GLUT1 expression, lymphocyte distribution and CD3+ T-cell metabolic subsets as predictive markers of response to immunotherapy in advanced melanoma

Glycolysis, commonly used by malignant tumors for energy production, results in acidification of the tumor microenvironment (TME) through the secretion and accumulation of lactic acid. Acidosis is a potent inhibitor of immune cell function and may therefore affect T-cell infiltration and the efficacy of immunotherapy. This study aimed to characterize the metabolic tumor microenvironment and its association with lymphocyte distribution in patients with advanced melanoma treated with immune checkpoint blockade (ICB) therapies. Pre-treatment formalin-fixed, paraffin-embedded metastatic melanoma specimens from 45 patients treated with anti-PD-1 ± anti-CTLA-4 ICB were included in this study. Patients with progression-free survival (PFS) ≥ 6mo were categorized as responders (n = 23), while non-responders had a PFS < 6mo (n = 22). Two custom multiplex immunofluorescence panels were developed to evaluate the expression and distribution of markers of a hypoxic microenvironment (CA9 and HIF1α), glycolysis (GLUT1 and GLUT3) and vessels (CD31) in relation to melanocytes (SOX10) and T lymphocytes (CD3). GLUT1 + melanoma regions contained significantly lower proportions of CD3+ T-cells than GLUT1- regions (p < 0.0001). Responders displayed significantly higher proportions of intratumoral T-cells expressing GLUT1 (p = 0.049) and GLUT3 (p = 0.043) compared to non-responders. CD3+ T-cells co-expressing hypoxia-associated markers were present in higher proportions significantly closer to GLUT1+ melanoma cells in responders compared to non-responders (p < 0.05). Patients with higher proportions of CD3+ T-cells and CD3+CA9+ T-cells within the 20 µm distance to GLUT1+ melanoma cells had significantly longer progression-free survival (p = 0.0133 and p = 0.0378, respectively). Together, these findings support the hypothesis that the presence of glycolysis in melanoma (as inferred by increased GLUT expression) may affect the ability of T-cells to infiltrate tumors and function effectively. The results also suggest that the overall proportion and spatial distribution of GLUT+ T-cells, including those displaying evidence of adaptation to a hypoxic/acidic TME, may be relevant for responses to ICB therapy.