Altered response to dacarbazine by murine melanoma cells cultured in type I collagen gel

Abstract

The effects of antitumor agents are usually assayed on soft agar culture or monolayer culture on plastic. However, such an environmental condition cannot be found in vivo and could be too severe for cells to survive. Recently, studies have indicated that culture of cells in or on a collagen gel provides a matrix-like environment and enables the cells to exhibit fully their differentiating functions not observed on plastic [1, 4, 9, 11, 12]. To evaluate the effect of antitumor drugs under the conditions resembling those occurring in the process of infiltrating the dermis in vivo as much as possible, we cultured B16 melanoma cells in type I collagen gel and compared the effects of dacarbazine (DTIC) [3, 8] and all-trans-retinoic acid (RA) [7] with those on the conventional plastic dishes. Three-dimensional culture in type I collagen gel was performed according to the method of Elsdale and Bard [4] after modification as described previously [6]. Briefly, 0.3% pepsinized type I collagen solution (derived from porcine tendon, Nitta Gelatine, Osaka, Japan), HEPES/ NaOH neutralizing solution, concentrated Eagle's medium, cell suspension, and fetal calf serum (FCS) were mixed at below 4 ~ C. One millilitre of the mixture was poured into a 35-ram bacteriological dish (Falcon) and incubated at 37 ~ C. The final collagen concentration was 2.0 mg/ml and the initial cell density was 2 x 10 s cells/ml. After gelling, 2 ml of Eagle's medium supplemented with 10% FCS was added. Addition of DTIC or RA was started on day 4. DTIC was activated with exposure to white light [8]. Culture medium supplemented with FCS and these reagents was changed every other day. At indicated times gels were digested by collagenase at 37 ~ C for 40 rain and released viable cells were counted with a hemocytometer using the dye exclusion test.