Journal of Experimental & Clinical Cancer Research

The biology centered around the TGF-{beta} type I receptor ALK1 (encoded by ACVRL1) has been almost exclusively based on its reported endothelial expression pattern since its first functional characterization more than two decades ago. Here, in efforts to better define the therapeutic context in which to use ALK1 inhibitors, we uncover a population of tumor-associated macrophages (TAMs) that, by virtue of their unanticipated Acvrl1 expression, are effector targets for adjuvant anti-angiogenic immunotherapy in mouse models of metastatic breast cancer. The combinatorial benefit depended on ALK1-mediated modulation of the differentiation potential of bone marrow-derived granulocyte-macrophage progenitors, the release of CD14+ monocytes into circulation, and their eventual extravasation. Notably, ACVRL1+ TAMs coincided with an immunosuppressive phenotype, and were over-represented in human cancers progressing on therapy. Accordingly, breast cancer patients with a prominent ACVRL1hi TAM signature exhibited a significantly shorter survival. In conclusion, we shed light on an unexpected multimodal regulation of tumorigenic phenotypes by ALK1 and demonstrate its utility as a target for anti-angiogenic immunotherapy. Graphical abstractSee submitted file

Examining LncRNA-driven cancer biology, our study represents the first comprehensive investigation of the LncRNA CDKN2B-AS1 across multiple cancer types, revealing its genetic basis for cancer susceptibility, and associations with thromboembolic risks and immunological dynamics. Stratification across hematological and non-hematological cancers revealed predominant prevalence rates of the CDKN2B-AS1 C-allele, notably higher in solid tumor malignancies, peaking at 86.8% in colorectal carcinoma (CRC). CRC-C-carriers exhibited worse outcomes, marked by reduced overall survival, increased venous-thromboembolism, elevated frequencies of thromboembolic-associated gene polymorphisms and BRAF mutations, and decreased MSI-H. Key tumor progression aspects in CRC-C-carriers included an inflammatory profile (reduced Th2 and increased Th1/Th17 cells), angiogenesis and endothelial activity (elevated CD14+, CD31+, CD144+, and VEGFR2+ cells), decreases in tumor suppressor-CD146+ cells (elevated tissue-adherent TA-EPCs lacking CD146), and marked elevation in tumor activity markers (Ki-67, CEA, CA 19-9, EGFR, VEGF-A, PD-1, and CTLA-4). Palbociclib significantly improved progression-free survival relative to baseline, primarily through suppression of tumor cell proliferation, with a more pronounced clinical and biomarker response observed in CDKN2B-AS1 non-C-allele carriers, whereas C-allele carriers exhibited an attenuated response consistent with relative treatment resistance. This underscores the multifaceted role of LncRNA CDKN2B-AS1, presenting it not just as a genetic determinant but also as a potential prognostic biomarker in cancer dynamics, paving the way for targeted personalized interventions, representing promising advancements in cancer therapeutics.

Primary resistance to anti-PD-1 therapy in metastatic melanoma remains a clinical challenge. This study reanalyzed the GSE168204 dataset to elucidate molecular mechanisms of resistance and response, incorporating batch correction to address limitations in our prior preprint[6]. RNA-seq data from 25 melanoma biopsies (9 responders, 16 non-responders) were analyzed using DESeq2 with surrogate variable analysis[9,10]. We identified 3,247 differentially expressed genes, revealing a "cell cycle shield" signature in non-responders characterized by upregulation of CDK1, CCNB1, E2F1, and HSP90AA1 enriched for proliferation and DNA repair pathways, suggesting immune evasion through rapid tumor growth. Responders exhibited upregulation of EP300, CREBBP, FCGR2B, and histone genes enriched for chromatin organization and systemic lupus erythematosus pathways, indicating immune activation and autoimmune-like transcriptional programs. Notably, batch correction reversed the roles of EP300 and FCGR2B from non-responders to responders[6], highlighting their context-dependent functions in immune engagement. The "cell cycle shield" suggests targeting CDK1 or HSP90AA1 to overcome resistance[13,14], while the SLE signature may serve as a response biomarker reflecting immune activation states[7]. Validation in larger cohorts and experimental models is needed to translate these findings into personalized immunotherapy strategies.

