We proposed to use cellular spheroids of co-cultured lung cancer cells and fibroblasts as a platform to evaluate the efficacy of anti-cancer drug combinations. We labelled the cancer cells and fibroblast with dyes of different colors, and employed selective plane illumination microscopy (SPIM) [1, 2] to provide a three-dimensional (3D) perspective of relative positions and amounts of the co-cultured cells. Therefore we were able to evaluate the drug effects on individual types of cells in the 3D co-culture environment.

The size of the spheroids strongly influences the evaluation of the drug effects. In order to unify the spheroid size, we used a microfluidic culture device that contained cubic chambers for confining the cellular spheroids [3]. Figure 1 shows the scheme and photo of the device used in the present work. The side wall of these culture chambers was flat such that the illuminating light sheet could propagate through without distortion. In the present work, the spheroids were kept in cubic chambers with a side length of 250 µm.

We found that the co-culture of CL1-0 lung cancer cells and MRC-5 fibroblasts could form a spheroid (diameter ~ 200 µm) much easier than the cancer cell alone. The fibroblasts were enclosed by the cancer cells in a spheroid, regardless of the seeding sequences. In contrast, while the cancer cells were co-cultured with bronchial epithelial cells BEAS-2B, the latter did not invade into the cancer cell aggregation (Fig. 2). This result implied that fibroblasts could play an essential role in the early stage of tumor formation.

Next, we used the co-culture spheroids to test the efficacy of a common anti-cancer drug cisplatin (CDDP) in combination with chloroquine (CQ), an inhibitor of cellular autophagy. We first used the total intensity of the fluorescent dye-labelled cells as a parameter to judge the drug efficacy. The addition of CQ enhanced the CDDP efficacy at 0.2 and 2.0 µM. With the SPIM images, we further realized that the survival rate of cancer cells in the co-culture spheroids was reduced by the addition of CQ, in comparison with the cases with CDDP only. In other words, CQ might selectively enhance the injury to the cancer cells in the co-culture spheroids. This result could not be revealed with the conventional cell viability test on the whole spheroid. We will report the results of other drug combinations in the conference.

The co-culture spheroids consist of cancer cells and stromal cells combined with 3D SPIM imaging could serve as a useful platform to investigate the tumor formation process and to test the drug combination efficacy. For the evaluation of therapeutic strategies, the results from experiments in 3D microenvironments could be more relevant than those from 2D experiments.

REFERENCES:

1. Huisken et al., "Optical sectioning deep inside live embryos by selective plane illumination microscopy," Science 2004, 305, 1007-1009.
2. J. Verveer et al., "High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy," Nat. Methods 2007, 4, 311-313.
3. Patra et al., "Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes," Biomicrofluidics 2014, 8, 052109.