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Single-cell technologies have enabled the characterization of the tumour microenvironment at unprecedented depth and have revealed vast cellular diversity among tumour cells and their niche. Anti-tumour immunity relies on cell–cell relationships within the tumour microenvironment1,2, yet many single-cell studies lack spatial context and rely on dissociated tissues3. Here we applied imaging mass cytometry to characterize the immunological landscape of 139 high-grade glioma and 46 brain metastasis tumours from patients. Single-cell analysis of more than 1.1 million cells across 389 high-dimensional histopathology images enabled the spatial resolution of immune lineages and activation states, revealing differences in immune landscapes between primary tumours and brain metastases from diverse solid cancers. These analyses revealed cellular neighbourhoods associated with survival in patients with glioblastoma, which we leveraged to identify a unique population of myeloperoxidase (MPO)-positive macrophages associated with long-term survival. Our findings provide insight into the biology of primary and metastatic brain tumours, reinforcing the value of integrating spatial resolution to single-cell datasets to dissect the microenvironmental contexture of cancer.

The native sIGFIR decoy protein blocks IGF-I and IGF-II mediated functions in vitro

This documents contains the legends of Figures 1 to S6

This documents contains the materials and methods of experiments described in supplementary data

This document contains Table S1 to S3

Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer.Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent.Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate.Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

Supplementary Fig. S1. Heatmap of mRNA expression for individual melanosomal differentiation antigens and a MITF transcriptional signature using a published dataset of melanoma samples (2). Supplementary Fig. S2. GPNMB expression is induced in multiple BRAF-mutant cells in response to MAPK pathway inhibition. Supplementary Fig. S3. Gene expression of MITF and GPNMB in melanoma cells following MAPK pathway inhibition. Supplementary Fig. S4. Dabrafenib and Trametinib stimulate MITF nuclear localization. Supplementary Fig. S5. Correlation of MITF and GPNMB mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway 7 inhibitors. RT-qPCR data for MITF and GPNMB was expressed as the ratio relative to pretreatment levels. Supplementary Fig. S6. Correlation between the expression of individual melanosomal genes with MITF mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway inhibitors. RT-qPCR data for each gene and MITF was normalized to GAPDH and expressed as the ratio relative to pre-treatment levels. Supplementary Fig. S7. MAPK pathway inhibition results in elevated levels of soluble GPNMB released from melanoma cells. Table S1: Clinical characteristics of patients biopsied for evaluation of MITF and GPNMB mRNA expression Table S2: Correlation coefficients between individual gene expression and MITF or a MITF Signature Table S3: Complete Responses Following Trametinib Treatment in Cohorts Injected with A375 or WM-2664 Melanoma Cells. Table S4: List of Primers for RT-qPCR Analyses

Supplementary legends and methods

Contains supplementary methods, references and figure legends.

Purpose: To determine if BRAF and/or MEK inhibitor–induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB.Experimental Design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth.Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor–treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone.Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088–98. ©2016 AACR.

Supplementary Data from Glycoprotein Nonmetastatic B Is an Independent Prognostic Indicator of Recurrence and a Novel Therapeutic Target in Breast Cancer

FIGURE S1: LY3009120 is an effective inhibitor of BRAF class II mutant melanoma cells. FIGURE S2: Bioinformatic characterization of PDX models. FIGURE S3: Inhibitory effects of BRAF and MEK inhibitors in melanoma cells. FIGURE S4: Representative images from cell growth clonogenic assays. FIGURE S5: Encorafenib and binimetinib are an effective combination for class II BRAF mutant melanoma. FIGURE S6: Dual MAPK inhibition slows progression of class II BRAF mutant melanoma brain metastases.

Purpose:Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi.Experimental Design:Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi.Results:BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi.Conclusions:Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.See related commentary by Johnson and Dahlman, p. 6107.

Supplementary Figure S8. Ribavirin reduces MMP production in 66cl4 tumors in vivo.

Legend for Supplementary Figures S1-S8.

