Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy

Abstract

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA + MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA + Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.

LDH-A Knockdown: Changes in The LDH Isoenzyme Profile and Variability in Glioma Response

“…4Ab,c and 4Db,c). A Weka analysis (28) showed signi cantly greater LDH-A staining in both GL261 and CT2A NC tumors compared to the LDH-A KD tumors (Fig. 4B, 4E, Table S-1), consistent with the Western blot and LDH enzyme results (Fig.…”

“…Stably transduced clones (KD cell lines) were developed, along with a control (NC) cell line bearing a scrambled shRNA. Based on the previous experience (12,28), we decided to use the most effective shRNAs (shRNA-2) to develop LDH-A KD cells in murine glioma cells. The transfection of GL261 cancer cells with shRNA-2 resulted in a signi cant knock-down effect for LDH-A (approximately 10% of that in wild type cells), while bulk CT2A and ALTS1C1 cells transfected with shRNA-2 had a signi cantly less level of LDH-A knock-down (40-60%).…”

“…We chose three murine brain tumor models to understand the impact of LDH-A downregulation on tumor phenotype and growth potential: GL261 (35), CT2A (26) ,and ALTS1C1 (25). The wild-type cell lines were transduced with two shRNA retroviral vectors: i) shRNA-2 was used for targeting LDH-A, it was found to provide effective knockdown, based on our previous work (28,36), and ii) one scrambled control (11). In vitro comparisons between the LDH-A knockdown (KD) and the scrambled control (NC) cell lines are shown in Figures 1 and 2, S-1.…”

Tinkering under the Hood: Metabolic Optimisation of CAR-T Cell Therapy

“…Whilst little is known about the direct influence of lactate on CAR-T cell metabolism and function, in an attempt to overcome the suppressive properties of lactate, studies have begun to investigate the mechanistic effect of lactate dehydrogenase (LDH) inhibition. Specifically, the inhibition of LDH enhances the CAR-T cell function [ 47 , 48 ], in particular one study identified a synergistic effect with IL-21 to promote CAR-T memory and enhanced downstream anti-tumour immunity [ 48 ].…”

Microenvironmental influences on T cell immunity in cancer and inflammation

“…Treg are oxidative and can take up and consume lactate in the TME [ 152 , 153 ]. Clinically, levels of lactate dehydrogenase A (LDH-A), which converts pyruvate into lactate, correlate with worse clinical outcomes and fewer infiltrating T cells in multiple cancer types [ 148 , 154 , 155 ].…”