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Background Durable clinical responses to immune checkpoint blockade (ICB) occur in a limited fraction of patients. We thus hypothesized that the characteristic tumor metabolic switch towards aerobic glycolysis could contribute to ICB resistance. High glucose consumption and lactate production by tumor cells can indeed restrict nutrient availability for tumor-infiltrating T cells, which also rely on glycolysis to proliferate and function. Therefore, we investigated whether targeting tumor glucose metabolism potentiates ICB anti-tumor activity. Methods We modeled tumor-selective glycolysis inhibition by knocking down the critical glycolytic enzyme lactate dehydrogenase A (LDHA-KD) in the murine metastatic breast carcinoma 4T1 and melanoma B16, which are known immune-refractory tumor models. Anti-CTLA-4 and anti-PD-1 were tested in immunocompetent mice orthotopically implanted with control vs. LDHA-KD tumor cells. Changes in glucose metabolism were assessed by Seahorse and fluorescent-glucose flow-cytometry staining. Changes in immune cells were measured by multiparameter flow cytometry. Glucose-dependent effects of anti-CTLA-4 in regulatory T cells (Tregs) were tested in standard suppression assays with increasing glucose concentration (0.5–10 mM). Pearson correlations between glycolysis and intra-tumor immune-cell infiltration by CIBERSORT immune-deconvolution method were analyzed in bulk RNA-sequencing data sets from human and murine tumors treated with ICB. Results Comparison of ICB activity in LDHA-KD vs. control tumor-bearing mice revealed improved anti-tumor effects and overall survival in the setting of glycolysis-defective tumors specifically upon CTLA-4 blockade. Anti-tumor CD8+ T-cell responses correlated with Treg phenotypic and functional destabilization in anti-CTLA-4-treated LDHA-KD tumors. CTLA-4 blockade led to CTLA-4 and CD25 downregulation associated with increased IFN-gamma and TNF-alpha production in Tregs from glycolysis-defective vs. control tumors. We next mimicked high- vs. low-glycolysis tumor microenvironment (TME) in vitro using control vs. LDHA-KD tumor co-cultures with Tregs, control vs. LDHA-KD tumor-conditioned media or directly modulating glucose concentrations. In these assays, we observed that CTLA-4 blockade promotes IFN-gamma±TNF-alpha production and glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. Lastly, by interrogating transcriptomic data from human melanoma and murine 4T1 tumors, we found that CTLA-4 blockade promotes immune-cell infiltration and metabolic fitness especially in glycolysis-defective tumors. Conclusions Our findings indicate that increasing glucose availability in the TME may improve anti-CTLA-4 therapeutic activity and reveal a new mechanism through which CTLA-4 blockade interferes with Treg immunosuppression in a glucose-dependent manner. These results suggest that CTLA-4 blockade can be more effective in tumors with low glycolysis and/or can be best exploited in combination with inhibitors of tumor glycolysis.

Abnormal vessel formation plays a major role in tumor progression. Previously, we showed that lactate dehydrogenase A (LDH-A) knockdown (KD) reduces orthotopic 4T1 breast tumor lactate, and has a robust anti-tumor effect through changes in the tumor microenvironment (TME) [1, 2]. Tumors with LDH-A downregulation have lower VEGF-A secretion and reduced formation of new blood vessels [2]. In addition, LDH-A KD cells/tumors displayed reduced HIF-1 signaling, as assessed by BLI using HRE-exGLuc reporter. Changes in the bioluminescence signal corresponded with changes in HIF-1α staining. LDH-A KD tumors also showed less HIF-1α staining compared to control tumors, consistent with less pimonidazole (hypoxia) staining [2]. Murine 4T1 breast cancer cells and tumors with LDH-A knockdown were developed and described [2]. To evaluate the degree of tumor neovascularization and its normalization, we stained tumors for CD31 (endothelial marker) and NG2 (pericytes marker). For pericyte coverage quantification, vessels were counted, and percentage of vessels covered by pericytes were calculated. Blood vessel permeability was assessed by intravenous injection of Evans Blue dye and tumor dye extravasation was quantified. To assess in vivo vascular network over time, a raster-scan optoacoustic mesoscopy (RSOM) was performed. Vessel structure in 4T1 murine tumors changes significantly following LDH-A KD. Normalized LDH-A KD tumor neovascularization was shown using the double immunofluorescence staining for CD31 and NG2, including a more normal vascular morphology, with shorter, more linear (less branched) vessel structure, with a higher percentage of vessels covered by pericytes compared to control tumors. Evans Blue dye extravasation was significantly less (lower vascular permeability) in LDH-A KD tumors compared to control tumors. Applying RSOM to follow the development of the vascular network during tumor growth, we showed better vascularization of LDH-A KD tumors. Furthermore, the number of circulating tumor cells (CTCs) was also significantly lower in blood collected from mice bearing LDH-A KD tumors. We show that LDH-depletion leads to tumor vessel normalization that includes several unique features: structural changes, greater coverage of blood vessel by pericytes and reduced vessel leakiness, leading to changes in the tumor microenvironment, inhibition of metastases formation and prolongation of survival. 1. Rizwan, A., et al., Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors. Clin Cancer Res, 2013. 19(18): p. 5158-69. 2. Serganova, I., et al., LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLOS ONE, 2018. 13(9): p. e0203965. Citation Format: Inna Serganova, Kiranmayi Vemuri, Ivan Cohen, Matthew Lubin, Sheryl Roberts, Masatomo Maeda, Mayuresh Mane, Henry Mann, Thomas Reiner, Eric Chan, Dmitry Yarilin, Katia Manova-Todorova, Roberta Zappasodi, Taha Merghoub, Jason Koutcher, Ronald Blasberg. Vessel normalization following LDH-A knockdown in murine breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1476.

