The peritoneal tumour microenvironment of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3(+) T cells and CD68(+) macrophages, with occasional neutrophils. Alpha-smooth muscle actin(+) (α-SMA(+) ) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6-48 h) with an extensive CD45(+) leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3(+) T lymphocytes and CD68(+) macrophages with α-SMA(+) cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2-3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease.

CD44 is overexpressed in basal-like breast cancers but is not a driver of 11p13 amplification.

Overexpression and alternative splicing of CD44 have been implicated in tumour progression. Here we describe the identification of a high level amplification of human 11p13, encompassing the CD44 gene, in primary breast cancers and cell lines and test whether CD44 acts as the driver of this amplicon. aCGH analysis revealed 11p13 amplification in 3% (3/100) of primary breast carcinomas and in two cell lines. The minimal region of amplification was 34.38-37.62 Mb. Amplification was confirmed by dual-colour FISH in these cell lines and further validated by CISH in an independent tumour cohort. CD44 expression in primary breast cancers was significantly associated with features of basal-like breast cancer. Detection of CD44 expression in breast cancer cell lines confirmed moderate to high expression in basal-like cell lines and minimal expression in luminal cell lines. In both, primary breast cancers and cell lines, 11p13 amplification was associated with high levels of CD44 mRNA expression. CD44 alternative splicing was detected in four of nine cell lines and in tumour samples, irrespective of the amplification status. RNAi mediated knock down of CD44 failed to reveal an increased dependence on CD44 expression for proliferation or survival in amplified cell lines. Given that expression of CD44 is not an absolute requirement for the survival of cells harbouring CD44 gene amplification, CD44 is unlikely to be a driver of the 11p13 amplicon.

Integrated transcriptomic and proteomic analysis identifies protein kinase CK2 as a key signaling node in an inflammatory cytokine network in ovarian cancer cells.

We previously showed how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in ovarian cancer. This network, which we named the "TNF network", has paracrine actions within the tumor microenvironment, influencing angiogenesis and the immune cell infiltrate.The aim of this study was to identify critical regulators in the signaling pathways of the TNF network in ovarian cancer cells that might be therapeutic targets. To achieve our aim, we used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis. Among the potential therapeutic kinase targets identified was the protein kinase Casein kinase II (CK2).Knockdown of CK2 expression in malignant cells by siRNA or treatment with the specific CK2 inhibitor CX-4945 significantly decreased Notch signaling and reduced constitutive cytokine release in ovarian cancer cell lines that expressed the TNF network as well as malignant cells isolated from high grade serous ovarian cancer ascites. The expression of the same cytokines was also inhibited after treatment with CX-4945 in a 3D organotypic model. CK2 inhibition was associated with concomitant inhibition of proliferative activity, reduced angiogenesis and experimental peritoneal ovarian tumor growth.In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer.

Neoadjuvant Chemotherapy Modulates the Immune Microenvironment in Metastases of Tubo-Ovarian High-Grade Serous Carcinoma.

PURPOSE: The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSC), and its relationship to treatment response. EXPERIMENTAL DESIGN: We obtained pre- and posttreatment omental biopsies and blood samples from a total of 54 patients undergoing platinum-based NACT and 6 patients undergoing primary debulking surgery. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC, flow cytometry, electrochemiluminescence assays, and RNA sequencing and related our findings to the histopathologic treatment response. RESULTS: There was evidence of T-cell activation in omental biopsies after NACT: CD4(+) T cells showed enhanced IFNγ production and antitumor Th1 gene signatures were increased. T-cell activation was more pronounced with good response to NACT. The CD8(+) T-cell and CD45RO(+) memory cell density in the tumor microenvironment was unchanged after NACT but biopsies showing a good therapeutic response had significantly fewer FoxP3(+) T regulatory (Treg) cells. This finding was supported by a reduction in a Treg cell gene signature in post- versus pre-NACT samples that was more pronounced in good responders. Plasma levels of proinflammatory cytokines decreased in all patients after NACT. However, a high proportion of T cells in biopsies expressed immune checkpoint molecules PD-1 and CTLA4, and PD-L1 levels were significantly increased after NACT. CONCLUSIONS: NACT may enhance host immune response but this effect is tempered by high/increased levels of PD-1, CTLA4, and PD-L1. Sequential chemoimmunotherapy may improve disease control in advanced HGSC. Clin Cancer Res; 22(12); 3025-36. ©2016 AACR.

Adaptive Upregulation of EGFR Limits Attenuation of Tumor Growth by Neutralizing IL6 Antibodies, with Implications for Combined Therapy in Ovarian Cancer.

Excess production of the proinflammatory IL6 has both local and systemic tumor-promoting activity in many cancers, including ovarian cancer. However, treatment of advanced ovarian cancer patients with a neutralizing IL6 antibody yielded little efficacy in a previous phase II clinical trial. Here, we report results that may explain this outcome, based on the finding that neutralizing antibodies to IL6 and STAT3 inhibition are sufficient to upregulate the EGFR pathway in high-grade serous and other ovarian cancer cells. Cell treatment with the EGFR inhibitor gefitinib abolished upregulation of the EGFR pathway. Combining neutralizing IL6 antibodies and gefitinib inhibited malignant cell growth in 2D and 3D culture. We found that ErbB-1 was localized predominantly in the nucleus of ovarian cancer cells examined, contrasting with plasma membrane localization in lung cancer cells. Treatment with anti-IL6, gefitinib, or their combination all led to partial restoration of ErbB-1 on the plasma membrane. In vivo experiments confirmed the effects of IL6 inhibition on the EGFR pathway and the enhanced activity of a combination of anti-IL6 antibodies and gefitinib on malignant cell growth. Taken together, our results offer a preclinical rationale to combine anti-IL6 and gefitinib to treat patients with advanced stage ovarian cancer.