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

Estrogen receptor-negative (ER-) breast cancer is an aggressive breast cancer subtype with limited therapeutic options. Upregulated expression of both inducible nitric oxide synthase (NOS2) and cyclo-oxygenase (COX2) in breast tumors predicts poor clinical outcomes. Signaling molecules released by these enzymes activate oncogenic pathways, driving cancer stemness, metastasis, and immune suppression. The influence of tumor NOS2/COX2 expression on the landscape of immune markers using multiplex fluorescence imaging of 21 ER-breast tumors were stratified for survival. A powerful relationship between tumor NOS2/COX2 expression and distinct CD8+ T cell phenotypes was observed at 5 years post-diagnosis. These results were confirmed in a validation cohort using gene expression data showing that ratios of NOS2 to CD8 and COX2 to CD8 are strongly associated with poor outcomes in high NOS2/COX2-expressing tumors. Importantly, multiplex imaging identified distinct CD8+ T cell phenotypes relative to tumor NOS2/COX2 expression in Deceased vs Alive patient tumors at 5-year survival. CD8+NOS2-COX2-phenotypes defined fully inflamed tumors with significantly elevated CD8+ T cell infiltration in Alive tumors expressing low NOS2/COX2. In contrast, two distinct phenotypes including inflamed CD8+NOS2+COX2+ regions with stroma-restricted CD8+ T cells and CD8-NOS2-COX2+ immune desert regions with abated CD8+ T cell penetration, were significantly elevated in Deceased tumors with high NOS2/COX2 expression. These results were supported by applying an unsupervised nonlinear dimensionality-reduction technique, UMAP, correlating specific spatial CD8/NOS2/COX2 expression patterns with patient survival. Moreover, spatial analysis of the CD44v6 and EpCAM cancer stem cell (CSC) markers within the CD8/NOS2/COX2 expression landscape revealed positive correlations between EpCAM and inflamed stroma-restricted CD8+NOS2+COX2+ phenotypes at the tumor/stroma interface in deceased patients. Also, positive correlations between CD44v6 and COX2 were identified in immune desert regions in deceased patients. Furthermore, migrating tumor cells were shown to occur only in the CD8-NOS2+COX2+ regions, identifying a metastatic hot spot. Taken together, this study shows the strength of spatial localization analyses of the CD8/NOS2/COX2 landscape, how it shapes the tumor immune microenvironment and the selection of aggressive tumor phenotypes in distinct regions that lead to poor clinical outcomes. This technique could be beneficial for describing tumor niches with increased aggressiveness that may respond to clinically available NOS2/COX2 inhibitors or immune-modulatory agents.

The high mortality rate of colorectal cancer (CRC) combined with the lack of non-toxic and efficient personalized treatments makes it urgent to develop new targeted therapies for this disease. B7-H3 appears to be a good target as it is overexpressed in tumor tissue compared to normal tissue. However, B7-H3 is a molecule with ambivalent functions and is expressed by different cell types. This complexity contributed to the delay in identifying cell subtypes that express B7-H3 and their potential role in colorectal oncogenesis. In this study, we used in silico bulk, single-cell, and spatial transcriptomic data to investigate the clinical and biological characteristics of tumors with high B7-H3 expression, the precise nature of cells expressing high level of B7-H3, and their temporal appearance during colorectal oncogenesis. We found that tumors with high B7-H3 expression corresponded to tumors with a predominant stroma composed mainly of fibroblasts. Among them, two subtypes of ecm-myCAF fibroblasts and pro-fibrotic pericytes specifically expressed high levels of B7-H3, the former being an independent factor for poor prognosis in CRC. Finally, by examining precancerous lesions, we report that fibroblast subtypes with high levels of B7-H3 appear early during oncogenesis, especially at the inflamed stage. We also shed light on the fact that anti-B7-H3 immunotherapies might therefore preferentially target cells from the microenvironment rather than the tumor cells. This is particularly important to understand the mode of action of the anti-B7-H3 antibody-drug conjugate, which is currently being tested in clinical trials in several solid tumors. HighlightsO_LIHigh level of B7-H3 expression is associated with poor prognosis in colorectal cancer C_LIO_LIB7-H3 is recurrently found expressed by two subtypes of cancer-associated fibroblasts (CAF): pro-fibrotic pericytes and ecm-myCAF C_LIO_LIB7-H3high ecm-myCAF subtype is an independent poor prognosis for survival C_LIO_LIB7-H3high ecm-myCAF is detectable in pre-cancerous inflamed colonic tissues C_LI

