Treatment of established tumor is associated with ICAM-1 upregulation and reversed by CD8 depletion in a tumor necrosis factor-alpha gene transfected mouse mammary tumor

Introduction: We have performed TNF-alpha gene transfection in a mouse mammary cancer line and found significant antitumor effects. We hypothesize that the antitumor effects observed in this model are mediated by ICAM-1 and by the recruitment of CD4+ and CD8+ T cells. In vivo (Balb/c mice) tumor growth inhibition, treatment of established tumor and the effects of ICAM-1 and CD4+ and CD8+ T cells were evaluated.

Methods and results: Gene transfection with highly efficient vectors resulted in secretion of large amounts of TNF-alpha (ELISA). In vivo antitumor effects were tested. The number of cells required to generate palpable tumor 7-10 days after subcutaneous injection was determined (1 x 10(6)). The same number of transfected cells were injected subcutaneously and compared to nontransfected controls. Tumors were measured blindly and size was analyzed on day 30 by the Wilcoxon rank sum test. Mean tumor size after injection of transfected cells is compared to that of controls. Control tumors reached the maximum allowable size by day 30 (4 cm(2)). On day 30 EMT6-TNF-alpha tumors were 0.48 cm(2) (p < 0.05). The effect of repeat injection (challenge was also tested. Animals were injected with transfected cells or wild-type control on day -6 and challenged with the same number of wild-type tumor cells on day 0. Significant immune protection against subsequent challenge was seen after first time injection with EMT6-TNF-alpha but not after first time EMT6 wild-type injection (1.62 vs. 4 cm(2)). Treatment of 6-day-old tumor was also evaluated. On day 30, mean tumor size in animals treated with EMT6-TNF-alpha was 0.9 cm(2) compared to 4 cm(2) for controls. In all experiments, CD8+ T cell depletion and CD4+ T cell depletion caused a reversal of TNF-alpha-induced inhibitory effects. In addition, in vivo antibody blocking of ICAM-1 in tumor growth experiments reversed antitumor effects (control 4 cm(2), TNF-alpha 0.2 cm(2) and ICAM-1 blocking 3.14 cm(2)). Using flow cytometry, MHC class I and II and ICAM-1 adhesion molecule expression of transfected tumor was tested. ICAM-I expression was significantly upregulated. MHC class II antigen expression was also increased. TNF-alpha-transfected human breast cancer was also evaluated. Three cell lines and fresh tumor were transfected to express TNF-alpha. In vitro analysis revealed ICAM-1 upregulation following transfection. Histologic analysis and immunohistochemical staining revealed TNF-alpha and ICAM-1 in transfected tumors and not in wild-type tumors.

Conclusion: Highly significant in vivo tumor growth inhibition and immune protection after injection with TNF-alpha-transfected tumors appears to be mediated predominantly by CD8+ T cells and ICAM-1 upregulation. These findings suggest that TNF-alpha increases recruitment and adhesion of effector T cells.