Stromal Oncostatin M axis promotes breast cancer progression

Cancer cells are constantly communicating with the surrounding tumour microenvironment (TME) and they hijack physiological cell interactions to overcome immune system surveillance and promote cancer progression1,2. However, the contribution of stromal cells to the reprogramming of the TME is not well understood. In this study we provide unprecedented evidence of the role of the cytokine Oncostatin M (OSM) as central node for multicellular interactions between immune and non-immune stroma and the epithelial compartment. We show that stromal expression of the OSM:Oncostatin M Receptor (OSMR) axis plays a key role in breast cancer progression. OSMR deletion in a multistage breast cancer model delays tumour onset, tumour growth and reduces metastatic burden. We ascribed causality to the stromal function of OSM axis by demonstrating reduced tumour burden of syngeneic tumours implanted in mice. Single-cell and bioinformatic analysis of murine and human breast tumours revealed that the expression of OSM signalling components is compartmentalized in the tumour stroma. OSM expression is restricted to myeloid cells, whereas OSMR expression is detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprograms fibroblasts to a more contractile and pro-tumorigenic phenotype, elicits the secretion of VEGF and pro-inflammatory chemokines (e.g. CXCL1 and CXCL16), leading to increased neutrophil and macrophage recruitment. In summary, our work sheds light on the mechanism of immune regulation by the tumour microenvironment, and supports that targeting OSM:OSMR interactions is a potential therapeutic strategy to inhibit tumour-promoting inflammation and breast cancer progression.


Abstract
Cancer cells are constantly communicating with the surrounding tumour microenvironment (TME) and they hijack physiological cell interactions to overcome immune system surveillance and promote cancer progression 1,2 . However, the contribution of stromal cells to the reprogramming of the TME is not well understood. In this study we provide unprecedented evidence of the role of the cytokine Oncostatin M (OSM) as central node for multicellular interactions between immune and non-immune stroma and the epithelial compartment. We show that stromal expression of the OSM:Oncostatin M Receptor (OSMR) axis plays a key role in breast cancer progression. OSMR deletion in a multistage breast cancer model delays tumour onset, tumour growth and reduces metastatic burden. We ascribed causality to the stromal function of OSM axis by demonstrating reduced tumour burden of syngeneic tumours implanted in mice. Single-cell and bioinformatic analysis of murine and human breast tumours revealed that the expression of OSM signalling components is compartmentalized in the tumour stroma. OSM expression is restricted to myeloid cells, whereas OSMR expression is detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprograms fibroblasts to a more contractile and pro-tumorigenic phenotype, elicits the secretion of VEGF and pro-inflammatory chemokines (e.g. CXCL1 and CXCL16), leading to increased neutrophil and macrophage recruitment. In summary, our work sheds light on the mechanism of immune regulation by the tumour microenvironment, and supports that targeting OSM:OSMR interactions is a potential therapeutic strategy to inhibit tumourpromoting inflammation and breast cancer progression.

Main text
The tumour microenvironment (TME), composed by different cell types (e.g. fibroblasts, adipocytes, endothelial and infiltrating immune cells), harbours complex cell interactions that are often manipulated and hijacked by tumour cells in with every step of cancer progression 2 .
However, the contribution of stromal cells to the reprogramming of the tumour microenvironment is poorly understood. Here, we discovered that the cytokine Oncostatin M (OSM) acts as a central regulator of the crosstalk between immune stroma and non-immune stroma, favouring breast cancer progression and metastasis. First, we set to study the contribution of OSM signalling in the genetic mouse model MMTV-PyMT, widely used to study breast cancer progression in a fully competent tumour microenvironment and immune in OSMR-deficient mice showed decreased levels of the extracellular matrix protein fibronectin, predominantly produced by CAFs 4 (Fig 1f), increased levels of apoptosis, but similar degree of proliferation (Extended Data Fig. 1e). Finally, OSMR deficiency produced a remarkable reduction in the percentage of animals with lung metastases (Fig. 1g,h).
These results show that OSM signalling is causally associated with tumour aggressiveness but, surprisingly, by using syngeneic cancer models, we found that this association requires, at least in part, the presence of the OSM:OSMR axis in the tumour stroma. We injected TS1 cells, derived from a MMTV-PyMT tumour 5 , orthotopically into the mammary gland of syngeneic OSMR deficient (KO) and wild-type (WT) control mice (Fig. 1i). This model allows the assessment of the contribution of stromal OSMR signalling to cancer progression as OSMR is only depleted in the tumour microenvironment while TS1 cancer cells express OSMR that can be activated by host-derived OSM (Extended Data Fig. 1f,g). Depletion of OSMR in the tumour microenvironment resulted in delayed tumour onset and tumour growth ( Analysis of published gene expression profiles of breast cancer demonstrated that both OSM and OSMR are increased in human breast cancer stroma, compared to cancer epithelial compartment and healthy stroma (Fig. 1m). A similar pattern of OSM:OSMR expression was observed in other cancer types including colorectal and ovarian cancers (Extended Data Fig.   1h). We also observed that increased OSM mRNA levels associated with decreased diseasefree survival (Extended Data Fig.1i) in the Metabric 6 and Wang 7 breast cancer datasets.
