Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response

Abstract

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68⁺ CD163⁺ CD206⁺ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet⁺ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)⁺ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Keywords: T-bet; immune cell markers; immunotherapy; matched pairs; metastatic renal cell carcinoma; tumor immune microenvironment; tumor-associated macrophages.

Fig. 1

Differences in the distribution of immune cell markers across regions of interest in primary tumors.

Fig. 2

Heat‐map showing differences in immune cell marker density between primary and metastatic tumors.

Fig. 3

Differences in CD68⁺CD163⁺ macrophage marker (yellow) distribution across three regions of interest shown on immunofluorescence and box‐plot graphs.

Fig. 4

Correlations with marker density and survival following immunotherapy for CD68 in primary tumors and forkhead box protein 3 (FoxP3) in metastatic tumors estimated using Cox regression and Kaplan–Meier analyses

Fig. 5

Distribution of T‐box protein expressed in T cells (T‐bet)⁺ cells (red) at the pan‐cytokeratin‐stained tumor‐ (cyan) and 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained stroma (dark blue) interface shown on immunofluorescence and correlation with survival after first dose of immunotherapy estimated using Kaplan–Meier survival analyses.