We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253.

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Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.

Prognostic factors aid in the stratification and treatment of cancer. This study evaluated prognostic importance of tumor infiltrating immune cell in patients with oral squamous cell carcinoma. Profiles of infiltrating immune cells and clinicopathological data were available for 78 OSCC patients with a median follow-up of 48 months. The infiltrating intensity of CD8, CD4, T-bet, CD68 and CD57 positive cells were assessed by immunohistochemistry. Chi-square test was used to compare immune markers expression and clinicopathological parameters. Univariate and multivariate COX proportional hazard models were used to assess the prognostic discriminator power of immune cells. The predictive potential of immune cells for survival of OSCC patients was determined using ROC and AUC. The mean value of CD8, CD4, T-bet, CD68 and CD57 expression were 28.99, 62.06, 8.97, 21.25 and 15.75 cells per high-power field respectively. The patient cohort was separated into low and high expression groups by the mean value. Higher CD8 expression was associated with no regional lymph node metastasis (p = 0.033). Patients with more abundant stroma CD57+ cells showed no metastasis into regional lymph node (p = 0.005), and early clinical stage (p = 0.016). The univariate COX regression analyses showed that no lymph node involvement (p < 0.001), early clinical stage (TNM staging I/II vs III/IV, p = 0.007), higher CD8 and CD57 expression (p < 0.001) were all positively correlated with longer overall survival. Multivariate COX regression analysis showed that no lymph node involvement (p = 0.008), higher CD8 (p = 0.03) and CD57 (p < 0.001) expression could be independent prognostic indicators of better survival. None of CD4, T-bet or CD68 was associated with survival in ether univariate or multivariate analysis. ROC and AUC showed that the predictive accuracy of CD8 and CD57 were all superior compared with TNM staging. CD57 (AUC = 0.868; 95% CI, 0.785–0.950) and CD8 (AUC = 0.784; 95% CI, 0.680–0.889) both provided high predictive accuracy, of which, CD57 was the best predictor. Tumor stroma CD57 and CD8 expression was associated with lymphnode status and independently predicts survival of OSCC patients. Our results suggest an active immune microenvironment in OSCC that may be targetable by immune drugs.

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Prognostic significance of tumor infiltrating immune cells in oral squamous cell carcinoma

Cartilage oligomeric matrix protein: COMPopathies and beyond.

Keloids are considered as benign fibroproliferative skin tumors growing beyond the site of the original dermal injury. Although traditionally viewed as a form of skin scarring, keloids display many cancer-like characteristics such as progressive uncontrolled growth, lack of spontaneous regression and extremely high rates of recurrence. Phenotypically, keloids are consistent with non-malignant dermal tumors that are due to the excessive overproduction of collagen which never metastasize. Within the remit of keloid pathobiology, there is increasing evidence for the various interplay of neoplastic-promoting and suppressing factors, which may explain its aggressive clinical behavior. Amongst the most compelling parallels between keloids and cancer are their shared cellular bioenergetics, epigenetic methylation profiles and epithelial-to-mesenchymal transition amongst other disease biological (genotypic and phenotypic) behaviors. This review explores the quasi-neoplastic or cancer-like properties of keloids and highlights areas for future study.

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Understanding Keloid Pathobiology From a Quasi-Neoplastic Perspective: Less of a Scar and More of a Chronic Inflammatory Disease With Cancer-Like Tendencies.

