Massimiliano Mellone

Abstract Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 “normalized” CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. Significance: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion.

// Christopher J. Hanley 1 , Fergus Noble 1 , Matthew Ward 1 , Marc Bullock 1 , Cole Drifka 3 , Massimiliano Mellone 1 , Antigoni Manousopoulou 2 , Harvey E. Johnston 2 , Annette Hayden 1 , Steve Thirdborough 1 , Yuming Liu 3 , David M. Smith 1 , Toby Mellows 1 , W. John Kao 3 , Spiros D. Garbis 1, 2 , Alex Mirnezami 1 , Tim J. Underwood 1 , Kevin W. Eliceiri 3, * , Gareth J. Thomas 1, * 1 Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK 2 Clinical and Experimental Sciences Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK 3 Laboratory for Optical and Computational Instrumentation (LOCI), Department of Biomedical Engineering, University of Madison, Wisconsin 53706, USA * These authors contributed equally to this work Correspondence to: Gareth J. Thomas, e-mail: g.thomas@soton.ac.uk Keywords: extracellular matrix, collagen, cancer associated fibroblasts, tumor microenvironment, second harmonic generation Received: July 22, 2015      Accepted: December 05, 2015      Published: December 23, 2015 ABSTRACT Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo . However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.