Only a limited fraction of patients derives durable clinical benefit upon immune checkpoint blockade. Deepening our understanding of mechanisms of response and resistance to these therapies is thus needed to optimize their employment in rational combinations. Cellular energy metabolism reprogramming is a critical hallmark of cancer. High glucose consumption and lactate production by tumor cells restrict nutrient availability in the tumor microenvironment (TME) for effector T cells, which also rely on glycolysis to replicate and function. In addition, immune checkpoints and co-stimulatory molecules are emerging as important regulators of T cell metabolism. Exploiting the capacity of immune checkpoint blockade to perturb T cell metabolism in combination with inhibition of tumor glycolysis may thus be a rational and more effective anti-cancer approach. We investigated the link between tumor glycolysis and immune cell function using RNA sequencing data sets from patients treated with checkpoint blockade and in tumor:T cell co-culture systems. We then studied the effects of checkpoint blockade in syngeneic glycolysis-defective murine tumor models and explored mechanisms underlying anti-tumor activity. We found that expression of glycolysis-related genes is inversely correlated with infiltration of most immune cell types in melanomas from patients before CTLA-4 blockade. However, after treatment, immune-related and glycolysis-related genes were more often co-expressed, suggesting that anti-CTLA-4 may partially restore immune cell fitness in the glycolytic TME. To directly assess the effect of tumor metabolism on T cell function, we co-cultured activated T cells with the highly glycolytic murine mammary carcinoma 4T1 and observed that tumor cells, or similar non-toxic concentrations of exogenous lactate, significantly limited T cell activation and viability. We thus investigated whether inhibition of tumor glycolysis could potentiate the activity of CTLA-4 blockade. We found that neoadjuvant treatment with anti-CTLA-4 significantly prolonged survival in mice bearing glycolysis defective 4T1 tumors, where lactate dehydrogenase A (LDH-A) - the critical enzyme controlling lactate production in aerobic glycolysis - was knocked down (4T1-KD). Intriguingly, tumor protection was associated with intratumoral regulatory T cell (Treg) functional phenotypic destabilization towards IFN-gamma and TNF-alpha producing Tregs. By mimicking the LDH-A-KD and control 4T1 TME in vitro using higher and lower concentrations of glucose, respectively, we showed that CTLA-4 blockade promotes glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. These findings indicate that rebalancing glucose utilization in favor of immune cells in the TME may facilitate the activity of CTLA-4 blockade and reveal an additional mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose. Citation Format: Roberta Zappasodi, Inna Serganova, Ivan Cohen, Masatomo Maeda, Masahiro Shindo, Yasin Senbabaoglu, Avigdor Leftin, Rachana Maniyar, Svena Verma, Matthew Lubin, Myat Kyaw Ko, Mayuresh M. Mane, Arnab Ghosh, Ellen Ackerstaff, Jason A. Koutcher, Ping-Chih Ho, Greg M. Delgoffe, Ronald Blasberg, Jedd D. Wolchok, Taha Merghoub. CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3257.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells 1 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis 1 . Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8 + T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (T reg ) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of T reg cells is dependent on T reg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with T reg cell function in the presence of glucose.