Despite the fact that the role of endoglin on endothelial cells has been extensively described, its expression and biological role on (epithelial) cancer cells is still debatable. Especially its... Show moreDespite the fact that the role of endoglin on endothelial cells has been extensively described, its expression and biological role on (epithelial) cancer cells is still debatable. Especially its function on squamous cell carcinoma (SCC) cells is largely unknown. Therefore, we investigated SCC endoglin expression and function in three types of SCCs; head and neck (HNSCC), esophageal (ESCC) and vulvar (VSCC) cancers. Endoglin expression was evaluated in tumor specimens and 14 patient-derived cell lines. Next to being expressed on angiogenic endothelial cells, endoglin is selectively expressed by individual SCC cells in tumor nests. Patient derived HNSCC, ESCC and VSCC cell lines express varying levels of endoglin with high interpatient variation. To assess the function of endoglin in signaling of TGF-beta ligands, endoglin was overexpressed or knocked out or the signaling was blocked using TRC105, an endoglin neutralizing antibody. The endoglin ligand BMP-9 induced strong phosphorylation of SMAD1 independent of expression of the type-I receptor ALK1. Interestingly, we observed that endoglin overexpression leads to strongly increased soluble endoglin levels, which in turn decreases BMP-9 signaling. On the functional level, endoglin, both in a ligand dependent and independent manner, did not influence proliferation or migration of the SCC cells. In conclusion, these data show endoglin expression on individual cells in the tumor nests in SCCs and a role for (soluble) endoglin in paracrine signaling, without directly affecting proliferation or migration in an autocrine manner. Show less
BACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer... Show moreBACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression. METHODS: Primary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsy specimens of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3-dimensional co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole-transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions. RESULTS: In 3-dimensional multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and up-regulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically up-regulated in T1CAFs but not in fibroblasts from premalignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections. CONCLUSIONS: Already in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, cathepsin H-dependent ECM remodeling. RNA sequencing data of the 10 NF-T1CAF pairs can be found under GEO accession number GSE200660. Show less
BACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSION\nImproving clinical management of early-stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating... Show moreBACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSION\nImproving clinical management of early-stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression.\nPrimary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsies of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3D co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions.\nIn 3D multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and upregulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically upregulated in T1CAFs but not in fibroblasts from pre-malignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of Cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections.\nAlready in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, Cathepsin H-dependent ECM remodeling. Show less
Roelands, J.; Ploeg, M. van der; Ijsselsteijn, M.E.; Dang, H.; Boonstra, J.J.; Hardwick, J.C.H.; ... ; Miranda, N.F.C.C. de 2022
ObjectiveBiological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical... Show moreObjectiveBiological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical management of this disease. Here, we aimed to dissect the transcriptional and immunologic alterations that accompany malignant transformation in CRC and to identify clinically relevant biomarkers through spatial profiling of pT1 CRC samples. DesignWe employed digital spatial profiling (GeoMx) on eight pT1 CRCs to study gene expression in the epithelial and stromal segments across regions of distinct histology, including normal mucosa, low-grade and high-grade dysplasia and cancer. Consecutive histology sections were profiled by imaging mass cytometry to reveal immune contextures. Finally, publicly available single-cell RNA-sequencing data was analysed to determine the cellular origin of relevant transcripts. ResultsComparison of gene expression between regions within pT1 CRC samples identified differentially expressed genes in the epithelium (n=1394 genes) and the stromal segments (n=1145 genes) across distinct histologies. Pathway analysis identified an early onset of inflammatory responses during malignant transformation, typified by upregulation of gene signatures such as innate immune sensing. We detected increased infiltration of myeloid cells and a shift in macrophage populations from pro-inflammatory HLA-DR(+)CD204(-) macrophages to HLA-DR(-)CD204(+) immune-suppressive subsets from normal tissue through dysplasia to cancer, accompanied by the upregulation of the CD47/SIRP alpha 'don't eat me signal'. ConclusionSpatial profiling revealed the molecular and immunological landscape of CRC tumourigenesis at early disease stage. We identified biomarkers with strong association with disease progression as well as targetable immune processes that are exploitable in a clinical setting. Show less
