To understand the functional impairment of RCC TILs during advanced stages of the disease, various assays to measure the metabolic reprogramming of the T-cells, required for effector function, were utilised. First, to examine the capacity for glucose uptake by RCC TILs, we pulsed T-cells with the fluorescent glucose analog 2-NBDG at the end of culture with or without TCR (T-cell receptor) stimulation. Whilst the basal uptake of 2-NBDG by resting PD1 + RCC TILs was similar to resting control T-cells, following stimulation RCC TILs had defects in glucose uptake. This was evident across all stages of RCC but more evident from stage 3–4 and from the locally advanced and metastatic sites (IVC). This contrasted with the T-cells from the blood of RCC patients that were able to increase glucose uptake upon stimulation (Fig. 3).
To further probe the metabolic phenotype of RCC-derived TILs, we measured cellular oxygen consumption rates (OCRs) during a mitochondrial stress test. The spare respiratory capacity (SRC), calculated as change in mean oxygen consumption rate upon treatment with FCCP (fluorocarbonyl cyanide phenylhydrazone), of sorted CD3 + TILs from RCC tumours varied. Most stage 1/2 RCC tumours displayed TILs with a high SRC whilst the TILs from tumours exhibiting invasive morphology ( > = pT3/IVC thrombus sites) often had a low/minimal SRC. This inability to generate additional energy through oxygen consumption in situations of metabolic stress was specific to TILs from advanced stage cases and not observed in the early-stage tumours or corresponding blood of RCC patients (Fig. 4).
As depleted mitochondrial mass may be one cause of decreased respiration, MitoTracker Green (MTG) staining of RCC-derived PD1 + TILs was carried out. This analysis revealed that whilst the TILs from early stage (p1-2) RCC cases had an equivalent mass to T-cells from the blood of RCC patients, the TILs from advanced stage disease showed a decreased mitochondrial mass (Fig. 5A).
We next investigated mitochondrial function by analysing the production of mitochondrial ROS levels in TILs from RCC patients using the mt-superoxide-specific dye MitoSOX Red and compared them with those of T-cells from the blood of the patients and healthy controls. The mitochondrial superoxide content was similar in unstimulated TILs from all stages of RCC as compared to the T-cells from the blood of patients or healthy controls except for 2 cases of IVC thrombus and a stage 3 tumour which displayed very high levels of basal ROS. Upon anti-CD3 stimulation, superoxide levels consistently increased in T-cells from the blood of control donors and from TILs from stage 1/2 RCC patients, whereas a mixed response with most samples exhibiting a decline or no change of superoxide levels was observed in TILs from stage 3/4 RCC patients and from IVC thrombus and metastatic sites (Fig. 5B).
Further investigation of the mitochondrial function of RCC TILs was carried out by examining the mitochondrial membrane potential (MMP) in freshly isolated TILs from different stages of RCC, after overnight stimulation with anti-CD3 antibody, using the lipophilic cation JC-1. JC-1 is mitochondria selective and forms aggregates in polarized mitochondria that result in a green-orange emission after excitation. However, the monomeric form present in cells with depolarized mitochondrial membranes emits only green fluorescence. After anti-CD3 stimulation, TILs from stage 3–4 RCC patients and from locally advanced (IVC thrombus) and metastatic sites displayed an increased percentage of cells with mitochondrial depolarization as compared to TILs from stage 1–2 RCC patients or T-cells from the peripheral blood of patients or healthy controls (Fig. 5C). This decreased MMP together with the inability to increase ROS levels upon stimulation further supports the existence of dysfunctional depolarized mitochondria within advanced RCC TILs.
