The prevalence and incidence of BLCA are seeing an unprecedented rise worldwide (23). BLCA can be classified into NMIBC and MIBC based on tumor invasion depth. MIBC is a lethal type, which warrants definitive treatment. It is approximated that only half of MIBC patients undergo definitive treatment, including surgery rate for fewer than 20% (24). The ease of metastasis in MIBC may explain the low surgical rate in patients with MIBC. Many patients with MIBC show metastases at diagnosis, therefore, are thus deprived of surgery. However, this group of patients is managed using a combination of medical therapies, including platinum-based chemotherapy. For many years, researchers are yet to make significant progress in the treatment of metastatic BLCA, especially after the failure of platinum-based chemotherapy. Recently, the advent of immune checkpoint inhibitors has provided new treatment options for patients with advanced BLCA. Several clinical pieces of evidence have validated the remarkable effectiveness of immune checkpoint inhibitors in the management of advanced BLCA. In this view, immune checkpoint inhibitors are now guideline-recommended therapy for advanced BLCA that has failed prior chemotherapy. Nevertheless, immune checkpoint inhibitors still face problems of low responsiveness and frequent adverse effects, which warrants urgent exploration of the strategies to increase the responsiveness of BLCA immunotherapy.
As the tumor cells begin to proliferate and invade, they initiate microenvironment remodeling by activating resident fibroblasts, which replace the adipocyte-rich stroma with CAFs (25). The contribution of the microenvironment to tumor progression is underpinned by autocrine and paracrine signaling, in which the secretome of CAFs and cancer cells plays a pivotal role (26). Recent advancement in single-cell RNA sequencing significantly enriches our understanding of the heterogeneity of the TME. There is a common view that the CAFs can be classified into subgroups, including iCAFs and myCAFs. The iCAFs, which earned profound secreting features and specifically expressed PDGFRA and CXCL12, are now considered to impact BLCA patients' prognosis essentially. Through single-cell RNA sequencing, iCAFs have been shown to exert essential functions in recruiting immune cells into the tumor microenvironment (14). Recent evidence also supports that the direct interactions between CAFs and T cells, mediated via coincident upregulation and engagement of PD-1 on T cells, drive T cell dysfunction and death within tumors (27). Besides, CAFs are critical players in shaping a tumor-permissive and immunosuppressive TME as they preferentially induce the tumor-promoting function of TAMs. The recruitment and pro-tumoral activation of both cell types are significantly induced via a combination of heterogeneous CAFs and TAMs through reciprocal interaction. Accordingly, this accelerates tumor progression and proves that therapies targeting both TAMs and CAFs, or those targeting the cell-cell interaction between TAMs and CAFs improve anti-tumor therapeutic efficacy (28).
In the present study, the constructed iCAFs signature represented the relative abundance of iCAFs in each patient. Our analysis demonstrated that the iCAF abundance was an unfavorable prognostic factor for BLCA, encompassing significant influences on TME components. Furthermore, a significant negative association between iCAFs scores and mutations in FGFR3 was reported. Previous studies suggested that mutations of FGFR3 were more common in low-stage urothelial cancers. Similarly, our results laterally confirmed a positive correlation between iCAFs scores and tumor stage.