BackgroundCancer immunotherapies for patients with colorectal cancer (CRC) continue to lag behind other solid cancer types with the exception of 4% of patients with microsatellite-instable tumors. Thus, there is an urgent need to broaden the clinical benefit of checkpoint blockers to CRC by combining conventional therapies to sensitise tumors to immunotherapy. However, the impact of conventional drugs on immunoediting, potentially promoting the positive selection of less immunogenic variants, and on the tumor immune contexture in CRC, remain elusive. MethodsWe performed comprehensive multimodal profiling using longitudinal samples from metastatic CRC patients undergoing neoadjuvant therapy with mFOLFOX6 and Bevacizumab. Exome-sequencing, RNA-sequencing and multiplexed immunofluorescence imaging was carried out on tumor samples obtained before and after therapy and the data was analysed using established methods. The results of the analysis were extrapolated to publicly available datasets (TCGA and CPTAC). In order to identify a surrogate marker, an explainable artificial intelligence method was developed using a transformer-based analytical pipeline for the identification of features in Hematoxylin and Eosin (H&E) images associated with specific biological processes, followed by manual evaluation of highly informative tiles by a pathologist. ResultsMutational profiles were highly modified and the level of genetic intertumoral heterogeneity between patients varied following treatment. Evolutionary analysis indicated eradication of some clones and dominant clonal prevalence of others, supporting the notion of pharmacologically-induced cancer immunoeditin. Post treatment samples showed upregulation of HLA class II genes, activation of differentiation and stemness pathways, and changes in the consensus molecular subtypes. The tumor immune contexture was characterised by increased densities of CD8+ and CD4+ T cells, but reduced T cell-tumor cell interactions (and increased T cell exhaustion. The AI-guided analyses of the H&E images pinpointed extracellular mucin deposits associated with stemness genes, suggesting a surrogate marker for routine pathological evaluation. ConclusionsConventional therapy induces immunoediting and modulates the immune contexture in metastatic CRC patients.

In solid cancers, inflammation and viral infections, two main fibroblast subtypes have been identified: myofibroblast-like fibroblasts and inflammatory fibroblasts. In the tumour microenvironment (TME), these cancer-associated fibroblast (CAF) subtypes are known as myCAFs, which generate a stiffened fibrotic extracellular matrix (ECM), and iCAFs, which secrete inflammatory cytokines to locally modulate the immune response. Yet, whether iCAFs contribute to shaping the ECM biochemical and biophysical properties remains unknown, mainly because robust in vitro models to generate fibroblast subtypes are lacking. Here, we established an in vitro cell culture system based on murine NIH3T3 fibroblasts and stimulation by TGF{beta} and IL1, alone or in combination, to induce fibroblast subtypes. Gene expression analysis of well-documented myCAF (Acta2/Tagln) and iCAF (Ccl2/Il6/Lif) markers revealed that TGF{beta} induced a myCAF-like phenotype, while a combination of TGF{beta} and IL1 induced an iCAF-like phenotype. We compared these in vitro subtypes to myCAFs and iCAFs from publicly available scRNAseq data of tumour tissues from cancer patients. We found that, similar to myCAFs, both tumour-associated and in vitro iCAFs express Acta2/Tagln as well as genes encoding for typical ECM proteins, which correlated in vitro with the ability to contract collagen. Furthermore, fluorescence microscopy and atomic force microscopy revealed that in vitro both subtypes generate thick, layered and stiff matrices with highly aligned ECM, demonstrating for the first time that iCAFs may also contribute to a pathological ECM. Finally, matrices generated from these in vitro fibroblast subtypes, but not from uninduced or IL1-only stimulated fibroblasts, enhanced the expression of the immune suppression marker Arg1 in co-cultured macrophages. Our study provides new insights in the contribution of inflammatory fibroblasts to ECM deposition and remodelling and puts forward a well-defined in vitro model to generate different fibroblast subtypes for future in-depth mechanistic studies of their roles in cancer and other pathologies.

