Mets and NETs: The Awakening Force.

In a recent study published in Science, Albrengues et al. (2018) unveil an intriguing mechanism whereby the release of neutrophil extra-cellular traps during chronic lung inflammation awakens dormant malignant cells and contributes to cancer progression.

Combining measures of immune infiltration shows additive effect on survival prediction in high-grade serous ovarian carcinoma.

BACKGROUND: In colorectal and breast cancer, the density and localisation of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations. METHODS: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. RESULTS: Epithelial CD8 [p = 0.027, hazard ratio (HR) = 0.83], stromal CD68 (p = 3 × 10-4, HR = 0.44) and stromal CD45RO (p = 7 × 10-4, HR = 0.76) were positively associated with survival and remained so when averaged across the tumour and stromal compartments. Using principal component analysis, we identified optimised multiparameter survival models combining information from all immune markers (p = 0.016, HR = 0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components. CONCLUSIONS: Combining measures of immune infiltration provided improved survival modelling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was associated with survival, underscoring the benefits evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Murine CD27(-) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages.

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17-deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27((-)) subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17-secreting CD27((-)) Vγ6((+)) γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17-dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.

Cancer cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts.

We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer.

Endothelial cell junctional adhesion molecule C plays a key role in the development of tumors in a murine model of ovarian cancer.

Junctional adhesion molecule C (JAM-C) is a transmembrane protein with significant roles in regulation of endothelial cell (EC) functions, including immune cell recruitment and angiogenesis. As these responses are important in promoting tumor growth, the role of EC JAM-C in tumor development was investigated using the ID8 syngeneic model of ovarian cancer. Within 10-15 wk, intraperitoneally injected ID8 cells form multiple tumor deposits and ascites that resemble human high-grade serous ovarian cancer. Compared to wild-type mice, survival in this model was increased in EC JAM-C knockouts (KOs; 88 vs. 96 d, P=0.04) and reduced in EC JAM-C transgenics (88 vs. 78.5 d, P=0.03), mice deficient in or overexpressing EC JAM-C, respectively. While tumor growth was significantly reduced in EC JAM-C KOs (87% inhibition at 10 wk, P<0.0005), this was not associated with alterations in tumor vessel density or immune cell infiltration. However, tumor microvessels from EC JAM-C-deficient mice exhibited reduced pericyte coverage and increased vascular leakage, suggesting a role for EC JAM-C in the development of functional tumor vessels. These findings provide evidence for a role for EC JAM-C in tumor growth and aggressiveness as well as recruitment of pericytes to newly formed blood vessels in a model of ovarian cancer.

Vaccinia virus induces programmed necrosis in ovarian cancer cells.

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis.

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf(-/-) mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α-mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf(-/-) mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf(-/-) mice were transferred to Rag2(-/-) mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf(-/-) mice was associated with increased IFN-γ and CD8(+) T cells in skin and a significant reduction in IL-10-producing B regulatory cells alongside an increase in IFN-γ-producing CD8(+) T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.

Cancer-related inflammation: common themes and therapeutic opportunities.

Inflammatory cells and mediators are an essential component of the tumor microenvironment. Inflammatory circuits can differ considerably in different tumors in terms of cellular and cytokine networks and molecular drivers. However, macrophages are a common and fundamental component of cancer promoting inflammation. Drivers of macrophage functional orientation include tumor cells, cancer-associated fibroblasts, T cells and B cells. Dissection of the diversity of cancer-related inflammation is instrumental to the design of therapeutic approaches that target cancer-related inflammation.

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFß-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

The chemokine system and cancer.

Chemokines (chemo-attractant cytokines) are a group of small proteins that act together with their cell surface receptors, in development, normal physiology and immune responses, to direct cells to specific locations throughout the body. Cancer cells acquire the ability to subvert the chemokine system, such that these molecules and their receptors become important regulators of cell movement into and out of the tumour microenvironment and major players in cancer biology.

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.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

Inhibition of the inflammatory cytokine TNF-α increases adenovirus activity in ovarian cancer via modulation of cIAP1/2 expression.

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.

Building invitro 3D human multicellular models of high-grade serous ovarian cancer.

Three-dimensional (3D), multicellular invitro models provide a useful platform for studying human cancer biology, particularly through deconvolution of the tumor microenvironment, or where animal models do not recapitulate the human condition. Here, we detail a protocol for building human multicellular models made of patient-derived primary cells and malignant cell lines, which recapitulate features of the tumor microenvironment. This protocol is optimized for building 3D models of high-grade serous ovarian cancer omental metastasis but can be adapted for modeling other cancers. For complete details on the use and execution of this profile, please refer to Delaine-Smith et al. (2021) and Malacrida et al. (2021).

Harnessing cytokines and chemokines for cancer therapy.