CXCL12 is now considered explicitly expressed by iCAFs in the TME (29). Studies have reported CAFs-driven modulatory effects of CXCL12 in immune suppression status in several cancer types (30, 31). However, reports on expression patterns and immune-modulating functions of CXCL12 in BLCA are unknown. The present work explored the roles of CXCL12 in BLCA through multi-omics bioinformatics analysis along with immunohistochemistry. We reported three exclusive characteristics of CXCL12 in BLCA: (i) the expression level of CXCL12 in BLCA tissues was significantly lower than that in normal tissues, and the decrease in the expression level of CXCL12 was highly accurate in the diagnosis of BLCA; (ii): methylation of CXCL12 has distinct effects on bladder cancer, with methylation of promoters tending to play a role in bladder carcinogenesis and methylation of the CpG sites in the gene body playing a vital role in bladder cancer prognosis. (iii) the expression of CXCL12 in bladder cancer increased with increasing tumor stage. Although these findings may seem contradictory, they implied intricate regulatory processes. Of note, the significant decrease in CXCL12 expression levels in tumors was potentially related to the hypermethylation of its promoter in tumors. While the increase in CXCL12 expression levels in tumors with tumor staging could be ascribed to several mechanisms, including gene body methylation(32) and increased tumor heterogeneity(33). However, we only found that CXCL12 gene body methylation was associated with the prognosis of bladder cancer patients, but not with CXCL12 expression levels. In view of the current evidence, we believe that the accumulation of CAFs during tumor progression may be one of the important reasons for the increase in CXCL12 expression.
Within the tumor patients, it is no doubt that CXCL12 is an adverse prognostic factor. Gene enrichment analysis suggested a significant role of CXCL12 in multiple TME modulating processes, including ECM organization, cytokine to cytokine interaction, epithelial to mesenchymal transition, hypoxia, angiogenesis, T cell receptors signaling pathway, and macrophage activation. Moreover, correlation analysis revealed that CXCL12 was significantly associated with the immune checkpoint-related gene expression, CD8 + T cells, macrophages infiltration, and the abundance CAFs. These results were consistent with the previous evidence that CXCL12, secreted by CAFs, was involved in regulating CD8 + T cells and macrophages, resulting in immunosuppression in the TME, thus promoting tumor progression(34). Using the TIDE algorithm, the impact of CXCL12 on T cell dysfunction and exclusion was revealed, and high CXCL12 expression was proved to influence the responsiveness of BLCA patients to ICB therapy.
Finally, by exploring the expression of CXCL12, PDGFRA, and CD8A in BLCA specimens, it was demonstrated that PDGFRA and CXCL12 were highly co-expressed in tumor stromal components. Meanwhile, a significant increase of CD8 + T-cell infiltration was reported in the stromal region. In contrast, a substantial decrease of intratumoral CD8 + T-cell infiltration was revealed in tumors positive for PDGFRA and CXCL12. Contrarily, intratumoral CD8 + T-cell infiltration was significantly elevated in tumor tissues negative for PDGFRA and CXCL12 expression. These findings mirrored the exclusive effect of CXCL12 and iCAFs on CD8 + T cells. In addition, CXCL12 protein was found globally expressed in MIBCs, whereas only a small subset of NMIBC expressed CXCL12. Moreover, the IMvigor210 cohort further validated that the iCAFs may impact patients' responsiveness to ICB therapy. These findings affirmed the results of bioinformatics analysis, strongly suggesting that CXCL12 and iCAFs play a part in the immunosuppression of BLCA and are potentially relevant factors in BLCA progression.
Although the present study revealed remarkable findings, a few limitations cannot be ignored. First, our research is mainly based on bioinformatics analysis, though Chen K et al. had confirmed the interaction of PDGFRA+CXCL12+ iCAFs with various immune cells in the TME (14). In this view, experimental data are still needed to verify the specific roles of CXCL12 methylation and TME remodeling in BLCA. Secondly, the number of clinical samples analyzed was limited; therefore, a clinical cohort of larger samples is needed to verify the accuracy of decreased CXCL12 levels in diagnosing BLCA and the effect of iCAFs and CXCL12 on how bladder cancer patients respond to immunotherapy.
In conclusion, this work demonstrates the effect of CXCL12 on the occurrence and development of BLCA at multiple levels through systematic multi-omics bioinformatics analysis along with immunohistochemical verification. Additionally, iCAFs and CXCL12 significantly impact the immunotherapy of BLCA and the prognosis of BLCA patients. The treatment targeting iCAFs may provide new ideas for advancing BLCA treatment in the future to improve patient's responsiveness to existing ICB treatments.