Growth and metastasis of human tumor cells in immunodeficient nude mice were improved when tumor cells were inoculated within a vascularized artificial polyurethane sponge matrix. The sponges had been implanted 7-10 days earlier and were vascularized at the time of cell injection. All cell lines tested, including colon carcinoma-derived lines from primary tumors (HT29, PT3 and PT4) or from liver metastasis (LM3), and a metastatic variant from a melanoma (MeWo-Met) grew in a high percentage (78-94%) of the inoculated sponge grafts. When growth in sponge grafts is compared with growth at a subcutaneous site, the sponge matrix appears to increase tumorigenicity, at least for some cell lines. Regular formation of metastases was observed when cells had been injected into sponges. Most metastases were found in a second sponge graft implanted at a contralateral site, but some were also found at other s.c. sites. In vivo depletion of NK cells by pre-treatment with cyclophosphamide could not further enhance the formation of metastasis. Tumor cells from fresh surgical specimens could be propagated in sponge matrix grafts and subsequently established as cell lines in tissue culture.

The activation of tumor-specific precursor cytotoxic T lymphocytes (CTLP) into cytotoxic T cells (CTL) was demonstrated in situ using the well-defined, highly metastatic ESb tumor as murine model system. Ten days after optimal immunization of syngeneic mice with a sublethal dose of live ESb tumor cells in the pinna, tumor-sensitized non-cytotoxic CTLP were recovered from the spleen and lymph nodes. These cells mature into tumor-specific CTL upon restimulation in vitro. Using a confined sponge matrix compartment, in combination with a specific tumor vaccine (autologous inactivated tumor cells), we induced a CD8+ (Lyt 2+) T-cell-mediated, highly cytotoxic anti-tumor immune response in situ in immunized mice. It was not possible to activate a similar response directly in lymphoid organs such as the spleen. The cytotoxic CD8+ T cells, recovered by simple mechanical pressing of the sponge, were active against the specific tumor cells in a 51Cr-release assay in vitro and also in a Winn neutralization assay in vivo. CTL activity was increased and remained in the non-adherent fraction when the cell mixture, squeezed out of the sponges, was passed over nylon wool. In a cell recruitment assay, the delayed-type hypersensitivity (DTH) potential of the activated sponge-infiltrating T cells was demonstrated by their capacity to recruit circulating host lymphocytes to sites of tumor-cell location in situ.