Levels of metabolites in RCC tumor interstitial fluid are similar to those found in normal kidney interstitial fluid.

(A) Schematic depicting study design whereby samples collected from patients with renal cell carcinoma (RCC) undergoing nephrectomy were used to derive tumor interstitial fluid (TIF), kidney IF (KIF), and plasma. Samples were then subjected to polar metabolomics and lipidomics analyses. See Table S1 for patient information, and Table S2 for metabolite concentrations. (B) Principal component analysis of polar metabolites measured from the indicated RCC patient samples (n = 55 patients). For each sample, absolute levels of 98 polar metabolites were quantified by LC/MS. Data represent 55 TIF, 46 KIF, and 27 plasma samples. The 95% confidence interval is displayed. (C) Principal component analysis of lipid species measured from the indicated RCC patient samples (n = 38 patients). For each sample, relative levels of 195 lipids were assessed by LC/MS. Data represent 34 TIF, 25 KIF, and 18 plasma samples. The 95% confidence interval is displayed. (D) T-test analysis of polar metabolites (n = 98) that do or do not significantly differ in concentration between each site from all RCC patient samples (n = 55 patients). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to determine significant metabolites. p-values in the plot are derived from chi-squared statistical analysis. (E) T-test analysis of lipids (n = 195) that do or do not significantly differ in concentration between each site from all RCC patient samples (n = 38 patients). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to determine significant metabolites. p-values in the plot are derived from chi-squared statistical analysis.

Assessment of metabolites that differ between RCC interstitial fluid and normal kidney interstitial fluid.

(A-B) Volcano plots depicting the log2 fold change in polar metabolite concentration. (A) or relative lipid levels (B) between TIF and KIF from RCC patients (n = 55 patients in [A], n = 38 patients in [B]). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to select significantly altered metabolites. Metabolites or lipids highlighted in red and blue are significantly higher and lower in TIF compared to KIF, respectively. Full names of selected lipids: PC(O-34:2), PC(P-34:1)/PC(O-34:2); PC(O-40:7), PC(P-40:6)/PC(O-40:7); LPC(O-18:1), LPC(P-18:0)/LPC(O-18:1); PC(O-34:1), PC(P-34:0)/PC(O-34:1); PC(O-34:4), PC(P-34:3)/PC(O-34:4); PC(O-36:1), PC(P-36:0)/PC(O-36:1). (C-E) Levels of selected metabolites measured by LC/MS in plasma, TIF, and KIF from matched RCC patients (n = 10 patients). Each point represents a value measured from one patient, and the red line represents the mean across all patients considered. p-values were derived from either mixed-effects analysis (kynurenine, glutathione, glucose) or repeated measures one-way ANOVA (2-hydroxyglutarate, lactate, arginine, citrulline, ornithine), depending on whether missing values were present, and were Tukey multiple comparisons corrected (ns, not significant; * p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (F) Normalized peak area values of cholesterol measured by LC/MS in plasma, TIF, and KIF from matched RCC patients (n = 6 patients). Each point represents a sample, and the red line represents the mean across all patients considered. p-values were derived from repeated measures one-way ANOVA with Tukey multiple comparisons correction (ns, not significant; ****p < 0.0001). (G-H) Relative abundance of cholesterol (G) or cholesteryl esters (H) in TIF compared to KIF from matched RCC patients (n = 20 patients). The mean is presented ± SEM, and the black dotted line indicates a ratio of 1, representing no difference in lipid levels between TIF and KIF. p-values were derived from a one sample t-test compared to 1 (*p < 0.05).

Levels of metabolites in clear cell RCC interstitial fluid are similar to those found in normal kidney interstitial fluid.

(A) Principal component analysis of polar metabolites measured from the indicated clear cell RCC patient samples (n = 41 patients). For each sample, absolute levels of 98 polar metabolites were quantified by LC/MS. Data represent 36 TIF, 28 KIF, and 20 plasma samples. The 95% confidence interval is displayed. (B) Principal component analysis of lipid species measured from the indicated clear cell RCC patient samples (n = 28 patients). For each sample, relative levels of 195 lipids were assessed by LC/MS. Data represent 25 TIF, 18 KIF, and 15 plasma samples. The 95% confidence interval is displayed. (C) T-test analysis of polar metabolites (n = 98) that do or do not significantly differ in concentration between each site from clear cell RCC patient samples (n = 41 patients). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to determine significant metabolites. p-values in the plot are derived from chi-squared statistical analysis. (D) T-test analysis of lipids (n = 195) that do or do not significantly differ in concentration between each site from all clear cell RCC patient samples (n = 28 patients). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to determine significant metabolites. p-values in the plot are derived from chi-squared statistical analysis.

Assessment of metabolites that differ between plasma in patients with RCC with plasma from normal individuals and from patients with NSCLC.

(A-B) Volcano plots depicting the log2 fold change in polar metabolite concentration measured in plasma from patients with RCC (n = 27) compared to that measured in plasma from healthy adults (n = 10) (A), or measured in plasma from patients with RCC compared to that measured in patients with non-small cell lung cancer (NSCLC) (n = 20). Cutoffs of | log2 fold change | > 1 and adjusted p-value (FDR-corrected) < 0.05 were used to select significantly altered metabolites. Metabolites highlighted in red and blue are significantly higher and lower in RCC plasma, respectively. (C) Concentration of cystine as reported in the Human Metabolome Database (HMDB) (n = 5) or measured in plasma from healthy adults (normal, n = 10), from RCC patients (n = 27), or NSCLC patients (n = 20). Each point represents a sample from patient, and the red line represents mean across all samples considered. p-values were derived from Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons correction (ns, not significant; ***p < 0.001; ****p < 0.0001). (D) Concentration of cystine measured by LC/MS in plasma from an overnight (>9.5 hr) fasted healthy adult and plasma from the same adult ∼4 hours after eating (fed).