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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session E

Poster K6 - Three-dimensional Co-culture of Adipose-derived Stem Cells with Estrogen Receptor Positive Breast Cancer Cells Using a Microfluidic Droplet Trapping Array to Study Endocrine Resistance

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Estrogen receptor positive (ER+) breast cancer affects 1 in 8 women in the United States and is commonly treated with endocrine therapies. However, endocrine resistance has been found at both the primary and metastatic sites, which is attributed to the complex nature of the tumor microenvironment (TME). The TME is an intricate system containing malignant cells, stem cells, and the extracellular matrix (ECM). Crosstalk between cancer cells and other cells found in the TME has been identified as a potential driving factor in cancer progression and drug resistance. For example, adipose-derived stem cells (ASCs), stromal cells in the TME, have demonstrated their ability to repair and enhance regional tissues. Additionally, ASCs modify the ECM by secreting proteins to organize cells, enhancing cancer cell proliferation. Previous ASC-breast cancer crosstalk studies relied on established two-dimensional (2D) culture systems, which fail to imitate in vivo conditions. Three-dimensional (3D) culture systems utilizing hydrogel scaffolds and droplet microfluidics have proven to be ideal approaches to creating 3D spheroids as they more accurately depict the elemental crosstalk of TME components and the cellular response to therapeutics. This study aims to leverage an established droplet microfluidic platform that utilizes a thiol-acrylate (TA) hydrogel scaffold to investigate cancer-ASC crosstalk in 3D co-culture spheroids. This will be accomplished by studying how the presence of ASCs alters the proliferative behavior and endocrine response in two model ER+ breast cancer cell lines.

Materials and

Methods:  The 3D co-cultured spheroids were generated using a droplet microfluidic trapping array (Figure 1A). The microdevice generates droplets containing cells and unpolymerized TA hydrogel in a continuous oil phase, which are trapped in a 300-member downstream circular trapping array. Two different ER+ breast cancer cell lines (MCF-7 and Zr-75) were single- or co-cultured with ASCs through encapsulation and trapping in the device. The device’s gravity-driven flow system continually infuses media and/or endocrine therapy (fulvestrant) without backflow or contamination (Figure 1B). Spheroids are grown for 3 days with full media, followed by 24 hours of stripping, 9 hours of fulvestrant induction, and 48 hours of estrogen exposure. Terminal immunostaining was performed using a fluorescent inverted microscope (Leica DMi8) equipped with an FITC filter, DAPI filter, and brightfield. The dataset, which contained numerous images, was quantified using FIJI software to measure the fluorescence intensity of the proliferative marker (Ki67), monitor spheroid formation, and track size changes within the brightfield images. Metrics, including the perimeter of the whole spheroid, the core spheroid perimeter, and the background fluorescence, were measured using FIJI software, which calculates the area and mean fluorescence intensity within an outlined measurement (Figure 2A). The spheroid measurements are then standardized by removing the background fluorescence intensity, which allows for a more accurate analysis. This rich dataset was clustered using K-means and HDBSCAN methods to identify subpopulations of cells based on area and normalized fluorescence intensity.

Results, Conclusions, and Discussions: Initial studies of single-cultured MCF-7 3D spheroids treated with fulvestrant endocrine therapy exhibited diminished proliferation. Additionally, a higher dose of fulvestrant was required to inhibit proliferation in 3D spheroids versus 2D-cultured cells. Past works suspected that the presence of ASCs in 3D spheroids could further enhance resistance to endocrine therapies. To investigate this, 3D co-cultured spheroids were allowed to grow under one of three conditions: (1) no treatment, (2) treatment with estrogen (E2) to induce proliferation, or (3) treatment with fulvestrant (ICI) followed by treatment with E2 to determine the inhibitory effect of the drug on the estrogenic response. The mean fluorescence intensity of the Ki-67 marker assesses alteration in estrogen-mediated proliferation. The co-cultured 3D spheroids exhibited an interesting response under the three conditions. The ASC:MCF-7 3D spheroids did not show a significant difference between the no treatment and E2 treatment conditions, suggesting that ASC presence replaces the need for exogenous E2 to induce proliferation. Interestingly, we observed enhanced proliferation in the ASC:MCF-7 spheroids in the E2+ICI treatment, suggesting that ASCs not only confer endocrine resistance but also enhance proliferation in the presence of the drug. This trend was not observed in the ASC:Zr75 3D spheroids, where the no treatment condition showed the highest level of proliferation, followed by the E2 treatment and then the E2+ICI treatment. Both co-cultures’ vehicle and E2 treatment groups show remarkably similar proliferation levels despite the added estrogen in the E2 treatment. These treatments respond uniformly because the 3D co-culture of ER+ breast cancer and ASC mimics extracellular stimulation of proliferation similar to estrogen (E2) induction. The ICI+E2 treatment groups result in a conflicting endocrine response with enhanced resistance and proliferation in MCF-7 cells and diminished resistance and proliferation in Zr-75 cells. These findings provide new insight into the role of ASCs in the proliferative nature of ER+ breast cancer, as well as ASCs’ response to endocrine therapies. Ongoing work is underway to further characterize this response and to study how different ASC attributes (e.g., patient age or BMI) affect endocrine response in ER+ breast cancer.

Acknowledgements (Optional):  LSU Discover Undergraduate Research Program