VHL-dependent alterations in the secretome of renal cell carcinoma: Association with immune cell response?

Abstract

Secreted proteins could modulate the interaction between tumor, stroma and immune cells within the tumor microenvironment thereby mounting an immunosuppressive tumor microenvironment. In order to determine the secretome-mediated, von Hippel Lindau (VHL)-regulated cross-talk between tumor cells and T lymphocytes peripheral blood mononuclear cells (PBMC) from healthy donors were either cultured in conditioned media obtained from normoxic and hypoxic human VHL-deficient renal cell carcinoma (RCC) cell line (786-0VHL-) and its wild type (wt) VHL-transfected counterpart (786-0VHL+) or directly co-cultured with both cell lines. An increased T cell proliferation was detected in the presence of 786-0VHL+-conditioned medium. By applying a quantitative proteomic-based approach using differential gel electrophoresis followed by mass spectrometry fourteen proteins were identified to be differentially expressed within the secretome of 786-0VHL- cells when compared to that of 786-0VHL+ cells. All proteins identified were involved in multiple tumor-associated biological functions including immune responses. Functional studies on manganese superoxide dismutase 2 (MnSOD2) demonstrated that it was a regulator of T cell activation-induced oxidative signaling and cell death. Direct effects of soluble MnSOD2 on the growth properties and interleukin 2 (IL-2) secretion of T cells could be demonstrated underlining the critical role of extracellular MnSOD2 levels for T cell proliferation and activation.

Keywords: VHL; manganese superoxide dismutase 2; renal cell carcinoma; secretome; tumor microenvironment.

Figure 1. Reduction of T cell proliferation and CD25 expression within 786-0^VHL− conditioned media

PBMC from healthy donors were prepared by Ficoll density gradient centrifugation and stimulated directly with anti-CD3 and anti-CD28 antibodies (mouse anti-human, BD, 1 μg/ml each) or PHA-M (Sigma-Aldrich, 4 μg/ml) and IL-2 (100 U/ml). “Cells were grown up to 6 days in 786- 0VHL- or 786-0VHL+ conditioned media (A) or in the presence of 786-0VHL-/VHL+ cells (B), respectively. Cells were analyzed via flow cytometry using covalent CFSE staining (Invitrogen) according to the manufacturer's protocol.” Cells were grown for 6 days under normoxic conditions and analyzed via flow cytometry for lymphocyte markers using seven-color staining panels (C) Data are represented as mean percentage (%) ± SD based on five independent experiments.

Figure 2. Comparative profiling of cell culture supernatants upon reconstitution of VHL expression in the 786-0 cell line

(A) Representative protein expression profiles of media conditioned by the parental cell line 786-0^VHL− (left panel) and its VHL-transfected variant (786-0^VHL+, right panel) under normoxic conditions (21% O₂, 48 h) is shown. The respective protein pools were individually labeled with distinct fluorescence dyes (DIGE technology) and than co-separated by 2-DE as described in the Materials and methods section. Differentially expressed proteins are labeled with numbers. Figure B and C show an overview of the conditioned media profiling results. The Venn diagrams display the distribution pattern of all differentially secreted proteins under normoxic and hypoxic conditions (B) as well as the repeatedly detected secreted proteins (C).

Figure 3. Classification of the differentially secreted proteins according to their biological processes (A), cellular components (B) and their molecular functions (C)

Figure 4. Representative qPCR-mediated target validation

786-0^VHL− and 786-0^VHL+ cells were cultured under normoxic (21% O₂, 48 h) and hypoxic (1% O₂, 48 h) conditions and subjected to quantitative real-time PCR analysis to determine mRNA levels of B2M, MnSOD2, plasminogen activator inhibitor 1 (SERPINE1), and ubiquitin-conjugating enzyme E2 N (UBE2N) using oligo-(dT)18 primed cDNA. Mean expression of HPRT and PPIA were used for normalization. Data were represented as mean percentage (%) ± SD based on three independent experiments.

Figure 5. VHL-mediated up-regulation of B2M in the supernatant of 786-0VHL+ cells

The up-regulation of B2M within the cell culture supernatant was verified at the protein level (A) as described within the Materials and Methods section. Blots were probed with an anti-B2M mAb, whereas the immunostaining with the anti-GAPDH mAb served as a loading control. In addition, the intracellular protein levels of B2M as well as HLA-ABC of 786-0^VHL− and 786-0^VHL+ cells were analyzed by flow cytometry after intracellular staining using FITC-conjugated anti-B2M or FITC-conjugated anti-HLA-ABC antibodies (BD) (B) Data are represented as mean percentage (%) ± SD based on five independent experiments.

Figure 6. VHL-mediated down-regulation of extracellular superoxide dismutase (MnSOD2) level and activity

Down-regulation of superoxide dismutase (MnSOD2) within the cell culture supernatant was also verified at the protein level (A) as described within the Materials and Methods section. Blots were probed with anti-MnSOD2 antibodies, whereas the immunostaining with the anti-β-actin mAb served as loading control. In addition, MnSOD2 activity was assessed using the Superoxide Dismutase Activity Colorimetric Assay Kit as described in the Materials and Methods section (B).

Figure 7. MnSOD2-induced reduction of T cell proliferation and IL-2 secretion

PBMC from healthy donors were prepared by Ficoll density gradient centrifugation, labeled with CFSE according to the manufacturer 's instructions, and stimulated directly with anti-CD3 and anti-CD28 antibodies (mouse anti-human, BD, 1 μg/ml each) and IL-2 (100 U/ml). Cells were grown in the absence or presence of MnSOD2 (1U/ml) for 20 and 40 h prior to flow cytometry (A). IL-2 secretion was analyzed using commercial enzyme-linked immunosorbent assay (ELISA) (eBioscience, Austria) using “day 6” T cells at a density of 1 × 10⁵ cells/well onto 96-well culture plates in the absence and presence of 1 U/ml MnSOD2 as described in Materials and Methods section (B). Data are represented as mean percentage (%) ± SD based on five independent experiments. *p-value < 0.05