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. 2024 Apr 15;134(8):e173934.
doi: 10.1172/JCI173934.

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis

Melissa M Wolf  1   2 , Matthew Z Madden  1   3 , Emily N Arner  1 , Jackie E Bader  1 , Xiang Ye  1 , Logan Vlach  1   2 , Megan L Tigue  1   2   3 , Madelyn D Landis  4 , Patrick B Jonker  5 , Zaid Hatem  1 , KayLee K Steiner  1   2 , Dakim K Gaines  6   7 , Bradley I Reinfeld  2   3   4 , Emma S Hathaway  1   2 , Fuxue Xin  8   9 , M Noor Tantawy  8   9 , Scott M Haake  4   7 , Eric Jonasch  10 , Alexander Muir  5 , Vivian L Weiss  1   7 , Kathryn E Beckermann  4   7 , W Kimryn Rathmell  4   7   11 , Jeffrey C Rathmell  1   7   11
Affiliations

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis

Melissa M Wolf et al. J Clin Invest. .

Abstract

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Keywords: Cancer; Macrophages; Metabolism; Oncology; T cells.

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Conflict of interest statement

Conflict of interest: Within the past 2 years, WKR has received clinical research support from Merck and Incyte. JCR has held stock equity in Sitryx and Caribou Bioscience and within the past 2 years has received research support, travel, and honoraria from Sitryx, Caribou, Nirogy, Kadmon, Calithera, Tempest, Merck, Mitobridge and Pfizer. KEB receives funding from BMS-LCFA-IASLC, Aravive, Pionyr, Arsenal Biosciences, Exelexis, Merck, Aveo, and Arrowhead.

