Abstract
Previous studies show that LDH-A knockdown reduces orthotopic 4T1 breast tumor lactate and delays tumor growth and the development of metastases in nude mice. Here, we report significant changes in the tumor microenvironment (TME) and a more robust anti-tumor response in immune competent BALB/c mice. 4T1 murine breast cancer cells were transfected with shRNA plasmids directed against LDH-A (KD) or a scrambled control plasmid (NC). Cells were also transduced with dual luciferase-based reporter systems to monitor HIF-1 activity and the development of metastases by bioluminescence imaging, using HRE-sensitive and constitutive promoters, respectively. The growth and metastatic profile of orthotopic 4T1 tumors developed from these cell lines were compared and a primary tumor resection model was studied to simulate the clinical management of breast cancer. Primary tumor growth, metastasis formation and TME phenotype were significantly different in LDH-A KD tumors compared with controls. In LDH-A KD cells, HIF-1 activity, hexokinase 1 and 2 expression and VEGF secretion were reduced. Differences in the TME included lower HIF-1α expression that correlated with lower vascularity and pimonidazole staining, higher infiltration of CD3+ and CD4+ T cells and less infiltration of TAMs. These changes resulted in a greater delay in metastases formation and 40% long-term survivors (>20 weeks) in the LDH-A KD cohort following surgical resection of the primary tumor. We show for the first time that LDH-depletion inhibits the formation of metastases and prolongs survival of mice through changes in tumor microenvironment that modulate the immune response. We attribute these effects to diminished HIF-1 activity, vascularization, necrosis formation and immune suppression in immune competent animals. Gene-expression analyses from four human breast cancer datasets are consistent with these results, and further demonstrate the link between glycolysis and immune suppression in breast cancer.
Highlights
Lactate dehydrogenase A (LDH-A) is required for the maintenance and progression of many tumors [1,2,3,4], and inhibition of LDH-A has an anti-proliferative effect [2, 5,6,7,8,9,10,11]
Given the importance of glycolytic metabolism and its metabolites on the immune response [12, 13] and on tumor progression [2, 14,15,16,17], and the effect of lactate on the activation of Hypoxia-Inducible Factor 1 alpha (HIF-1α) [18,19,20], we have explored the effect of LDH-A knockdown on HIF-1 responses by introducing HIF-1 reporters into tumor cells
4T1 cells were transduced with a retroviral vector, where a hypoxia response element (HRE) [37] drives the expression of exGaussia Luciferase (exGLuc) [38] and GFP (Panel A in S1 Fig)[33, 37, 39]
Summary
Lactate dehydrogenase A (LDH-A) is required for the maintenance and progression of many tumors [1,2,3,4], and inhibition of LDH-A has an anti-proliferative effect [2, 5,6,7,8,9,10,11]. Given the importance of glycolytic metabolism and its metabolites on the immune response [12, 13] and on tumor progression [2, 14,15,16,17], and the effect of lactate on the activation of Hypoxia-Inducible Factor 1 alpha (HIF-1α) [18,19,20], we have explored the effect of LDH-A knockdown on HIF-1 responses by introducing HIF-1 reporters into tumor cells. Tumor hypoxia and high HIF-1 activity promote an immunosuppressive phenotype (involving both tumor cells and infiltrating immune cells) that has a direct effect on metastatic tumor progression [27]. Understanding the mechanisms by which HIF-signaling promotes immunosuppression is under investigation and may have important therapeutic implications in the treatment of metastatic disease [28, 29]
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