Cartilage oligomeric matrix protein (COMP) is known to promote fibrosis in skin, lung and liver Emerging evidence shows that COMP plays critical roles in tumor development, including breast cancer, colon cancer and hepatocellular carcinoma (HCC) Nevertheless, the role of COMP in HCC proliferation and metastasis and its underlying mechanisms remain fully unclear Serum COMP was determined by ELISA Cell Counting Kit-8 and plate colony formation were performed to evaluate cell proliferation Wound healing and transwell assays were used to determine migration and invasion of HCC cells Western blotting and immunofluorescence were carried out for detection of epithelial-to-mesenchymal transition (EMT) markers and MMPs in HCC cells The in vivo role of COMP was evaluated using mouse models We also measured effects of hepatic stellate cells (HSCs)-conditioned medium (CM) on HCC progression using transwell coculture system Here, we found that serum COMP levels in HCC patients were significantly higher than those in healthy controls Accordingly, high serum COMP levels in HCC patients significantly correlated with malignant clinical characteristics and poor clinical outcomes Next, we investigated that recombinant human COMP protein (rCOMP) treatment resulted in increased abilities of proliferation, invasion and migration of HCC cells Furthermore, rCOMP treatment enhanced proliferative and metastatic colonization of HCC cells in vivo Mechanistically, CD36 receptor played an essential role in COMP-mediated HCC cell proliferation and metastasis Functionally, COMP/CD36 signaling caused phosphorylation of ERK and AKT, resulting in the upregulation of tumor-progressive genes such as EMT markers, MMP-2/9, Slug and Twist in HCC cells Interestingly, we revealed that COMP was secreted by HSCs CM of LX2 cells with COMP knockdown showed weaker effects on the activation of MEK/ERK and PI3K/AKT signaling pathways in HCC cells compared to control CM Our findings indicated that HSCs-derived COMP collaborated with CD36 and subsequently played an essential role in MEK/ERK and PI3K/AKT-mediated HCC progression COMP might act as a promising target for the diagnosis and treatment of aggressive HCC

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HSCs-derived COMP drives hepatocellular carcinoma progression by activating MEK/ERK and PI3K/AKT signaling pathways.

Cartilage oligomeric matrix protein (COMP) is a soluble pentameric protein expressed in cartilage and involved in collagen organization. Tissue microarrays derived from two cohorts of patients with breast cancer (n=122 and n=498) were immunostained, revealing varying expression of COMP, both in the tumor cells and surrounding stroma. High levels of COMP in tumor cells correlated, independently of other variables, with poor survival and decreased recurrence-free survival. Breast cancer cells, MDA-MB-231, stably expressing COMP were injected into the mammary fat pad of SCID (CB-17/Icr-Prkdcscid/Rj) mice. Tumors expressing COMP were significantly larger and were more prone to metastasize as compared with control, mock-transfected, tumors. In vitro experiments confirmed that COMP-expressing cells had a more invasive phenotype, which could in part be attributed to an upregulation of matrix metalloprotease-9. Furthermore, microarray analyses of gene expression in tumors formed in vivo showed that COMP expression induced higher expression of genes protecting against endoplasmic reticulum stress. This observation was confirmed in vitro as COMP-expressing cells showed better survival as well as a higher rate of protein synthesis when treated with brefeldin A, compared with control cells. Further, COMP-expressing cells appeared to undergo a metabolic switch, that is, a Warburg effect. Thus, in vitro measurement of cell respiration indicated decreased mitochondrial metabolism. In conclusion, COMP is a novel biomarker in breast cancer, which contributes to the severity of the disease by metabolic switching and increasing invasiveness and tumor cell viability, leading to reduced survival in animal models and human patients.

Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer.

Cartilage oligomeric matrix protein (COMP) was recently implicated in the progression of breast cancer Immunostaining of 342 prostate cancer specimens in tissue microarrays showed that COMP expression is not breast cancer-specific but also occurs in prostate cancer The expression of COMP in prostate cancer cells correlated with a more aggressive disease with faster recurrence Subcutaneous xenografts in immunodeficient mice showed that the prostate cancer cell line DU145 overexpressing COMP formed larger tumors in vivo as compared to mock-transfected cells Purified COMP bound to and enhanced the invasion of DU145 cells in vitro in an integrin-dependent manner In addition, intracellular COMP expression interfered with cellular metabolism by causing a decreased level of oxidative phosphorylation with a concurrent upregulation of lactate production (Warburg effect) Further, expression of COMP protected cells from induction of apoptosis via several pathways The effect of COMP on metabolism and apoptosis induction was dependent on the ability of COMP to disrupt intracellular Ca2+ signalling by preventing Ca2+ release from the endoplasmic reticulum In conclusion, COMP is a potent driver of the progression of prostate cancer, acting in an anti-apoptotic fashion by interfering with the Ca2+ homeostasis of cancer cells

Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis

The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

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The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERβ) as a new target in DLBCL. Here, we demonstrate that ERβ is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERβ plays a role in the protection against apoptosis in DLBCL. Targeting of the ERβ with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERβ knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients. 

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Bart Reitsma

Papers

Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer.

Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma