and invasion.In this study, label‐free high resolution LC‐MS/MS was used to determine the proteomic profiles of FFPE tissue sections from 10 infiltrative vSCC with FMX stroma and 10 pushing vSCC with LPC stroma. Of these 20 tumors, 11 were associated with recurrence and 8 contained lymph node metastases. Proteomic profiles were associated with morphologic tumor features, tumor recurrence, and nodal metastases to identify proteins linked to increased risk of adverse outcomes.When comparing infiltrative and pushing tumors, 205 proteins exhibited significant expression changes between the two groups. Importantly, 42 proteins were uniquely identified in infiltrative tumors. Included in these was thrombospondin‐4, a glycoprotein that mediates cell‐matrix interactions and may play a role in macrophage recruitment. In addition, 96 proteins were identified only in the pushing tumors, including STAT1, which may play a significant role in mediating effects of the inflammatory stromal response through IFNγ signaling. Expression of 105 proteins was either significantly different or uniquely identified in recurrent tumors, and the expression of 115 proteins was either uniquely identified or significantly varied in tumors with nodal metastases. Included in these 115 significant proteins was SRPX2, an important regulator of FAK signaling and cell motility, which could contribute to increased metastatic potential in these tumors.Overall, our results identify a proteomic profile in vSCC that implicates a role for specific pathways in differentiating aggressive vulvar squamous cell carcinoma as defined by morphologic tumor features, recurrence, and nodal metastases.Support or Funding InformationUAMS TRI grants UL1TR000039 and KL2TR000063; SPaT institutional training grant T32GM106999

Biological basis of personalized anticoagulation in cancer: oncogene and oncomir networks as putative regulators of coagulopathy

Activation of stromal response pathways in cancer is increasingly viewed as both a local and systemic extension of molecular alterations driving malignant transformation. Rather than reflecting passive and unspecific responses to anatomical abnormalities, the coagulation system is a target of oncogenic deregulation, impacting the role of clotting and fibrinolytic proteins, and integrating hemostasis, inflammation, angiogenesis and cellular growth effects in cancer. These processes signify, but do not depend on, the clinically manifest coagulopathy and thrombosis. In this regard, the role of driver mutations affecting oncoprotein coding genes such as RAS, EGFR or MET and tumour suppressors (PTEN, TP53) are well described as regulators of tissue factor (TF), protease activated receptors (PAR-1/2) and ectopic coagulation factors (FVII). Indeed, in both adult and pediatric brain tumours the expression patterns of coagulation and angiogenesis regulators (coagulome and angiome, respectively) reflect the molecular subtypes of the underlying diseases (glioblastoma or medulloblastoma) as defined by their oncogenic classifiers and clinical course. This emerging understanding is still poorly established in relation to the transforming effects of non-coding genes, including those responsible for the expression of microRNA (miR). Indeed, several miRs have been recently found to regulate TF and other effectors. We recently documented that in the context of the aggressive embryonal tumour with multilayered rosettes (ETMR) the oncogenic driver miR (miR-520g) suppresses the expression of TF and correlates with hypocoagulant tumour characteristics. Unlike in adult cancers, the growth of pediatric embryonal brain tumour cells as spheres (to maintain stem cell properties) results in upregulation of miR-520g and downregulation of TF expression and activity. We postulate that oncogenic protein and miR coding genes form alternative pathways of coagulation system regulation in different tumour settings, a property necessitating more personalised and biologically-based approaches to anticoagulation.

Hif1a Deletion Reveals Pro-Neoplastic Function of B Cells in Pancreatic Neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths worldwide, with an exceedingly low 5-year survival rate. PDAC tumors are characterized by an extensive desmoplastic stromal response and hypovascularity, suggesting that tumor hypoxia could regulate PDAC initiation and/or progression. Using a well-defined, autochthonous Kras(G12D)-driven murine model, as well as human tumors, we demonstrate that hypoxia and stabilization of hypoxia-inducible factor 1α (HIF1α), a principal mediator of hypoxic adaptation, emerge early during preinvasive stages of PDAC. Surprisingly, pancreas-specific Hif1a deletion drastically accelerated Kras(G12D)-driven pancreatic neoplasia and was accompanied by significant increases in intrapancreatic B lymphocytes, featuring prominent influx of a rare "B1b" B-cell subtype. Finally, treatment of HIF1α-deficient mice with B cell-depleting αCD20 monoclonal antibodies inhibited progression of pancreatic intraepithelial neoplasia (PanIN). Our data reveal a previously unrecognized role for B cells in promoting pancreatic tumorigenesis and implicate HIF1α as a critical regulator of PDAC development. We show here that pancreas-specific Hif1a deletion promotes PDAC initiation, coincident with increased intrapancreatic accumulation of B cells, and that B-cell depletion suppresses pancreatic tumorigenesis. We therefore demonstrate a protective role for HIF1α in pancreatic cancer initiation and uncover a previously unrecognized function of B cells.