Analysis of TCGA data by Kaplan-Meier Plotter 8 showed that high OSM levels were significantly associated with worse overall survival in other cancer types (Extended Data Fig. 1j).
As we found an unexpected contribution of stromal OSM: OSMR axis to breast cancer progression, we performed single cell RNA-seq analysis of mammary tumours from the MMTV-PyMT model to decipher which cells were responsible to produce OSM and to express OSMR in the breast cancer context (Fig. 2a). Our data indicate that the ligand OSM is almost exclusively expressed by the myeloid cell population, while the receptor OSMR is mainly expressed by the fibroblasts and some of the cancer epithelial clusters (Fig. 2a-c). The OSM:OSMR signalling module exhibits a distinct microenvironment-restricted expression and it differs from the one observed for other cytokine-receptor pairs of the same family such as IL6:IL6R (Il6ra) and LIF:LIFR (Fig. 2b,c), supporting that OSM exerts distinct and unique functions from other members of the family 9 . Il6st (GP130) is the common subunit receptor for OSM, IL6, LIF and other cytokines of the family and is ubiquitously expressed (Fig. 2b,c) Fig. 2d). To prove the relevance of our previous findings in human cancer clinical data, we used the TIMER 12 and xCell 13 web resources to analyse the association between OSM and OSMR expression and TME composition in two different clinical breast cancer datasets 8,14 . TIMER analysis showed that OSMR mRNA expression significantly correlates with fibroblast enrichment in human breast cancer, while OSM mRNA levels show the most significant associations with myeloid macrophage and neutrophil signatures (Fig. 2e). This analysis also showed that OSM and OSMR mRNA expression inversely correlated with tumour cell purity. The OSMR and OSM associations with fibroblasts and myeloid cell infiltration respectively, were validated by xCell in a different clinical dataset (Fig. 2f). A similar pattern of OSM:OSMR expression was observed in FACSsorted colorectal tumours (Extended data Fig. 2e). Altogether, our data reveal that OSM and OSMR are stroma-expressed molecules, and point to paracrine OSM:OSMR signalling in cancer, as ligand and receptor are expressed by different cell types in the tumour microenvironment.
As we previously observed that fibroblasts were the cell population with higher levels of OSMR within the tumour (  Fig. 3k), suggesting reduced levels of CAFs in this experimental group, probably due to impaired CAF proliferation upon OSMR reduction, in line with the increased size of CAF spheres observed after OSMR activation (Fig. 3b). Together, our data prove that OSM:OSMR signalling activates CAFs and that this contributes to cancer progression.
In an attempt to understand how OSMR activation in the stroma was inducing malignancy we deepened into our transcriptomic data of CAFs (CAF-173) treated with OSM. Microarray data indicated that pathways and signatures related to leukocyte chemotaxis and inflammatory response were significantly enriched by OSM (Fig. 4a,b). Interestingly, transcriptomic analysis of breast cancer cells (MDA-MB-231) activated by OSM showed enrichment of similar pathways (Extended Data Fig. 4a,b). These data suggested that, upon OSMR activation by OSM, both CAFs and cancer cells could be involved in shaping the tumour microenvironment by recruiting leukocytes to the tumour site. Analysis of a panel of 31 chemokines by antibody array showed that OSM induced expression of important chemoattractants ( Fig. 4c and Extended Data Fig. 4c,d). Some of these factors were exclusive of CAFs (mainly CXCL10 and CXCL12), others of cancer cells (mainly CXCL7 and CCL20) and some factors, such as CCL2, were common for both cell types. Vascular endothelial growth factor (VEGF) can also modulate tumour immunity by inducing macrophage and myeloid-derived suppressor cells (MDSCs) recruitment 18 and we previously showed that it is an OSMR target 16 . As seen in Fig. 4d and Extended Data Fig. 4e, VEGF levels were increased upon OSM treatment both in CAFs and tumour cells. As some of the OSM-induced chemokines are potent myeloid chemoattractants (e.g. VEGF, CCL2, CXCL12) 19,20 , we sought to determine whether myeloid cell populations were altered in tumours after OSMR signalling abrogation. We performed immunostaining of macrophages and neutrophils, assessed by F4/80 and Ly6G positivity respectively 2,21,22 , and we observed that these two populations were reduced in MMTV-PyMT: OSMR KO tumours compared to OSMR WT tumours (Fig. 4e). Interestingly, VEGF, CXCL1 and CXCL16 levels were reduced in the serum of tumour bearing MMTV-PyMT: OSMR KO mice compared to control mice (Fig. 4f), all factors being involved in myeloid cell recruitment 18,23,24 . In summary, these results show that OSM:OSMR signalling in stroma and cancer cells induces cytokine secretion and myeloid cell recruitment. Our findings are clinically relevant as VEGF, CXCL1 and CXCL16 mRNA expression is associated with decreased recurrence-free survival and with OSM and/or OSMR levels in breast cancer patients (Fig. 4g,h). As OSM is mainly expressed by myeloid cells  Fig. 4f). We did not observe this effect with conditioned media from OSM-activated CAFs or with OSM itself. These results suggest that OSMR activation in cancer cells, not only increases the secretion of chemokines involved in myeloid cell recruitment, but also induces OSM expression in these myeloid cells, doubly contributing to the aforementioned feed-forward loop.