The translation initiation factor eIF4E is an oncogene that is commonly overexpressed in primary breast cancers and metastases. In this article, we report that a pharmacologic inhibitor of eIF4E function, ribavirin, safely and potently suppresses breast tumor formation. Ribavirin administration blocked the growth of primary breast tumors in several murine models and reduced the development of lung metastases in an invasive model. Mechanistically, eIF4E silencing or blockade reduced the invasiveness and metastatic capability of breast cancer cells in a manner associated with decreased activity of matrix metalloproteinase (MMP)-3 and MMP-9. Furthermore, eIF4E silencing or ribavirin treatment suppressed features of epithelial-to-mesenchymal transition, a process crucial for metastasis. Our findings offer a preclinical rationale to explore broadening the clinical evaluation of ribavirin, currently being tested in patients with eIF4E-overexpressing leukemia, as a strategy to treat solid tumors such as metastatic breast cancer. Cancer Res; 75(6); 1102–12. ©2015 AACR.

Supplementary Table S1. Ribavirin concentrations measured in mouse plasma.

We updated our data set, please use thenew version(md5:ece00981be0f2616c1c0a57d10f3176e). All data supporting the findings of the publication "Single-cell spatial immune landscapeof primary and metastatic brain tumours", including masks of high-dimension tif images, single-cell segmentation, single-cell cell types, and patient data. The code used to produce the results of this study is available at https://github.com/walsh-quail-labs/IMC_Brain. Raw primary imaging data can be obtained from the authors directly upon reasonable request. Glioma channel index names: Channel Name Channel Index CD117 1 CD11c 2 CD14 3 CD163 4 CD16 5 CD20 6 CD31 7 CD3 8 CD4 9 CD68 10 CD8 11 CD94 12 DNA1 13 FoxP3 14 GFAP 15 HLA-DR 16 MPO 17 Olig2 18 P2PY12 19 Sox2 20 Sox9 21 BrM channel index names: Channel Name Channel Index CD117 1 CD11c 2 CD14 3 CD163 4 CD16 5 CD20 6 CD31 7 CD3 8 CD4 9 CD68 10 CD8a 11 CD94 12 DNA1 13 FoxP3 14 GFAP 15 HLA-DR 16 MPO 17 MeLanA 18 P2PY12 19 PMEL 20 PanCK 21

We updated our data set, please use the new version (md5:e33079d5299cdc17f37b9f5e4c08be12). All data supporting the findings of the publication "Single-cell spatial landscapes of the lung tumour immune microenvironment", including masks of high-dimension tif images, single-cell segmentation, single-cell cell types, and patient data. The code used to produce the results of this study is available athttps://github.com/walsh-quail-labs/IMC-Lung. Channel index names: Channel Name Channel Index CD117 1 CD11c 2 CD14 3 CD163 4 CD16 5 CD20 6 CD31 7 CD3 8 CD4 9 CD68 10 CD8a 11 CD94 12 DNA1 13 FoxP3 14 HLA-DR 15 Histone H3 16 MPO 17 Pancytokeratin 18 TTF1 19

Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.

Brain metastases (BM) are associated with significant morbidity and mortality in patients with advanced cancer. Despite significant advances in surgical, radiation, and systemic therapy in recent years, the median overall survival of patients with BM is less than 1 year. The acquisition of medical images, such as computed tomography (CT) and magnetic resonance imaging (MRI), is critical for the diagnosis and stratification of patients to appropriate treatments. Radiomic analyses have the potential to improve the standard of care for patients with BM by applying artificial intelligence (AI) with already acquired medical images to predict clinical outcomes and direct the personalized care of BM patients. Herein, we outline the existing literature applying radiomics for the clinical management of BM. This includes predicting patient response to radiotherapy and identifying radiation necrosis, performing virtual biopsies to predict tumor mutation status, and determining the cancer of origin in brain tumors identified via imaging. With further development, radiomics has the potential to aid in BM patient stratification while circumventing the need for invasive tissue sampling, particularly for patients not eligible for surgical resection.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using β-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer., Complex I inhibition induces oxidative stress leading to cancer cell cytotoxicity. Here, the authors show, in breast cancer models, that inflammatory mediators can potentiate complex I inhibitor phenformin cytotoxicity through STAT1-mediated downregulation of the reactive oxygen species scavenger NQO1.

