The dysfunctional immune response in renal cell carcinoma correlates with changes in the metabolic landscape of ccRCC during disease progression

Abstract

Renal cell carcinoma is an immunogenic tumour with a prominent dysfunctional immune cell infiltrate, unable to control tumour growth. Although tyrosine kinase inhibitors and immunotherapy have improved the outlook for some patients, many individuals are non-responders or relapse despite treatment. The hostile metabolic environment in RCC affects the ability of T-cells to maintain their own metabolic programme constraining T-cell immunity in RCC. We investigated the phenotype, function and metabolic capability of RCC TILs correlating this with clinicopathological features of the tumour and metabolic environment at the different disease stages. Flow cytometric analysis of freshly isolated TILs showed the emergence of exhausted T-cells in advanced disease based on their PD-1^high and CD39 expression and reduced production of inflammatory cytokines upon in vitro stimulation. Exhausted T-cells from advanced stage disease also displayed an overall phenotype of metabolic insufficiency, characterized by mitochondrial alterations and defects in glucose uptake. Nanostring nCounter cancer metabolism assay on RNA obtained from 30 ccRCC cases revealed significant over-expression of metabolic genes even at early stage disease (pT1-2), while at pT3-4 and the locally advanced thrombi stages, there was an overall decrease in differentially expressed metabolic genes. Notably, the gene PPARGC1A was the most significantly down-regulated gene from pT1-2 to pT3-4 RCC which correlated with loss of mitochondrial function in tumour-infiltrating T-cells evident at this tumour stage. Down-regulation of PPARGC1A into stage pT3-4 may be the 'tipping-point' in RCC disease progression, modulating immune activity in ccRCC and potentially reducing the efficacy of immunotherapies in RCC and poorer patient outcomes.

Keywords: Renal Cell carcinoma; T-cell exhaustion metabolic.

Fig. 1

Increase in exhausted phenotype of RCC TILs in advanced stages of disease a Flow cytometric analysis of PD1 expression on CD4 and CD8 T-cells derived from healthy control donor bloods (n = 11), RCC patient bloods (n = 14), normal kidney tissues (n = 29), benign kidney tumours (n = 3), and RCC tumours at stage pT1-2 (n = 18), pT3-4 (n = 18), IVC thrombi (n = 9) and RCC metastatic (n = 3). b Multicolour flow cytometric analysis of CD4+PD1+ and CD8+PD1+ T-cells for their co-expression of CD39. c Flow cytometric analysis of PD1 expression on CD4 and CD8 T-cells derived from multiple biopsy sites within the same tumour from n = 9 advanced pT3-4 RCC tumours. Significant differences between sample types was determined by two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001

Fig. 2

CD8+RCC TILs at advanced stages of the disease show a significantly reduced capability to produce effector cytokines upon stimulation. Freshly dissociated single-cell suspensions of RCC tumours at different stages of the disease (pT1-2 n = 5, pT3-4 n = 8, IVC thrombi n = 3 and RCCmet n = 2) along with control PBMC (n = 9) and RCC PBMC (n = 7) were stimulated in vitro with and without αCD3 and αCD28 and the production of effector cytokines, IFNϒ and TNFα, by CD4 and CD8T cells assessed by intracellular cell staining and flow cytometry. Background levels of cytokines detected within unstimulated controls were subtracted from the results obtained from stimulated samples. Significant differences between sample types were determined by two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001

Fig. 3

RCC TILs show decreased ability for glucose uptake freshly isolated PBMC from healthy donors (n = 11) or RCC patients (n = 5) or cell suspensions from dissociated normal kidney (n = 3) or RCC tumour tissues at different disease stages (pT1-2 n = 5, pT3-4 n = 4, IVC thrombi n = 3, RCCmet n = 2) was stimulated with or without plate-bound αCD3 for 48 h. The cells then underwent a short-time incubation in a glucose-free media (to normalize the glucose uptake rate across groups) followed by a 20–30-min incubation with 2NBDG. The cells were washed, stained for cell surface markers and the 2NBDG measured in the FL-1 (FITC) channel as a fluorescent indicator for direct glucose uptake measurement. For comparison between unstimulated and stimulated groups, paired Student t tests were used. Representative histogram plots are shown for CD4+PD1+ and CD8+PD1+ T-cells from PBMC and RCC tumours at different stages of disease