Metabolic profiling of the RCC tumour microenvironment reveals metabolic reprogramming from an early stage of disease
To explore whether the observed emergence of T-cell dysfunction in ccRCC at advanced stages of disease is associated with metabolic alterations within the tumour microenvironment a Nanostring nCounter cancer metabolism panel assay was performed on RNA obtained from 30 of the ccRCC cases studied above. Differential gene expression analysis was determined for each stage of the disease (p1-2, p3-4, IVC thrombi and RCCmet) comparing P1-2 to the normal baseline control (normal kidney), P3-4 to stage p1-2 and thrombi and RCCmet to stage P3-4. (Fig. 6). This analysis revealed that even during early stages of the disease (P1-2) there was over-expression of genes (LDHA, SLC2A1, HK2, SLC16A1, HK1) corresponding to proteins associated with glucose metabolism and solute transport functions that could potentially render the cancer cells more fit whilst at the same time imposing metabolic effects (nutrient deficiency and waste product toxicity) that restrict the TILs. In addition, PDK1, a gene known to regulate RCC cell proliferation, migration, invasion and epithelial mesenchymal transition, as well as HIF-responsive genes, ENO2 and EGLN3, were also over-expressed.
Similarly, we observed down-regulation of particular metabolic genes (ALDOB, AC01, FBP1, EGF, SUCLG1 and PDGFRA) also from an early (p1-2) stage of disease. These findings were in keeping with previous reports showing that ALDOB and FBP1 were some of the most down-regulated genes in RCC[15, 16].
Progression into p3-4 stage disease compared to p1-2 and thrombi compared to p3-4 showed an overall down-regulation of metabolic genes. Of note there was a significant down-regulation of PPARGC1A in p3-4 stage disease, a gene that encodes peroxisome proliferator-activated receptor gamma coactivator-1, consistent with previous reports of low expression of PPARGC1A in ccRCC tissues[17]. In thrombi compared to p3-4 stage disease there was a significant down-regulation of the kidney-specific NADPH oxidase isoform 4 (NOX4) which produces considerable amounts of ROS in ccRCC. In the most advanced stage of the disease, the RCCmets compared to p3-4 stage showed an up-regulation of genes (FASN, PFKM, PLD1, AKT2, Citrate synthase (CS), MDH2) that are associated with aggressive cell proliferation, migration, apoptosis, lipid droplet formation and regulate metabolic disorders of the ccRCC microenvironment. Similar to the thrombi, RCCmet showed a significant down-regulation of NOX4 along with aldolase B (ALDOB) and the glucose solute carrier, SLC2A2.
The most significantly up- or down-regulated genes from each stage comparison were then selected for analysis of associations with overall survival (OS) using mRNA expression data from the TCGA database (Sup Fig. 2). For genes differentially expressed in stage p1-2 relative to normal tissue, the results demonstrated that for the up-regulated genes increased mRNA expression level of PDK1 was associated with improved OS whereas the increased expression of ENO2 was linked to poor OS. For the down-regulated genes SUCLG1, FBP1, AC01 and ALDOB, low expression was associated with a poor OS. For the majority of the genes down-regulated at stage 3–4 (PRKAA1, DLST, PPARGC1A, SUCLA2 and AKT3) low expression was significantly associated with a poor OS and only for SLC2A3 and FASN low expression was linked with an improved OS. As published work had shown that PPARGC1A was involved in shifting the TME from immune-dominant to metabolic-dominant[19], we explored further the association of PPARGC1A expression and survival within different disease stages (Fig. 7). TCGA data revealed that low expression of this gene is associated with poor survival in all stages of RCC with our own data showing a significant down-regulation of this gene in stage p3-4 tumours compared to stage p1-2 (Fig. 6).
For genes down-regulated in the thrombi relative to stage p3-4, low expression of FOXO3 was associated with a poor outcome whereas low expression of G6PD and RAC2 was linked to improved OS. For genes differentially expressed in advanced metastatic stage, relative to stage p3-4, high expression of many of the up-regulated genes was associated with improved OS, however, high expression of FASN was linked to a poor outcome. Low expression of down-regulated genes SLC2A2 and ALDOB was associated with a poor OS.