Digital Spatial Profiling Identifies the Tumor Periphery as a Highly Active Biological Niche in Clear Cell Renal Cell Carcinoma

SIMPLE SUMMARY: Clear cell renal cell carcinoma is characterized by a high degree of genomic intratumoral heterogeneity. The extent of spatial and functional heterogeneity, however, is much less understood. In the present study, digital spatial profiling identified the tumor periphery as a highly active biological niche with the upregulation of a number of proteins involved in tumorigenic signaling nodes. One of the most significantly upregulated proteins was granzyme B, which plays an important role in anti-cancer immune responses. Patients exhibiting such an overexpression in the tumor periphery had a significantly worse cancer-specific survival. It is possible that high granzyme B expression creates selection pressure for tumor cells to escape antitumoral host responses. Results from this study highlight the role of the tumor periphery as a hotspot for various signaling activities and tumor evolution. Our findings have important implications for the future development of predictive and prognostic biomarkers to improve patient stratification. ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of intratumoral heterogeneity (ITH). Besides genomic ITH, there is considerable functional ITH, which encompasses spatial niches with distinct proliferative and signaling activities. The full extent of functional spatial heterogeneity in ccRCC is incompletely understood. In the present study, a total of 17 ccRCC tissue specimens from different sites (primary tumor, n = 11; local recurrence, n = 1; distant metastasis, n = 5) were analyzed using digital spatial profiling (DSP) of protein expression. A total of 128 regions of interest from the tumor periphery and tumor center were analyzed for the expression of 46 proteins, comprising three major signaling pathways as well as immune cell markers. Results were correlated to clinico-pathological variables. The differential expression of granzyme B was validated using conventional immunohistochemistry and was correlated to the cancer-specific patient survival. We found that a total of 37 proteins were differentially expressed between the tumor periphery and tumor center. Thirty-five of the proteins were upregulated in the tumor periphery compared to the center. These included proteins involved in cell proliferation, MAPK and PI3K/AKT signaling, apoptosis regulation, epithelial-to-mesenchymal transition, as well as immune cell markers. Among the most significantly upregulated proteins in the tumor periphery was granzyme B. Granzyme B upregulation in the tumor periphery correlated with a significantly reduced cancer-specific patient survival. In conclusion, this study highlights the unique cellular contexture of the tumor periphery in ccRCC. The correlation between granzyme B upregulation in the tumor periphery and patient survival suggests local selection pressure for aggressive tumor growth and disease progression. Our results underscore the potential of spatial biology for biomarker discovery in ccRCC and cancer in general.