The tumor microenvironment (TME) and its multifaceted interactions with cancer cells are major targets for cancer treatment. Single-cell technologies have brought major insights into the TME, but the resulting complexity frequently precludes conclusions on function. Therefore, we combined single-cell RNA sequencing and spatial transcriptomic data to explore the relationship between different cancer-associated fibroblast (CAF) populations and immune cell exclusion in breast tumors. Our data show for the first time the degree of spatial organization of different CAF populations in breast cancer. We found that IL-iCAFs, Detox-iCAFs, and IFN{gamma}-iCAFs tended to cluster together, while Wound-myCAFs, TGF{beta}-myCAFs, and ECM-myCAFs formed another group that overlapped with elevated TGF-{beta} signaling. Differential gene expression analysis of areas with CD8+ T-cell infiltration/exclusion within the TGF-{beta} signaling-rich zones identified elastin microfibrillar interface protein 1 (EMILIN1) as a top modulated gene. EMILIN1, a TGF-{beta} inhibitor, was upregulated in IFN{gamma}-iCAFs directly modulating TGF{beta} immunosuppressive function. Histological analysis of 74 breast cancer samples confirmed that high EMILIN-1 expression in the tumor margins was related to high CD8+ T-cell infiltration, consistent with our spatial gene expression analysis. High EMILIN-1 expression was also associated with better prognosis of patients with breast cancer, underscoring its functional significance for the recruitment of cytotoxic T cells into the tumor area. In conclusion, our data show that correlating TGF-{beta} signaling to a CAF subpopulation is not enough because proteins with TGF-{beta}-modulating activity originating from other CAF subpopulations can alter its activity. Therefore, therapeutic targeting should remain focused on biological processes rather than on specific CAF subtypes.

In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into "brisk", "non-brisk" and "absent" entailing a functional association that has never been proved. Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells. Here, we characterize the immune landscape in primary melanoma by high-dimensional single cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics. We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted. The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value than that of the brisk classification. Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.

Ovarian high grade serous carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesised aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a novel targeting strategy. We used both murine models and HGSC patient samples to study the impact of loss of Pten, a negative regulator of PI3K pathway signalling. We identified populations of resident macrophages specifically in Pten null omental tumours. These macrophages derive from peritoneal fluid macrophages and have a unique gene expression programme, marked by high levels of HMOX1 expression, the gene for the enzyme heme oxygenase-1. Targeting resident peritoneal macrophages prevents appearance of HMOX1hi macrophages and in doing so reduces tumour growth. Furthermore, direct inhibition of HMOX1 extends survival in vivo. HMOX1hi macrophages with corresponding gene expression programmes are also identified in human HGSC tumours and their presence correlates with activated tumoural PI3K pathway/mTOR signalling and poor overall survival in HGSC patients. In contrast, tumours with low number of HMOX1hi macrophages are marked by increased adaptive immune response gene expression. Our data suggest that HMOX1hi macrophages represent a potential therapeutic target and biomarker for poor prognosis HGSC.

In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). In this work, we performed a meta-analysis of 1700 CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested several CMSs in vitro models with crizotinib (CZB) or alectinib (ALC), potent ALK inhibitors, already approved for clinical use. ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D spheroids, which was impaired by the pharmacological inhibition of ALK. Consistently, CZB was responsible for the dampened activation of ALK along with the downstream AKT cascade. Mechanistically, we found a specific pro-apoptotic effect of ALK inhibition in CMS1 cell lines, both in 2D and 3D. Confocal analysis suggests that inhibition in CMS1 cells enhances cell-cell adhesion when growing in 3D. In agreement with our findings, an ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, the efficacy of ALK inhibition treatment was demonstrated in patient-derived organoids. Collectively, our findings suggest that ALK inhibition may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