Background Sixty percent of surgically resected brain metastases (BrM) recur within 1 year. These recurrences have long been thought to result from the dispersion of cancer cells during surgery. We tested the alternative hypothesis that invasion of cancer cells into the adjacent brain plays a significant role in local recurrence and shortened overall survival. Methods We determined the invasion pattern of 164 surgically resected BrM and correlated with local recurrence and overall survival. We performed single-cell RNA sequencing (scRNAseq) of >15,000 cells from BrM and adjacent brain tissue. Validation of targets was performed with a novel cohort of BrM patient-derived xenografts (PDX) and patient tissues. Results We demonstrate that invasion of metastatic cancer cells into the adjacent brain is associated with local recurrence and shortened overall survival. scRNAseq of paired tumor and adjacent brain samples confirmed the existence of invasive cancer cells in the tumor-adjacent brain. Analysis of these cells identified cold-inducible RNA-binding protein (CIRBP) overexpression in invasive cancer cells compared to cancer cells located within the metastases. Applying PDX models that recapitulate the invasion pattern observed in patients, we show that CIRBP is overexpressed in highly invasive BrM and is required for efficient invasive growth in the brain. Conclusions These data demonstrate peritumoral invasion as a driver of treatment failure in BrM that is functionally mediated by CIRBP. These findings improve our understanding of the biology underlying postoperative treatment failure and lay the groundwork for rational clinical trial development based upon invasion pattern in surgically resected BrM.

The immune system is comprised of both innate and adaptive immune cells, which, in the context of cancer, collectively function to eliminate tumor cells. However, tumors can actively sculpt the immune landscape to favor the establishment of an immunosuppressive microenvironment, which promotes tumor growth and progression to metastatic disease. Glycoprotein-NMB (GPNMB) is a transmembrane glycoprotein that is overexpressed in a variety of cancers. It can promote primary tumor growth and metastasis, and GPNMB expression correlates with poor prognosis and shorter recurrence-free survival in patients. There is growing evidence supporting an immunosuppressive role for GPNMB in the context of malignancy. This review provides a description of the emerging roles of GPNMB as an inducer of immunosuppression, with a particular focus on its role in mediating cancer progression by restraining pro-inflammatory innate and adaptive immune responses.

Neutrophils represent the immune system’s first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases., Tabariès et al. describe that claudin 2 is a promoter of colorectal cancer liver metastasis. Furthermore, high Claudin-2 expression is associated with shorter time to liver-specific recurrence and is a biomarker of replacement type CRC liver metastases.

Colorectal cancer liver metastasis (CRCLM) has two major histopathological growth patterns: angiogenic desmoplastic and non-angiogenic replacement. The replacement lesions obtain their blood supply through vessel co-option, wherein the cancer cells hijack pre-existing blood vessels of the surrounding liver tissue. Consequentially, anti-angiogenic therapies are less efficacious in CRCLM patients with replacement lesions. However, the mechanisms which drive vessel co-option in the replacement lesions are unknown. Here, we show that Runt Related Transcription Factor-1 (RUNX1) overexpression in the cancer cells of the replacement lesions drives cancer cell motility via ARP2/3 to achieve vessel co-option. Furthermore, overexpression of RUNX1 in the cancer cells is mediated by Transforming Growth Factor Beta-1 (TGFβ1) and thrombospondin 1 (TSP1). Importantly, RUNX1 knockdown impaired the metastatic capability of colorectal cancer cells in vivo and induced the development of angiogenic lesions in liver. Our results confirm that RUNX1 may be a potential target to overcome vessel co-option in CRCLM., Miran Rada et al. identify a key role for RUNX1 in liver metastasis of colorectal cancer. They show that RUNX1 is necessary for driving blood vessel co-option from surrounding liver tissue and that regulation of this process relies on a signaling feedback loop between RUNX1 transcriptional target TSP1 in tumor cells and TGFβ1 in the liver.

Although combination BRAF/MEK inhibition has produced significant survival benefits for BRAF p.V600 mutant melanomas, targeted therapies approved for BRAF non-p.V600 mutant melanomas remain limited. Through the analysis of 772 cutaneous melanoma exomes, we reveal that BRAF non-p.V600 mutations co-occurs more frequently with NF1 loss, but not with oncogenic NRAS mutations, than expected by chance. We present cell signaling data, which demonstrate that BRAF non-p.V600 mutants can signal as monomers and dimers within an NF1 loss context. Concordantly, BRAF inhibitors that inhibit both monomeric and dimeric BRAF synergize with MEK inhibition to significantly reduce cell viability in vitro and tumor growth in vivo in BRAF non-p.V600 mutant melanomas with co-occurring NF1 loss-of-function mutations. Our data suggest that patients harboring BRAF non-p.V600 mutant melanomas may benefit from current FDA-approved BRAF/MEK inhibitor combination therapy currently reserved for BRAF p.V600 mutant patients.

Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation andde novoglutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.

There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-β signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-β signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-β pathway in SCC cells.