Carbonic Anhydrase IX (CAIX) Expressing Hypoxic Micro-environment Hampers CD8+ Immune Cell Infiltrate in Breast Carcinoma.

Abstract

Hypoxia and necrosis are common features of invasive cancer. The dynamic upregulation of carbonic anhydrase IX (CAIX), triggered by hypoxia-inducible factor 1 (HIF-1) is 1 of the mechanisms supporting cellular adaptation to hypoxia in solid tumors, including breast carcinoma. CAIX activity results in extracellular acidosis and in a profound reorganization of the tumor micro-environment, influencing biological behavior and prognosis. The main focus of our study was to evaluate the mass and distribution of the immune infiltrate, more specifically of CD8+ effector T-cells, in relation with tumoral CAIX expression. Formalin-fixed and paraffin-embedded breast carcinoma sections were analyzed following double immunohistochemical staining for CAIX and CD8. Scanned digital slides were evaluated for both labelings, and CD8-related signal was determined within and outside CAIX-positive tumor areas using the HistoQuant (3DHistech) image analysis software. Statistical analysis was performed using GraphPad Prism software. Of the 34 breast carcinomas, 18 tested partially positive for CAIX. The remaining 16 cases were used as the CAIX-negative control group. Necrotic foci were generally associated with CAIX overexpression, and tumors exhibiting signs of necrosis had a significantly higher rate of relative CAIX expression compared with samples without necrosis (11.47±5.505 vs. without necrosis 3.765±3.5 P-value=0.0216). On the other hand, no statistically significant difference was found when comparing relative CD8+ lymphocyte counts in cases with necrosis as opposed to those where necrosis was absent (134.7±55.7 vs. 97.70±57.25; P value=0.1579). No difference in gross CD8+ T-lymphocyte infiltrate could be measured between CAIX positive and negative samples (98.48±37.32 vs. 95.99±50 P value=0.5928). However, in CAIX-expressing tumors a statistical correlation between the CD8+ T-lymphocyte infiltrate and the extent of CAIX-positive areas was observed. Within the same tumor, CD8+ T-lymphocyte counts showed a significant difference betweeen CAIX+ and CAIX- areas (13.06±9.4 vs. 135.6±62.2 P value <0.0001). Our measurements demonstrate for the first time that tumor areas with CAIX expression potentially hamper CD8+ T-lymphocyte infiltration in breast carcinoma. The hypoxia-driven adaptive micro-environment likely interferes with the specific response to biological and immune therapies requiring intact effector T-cell response.