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Summary In an attempt to enhance the anti-tumour immune response, the co-stimulatory molecules B7-1 or B7-2 were expressed on the surface of B16 melanoma cells. B7-expressing tumours grew more slowly in both syngeneic immunocompetent mice and athymic T cellimmunodeficient nude mice. The delay in growth of B7-expressing tumours was dependent on natural killer (NK) cells, as reductions in tumour growth rates were minimized in mice depleted of NK cells. Systemic immunity to B16 melanoma was examined by vaccination with irradiated tumour cells. Inoculation with irradiated B16 B7-1 cells failed to protect against a subsequent challenge with live parental B16 cells. but conferred partial protection against challenge with live B16 B7-1 cells. In contrast to the local anti-tumour reaction. this protective response was dependent on T cells. The results presented here reveal some of the mechanisms involved in the in vivo response to a poorty immunogenic tumour modified to express co-stimulatory molecules.

The adhesive behaviour of a series of human melanoma cell lines, of varying metastatic potential, to basement membrane and stromal components was investigated in vitro. Experimental metastatic propensity was assessed from the number of pulmonary nodules formed after i.v. injection of cells into BALB/c nude mice. All cell lines showed similar kinetics of attachment when tested on plastic, type-I collagen films, type-I collagen hydrated gels, fibronectin, laminin type-IV collagen substrates and bovine aortic endothelial monolayers. Fibronectin-coated plastic compared to plastic alone produced increased cell attachment and spreading to the same extent in all the cell lines. The melanoma lines attached preferentially to cryostat sections of lung compared to other organs reflecting the pattern of organ involvement of metastasis in vivo. However, no significant quantitative differences in attachment to lung sections were seen between melanoma variants of differing metastatic capacities. Cells labelled with [125I]iododeoxyuridine to determine their initial organ distribution following i.v. injection showed that tumour-cell arrest was not significantly changed enough to explain the differing metastatic capacities. Thus it appears that adhesive properties of these melanoma cells are not correlated with their capacity to form metastases in vivo.

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The integrin avβ6 promotes migration, invasion and survival of cancer cells, but the biological relevance has yet to be ascertained in breast cancer. Our immunhistochemical analysis of over 2000 breast cancers has revealed that high expression of the protein for the integrin subunit beta6 (β6) is associated with very poor survival (HR = 1.99, P = 2.9×10-6) and increased metastases to distant sites (P = 0·02). This correlation was confirmed at the mRNA level via bioinformatic analysis of the 2000 women in the METABRIC cohort. Furthermore, co-expression of HER2 gave a significantly worse prognosis (HR = 3.43, P = 4×10-12), which we investigated further. We report from in vitro studies that HER2-driven invasion is mediated by αvβ6 in an Akt2-dependent manner. Using the well-tolerated αvβ6-blocking antibody 264RAD in vivo we show that antibody-blockade of this integrin suppressed growth of BT-474 and MCF-7/HER2-18 human breast cancer xenografts similarly to trastuzumab alone (P<0.001), the antibody used for treating HER2-positive cancers (both 10mg/kg, bi-weekly). Moreover, when 264RAD was co-administered it significantly enhanced the ability of trastuzumab to suppress BT-474 tumor growth with a reduction in mean tumor volume of 94.8%+/-1.18% compared to 70.8%+/-5.98% observed with trastuzumab alone (P<0.0001) after 2 weeks treatment. This trend was reproduced even in the MCF-7/HER2-18 trastuzumab-resistant breast cancer tumors where a 76.24%+/-10.15% reduction was observed with combination therapy (P<0.0001) compared with only 44.62%+/-10.43% (P = 0.0006) and 46.6%+/-14.71% (P = 0.0004) reductions in final volume with 264RAD and trastuzumab respectively. The combination therapy was so effective it almost eradicated 100mm3 BT-474 tumors and completely eliminated small (10-20mm3) MCF-7/HER2-18 tumors. 264RAD or trastuzumab prolonged survival to a similar degree (14.3% and 33.33% treated mice alive after 100d, respectively, no significant difference) but again, when both drugs were combined 85.7% of mice were alive after 100d, a highly significant response compared with PBS (P<0.0001) or monotherapies (264RAD: P<0.0001, trastuzumab: P<0.0001). Post-therapy biochemistry revealed residual tumors expressed significantly reduced αvβ6, HER2, HER3 and downstream signaling molecules including Akt2 and Smad2, essentially a much lower ‘grade’ tumour. Since 70% of women treated with trastuzumab either have, or develop resistance, we suggest combined targeting of αvβ6 and HER2 could provide an important novel therapy for thousands of women with breast cancer. In fact, over 39,000 American women annually (NIH statistics) will develop HER2+ breast cancers for which no specific therapies exist. Our data shows that in excess of 40% of these women with trastuzumab-resistant disease are also likely to express high levels of αvβ6. Our data also suggest that routine determination of the level of expression of αvβ6 on breast cancers would be a valuable clinical tool as it identifies novel high-risk groups of women that require enhanced therapeutic intervention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-01.