Undergraduate Research Assistant Biomedical Engineering Society Pittsburgh, Pennsylvania, United States
Introduction: Breast cancer is the most often diagnosed and second deadliest form of cancer in women and will account for an estimated 42,000 deaths in 2024. HER2-positive (HER2+) breast cancer is a subtype of breast cancer characterized by the overexpression of human epidermal growth factor receptor 2 (HER2) protein. Studies suggest that patients with advanced HER2+ breast cancer have a 30-50% chance of disease recurrence, which indicates a need to discover new therapeutic strategies. The tumor microenvironment, most abundant with stromal fibroblasts, plays a critical role in the progression, metastasis, and therapy response of breast cancer. Previous studies show that fibroblasts communicate through paracrine signaling and reduce the sensitivity of HER2+ breast tumor cells to the HER2-targeted therapy lapatinib through protective signaling. Complex and dynamic communication between tumor cells and fibroblasts primarily occurs through paracrine or juxtacrine (adjacent) signaling mechanisms. To further dissect these mechanisms and understand how they play a role between tumor cells and fibroblasts, we measured the drug response of tumor cells cultured with fibroblasts or fibroblast-conditioned medium in a 2D and 3D system and performed endpoint staining to probe pathways altered by these factors. We will compare the effects of paracrine and juxtracrine signaling by designing conditions that separate signaling mechanisms in a 2D system. A more comprehensive understanding of the communication between stromal fibroblasts and tumor cells in the tumor microenvironment could open new insights and strategies to overcome fibroblast-mediated therapy resistance.
Materials and
Methods: Protein expression was measured using reverse phase protein arrays. Breast cancer cell lines were cultivated in RPMI with 10% fetal bovine serum (FBS), while AR22 mammary fibroblasts were grown in DMEM with 10% FBS cultured at 5% CO2 and 37°C conditions. UACC812 and BT474 cell lines were modified to express H2B-GFP and mCherry, respectively. To prepare conditioned medium, AR22 fibroblasts were cultured in DMEM for five days, followed by four days in RPMI before harvesting and filtering. This medium was diluted (1:3) in RPMI for experiments. In 2D experiments, cancer cells were seeded at 12000 cells/well in RPMI or conditioned medium in 24-well plates. Fibroblasts were either directly cocultured with tumor cells or placed in the transwell compartment at 12000 cells/well. Plates were processed for immunofluorescence imaging using primary and secondary antibodies, and images were captured with a Nikon Ti2 microscope. After 48 hours, cells were treated with lapatinib (0-1.0 µM). For 3D experiments, cancer cells were seeded in 3 µL collagen type I (2mg/mL) droplets (40x10^6 cells/mL) in 96-well plates using RPMI or conditioned medium. In coculture conditions, fibroblasts were included in the droplet. Following 48 hours, lapatinib treatment (0-3.0 µM) was applied. Sytox Deep Red (50 nM) was added to identify dead cells, and cell viability was measured using widefield fluorescence (2D) or confocal images (3D) from an ImageXpress Micro confocal. Data analysis for immunofluorescence and cell viability was performed using CellProfiler.
Results, Conclusions, and Discussions: To test the impact of paracrine and juxtracrine signaling, UACC812 and BT474 tumor cells were cultured under various conditions (monoculture, conditioned media, coculture, and transwell) in 2D. Fractional viability, measuring the ratio of live cells to the total number of cells, revealed differences in drug response. UACC812 cells showed a unique response to lapatinib, with transwell conditions yielding higher fractional viability and a distinct response curve compared to direct coculture and conditioned media, indicating differences in signaling mechanisms and drug response (Fig.1A). BT474 cells showed similar drug response curves for coculture and transwell at 0.1 uM, but the endpoint fractional viability was 0 for both conditions at 1.0 uM, making it difficult to differentiate the mechanisms leading to cell death (Fig.1A). Culturing UACC812 cells in 3D showed expected growth inhibition between untreated and treated conditions (Fig.1B). Given the differences in drug response between transwell and coculture conditions, Reverse Phase Protein Arrays indicated that DDR1 levels are higher in UACC812 cells cultured in treated conditioned media (Fig.1C). Based on drug response and RPPA data, immunofluorescence staining for PCNA, pDDR1, pHER2, and pERK markers was performed on UACC812 cells cultured in 2D (Fig.1D). The staining revealed that PCNA, pDDR1, and pERK are expressed at higher levels in direct coculture treated with lapatinib (Fig.1D). pHER2 staining showed expected lower levels across al l treated conditions, verifying drug efficacy (Fig.1D). Evaluated drug responses revealed that UACC812 cancer cells show a different response in treated conditions when compared to transwell and coculture in 2D. Drug response assays in 3D exhibited expected cell viability in untreated and treated conditions. Immunofluorescence staining revealed that cell proliferation and cancer metastasis (PCNA), collagen-mediated signal mechanisms (pDDR1), and overall cellular activity and growth (pERK) are upregulated in direct coculture when treated with lapatinib. This suggests that direct coculture induces juxtracrine signaling pathways that may lead to increased cancer cell survivability and proliferation. Understanding the mechanisms of communication between tumor cells and fibroblasts could lead to new therapeutic strategies. Blocking fibroblast communication with tumor cells might overcome fibroblast-mediated therapy resistance.