Adipose mesenchymal stromal cells response to ionizing radiation

Background aimsThis study evaluates the biological response of adipose tissue–derived mesenchymal stromal cells (aMSCs) to ionizing radiation (IR). MethodsIrradiated BALB/c mice aMSCs were characterized for functionality and phenotype. The clonogenic capacity of irradiated aMSCs was assessed and compared with those of metastatic breast cancer cell line (4T1) and normal mouse fibroblasts (NIH3T3-wt). We investigated the IR-induced DNA damage response, apoptosis, changes in cell cycle (CC) dynamics and protein and gene expression. ResultsIrradiated and non-irradiated aMSCs were able to differentiate into adipocytes, chondrocytes and osteocytes with no significant difference. Irradiated aMSCs maintained the expression of mesenchymal stromal cells (MSCs) surface antigens and, as expected, were negative for hematopoietic stem cells (HSCs) surface antigens when tested up to 7 days after IR for all irradiation doses with no significant difference. Clonogenically, irradiated aMSCs had higher relative survival fraction and plating efficiency than 4T1 and NIH3T3-wt. Irradiated aMSCs expressed higher □H2AX and significantly showed faster and more time-efficient IR-induced DNA damage response evident by up-regulated DNA-PKcs and RAD51. Two hours after IR, most of aMSCs DNA damage/repair-related genes showed up-regulation that disappeared within 6 h after IR. Irradiated aMSCs showed a significant rise and an earlier peak of p-ATM-dependent and -independent (p84/5E10-mediated) G2/M CC arrest compared with 4T1 and NIH3T3-wt. ConclusionsAfter IR exposure, aMSCs showed a robust and time-efficient radiation-induced DNA damage repair response, stable phenotypical characteristics and multi-lineage differentiation potential, suggesting they may be reliable candidates for cell therapy in radiation oncology regenerative medicine.

Abstract A31: High-grade serous ovarian cancer subtyping identifies pathways for targeted therapy.

Abstract Introduction: We recently identified three prognostic groups for high-grade serous ovarian cancers (HGS-OvCa) patients treated with platinum and taxane using transcriptomics and clinical data (Chen, Huhtinen, et al. Cancer Res. 2015). In this contribution we first use pathway analysis methods to identify transcript markers and pathways that drive resistance to platinum and experimentally inhibit of the key pathways to restore sensitivity to platinum. Two of the prognostic groups (Poor I and Poor II) were associated with poor survival but have distinct expression profiles. Poor II express high stromal response genes and confirms the C1 subtype characterized by Tothill et al. (2008), whereas Poor I is previously unidentified and characterized by genome-wide hypermethylation. Our objectives here are to: 1) identify key pathways for Poor I and Poor II based on transcriptomics data (data unpublished), 2) inhibit the herein identified pathways to suggest combinatorial therapeutics options for platinum resistance HGS-OvCa patients (data unpublished), 3) test the impact of hypermethylation drug Decitabine to platinum resistance in vitro (data partially unpublished). Methods: Expression levels were qRT-PCR validated using well described prospective patient cohort. The role of hypermethylation and specific pathways on cell viability were studied in vitro using commercial (CAOV4, NIHOVCAR3, OVCAR8, TYKnu) and spheroidal primary HGS-OvCa cell lines. Results: Poor I subtype presents with significantly higher methylation of CpG loci (p < 0.001) as compared with Good or Poor II subtypes. We observed significant up-regulation of three isoforms of DNA methyltransferase 3B (DNMT3B) in Poor I samples. The role of hypermethylation was further validated in vitro using patient derived cell lines with platinum resistance. Inhibition of methylation by Decitabine exhibited strong effect on cell viability specifically on Poor I characteristic HGS-OvCa cell lines. The data suggests that clinical trials testing the effect of Decitabine should be focused to patients with Poor I disease. To evaluate signaling pathways that are associated with platinum-taxane resistance we evaluated common pathways enriched in Poor I and Poor II using the transcriptomics data available at The Cancer Genome Atlas repository. The most prominent pathway is MAPK signaling pathway of which four genes (FAS, MAPK1, AKT and NR4A1) emerged from the analysis. Pathway analysis further suggested ERK5 and AKT as upstream regulators for NR4A1 in platinum-taxane resistance in HGS-OvCa. The impact of ERK5 and AKT pathways on platinum resistance was evaluated in vitro using commercial and primary HGS-OvCa cell lines with wild-type BRCA1/2. The combination of AKT-inhibitor API-2 and Cisplatin resulted in a marked decrease in cell viability over Cisplatin alone whereas AKT-inhibitor alone had only little effect. In contrast, ERK5 inhibition with XMD8-92 combined with Cisplatin did not have an effect on HGS-OvCa cell viability. However, XMD8-92 enhanced the effect of API-2 in subset of the cell lines. Thus, we tested whether combination of the three drugs results in better response. In general, the addition of XMD8-92 did not improve the effect of API-2 and Cisplatin. However, in two highly platinum resistant cell lines, CAOV-4 and M022i primary cell line, the combination of API-2, XMD8-92 and Cisplatin produced the highest reduction in cell viability. In conclusion, we have identified pathways that are likely driving drug resistance in HGS-OvCa and tested the impact of platinum combined with targeted AKT and ERK5 inhibition in vitro. We further show that the Poor I type HGS-OvCa cells are sensitive to Decitabine. Thus, Decitabine may be an effective (combinatorial) treatment option for patients with the Poor I subtype disease. Combination of AKT and ERK5 inhibitors with platinum can also be effective for a subset of platinum resistant ovarian cancer patients. Citation Format: Kaisa Huhtinen, Ping Chen, Katja Kaipio, Piia Mikkonen, Viljami Aittomäki, Rony Lindell, Johanna Hynninen, Annika Auranen, Seija Grenman, Rainer Lehtonen, Sampsa Hautaniemi, Olli Carpén. High-grade serous ovarian cancer subtyping identifies pathways for targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A31.