The complexity of the ER-negative subtype of breast cancer arises due to the heterogeneous nature of the disease rendering them more aggressive and this poses a challenge to effective treatment and eventually the prognosis of the patients. We have explored the miRNA regulation of altered molecular signatures and the effect on tumour progression in ER-negative breast cancer. Using breast tumour specimens, gene expression data from public datasets and in-vitro and in-vivo model systems we have shown that low-levels of miR-18a in ER-negative tumours drives enrichment of hybrid Epithelial/Mesenchymal (E/M) cells with luminal attributes. On inhibition of miR-18a in ER-negative breast cancer cell lines, the cells showed traits of increased migration, stemness and drug-resistance. miR-18a/low tumours were also associated with increased expression of genes associated with EMT, stemness, drug resistance and immune-suppression. Further analysis of the miR-18a targets pointed out at a possible HIF-1 mediated signalling in these tumours. HIF-1 inhibition reduced the enrichment of the hybrid E/M cells and decreased the migratory ability of miR-18a/low cells. Our study reports for the first time a dual role of miR-18a in breast cancer that is subtype specific based on hormone receptor expression and a novel association of low miR-18a levels and enrichment of hybrid E/M cells. The results highlight the possibility of stratifying the ER-negative disease into clinically relevant groups by analysing epigenetic signatures.

Sarcomas are comprised of diverse bone and connective tissue tumors with few effective therapeutic options for locally advanced unresectable and/or metastatic disease. Recent advances in immunotherapy, in particular immune checkpoint inhibition (ICI), have shown promising outcomes in several cancer indications. Unfortunately, ICI therapy has provided only modest clinical responses and seems moderately effective in a subset of the diverse subtypes. To explore the immune parameters governing ICI therapy resistance or immune escape, we performed whole exome sequencing (WES) on tumors and their matched normal blood, in addition to RNA-seq from tumors of 31 sarcoma patients treated with pembrolizumab. We used advanced computational methods to investigate key immune properties, such as neoantigens and immune cell composition in the tumor microenvironment (TME). A multifactorial analysis suggested that expression of high quality neoantigens in the context of specific immune cells in the TME are key prognostic markers of progression-free survival (PFS). The presence of several types of immune cells, including T cells, B cells and macrophages, in the TME were associated with improved PFS. Importantly, we also found the presence of both CD8+ T cells and neoantigens together was associated with improved survival compared to the presence of CD8+ T cells or neoantigens alone. Interestingly, this trend was not identified with the combined presence of CD8+ T cells and TMB; suggesting that a combined CD8+ T cell and neoantigen effect on PFS was important. The outcome of this study may inform future trials that may lead to improved outcomes for sarcoma patients treated with ICI.

Cancer-associated fibroblasts (CAFs) are the dominant cell type in the stroma of solid organ cancers, including non-small cell lung cancer (NSCLC). Fibroblast heterogeneity is widely recognised in many cancers, with subpopulations of CAFs being identified and potentially being indicative of prognosis and treatment efficacy. Here, the subtypes displayed by CAFs isolated from human NSCLC resections are initially identified by flow cytometry, using the markers FAP, CD29, SMA, PDPN, CD90, FSP-1 and PDGFR{beta}, showing five distinct subpopulations, CAF-S1-S5. Our findings show that when comparing fibroblasts from tumour tissue with that from adjacent lung tissue, CAF-S2 and CAF-S3 are found in the normal tissue and marker expression suggests a less activated phenotype whereas CAF-S1, CAF-S4 and CAF-S5 are predominantly found in the tumour tissue and are positive for a combination of markers of fibroblast activation. We focus on these subtypes most associated with fibroblast activation, primarily focussing on a previously unreported CAF-S5 subtype, and comparing to the previously identified CAF-S1. Both these subsets express FAP and PDPN as markers of fibroblast activation, but CAF-S5 lacks expression of the common activation marker SMA. The spatial relevance of these subtypes in a cohort of 163 NSCLC patients was then investigated by multiplex immunofluorescence on a tumour micro-array of patient samples, revealing CAF-S5 are found further from tumour regions than CAF-S1. To understand the functional role of CAF-S5, scRNA sequencing data was used to compare the subset to the previously identified CAF-S1, finding that CAF-S5 displays an inflammatory phenotype, whereas CAF-S1 displays a contractile phenotype. We demonstrate that presence of either the CAF-S1 or CAF-S5 subtype is correlated to worse survival outcome in NSCLC, highlighting the importance of the identification of CAF subtypes in NSCLC.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. SignificanceEvidence of the value of ex vivo 3D models obtained by combining patient-derived extracellular matrices depleted of cellular components and organoids to mimic the metastatic niche, to be used as a tool to develop new therapeutic strategies in a biologically relevant context, to personalize treatments and increase their efficacy.

