(H-288) Investigating the Effect of Increased Adipocyte Density on a 3D Human Breast Tumor Spheroid Model

Introduction:: Obesity has been reported to increase the risk of breast cancer. Those with obesity who have breast cancer also have a poor prognosis with increased rates of metastasis and decreased rates of cancer-free survival. Current obesity models^1,2 do not use adipocytes and instead utilize adipose-derived stem cells or are 2D models that don’t recapitulate the 3D environment of the human body. Mice are often used to model obesity but lack human physiology. While the impact of obesity on breast cancer risk is clinically clear, the effect of an increased adipocyte density within the breast tissue, which occurs in the case of severe obesity, has not yet been investigated.

Materials and Methods:: To create the 3D obese breast tumor model, we first differentiated human adipose-derived stem cells (hASCs) into adipocytes. We created tumor spheroids by culturing MDA-MB-231 cells within Matrigel. Adipocytes were printed within a collagen gel then tumor spheroids were aspirated and printed within the adipocyte-laden collagen gel and allowed to gelate before culture. The models were first validated through live/dead staining and Nile red staining to confirm that the cells were alive within the gels and that two different adipocyte densities were present within our models. Then, the models were cultured for two weeks while performing an Alamar blue assay every third day. After the culture period, the models were sectioned and stained for Ki67, MMP-2, MMP-9, ZEB2, and E-Cadherin.

Results, Conclusions, and Discussions::
After 3D bioprinting the models, we first characterized it through brightfield imaging, live/dead staining, and Nile red staining to confirm its viability. We showed that the cells had over 66% viability and the Nile red staining confirmed that we had two models with different adipocyte densities. Then, we investigated the effect of an increased adipocyte density on the tumor spheroid within our model. We saw an increase in the proliferation of the tumor spheroid within the higher-density adipocyte model through an Alamar blue assay and Ki67 staining (Figs. 1A and 1B). Finally, we showed that the tumor spheroid (expressing GFP) within the higher density adipocyte model has an increase in tumor progression factors MMP-2 (Fig. 1C), MMP-9 (Fig. 1D), and ZEB2 (Fig. 1E), and a decrease in tumor suppression factor E-Cadherin (Fig. 1F).
Our study shows that increasing adipocyte density within our obese model causes an increase in proliferation and tumor progression factors and a decrease in a tumor suppression factor. These results suggest that the increased adipocyte density is another contributing factor to how obesity supports breast cancer progression. This work is the first of its kind to utilize 3D bioprinting of fat tissue for breast cancer research. This model will allow for the recapitulation of a more human replicative environment, through utilizing human cells and collagen, and can contribute to the field of breast cancer research through testing novel breast cancer drugs on the model in the future. The 3D bioprinted obese breast cancer model better replicates the severe obese microenvironment and helps to solve issues of previous models.





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References (Optional): :
1. Springer NL, et al. Am J Pathol; 2019, Vol. 189, Iss. 10, Pages 2019-2035




2. Fischbach C, et al. Exp Cell Res; 2004, Vol. 300, Iss. 1, Pages 54-64