Fig. 1. Schematic representation of the experimental setup for the 3D collagen-fibrin gel killing assay.

(A to D) Illustration and representation of the model and technique used in this study. (A) Melanoma tumors expressing the T cell antigens ovalbumin and Pmel-1 (B16-OVA) are excised from C57BL/6 mice 10 days after implantation and dissociated into single-cell suspensions. Collagen-fibrin gels are prepared in 48-well tissue culture plates containing B16-OVA cells from in vitro culture or B16-OVA cells from the dissociated tumors in the presence or absence of antigen-specific CD8⁺ T cells. The gels are lysed daily with collagenase and trypsin and the numbers of remaining viable B16-OVA cells are assessed with a clonogenic assay as previously described (11). (B to D) Illustration of the use of the 3D collagen-fibrin gel killing assay to qualitatively measure the suppression of T cell killing by the tumor microenvironment with hypothetical representation of semi-log plots showing the expected numbers of viable B16 cells recovered from collagen-fibrin gels in which T cell–mediated killing (B) or immunosuppression of killing (C) occurred. (D) Equation modeling the T cell–mediated killing of tumor cells in collagen-fibrin gels to calculate the killing efficiency, k as previously described (11).