Background
Bladder cancer is the most widespread and complex urinary tract malignancy all over the world with high-risk mortality, 25% of which are diagnosed as muscle-invasive bladder cancer (MIBC) [
1]. MIBC is a more aggressive stage with unfavorable prognosis [
2]. Cisplatin-based adjuvant chemotherapy (ACT) after radical cystectomy (RC) is commonly considered as the mainstay of current treatment for MIBC [
3]. Unfortunately, the therapeutic efficacy is still far from satisfactory for advanced patients with metastases [
4]. With the approval of nivolumab (anti-PD-1) from the US Food and Drug Administration (FDA) in MIBC [
5], immune checkpoint inhibitors (ICIs) have shown a resounding success among cancer therapeutic strategies, of which regulatory molecules in B7 family are promising targets [
6,
7]. Nevertheless, existing biomarker failed to cover all the responders, which could be partly attributed to the heterogeneity of tumor microenvironment (TME) [
8,
9]. Consequently, there is an urgent need to seek predictive biomarkers for existing treatment and novel therapeutic paradigm for non-responders.
V domain immunoglobulin suppressor of T cell activation (VISTA), also known as VSIR, PD-1H, C10orf54, Dies1, DD1α and Gi24, belongs to the immune checkpoint proteins of B7 family, which is homologous to PD-L1 [
10,
11]. VISTA serves a crucial function in regulating the immune system, preserving the stability of the intracellular environment [
12]. Nonetheless, cancer cells could exploit VISTA to evade immune defenses in MIBC. Therefore, the development of alternative treatment strategies to the evasion mechanisms of MIBC are imperative to maximize anti-tumor efficacy. Investigating how VISTA interact between immune cells (ICs) and tumor cells (TCs) could facilitate the development of personalized therapies for MIBC patients.
VISTA has provided prognostic value and demonstrated the potential as an immunotherapy target for the patients. Elevated VISTA was associated with unfavorable outcomes across multiple malignancies [
13‐
15]. Paradoxically, VISTA in TCs, but not in ICs, was significantly associated with prolonged survival in pancreatic cancer, hepatocellular carcinoma and high-grade serous ovarian cancer [
16‐
18]. Furthermore, previous studies suggested that VISTA-positive ICs correlated with shorter recurrence-free in non-muscle-invasive bladder cancer (NMIBC) [
19], however, its predictive value in MIBC remains unclear. The study of VISTA in MIBC could contribute to improved risk stratification and personalized treatment options.
In this work, we found that VISTA+ ICs infiltration indicated miserable clinical outcomes and poor responsiveness to ACT. Nonetheless, VISTA+ ICs infiltration in turn possessed a superior responsiveness to ICIs. In-depth transcriptomic and histological studies uncovered the immunosuppressive TME and presented basal-squamous and luminal-infiltrated subtype in VISTA+ ICs high subgroup, which might account for prognosis and therapeutic response. Our study unraveled the potential of VISTA as a novel candidate biomarker for MIBC patients.
Methods
Study patients
This study enrolled three independent cohorts, Zhongshan Hospital (ZSHS) Cohort, The Cancer Genome Atlas (TCGA) Cohort and IMvigor210 Cohort. The selecting procedure of studying cohorts was summarized in Supplementary Figure
1. The clinicopathological characteristics of patients were listed in Supplementary Table
1-
3.
For ZSHS Cohort, 215 patients who received radical cystectomy (RC) at Zhongshan Hospital from 2002 to 2014 were followed up regularly till July 2016. 80 patients were ruled out due to the exclusion criteria: (1) postoperative histopathological diagnosis of non-urothelial carcinoma (UC) (n=13) or NMIBC (n=60), (2) unavailable in paraffin-embedded tumor tissues (n=7). 135 cases were enrolled in this study ultimately. The pathological type of all these patients was pure UC. Among them, 65 patients received ACT for at least one therapeutic cycle. The follow-up protocol was instructed by European Association of Urology guidelines for MIBC. Overall survival (OS) was calculated as the time from the date of RC to the date of death from all causes, or to the last follow-up.
TCGA Cohort enrolled 412 bladder cancer patients whose clinical information was downloaded from
http://www.cbioportal.org/ in July 2021, 21 patients were excluded because of: (1) missing survival time (
n=3), (2) unaccessible sequencing data (
n=4), (3) accepted neoadjuvant chemotherapy (
n=10), (4) postoperative histopathological diagnosis of NMIBC (
n=4). 391 patients were enrolled according to the inclusion criteria of TCGA Cohort. Among them, 335 patients were classified as pure UC histology. 51 patients had UC with variant histology. 5 additional tumors were included: 1 bladder adenocarcinoma, 1 squamous cell carcinoma of non-bladder origin, and 3 pure squamous cell bladder carcinomas.
