Integrated Analysis Revealed an Inflammatory Cancer-Associated Fibroblast-Based Subtypes with Promising Implications in Predicting the Prognosis and Immunotherapeutic Response of Bladder Cancer Patients

Abstract

Inflammatory cancer-associated fibroblasts (iCAFs) are closely related to progression, anticancer therapeutic resistance, and poor prognosis of bladder cancer (BCa). However, the functional role of iCAFs in BCa has been poorly studied. In our study, two BCa scRNA-seq datasets (GSE130001 and GSE146137) were obtained and integrated by the Seurat pipeline. Based on reported markers (COL1A1 and PDGFRA), iCAFs were identified and the related signature of 278 markers was developed. Following unsupervised consensus clustering, two molecular subtypes of TCGA-BLCA were identified and characterized by distinct dysregulated cancer hallmarks, immunological tumor microenvironments, prognoses, responses to chemotherapy/immunotherapy, and stemness. Subsequently, the robustness of the signature-based clustering, in terms of prognosis and therapeutic response prediction, was validated in a GEO-meta cohort with seven independent GEO datasets of 519 BCa patients, and three immune checkpoint inhibitor (ICI)-treated cohorts. Considering the heterogeneity, re-clustering of iCAFs was performed and a subpopulation, named “LOXL2+ iCAFs”, was identified. Co-culture CM derived from LOXL2 overexpression/silencing CAFs with T24 cells revealed that overexpression of LOXL2 in CAFs promoted while silencing LOXL2 inhibited the proliferation, migration, and invasion of T24 cells through IL32. Moreover, the positive correlation between LOXL2 and CD206, an M2 macrophage polarization marker, has been observed and validated. Collectively, integrated single-cell and bulk RNA sequencing analyses revealed an iCAF-related signature that can predict prognosis and response to immunotherapy for BCa. Additionally, the hub gene LOXL2 may serve as a promising target for BCa treatment.

POLR3G promotes EMT via PI3K/AKT signaling pathway in bladder cancer

“…Conversely, POLR3G overexpression in RT4 and J82 cells reversed this pattern entirely (Figure 5A,B). Furthermore, we examined the association between POLR3G and EMT‐related genes in a previously reported GEO meta cohort 5 and observed a negative correlation between POLR3G and E‐cadherin, as well as a positive correlation with N‐cadherin, Vimentin, Snail, and MMP‐2/9 at the transcript level (Figure 5C). These findings suggested the role of POLR3G in inducing EMT in BLCA cells.…”

“…However, despite undergoing standard treatment, 31%–78% of NMIBC patients relapse within five years. Additionally, 10%–30% of NMIBC patients will ultimately progress to MIBC, which requires a more aggressive approach involving radical cystectomy and chemotherapy 3–6 . Unfortunately, some people with MIBC still face poor survival rates even with these treatments 7 .…”

“…Additionally, 10%-30% of NMIBC patients will ultimately progress to MIBC, which requires a more aggressive approach involving radical cystectomy and chemotherapy. [3][4][5][6] Unfortunately, some people with MIBC still face poor survival rates even with these treatments. 7 The increase in publicly available RNA-seq datasets and advanced bioinformatic toolkits have enabled the discovery of numerous new molecular biomarkers.…”

Leveraging a disulfidptosis-based signature to improve the survival and drug sensitivity of bladder cancer patients

“…The enrichment analysis (GO, KEGG, and gene set enrichment analysis [GSEA]) was employed via the “clusterProfiler” package as described previously ( 7 , 17 ). The activity of GO-BP and KEGG terms for each sample was quantified by the “GSVA” package ( 18 ).…”

“…These assays were performed according to our previous studies ( 7 , 17 , 24 ). The primer sequences for qRT-PCR analysis were listed in Table S3 .…”