Poster S17 - Engineering the 3D Melanoma Tumor Microenvironment In Vitro Using Extracellular Matrix Enhanced Spheroids

Introduction: In vitro cancer cell culture models, such as spheroids, are efficient and inexpensive ways to recapitulate tumors in vivo. Melanoma is the most aggressive form of skin cancer, yet many melanoma cell lines do not form 3D spheroids under standard non-adherent culture conditions in vitro, which limits modeling capabilities for drug discovery and mechanistic studies. We have found that cancer cell lines that are resistant to forming spheroids can be facilitated by addition of extracellular matrix (ECM) hydrogel precursors during the spheroid formation process (MatriSpheres). We hypothesized that we could successfully form spheroids from melanoma by matching an appropriate ECM milieu. We therefore examined ECM organization in an in vivo genetically engineered mouse tumor model to inspire ECM compositions that may facilitate 3D spheroid formation in the B16-F10 melanoma cell line.

Materials and

Methods: We first characterized the in vivo tumor ECM microenvironment of the Cdk4(R24C)/Hgf-tg genetically engineered melanoma allograft model via histology and immunofluorescent (IF) staining of collagen hybridizing peptide and the melanoma marker gp100. Collagen intensity was displayed via a heatmap look-up table. B16-F10 cells were cultured under standard conditions and used for spheroid optimization in ultralow attachment round bottom 96 well plates. Matrigel was used as a basement membrane (BM) rich ECM formulation that lacks type I collagen. Decellularized tissue ECM was prepared from porcine intestine (SIS-ECM) as a collagen-rich control. We tested collagen-rich ECM formulations diluted to 125 µg/mL and BM-rich ECM formulations diluted to 31.25 µg/mL, 62.5 µg/mL, and 125 µg/mL concentrations with RPMI cell culture media. The plate of spheroids was placed in a standard 37°C incubator and maintained for seven days, with media changes every three days. Spheroids were imaged on day 3 to day 7 via the Molecular Devices ImageXpress. The spheroids were analyzed via the ImageXpress by inverting the images and calculating the area of dense clusters of cells in the well. The area was then used to calculate the diameter of each spheroid.

Results, Conclusions, and Discussions: In vivo melanoma tumor IF staining showed a tumor microenvironment consisting of gp100+ melanocyte tumors and overall exhibited poor collagen deposition that was primarily limited to the tumor margin and necrotic areas (Fig. 1A,1B). Only very thin collagen fibers observed between melanoma cell nests (Fig. 1C,1D) suggesting a collagen rich matrix does not support their 3D formation. In vitro, B16-F10 cells formed intact MatriSpheres with the addition of 31.25 µg/mL and 62.5 µg/mL concentrations of Matrigel. Concentrations of 125 µg/mL SIS-ECM and 125 µg/mL B16-F10 spheroids did not fully aggregate. B16-F10 cells alone formed a flat sheet of cells at the bottom of the plate (Fig. 1E). The average diameter of the spheroids formed in the 31.25 µg/mL and 62.5 µg/mL concentrations of Matrigel were 1020.68 µm and 826.92 µm, respectively (Fig. 1F). Because melanoma tumors are collagen poor, B16-F10 MatriSpheres form well at low concentrations because the cells may prefer little to no collagen in their environment. Specific cell lines likely prefer conditions that resemble the in vivo TME.
Low concentrations of Matrigel are effective in the facilitation of B16-F10 MatriSpheres. Matrigel concentrations that are too high inhibited formation, as were collagen rich formulations suggesting that ECM composition must be carefully tuned in a tumor type specific manner. The ECM components added to cancer cells to form spheroids should reflect the structure of the in vivo TME. These methods enable 3D testing in spheroid-resistant cell lines for applications such as in vitro recapitulation of in vivo TME.

Acknowledgements (Optional):