IMvigor210 trial originated from 348 metastatic UC patients treated with anti-PD-L1 agent atezolizumab [
20]. In this study, we enrolled 195 bladder-derived urothelial cancer patients as IMvigor210 Cohort. The clinical and RNA-seq data were obtained through
http://research-pub.gene.com/IMvigor210CoreBiologies. All of these patients had UC that had either been histologically or cytologically proven to be locally advanced or metastatic, including metastasis from the renal pelvis, ureter, urinary bladder, or urethra.
Immunohistochemistry
All the bladder cancer tissues were obtained from the bladder specimens of 215 patients in ZSHS Cohort, which were subsequently formalin-fixed and paraffin-embedded. Before the construction of tissue microarray (TMA), 4 μm-thick sections were sliced from each tissue block. All samples were reviewed histologically by hematoxylin and eosin staining, and representative areas were marked on the paraffin blocks away from necrotic and hemorrhagic materials. Besides, each section of TMA was stained at the same time to guarantee an objective comparison between different samples. The protocol of immunohistochemistry (IHC) was executed as previously described [
21]. Antibodies for VISTA and other molecules were provided in Supplementary Table
4.
Assay methods
TMA slides were scanned under high-power magnification filed (HPF, 200 magnification) on NanoZoomer-XR (Hamamatsu) and scored by means of software ImageJ. All stained tissues were counted independently by two pathologists who were blind to the clinical and follow-up data. For the accurate purpose of statistical evaluation, we adopted as the mean value of cells infiltration in three representative fields (HPF, × 200 magnification). Consistent to previous reports [
13,
14,
16,
17], we adopted a method of counting VISTA expressed in ICs or TCs separately in the immunohistochemical scoring of MIBC patients in ZSHS Cohort. The cut-off value of VISTA
+ ICs in ZSHS Cohort was 37 cells/HPF, which determined by the R package survMisc (
https://CRAN.R-project.org/package=survMisc). Whether VISTA expressed in TCs could be divided into positive and negative subgroup. Patients were dichotomized into VISTA
+ ICs signature low and high subgroups in TCGA and IMvigor210 Cohorts also based on R package survMisc to identify the optimal cut-off values.
RNA-seq and data processing
RNA-seq data of both TCGA Cohort and IMvigor210 Cohort were normalized by the formula log
2(FPKM+1) before analysis. Based on the immune cell subsets expressing VISTA, ligand-receptor relationship and involved immune regulation process of VISTA, we used the average mean of the mRNA expression of
VSIR, IL6, IL10, IGSF11, SELPLG, VSIG8, ESAM, CD45 to constitute VISTA
+ ICs signature [
10,
22]. The infiltration of 22 ICs in TCGA Cohort was calculated by CIBERSORT algorithm, of which the sum was considered as the absolute score of each case. The involved signatures for gene set enrichment analysis (GSEA) were defined from previous studies or downloaded from
https://gsea-msigdb.org and showed in Supplementary Table
5.
Genomic analysis
Tumor mutation burden (TMB) is broadly identified as the number of somatic mutations per megabase of interrogated genome sequence (mut/Mb) [
23]. Generally, TMB≥10 mut/Mb is identified as TMB
hi [
24]. Gene alterations involved in signaling pathways were used to describe the genome pattern, which incorporated mutations and copy number variation (CNV) [
25,
26]. The types of mutation included nonsense, missense, splice site, in frame deletion, multi hit, frame shift insertion and frame shift deletion. The types of CNV included deletions and amplifications.
Statistical analysis
The relationship of VISTA+ ICs infiltration with patients’ clinicopathological parameters were conducted by Chi-square test. Analyses of the different cells infiltration between subgroups were dealt with Student’s t test. Overall survival (OS) was calculated from the date of operation until the date of death or last follow-up and disease-free survival (DFS) was calculated from the date of operation until the date of first recurrence or last follow-up. Kaplan-Meier curves for OS and DFS was evaluated by log-rank tests. Univariate and multivariate analyses were detected by constructing cox proportional hazard regression models. Gene Set enrichment analysis (GSEA) performed by three clusters of signaling signatures to identify the enrichment of exhausted CD8+ T cells in MIBC. In our study, all of data in the figure were shown as means ± SDs. P value of less than 0.05 was considered statistically significant. IBM SPSS Statistics 25.0 was utilized for all of the statistical analyses. Figures were visualized using MedCalc Statistical Software version 15.6.1, Graph Pad Prism Software version 7.0.1 and R software version 4.0.3.