Figures

Figure 1
Figure 1. Lymphocytes and macrophages are abundant in ccRCC.
(A) CD68, (B) CD8A, and (C) CD4 mRNA expression ranked in nonlymphoid solid tumors queried in TCGA. Kidney renal clear cell carcinoma (KIRC) (ccRCC) cells are highlighted with stage-specific expression. *P < 0.05, **P < 0.01, and ***P < 0.001, by Brown-Forsythe and Welch’s ANOVA tests corrected with Games-Howell. (D) Representative image of TMA staining identifying CD68+ and CD8+ cells from patients with ccRCC (scale bar: 50 μm) and quantification of the percentage of area stained with hematoxylin (HEM).
Figure 2
Figure 2. Vhl loss functionally upregulates HIF targets in the Renca cell model.
(A) Representative Western blot showing protein expression of VHL, HIF-1α, and actin in the indicated Renca cell lines. (B–E) Quantitative PCR of Glut1, Ldha, Pdk1, and Egln1 in the indicated cell lines relative to Vhl WT.2. Each data point represents a technical replicate from 2 independent experiments. Nuclear magnetic resonance (NMR) quantification of glucose uptake (F) and lactate secretion (G) from CM in Renca Vhl WT.1/KO.1 and Vhl WT.2/KO.7 paired cell lines. Each data point represents a technical replicate. (H) Representative images and quantification of CD31 IHC staining of Vhl WT.2 and Vhl-KO.7 subcutaneous tumors. Scale bars: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ordinary 1-way ANOVA and Bonferroni’s multiple-comparison test (B–G) and 2-tailed Student’s t test (H).
Figure 3
Figure 3. Vhl deletion slows tumor growth and increases immune infiltration.
(A) Average growth curve of all Vhl WT (black) and Vhl-KO (gray) tumors represented as tumor volume (mm3). Smaller graphs represent all biological replicates of paired Vhl WT and Vhl-KO tumors. Vhl WT.1 (n = 16) and Vhl-KO.1 (n = 12); Vhl WT.2 (n = 14) and Vhl-KO.7 (n = 14); Vhl WT.2 (n = 16) and Vhl-KO.32 (n = 16). D, day. (B) Final mass of each tumor. (C) Representative gating scheme for CD45+, CD11b+, and CD3+ T cell populations. See Supplemental Figure 3 for complete gating schemes. SSC, side scatter. (D) Quantification of CD45+ immune cell, CD11b+ myeloid cell, and CD3+ T cell infiltrate from each Vhl WT and Vhl-KO pair. WT and KO pairs are represented by matched symbol: Vhl WT.1/KO.1 (circle), Vhl WT.2/KO.7 (square), Vhl WT/KO.32 (triangle), Vhl rescue/KO.7 control (reverse triangle). ctrl, control. (E) Representative images of CD45+ IHC staining in Vhl WT.1, Vhl-KO.1, and Vhl-KO.32 whole tumors. Data are representative of experiments performed at least twice. Graph represents hematoxylin quantification. Each data point represents an individual mouse. Graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA and Šidák’s multiple-comparison test (A), unpaired, 2-tailed Student’s t test (B and E), and ordinary 1-way ANOVA with Bonferroni’s multiple-comparison test (D).
Figure 4
Figure 4. Increased TAM displaying proinflammatory properties reside in the Vhl-KO TME.
(A) Representative flow plots of myeloid cell populations in viable CD45+CD11b+ cell populations defined as PMN-MDSC (Ly6G+Ly6Clo-int), M-MDSC (Ly6G–Ly6Chi), overall TAMs (Ly6G–Ly6Clo), and the TAM subsets TAM1 (Ly6G–Ly6CloF4/80lo-int) and TAM2 (Ly6G–Ly6CloF4/80hi), and quantification of overall TAM, TAM1, and TAM2 infiltration as a percentage of viable cells in each Vhl WT and Vhl-KO pairs (pairs are represented by a matched symbol). See Supplemental Figure 3 for complete gating schemes. (B) Percentage of Phrodo+ cell populations as a fraction of viable CD45+CD11b+F4/80+ cells in Vhl WT.2 and Vhl-KO.7 tumors. Protein MFI quantification and representative histogram of (C) CD11c and (D) CD206 in overall TAMs from Vhl WT.2, Vhl-KO.7, Vhl rescue, and Vhl-KO.7 control tumors. (E) UMAP of CD45+ scRNA-Seq and mRNA expression levels of (F) Itgax (CD11c) and (G) Mrc1 (CD206) in TAMs/monocytes from Vhl WT.2 or KO.7 tumors. Each data point represents a biological replicate, and graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ordinary 1-way ANOVA with Bonferroni’s multiple-comparison test (A), 2-tailed Student’s t test (B, C, and D), and Wilcoxon rank-sum test with a threshold of q < 0.05 (F and G). Max, maximum.
Figure 5
Figure 5. Vhl loss promotes proinflammatory TAM transcriptional signatures.
(A) Heatmap of HALLMARK GSEA scores from flow-sorted cell populations of the indicated cell populations from 4 Vhl WT.1 and 4 Vhl-KO.1 tumors. Pathways outlined in dark red are enriched in TAM1 and TAM2 from Vhl-KO tumors; the pink outline highlights enriched pathways associated with TAM1 from Vhl-KO tumors; and the yellow outline highlights pathways enriched in M-MDSCs. (B) PCA of RNA-Seq of the indicated cell populations.
Figure 6
Figure 6. Myeloid cells in the Vhl-KO TME are more glycolytic.
(A) Experimental workflow for FDG PET imaging and characterization of FDG avid single-cell populations. (B) Representative FDG PET images of Vhl WT and Vhl-KO tumors, and quantification of whole tumor PET avidity of the indicated WT and KO pairs. (C) Representative flow cytometric gating of whole tumor and MDSC-enriched (anti-Gr1 microbead positive selection) and TAM-enriched (Gr1–, anti-CD11b microbead positive selection) populations. (D) Quantification of cellular FDG avidity in MDSC-enriched and TAM-enriched cell fractions from the indicated Vhl WT and Vhl-KO tumors. (E) Cellular FDG avidity of CD45– (anti-CD45 microbead negative selection) and CD3+ enriched (anti-CD4/CD8 microbead positive selection) T cells from the indicated Vhl WT or Vhl-KO tumors. (F) UMAP showing snRNA-Seq data from 3 human ccRCC patient tumors, and (G) KEGG pathway analysis for oxidative phosphorylation and glycolysis/gluconeogenesis in the designated cell populations. Each data point represents a biological replicate, and graphs show the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by unpaired, 2-tailed Student’s t test (B, D, and E) and Brown-Forsythe and Welch’s ANOVAs corrected with Games-Howell (G).
Figure 7
Figure 7. T cells residing in the Vhl-KO TME are dysfunctional.
(A) Quantification of the specified lymphocyte populations as the percentage of viable cells in each Vhl WT and Vhl-KO pair (pairs are represented by matched symbols). See Supplemental Figure 3 for the complete gating strategy. (B) Representative gating and quantification of the percentage of unstimulated and stimulated CD3+CD44+CD8+ T cells expressing TNF-α and IFN-γ in Vhl WT.2 or Vhl-KO.7 tumors. (C) Representative gating and quantification of the percentage of CD3+CD8+ T cells expressing PD-1 and TIM3 in Vhl WT.2, Vhl-KO.7, Vhl-KO control, and Vhl rescue tumors. (D) Representative gating and quantification of the percentage of CD3+CD4+ T cells expressing PD-1 in Vhl WT.2, Vhl-KO.7, Vhl-KO control, or Vhl rescue tumors. (E) Volume measurements (mm3) over time of IgG- or anti–PD-1–treated Vhl WT and Vhl-KO tumors. Each data point represents a biological replicate, and graphs show the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by ordinary 1-way ANOVA with Bonferroni’s multiple-comparison test (A), 2-tailed Student’s t test (B, C, and D), and 2-way ANOVA with Šidák’s multiple-comparison test (E).
Figure 8
Figure 8. The myeloid CX3CL1/CX3CR1 axis is augmented in Vhl-deficient tumors.
(A) Quantification of soluble CX3CL1 in Vhl WT.2, Vhl-KO.7, and Vhl CX3CL1-DKO CM by ELISA. Data represent technical replicates from 2 independent experiments and were normalized per 106 cells. (B) Relative monocyte migration stimulated by either SF media or CM from Vhl WT.2, Vhl-KO.7, or Vhl Cx3cl1-DKO cells. (C) Average growth curve of all Vhl-KO.7 (gray) and Vhl Cx3cl1-DKO (purple) tumors represented as tumor volume (mm3). Smaller graphs represent biological replicates. (D) Quantification of CD45+, CD11b+, and CD3+ immune infiltrate from Vhl WT, Vhl-KO, and Vhl Cx3cl1-DKO tumors. (E) Quantification of overall TAM, TAM1, and TAM2 infiltration as the percentage of viable cells in Vhl-KO and Vhl Cx3cl1-DKO tumors (F) Protein MFI quantification and representative histogram of CD11c and (G) CD206 in overall TAMs from Vhl WT.2, Vhl-KO.7, and Vhl Cx3cl1-DKO tumors. (H) Percentage of Phrodo+ cells as a fraction of viable CD45+CD11b+F4/80+ cells in Vhl WT.2 and Vhl-KO.7, and Vhl Cx3cl1-DKO tumors. (I) Ranked gene expression scores for CX3CL1 and (J) CX3CR1 across 30 nonlymphoid solid tumors queried in TCGA. Data represent biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Bonferroni’s multiple-comparison test (A, B, D, F, and G), 2-way ANOVA with Šidák’s multiple-comparison test (C), and 2-tailed Student’s t test (E and H). Graphs show the mean ± SEM.
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