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Notes: Summary The effect produced on tumor progression by the injection of either VCN-treated tumor cells or tumor cells mixed with VCN to dogs with spontaneous mammary tumors was investigated. Dogs of different breeds and ages with at least two palpable spontaneous mammary tumors were selected. One tumor was left in the animal for further clinical examination, whereas the other tumor(s) was (were) excised for histologic diagnosis and for preparation of a single-cell suspension. Autologous M-cells were treated with VCN, subsequently extensively washed and injected SC into the neck of the dog on the day of operation and on the next day or different numbers of autologous M tumor cells (105, 106, 107, 5×107, 108) were mixed with different amounts of VCN (0, 0.65, 6.5, 65 mU), and these various mixtures were injected ID at different sites to each dog on the day of operation. This procedure has been called chessboard vaccination (Seiler and Sedlacek, 1978). Altogether 79 dogs were blindly distributed into six groups in three consecutive studies. The results show that the therapeutic effect of the injection of VCN-treated autologous tumor cells depends on the number of tumor cells injected: injection of 2×107 tumor cells repeatedly induced regression of the residual tumor mass (Studies I, II, and III) in most dogs and prevention of metastasis (Study I), while the application of 1×108 tumor cells caused enhanced tumor proliferation in all and early metastasis in most of the dogs (Study I). The injection of 2×106 tumor cells induced only a transient regression, with subsequent progression of the residual mammary tumor (Study II). Repetition of the injection of 2×106 tumor cells three times every 4 weeks did not improve this effect (Study II). The chessboard vaccination proved to be at least as effective as the injection of 2×107 VCN-treated tumor cells (Study III), although 1×108 or more tumor cells had been injected; this number of cells caused tumor enhancement when the cells were treated with VCN only and injected SC (Study I). Moreover, the DTH reaction after ID injection of autologous tumor cells could be increased by the addition of VCN: low numbers of tumor cells and high amounts of VCN or high numbers of tumor cells and low amounts of VCN caused the most pronounced skin response. The relevance of these data to overcoming the risk of tumor enhancement after injection of an inadequate number of VCN-treated tumor cells and the possible diagnostic and therapeutic relevance of the DTH response after chessboard vaccination will be discussed.