Discussion
Nowadays, accurately predicting treatment responders is the main concern. Suitable biomarker for predicting treatment response will aid in identifying responders [
29]. Numerous reviews suggested that increasing the antigenicity of cancer cells and inducing a more immunogenic microenvironment after conventional chemotherapy [
30,
31]. However, our work reported that VISTA
+ ICs infiltration was associated with inhibitory TME, which suppressed the further expanded immune effects. That’s the possible reason why VISTA
+ ICs infiltration could be resistant to chemotherapy but favorable to derived more benefits from ICIs. TMB is a measure of the number of mutations in a cancer [
32]. The more mutations (i.e., the higher the TMB) reflected the greater the chances that some neo-antigens would be immunogenic and enable T cell recognition [
33,
34]. Recent research reported that TMB might not always correlate with ICIs responsiveness [
35]. Developing precision therapies could improve clinical significance by stratification of patients who are resistant or sensitive to ICIs. Herein, we identified VISTA
hiTMB
hi patients who possessed the outstanding responsiveness to ICIs based on integrating VISTA
+ ICs infiltration stratification with TMB, which probably accounted for immune enriched and increased immunogenicity. The exact mechanisms still need to be further explored.
Accumulating evidences that the success of chemotherapy and immunotherapy could be partially attributed to the immune landscape of TME [
36]. Our previous study showed that some specific subsets of ICs infiltration could influence the responsiveness of MIBC patients to chemotherapy [
37‐
39]. Given the evasion of immune control from the standpoint of VISTA
+ ICs infiltration, VISTA could promote the transformation of naive Foxp3
+CD4
+ T cells into adaptive Foxp3
+ Tregs and induce polarized M2 macrophages [
12,
40], Most Tregs and M2 macrophages generally released a high level of IL-10 and TGF-β, which exerted inhibitory influence on antigen-presenting capability of macrophages and T cells function [
41]. It has been reported that terminally exhausted CD8
+ T cells enrichment always accompanied with increasing immune checkpoints [
37,
42], which could support our finding that CD8
+ T cells played an exhausted role in VISTA
+ ICs infiltration. Moreover, targeting VISTA has been shown to induce inflammatory mediators and modulate exhausted CD8
+ T cells into antitumor effector T cells [
43], suggesting VISTA blockade had great value in treating MIBC patients and could be a novel method of further clinical treatment.
Resent research concluded that IFN-γ could induce the upregulation of suppressive receptors on tumor cell and TAMs, such as PD-L1, and further upregulated the expression of VISTA to suppress the degree of antitumor immune responses [
44,
45]. Similar to VISTA-positive ICs in NMIBC, VISTA
+ ICs infiltration was associated with poor prognosis in MIBC [
19], which indicated VISTA play a consistent and persistent role in bladder cancer disease progress. The expression of VISTA and PD-L1 in different ICs showed that they may have immunologic activities in NMIBC. Additionally, VISTA expression on antigen-presenting cells is distinct from the PD-1/PD-L1 in melanoma [
46]. In murine, VISTA promoted tumor growth via regulation of T cells activation independent of PD-1/PD-L1 pathway [
47]. In conclusion, VISTA demonstrated the valuable significance as a potential immunotherapy target, which aids the immune system in anti-tumor. Moreover, VISTA and PD-1 blockade could make more informed decisions regarding effective therapies and personalized treatment option for MIBC patients.
Conclusion
We aimed to investigate clinical significance of VISTA in MIBC. Our study evaluated VISTA+ ICs infiltration as a candidate biomarker for predicting survival outcomes and therapeutic responsiveness. Specifically, VISTA+ IC high subgroup was refractory to ACT while favorable to PD-L1 inhibitor. Further we deciphered VISTA+ IC infiltration was associated with inhibitory tumor environment characterzied by Tregs, M2 macrophages, mast cells and exhausted CD8+ T cells infiltration, along with increased IL-10 and TGF-β, but also elevated immune checkpoint expression, such as TIM-3, LAG-3 and TIGIT, which mediated the immune evasion in MIBC. In brief, VISTA+ ICs infiltration could be as a promising biomarker for guiding precision medicine strategies for MIBC patients.
Acknowledgements
We thank Dr. Lingli Chen (Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China) and Dr. Yunyi Kong (Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China) for their excellent pathological technology help.
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