Fig. 4

Low spare respiratory capacity of RCC TILs derived from invasive tumour sites. The metabolic phenotype of isolated RCC-derived CD3+ TILs a and healthy donor isolated CD3+ cells b was measured by assessing the cellular oxygen consumption rates during a mitochondrial stress test. c The spare respiratory capacity (SRC) was calculated as change in mean OCR upon treatment with FCCP (fluorocarbonyl cyanide phenylhydrazone) from basal respiration of CD3+ TILs from RCC primary tumour sites (n = 5) vs. locally advanced tumours exhibiting invasive morphology (IVC n = 2)) and healthy control CD3+T-cells (n = 3) as a control. The * indicates TILs derived from the primary tumour site and IVC thrombus site from the same patient (RCC 5)

Fig. 5

Dysfunctional depolarized mitochondria within advanced RCC TILs. a Freshly isolated PBMC from healthy controls (n = 16) or RCC patients (n = 3) and TILs derived from RCC tumours at different disease stages (pT1-2 n = 4, pT3-4 n = 3, IVC thrombi n = 2, RCCmet n = 1) were stimulated overnight with plate-bound αCD3 and 5 ng/ml IL-2. The cells were then stained with surface antibodies to identify CD4+PD1+ and CD8+PD1+ T-cells. Cells were then incubated with the MitoTracker^® Green dye and median fluorescence intensity (MFI) of the MitoTracker^® Green dye in CD4+ and CD8+ PD1+ T-cells measured by flow cytometry. b Mitochondrial superoxide content was detected in unstimulated and anti-CD3 stimulated TILs from >= pT3 tumours (pT3-4 n = 7, IVC thrombi n = 3, RCCmet n = 3) as compared to TILs from pT1-2 RCC (n = 3) or the T-cells from the blood of healthy controls (n = 13). Representative histogram plots are shown for unstimulated and anti-CD3 stimulated CD4+PD1+ and CD8+PD1+ T-cells from PBMC and from tumours at different stages of disease. c The fluorescent dye JC-1 was used to assess mitochondrial potential, a key readout of mitochondrial function. Following an overnight incubation with plate-bound anti-CD3 and 5 ng/ml IL-2 stimulation, mitochondrial depolarization of TILs from RCC tumours at different disease stages (pT1-2 n = 5, pT3-4 n = 10, IVC thrombi n = 4, RCCmet n = 3) and T-cells from the blood of healthy controls (n = 6) and RCC patients (n = 11) was indicated by a decrease in the red/green fluorescence intensity ratio. Representative FACS plots are shown for CD4+PD1+ and CD8+PD1+ T-cells from PBMC and from tumours at different stages of disease. Significant differences between sample types were determined by two-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001

Fig. 6

Differential metabolism gene expression analysis during RCC disease progression. Nanostring nCounter cancer metabolism panel assay was performed on RNA obtained from 30 ccRCC cases. Differential gene expression analysis was determined for each stage of the disease (pT1-2 n = 16), p3-4 (n = 10), IVC thrombi n = 5 and RCCmet n = 5) comparing pT1-2 to the normal baseline control (normal kidney), pT3-4 to stage pT1-2 and thrombi and RCCmet to stage pT3-4. For each of these disease stage comparisons, Volcano plots display each gene’s −log10(p value) and log2fold change with the highly statistically significant genes labelled and falling at the top of the plot above the horizontal lines (p value thresholds). The tables list the top 10 up- and down-regulated differentially expressed metabolism genes for each disease stage comparison

Fig. 7

PPARGC1A expression is associated with prognosis in clear cell renal cell carcinoma. Kaplan–Meier overall survival of patients stratified according to PPARGC1A gene expression as high (above 75%) and low (below 25%), in the TCGA-KIRC dataset, at different stages of renal cell carcinoma (left: stage pT1-2, centre: stage pT3-4 and right: all stages)