Brain metastases (BM) are one of the most serious clinical problems in breast cancer (BC) progression, associated with lower survival rates and a lack of effective therapies. Thus, to dissect the early stages of the brain metastatic process, we have searched for a brain-tropic metastatic signature on BC cells secretome, as a promising source for the discovery of new biomarkers involved in brain metastatic progression. Therefore, six specifically deregulated peptides were found to be enriched in the secretome of brain organotropic BC cells. Importantly, these secretomes caused significant blood-brain barrier (BBB) disruption, as well as microglial activation, in vitro and in vivo. We identified the VGF nerve growth factor inducible as a brain-specific peptide, promoting BBB dysfunction similar to the secretome of brain organotropic BC cells. Concerning microglial activation, a slight increase was also observed upon VGF treatment. In a series of human breast tumors, VGF was found to be expressed in both cancer cells and in the adjacent stroma. VGF-positive tumors showed a significant worse prognosis and were associated with HER2 overexpression and triple-negative molecular signatures. Finally, in a cohort including primary breast tumors and their corresponding metastatic locations to the lung, bone, and brain, we found that VGF significantly correlates with the brain metastatic site. In conclusion, we found a specific BC brain metastatic signature, where VGF was identified as a key mediator in this process. Importantly, its expression was associated with poor prognosis for BC patients, probably due to its associated increased risk of developing BM.

The tumor microenvironment (TME) is important for cancer growth and progression. While angiogenesis is an established hallmark of cancer, the role of nerve fibers is less studied. Here, we investigated neurogenesis and angiogenesis in breast cancer and found them to be closely associated. Single-cell based spatial mapping by imaging mass cytometry (IMC) indicated close proximity between neural and vascular structures. Subsequent validation by tissue-based markers of neurogenesis and angiogenesis, supported by proteomics and transcriptomics data of tissues and cell lines, supported a link between these processes. A consolidated neuro-angiogenic signature score was linked to high-grade breast cancer and reduced patient survival, also within the low-grade luminal tumor subgroup. Our findings support that neurogenesis and angiogenesis are related in aggressive breast cancer and might possibly improve tumor stratification and clinical management.

Cell line models for high grade serous ovarian cancer (HGSOC) are limited in number and are poorly clinically annotated. Consequently, existing models often fail to recapitulate common features of HGSOC, inhibiting mechanistic and therapeutic discovery. We generated and characterised three spontaneously immortalized continuous HGSOC cell lines named CIOV1, CIOV2, and CIOV3 and confirmed that each cell line retained the genomic and pathologic characteristics of its parental tumour. We show that subclonal cell populations present at initiation, expanded and contracted during the culturing process before converging on a stable immortalized line. These lines are new valuable models to study acquired chemoresistance of HGSOC.

The crosstalk between cancer and the tumour immune microenvironment (TIME) has attracted significant interest because of its impact on cancer evolution and response to treatment. Despite this, cancer-specific tumour-TIME interactions and their mechanisms of action are still poorly understood. Here we identified the interactions between cancer-specific genetic drivers and anti- or pro-tumour TIME features in individual samples of 32 cancer types. The resulting 477 TIME drivers are multifunctional genes whose alterations are selected early in cancer evolution and recur across and within cancer types. Moreover, the anti-tumour TIME driver burden is predictive of overall response to immunotherapy. Focusing on head and neck squamous cancer (HNSC), we rebuilt the functional networks linking specific TIME driver alterations to the TIME state. We showed that TIME driver alterations predict the immune profiles of HNSC molecular subtypes, and that deregulation of keratinization, apoptosis and interferon signalling underpin specific driver-TIME interactions. Overall, our study provides a comprehensive resource of TIME drivers giving mechanistic insights into their immune-regulatory role.