Conflict of interest: DG is named as an inventor on a patent application relating to the Mab203E1H5 (EP18155716) antibody filed by the University of Bergen.

Cancer‐associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen‐binding integrin α11β1 has been proposed to be upregulated in a pro‐tumorigenic subtype of cancer‐associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11β1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin‐fixed paraffin‐embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co‐expression patterns of integrin α11 in relation to αSMA and cytokeratin‐14 were also investigated. Integrin α11 was expressed to varying degrees in spindle‐shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co‐localized with αSMA in stromal cells, and with αSMA and cytokeratin‐14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple‐negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.

PurposeIt has been implied that the collagen binding integrin α11β1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11β1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model.MethodsTumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections.ResultsRM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups.ConclusionDeficiency of stromal integrin α11β1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis.

BackgroundCancer progression is influenced by a pro-tumorigenic microenvironment. The aberrant tumor stroma with increased collagen deposition, contractile fibroblasts and dysfunctional vessels has a major impact on the interstitial fluid pressure (PIF) in most solid tumors. An increased tumor PIF is a barrier to the transport of interstitial fluid into and within the tumor. Therefore, understanding the mechanisms that regulate pressure homeostasis can lead to new insight into breast tumor progression, invasion and response to therapy. The collagen binding integrin α11β1 is upregulated during myofibroblast differentiation and expressed on fibroblasts in the tumor stroma. As a collagen organizer and a probable link between contractile fibroblasts and the complex collagen network in tumors, integrin α11β1 could be a potential regulator of tumor PIF.MethodsWe investigated the effect of stromal integrin α11-deficiency on pressure homeostasis, collagen organization and tumor growth using orthotopic and ectopic triple-negative breast cancer xenografts (MDA-MB-231 and MDA-MB-468) in wild type and integrin α11-deficient mice. PIF was measured by the wick-in-needle technique, collagen by Picrosirius Red staining and electron microscopy, and uptake of radioactively labeled 5FU by microdialysis. Further, PIF in heterospheroids composed of MDA-MB-231 cells and wild type or integrin α11-deficient fibroblasts was measured by micropuncture.ResultsStromal integrin α11-deficiency decreased PIF in both the orthotopic breast cancer models. A concomitant perturbed collagen structure was seen, with fewer aligned and thinner fibrils. Integrin α11-deficiency also impeded MDA-MB-231 breast tumor growth, but no effect was observed on drug uptake. No effects were seen in the ectopic model. By investigating the isolated effect of integrin α11-positive fibroblasts on MDA-MB-231 cells in vitro, we provide evidence that PIF regulation was mediated by integrin α11-positive fibroblasts.ConclusionWe hereby show the importance of integrin α11β1 in pressure homeostasis in triple-negative breast tumors, indicating a new role for integrin α11β1 in the tumor microenvironment. Our data suggest that integrin α11β1 has a pro-tumorigenic effect on triple-negative breast cancer growth in vivo. The significance of the local microenvironment is shown by the different effects of integrin α11β1 in the orthotopic and ectopic models, underlining the importance of choosing an appropriate preclinical model.Electronic supplementary materialThe online version of this article (10.1186/s12885-019-5449-z) contains supplementary material, which is available to authorized users.

IntroductionCancer associated fibroblast (CAF) mediated crosstalk with other tumor compartments is shown to modulate cancer development and progression. Conventionally, CAFs are known to promote tumorigenesis either by secreting a wide range of growth factors, cytokines, and chemokines or by remodeling the underlying stroma. However, recent studies aimed at depleting CAFs have shown progression to more aggressive tumors leading to poorer outcomes. This may be attributed to the heterogeneity and plasticity of CAFs. Understanding CAF heterogeneity has been limited due to the lack of specific markers. Imaging mass cytometry (IMC) can analyze up to 40 parameters at subcellular resolution simultaneously and hence it can be an ideal tool to unravel the heterogeneity of CAFs and decipher the role of various CAF subsets in cancer development and progression. The aim of this study was to develop a panel of antibodies (ABs) for IMC to identify different CAF phenotypes and their spatial distribution in tissues and other tumor components in formalin-fixed paraffin-embedded (FFPE) archival tissues.MethodsA comprehensive panel of ABs were chosen based on known literature on CAFs and other tumor and microenvironment-related markers. ABs were conjugated to rare earth metals using the protocol provided by the supplier (Fluidigm, CA, USA). FFPE tissue samples from different organs/lesions known to express certain markers were used for making control tissue microarrays (cTMAs) for verification of conjugated ABs. Immunohistochemistry (IHC) with metal-conjugated ABs was used as the “gold standard” for optimization of IMC. Tissue images were segmented using ImcSegmentationPipeline (Ilastik and CellProfiler). Downstream analysis of single-cell data was performed using a newly developed bioinformatics pipeline as well as ImaCytE, an existing software solution.ResultsThe final panel of ABs was decided based on a comparable performance of conjugated ABs validated using IHC and IMC. Different cell types were identified including various subsets of CAFs and immune cells using cell segmentation and unsupervised clustering.ConclusionThis study establishes an extensive panel of IMC ABs on archival FFPE tissue material for unravelling CAF heterogeneity. In addition, the panel described here, the downstream analysis and the newly developed pipeline can be readily applied to different tissue types in context of cancer research or other fibrotic disease.