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. 2007 Feb 15;67(4):1832-41.
doi: 10.1158/0008-5472.CAN-06-3014.

Ectopic expression of vascular cell adhesion molecule-1 as a new mechanism for tumor immune evasion

Ken-Yu Lin  1 , Dan Lu, Chien-Fu Hung, Shiwen Peng, Lanqing Huang, Chunfa Jie, Francisco Murillo, Jesse Rowley, Ya-Chea Tsai, Liangmei He, Dae-Jin Kim, Elizabeth Jaffee, Drew Pardoll, T-C Wu
Affiliations

Ectopic expression of vascular cell adhesion molecule-1 as a new mechanism for tumor immune evasion

Ken-Yu Lin et al. Cancer Res. .

Abstract

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8(+) (CD8(+)) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8(+) T cells through its interaction with the alpha(4)beta(1) integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with alpha(4)beta(1) integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC.

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Figures

Figure 1
Figure 1
VCAM-1 is up-regulated in immune escape variant P3. A, a schematic diagram showing how a highly resistant escape variant P3 was generated from TC-1/P0 cell line. Filled circles, tumor outgrowth in the mouse. A representative mouse experiment showing the growth property of P0 and P3 cell lines in (B) naïve and (C) immunized mice. D, microrray data showing the most highly expressed genes in P3 by at least 10-fold (NCBI accession number GSE2774).
Figure 2
Figure 2
Characterization of VCAM-1 expression in P0 and P3. A, semiquantitative RT-PCR shows that VCAM-1 transcript is more abundant in P3 than P0. B, real-time quantitative RT-PCR shows that there is a 6.6-fold increase in VCAM-1 transcript in P3. C, flow cytometry analysis shows that P3 expresses a significantly higher level of VCAM-1 than does P0. Dotted lines, cells stained with isotype control antibody. D, flow cytometry analysis characterizes the expression of VCAM-1 in P0-VCAM-1 and P0-NoInsert cell lines.
Figure 3
Figure 3
VCAM-1 converts P0 from a susceptible cell line into a resistant cell line through an immune-mediated mechanism. A, in vivo tumor growth experiment in C57BL/6 mice. Both P0-VCAM-1 and P0-NoInsert cell lines were injected into naïve C57BL/6 mice (five per group) at a dose of 1 × 105 tumor cells per mouse. In addition, both types of cell lines were injected into C57BL/6 mice (five per group) immunized with vaccinia Sig/E7/LAMP-1 7 d after immunization. B, in vivo tumor growth experiment in immunocompetent C57BL/6 mice and in RAG1−/− mice. Both P0-VCAM-1 and P0-NoInsert cell lines were injected into immunocompetent C57BL/6 mice and in RAG1−/− mice (five per group) at a dose of 1 × 105 tumor cells per mouse. P0-VCAM-1 and P0-NoInsert tumors have similar growth kinetics and size in RAG1−/− mice. However, P0-VCAM-1 and P0-NoInsert tumors have significantly different growth kinetics, suggesting that their size difference in wild-type, naïve mice is lymphocyte mediated.
Figure 4
Figure 4
VCAM-1 expression promotes the migration of CD8+ T cells, resulting in a fewer number of apoptotic cells and a lower number of infiltrating CD8+ T cells. A, TUNEL assay. Frozen sections of P0-NoInsert and P0-VCAM-1 were stained for apoptotic cells using TUNEL assay. The number of apoptotic cells in P0-NoInsert and P0-VCAM-1 tumors was determined by counting TUNEL-positive cells in two representative frozen sections. B, left, representative staining of total CD8+ T cells by flow cytometry analysis. CD8+ T cells in single cell suspensions of P0-NoInsert and P0-VCAM-1 tumors were isolated 11 d after inoculation. B, right, bar graph depicting the number of total CD8+ T cells in the P0-NoInsert and P0-VCAM-1 tumors by flow cytometry analysis on days 7, 9, and 11. Five tumors were analyzed for each time point. C, bar graph depicting the number of CD4+ T cells and natural killer cells in the P0-NoInsert and P0-VCAM-1 tumors. C57BL/6 mice (five per group) were challenged with either P0-NoInsert or P0-VCAM-1 tumors. Tumor-infiltrating lymphocytes were isolated, and the numbers of CD4+ T cells and natural killer cells in the P0-VCAM-1 tumors and P0-NoInsert tumors were determined using flow cytometry analysis. D, in vitro transwell migration experiments. About 5 × 104 of total CD8+ T cells were put into wells that were coated with either VCAM-1-Fc or control IgG1-Fc (left ). Recombinant VCAM-1 can prompt the migration of freshly isolated tumor-infiltrating CD8+ T cells. In another experiment, 5 × 104 of total CD8+ T cells were put into wells that were coated with VCAM-1-Fc. The wells were added with antimouse α4 integrin antibody (PS/2) or control Rat IgG (right ). Blocking with antimouse α4 integrin antibody (PS/2) significantly retarded the migration of freshly isolated tumor-infiltrating CD8+ T cells in the transwell migration assay.
Figure 5
Figure 5
VCAM-1–mediated impairment of T cell effector functions is integrin dependent. A, two VCAM-1 mutants (VCAM-1/D40A and VCAM-1/D328A). The location of the amino acid change in domains 1 and 4 of VCAM-1 was indicated by A. Tm/Cy, the transmembrane and cytoplasmic domains. B, flow cytometry analysis that characterize the relative levels of VCAM-1 expression in P0-VCAM-1/D40A and P0-VCAM-1/D328A cell lines. Both P0-VCAM-1/D40A and P0-VCAM-1/D328A cell lines expressed similar levels of VCAM-1 compared with P0-VCAM-1. C, in vivo tumor growth experiment. C57BL/6 mice (five per group) were immunized with 1 × 107 pfu/mouse of vaccinia Sig/E7/LAMP-1. Seven days after the vaccination, immunized mice were challenged with the various tumor cell lines at a dose of 1 × 105 tumor cells per mouse. P0-VCAM-1/D40A and P0-VCAM-1/D328A have completely lost immune resistance.
Figure 6
Figure 6
Mutation of VCAM-1 in P0-VCAM-1 tumor cells restores the number of tumor-infiltrating CD8+ T cells and leads to the accumulation of E7-specific CD8+ T cells in the tumors. A, CD8+ T cells in single cell suspensions of P0-NoInsert, P0-VCAM-1/D40A, P0-VCAM-1/D328A, and P0-VCAM-1 tumors were isolated 11 d after inoculation. The number of CD8+ T cells within the various tumors was characterized by flow cytometry analysis. Bar graphs depicting the percentage of CD8+ T cells in tumors are shown. Five tumors were analyzed in each group. B, C57BL/6 mice (five per group) were challenged s.c. with P0-VCAM-1 tumors in the right hind leg and with either P0 tumor cells or P0-VCAM-1 D40A tumor cells in the left hind leg. One week later, tumor-bearing mice were injected with the luciferase-expressing E7-specific CD8+ T cells (5 × 106 per mouse) by tail vein. The intensity of luminescence in the T cell–challenged mice was monitored on days 0, 1, 3, and 7 after transfer of the T cells using the IVIS 200 system (Xenogen). Tumor-bearing mice, which did not receive E7-specific CD8+ T cells, were used as a negative control. An integration time of 5 min was used for luminescence image acquisition. C, line graph depicting the photon counts in T cell-challenged mice on days 0, 1, 3, and 7 after transfer of T cells.
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