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Cancer Technologies

Cancer Technologies - Poster Session D

Poster G9 - Investigation of Collagen-I-induced Endocrine Resistance in 3D Co-Cultured ER+ Breast Cancer and Fibroblasts Spheroids

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Estrogen receptor positive (ER+) breast cancer is the most common form of breast cancer and will respond to endocrine therapy through direct inhibition of the ER; however, some patients exhibit de novo or developed resistance. Traditional two-dimensional (2D) single-cell type culture approaches do not adequately recapitulate the complex nature of the cancer tumor microenvironment (TME). Cell-to-cell and cell-to-matrix interactions between cancer and stromal cells in the TME, such as the enhanced expression of the extracellular matrix (ECM) protein collagen-I, are suspected to drive endocrine resistance in ER+ breast cancer. This motivates the need for 3D co-culture techniques, such as the microfluidic generation of co-cultured spheroids, to provide a new approach to grow and evaluate cell-induced changes in the TME. A microfluidic co-culture approach leverages greater control and higher throughput in conjunction with a more realistic replication of the TME as an improved pre-clinical model that mimics potential in vivo results and patient outcomes. Within the tumors, stromal cells like fibroblasts and cancer-associated fibroblasts (CAFs) at the primary and secondary sites promote tumorigenic behaviors. The increased collagen-I production from the CAFs can interact with receptors on the cancer cells to trigger proliferation and metastasis. The instigation of pro-tumorigenic behaviors from increased collagen-I expression in the ECM can also create a resistance to various treatments, such as endocrine therapies.

Materials and

Methods:  This work leverages a droplet microfluidic trapping array (Fig. 1A) combined with a thiol-acrylate (TA) hydrogel to grow, interrogate, and evaluate the influence of fibroblasts on endocrine resistance in a model ER+ breast cancer cell line. The fibroblast and cancer cell-laden hydrogel droplets are isolated in a 450-member trapping array. Initially, the TA hydrogel has a low viscosity, allowing the formation of the cell-laden droplets within the trapping array. Such droplets are formed at a flow-focusing junction (Fig. 1B) in a continuous oil phase within the microfluidic device. Over time, the hydrogel polymerizes, creating a temporary scaffold that supports spheroid formation during their early stages of growth (Fig. 1C-D). After the gelation of the hydrogel, the oil is replaced with culture media via a gravity-driven infusion system to constantly supply growth media to prevent cell stagnation (Fig. 1E). The spheroids are cultured for seven days (Fig. 1F), followed by terminal immunostaining for collagen-I expression and the proliferative marker Ki67 . The fluorescent images are analyzed using Fiji with several quantitative measurements collected from the images, including the size of the spheroids and the sizes and intensities of the fluorescent signals to determine the distribution of collagen-I and Ki67expression within the spheroids. The spatial distributions of localized collagen-I expression were investigated using data clustering to identify distinct subpopulations of 3D co-cultured spheroids.

Results, Conclusions, and Discussions: Prior work has identified that two distinct cohorts exist with respect to collagen-I expression in 3D co-culture spheroids. Terminal immunostaining for collagen-I in 3D spheroids containing an ER+ breast cancer cell line (MCF7) and primary keloid fibroblasts identified two distinct cohorts based on what was termed ‘hotspots’ – localized regions of collagen-I expression within the spheroids. Cohort 1 (Fig. 2, 3rd row) exhibited a single, small hotspot with low fluorescence intensity correlating to low collagen-I expression, while cohort 2 (Fig. 2, 4th row) had multiple, larger hotspots with high fluorescence intensity. Interestingly, single MCF7 spheroids exhibited low homogeneous collagen-I levels (Fig. 2, 1st row), whereas single fibroblast spheroids exhibited high homogeneous collagen-I levels (Fig. 2, 2nd row), suggesting the unique spatial distributions are driven by the cell-to-cell communication between the two cell types. However, these studies did not evaluate how collagen-I alters the endocrine response or if there are spatial alignments between collagen-I and Ki67 expression. Moreover, prior studies have used keloid fibroblasts, which are not found at the metastatic site. This study aimed to address this through the 3D co-culture of MCF-7 cells with lung-derived fibroblasts (MRC-5) to recreate the metastatic TME. Using the microfluidic approach, we found that the MRC-5 line resulted in similar variations of collagen-I expression when compared to the primary keloid fibroblasts, suggesting a similar heterogeneity at the single spheroid level. Ongoing work using the co-culture approach should also reveal an increased proliferative rate within Cohort 2 compared to Cohort 1. This data will be a foundation for future endocrine resistance testing by exposing the co-cultured spheroids to an endocrine therapy (fulvestrant) and immunostaining for collagen-I and the proliferation marker Ki67. The hypothesis is that increased resistance to fulvestrant, indicated by enhanced Ki67 expression, is due to increased collagen-I expression in the spheroids. The findings from this study will provide new insight into companion therapeutic targets for ER+ breast and confirm that collagen-I production leads to increased endocrine resistance.

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