During the past 40 years, cytokines and cytokine receptors have been extensively investigated as either cancer targets or cancer treatments. A strong preclinical rationale supports therapeutic strategies to enhance the growth inhibitory and immunostimulatory effects of interferons and interleukins, including IL-2, IL-7, IL-12 and IL-15, or to inhibit the inflammatory and tumour-promoting actions of cytokines such as TNF, IL-1ß and IL-6. This rationale is underscored by the discovery of altered and dysregulated cytokine expression in all human cancers. These findings prompted clinical trials of several cytokines or cytokine antagonists, revealing relevant biological activity but limited therapeutic efficacy. However, most trials involved patients with advanced-stage disease, which might not be the optimal setting for cytokine-based therapy. The advent of more effective immunotherapies and an increased understanding of the tumour microenvironment have presented new approaches to harnessing cytokine networks in the treatment of cancer, which include using cytokine-based therapies to enhance the activity or alleviate the immune-related toxicities of other treatments as well as to target early stage cancers. Many challenges remain, especially concerning delivery methods, context dependencies, and the pleiotropic, redundant and often conflicting actions of many cytokines. Herein, we discuss the lessons learnt from the initial trials of single-agent cytokine-based therapies and subsequent efforts to better exploit such agents for the treatment of solid tumours.

Immune Mechanisms of Resistance to Cediranib in Ovarian Cancer.

This article investigates mechanisms of resistance to the VEGF receptor inhibitor cediranib in high-grade serous ovarian cancer (HGSOC), and defines rational combination therapies. We used three different syngeneic orthotopic mouse HGSOC models that replicated the human tumor microenvironment (TME). After 4 to 5 weeks treatment of established tumors, cediranib had antitumor activity with increased tumor T-cell infiltrates and alterations in myeloid cells. However, continued cediranib treatment did not change overall survival or the immune microenvironment in two of the three models. Moreover, treated mice developed additional peritoneal metastases not seen in controls. Cediranib-resistant tumors had intrinsically high levels of IL6 and JAK/STAT signaling and treatment increased endothelial STAT3 activation. Combination of cediranib with a murine anti-IL6 antibody was superior to monotherapy, increasing mouse survival, reducing blood vessel density, and pSTAT3, with increased T-cell infiltrates in both models. In a third HGSOC model, that had lower inherent IL6 JAK/STAT3 signaling in the TME but high programmed cell death protein 1 (PD-1) signaling, long-term cediranib treatment significantly increased overall survival. When the mice eventually relapsed, pSTAT3 was still reduced in the tumors but there were high levels of immune cell PD-1 and Programmed death-ligand 1. Combining cediranib with an anti-PD-1 antibody was superior to monotherapy in this model, increasing T cells and decreasing blood vessel densities. Bioinformatics analysis of two human HGSOC transcriptional datasets revealed distinct clusters of tumors with IL6 and PD-1 pathway expression patterns that replicated the mouse tumors. Combination of anti-IL6 or anti-PD-1 in these patients may increase activity of VEGFR inhibitors and prolong disease-free survival.

The Tumor Microenvironment of Clear-Cell Ovarian Cancer.

Some patients with advanced clear-cell ovarian cancer (CCOC) respond to immunotherapy; however, little is known about the tumor microenvironment (TME) of this relatively rare disease. Here, we describe a comprehensive quantitative and topographical analysis of biopsies from 45 patients, 9 with Federation Internationale des Gynaecologistes et Obstetristes (FIGO) stage I/II (early CCOC) and 36 with FIGO stage III/IV (advanced CCOC). We investigated 14 immune cell phenotype markers, PD-1 and ligands, and collagen structure and texture. We interrogated a microarray data set from a second cohort of 29 patients and compared the TMEs of ARID1A-wildtype (ARID1Awt) versus ARID1A-mutant (ARID1Amut) disease. We found significant variations in immune cell frequency and phenotype, checkpoint expression, and collagen matrix between the malignant cell area (MCA), leading edge (LE), and stroma. The MCA had the largest population of CD138+ plasma cells, the LE had more CD20+ B cells and T cells, whereas the stroma had more mast cells and αSMA+ fibroblasts. PD-L2 was expressed predominantly on malignant cells and was the dominant PD-1 ligand. Compared with early CCOC, advanced-stage disease had significantly more fibroblasts and a more complex collagen matrix, with microarray analysis indicating "TGFß remodeling of the extracellular matrix" as the most significantly enriched pathway. Data showed significant differences in immune cell populations, collagen matrix, and cytokine expression between ARID1Awt and ARID1Amut CCOC, which may reflect different paths of tumorigenesis and the relationship to endometriosis. Increased infiltration of CD8+ T cells within the MCA and CD4+ T cells at the LE and stroma significantly associated with decreased overall survival.