Analysis of OSM protein levels in 141 samples of early breast cancer samples confirmed the association between OSM expression and increased inflammation in a clinical setting (Fig. 4i,j).
Inflammation was assessed by the pathologist as infiltration of inflammatory cells from all lymphoid and myeloid subtypes. We observed that OSM was mainly expressed by myeloid-like cells as determined by their larger size and more irregular shape (Fig. 4i). Lymphoid cells, characterized by being smaller and round and by having a round nucleus with little cytoplasm, showed very low or negative OSM expression (Fig. 4i). Importantly, high OSM protein levels were associated with decreased overall survival in this dataset (P=0.029, Fig. 4k).
Cytokines are important players in inflammation, a process associated with tumour progression 1 . Even the cancers not directly associated with persistent infections or chronic inflammation, such as breast cancer, exhibit tumour-elicited inflammation, which has important consequences in tumour promotion, progression and metastasis 25,26 . Understanding how inflammatory signals orchestrate pro-malignant effects in the different cell compartments within the tumour microenvironment is key to design new therapeutic strategies to target tumour-promoting inflammation. Our study identifies the pro-inflammatory cytokine OSM as a crucial mediator of the crosstalk between different cell types within the tumour by activating an intriguing pro-tumoural "ménage-à-trois" between myeloid cells, CAFs and cancer cells (Extended Data Fig. 5). OSM:OSMR interactions could be blocked by antibody based inhibition, a strategy that has had a major impact on cancer 27 , which makes them a promising candidate for therapeutic targeting. Interestingly, anti-OSM humanized antibodies have proven to be safe and well tolerated 28  TMAs were generated by punching 1 mm spot of each sample. Complete histopathological information, date and cause of death, as well as date of local and/or distant relapse were available for all the patients. Tissue sections were subjected to a heat-induced antigen retrieval step prior to exposure to primary OSM antibody (1:50, HPA029814, Sigma-Aldrich).
Immunodetection was performed using the Roche Ventana BenchMark ULTRA IHC/ISH staining system, with DAB as the chromogen. Cases were reviewed by two independent pathologists and OSM staining was evaluated by the semiquantitative method of H-score (or "histo" score), used to assess the extent of immunoreactivity in tumour samples 29  Collagen gel contraction assays. To assess the collagen remodelling capacity 43 , fibroblasts were treated for 4 days with recombinant human OSM (R&D Systems) at 10 ng/μl or vehicle (PBS) before being embedded in collagen (2mg/ml, Corning). After polymerization, collagen gels were detached, and they were treated with OSM (10ng/ml) or vehicle. Pictures were taken 48 hours later, and the area of collagen disks was analysed using Fiji-Image J software. Histopathology and Immunohistochemistry (IHC) analysis. Histological analysis of murine tumours and lung metastasis was performed in formalin-fixed paraffin-embedded haematoxylin-eosin stained sections. Immunohistochemical staining was performed in formalin-fixed paraffin-embedded sections using the streptavidin-biotin-peroxidase complex method. Antigen retrieval was performed using boiling 10 mM citrate buffer, pH 6.0, for 15 min. Endogenous peroxidase activity was inactivated by incubation with 3% hydrogen peroxide in methanol (15 min, at room temperature). Tissue sections were incubated in a humidified chamber (overnight, 4 ºC) using the antibodies described in Supplementary Table 1  Statistical analyses. Statistical analyses were performed using GraphPad Prism or SPSS softwares. For Gaussians distributions, the student's t-test (paired or unpaired) was used to compare differences between two groups. Welch's correction was applied when variances were significantly different. One-way ANOVA was used to determine differences between more than two independent groups. For non-Gaussian distributions, Mann-Whitney's test was performed. Chi-square test was used to determine differences between expected frequencies.