Developing Collagen-Binding Integrin Knockdown Cell Lines for 3D Breast Cancer Spheroid Migration Models

Introduction: Distinguishing low- from high-risk early-stage breast cancer tumors is difficult due to the heterogeneity of cancer progression. The identification of biomarkers to differentiate invasive cancers may reduce breast cancer overtreatment and positively impact the sense of well-being, health, and life expectancy of patients. Therefore, it is essential to develop biomarkers to differentiate invasive cancers. The extracellular matrix (ECM) provides a milieu of pathways that either promote or impede tumorigenesis. Collagen is a dominant protein within the ECM and its organization impacts cancer cell migration and metastasis. Therefore, collagen organization is likely a critical biomarker; however, the process of collagen deposition and remodeling is not well understood. Integrins are a transmembrane protein eliciting signaling cascades that both impact collagen deposition and organization as well as internal cellular pathways critical for cellular migration. In this work, we are focused on examining the role of collagen-binding integrin α2β1. The expression levels and role of α2β1within breast cancer progression is conflicting. Some correlate increased α2β1 expression levels with increased cellular migration and metastasis while other aggressive breast cancers do not have elevated α2β1. Therefore, we are using the MCF10A cell series – multiple cell lines that mimic the progression of breast cancer from non-tumorigenic to metastatic – to develop 3D in-vitro integrin α2β1 knockdown spheroid models to understand the impact of α2β1 on cellular migration. The goal of this research is to understand if α2β1 may be both a potential biomarker as well as a potential therapeutic option for breast cancer patients.

Materials and

Methods: Knockdown (KD) and negative scrambled shRNA control (RNA-ctrl) cell lines from three cell lines in the MCF10A cell series [MCF10A (non-tumorigenic, M1), MCF10AT (pre-malignant, M2), and MCF10CA1a (metastatic, M4)] were generated via shRNA transduction using lentiviral particles targeting integrin α2β1 (protocol followed as supplied by Santa Cruz Biotechnology). COL1 adhesion assays (1 µg/cm2) were used to increase the KD efficiency to at least 60% compared to the RNA-ctrl, validated with fluorescent labeling and Western blotting. Spheroids were generated at an initial seeding density of 4,000 cells per well with 2.5% Matrigel using the centrifugation method. They were embedded 72 hours after generation in 150 µL of 2 mg/mL COL1 hydrogel in a clear, flat bottom 96 well plate (1 spheroid/well). The hydrogel contained an equal volume of acid-solubilized rat tail COL1 and neutralizing solution (100 mM HEPES, pH 7.3 in 2X PBS) with the correct collagen concentration achieved by adding 1X PBS. The gel was allowed to fully polymerize for 90 minutes in an incubator (37°C, 5% CO2) before the addition of 150 µL of serum/phenol red free DMEM/F12 cell media. Using a Cytation 5 Multimode Reader with environmental controls, a 3D stack of widefield brightfield images was captured every 12 hours for a total of 72 hours. A single image projection was produced using the focus stacking method for each time point. A custom FIJI and RStudio image analysis pipeline was created to quantify the migration distance and number of migrating cells on a single cell level.

Results, Conclusions, and Discussions: The M4 metastatic cell line was first explored (the M1 and M2 cell lines are in progress). After the shRNA transduction, there were varying levels of integrin α2β1 seen in the KD cell line. Therefore, adhesion assays were completed to isolate the KD population (Figure 1). Furthermore, a time course series using fluorescent labeling of integrin α2β1 was completed to ensure KD efficiency remained above 60% for all subsequent experimentation compared to the negative scrambled shRNA control (RNA-ctrl) cells (Figure 1b). Current results demonstrate significant reduction of the KD compared to the RNA-ctrl for at least 9 passages (~ 4-5 weeks) after the adhesion assay (Figure 1b). Once KD efficiency was validated, 3D in-vitro spheroid models were generated for cellular migration experiments. To be a viable pre-clinical model, spheroid screening is a must to ensure uniformity and reproducibility. Therefore, widefield imaging was used to screen and omit spheroids prior to experimentation based on size and morphology (diameter, circularity, and roundness). In our cellular migration trials, there were significant reductions in both cellular migration distance and the number of migrating cells in the integrin α2β1 KD spheroids compared to the RNA-ctrl spheroids at all time points evaluated (Figure 2). These initial trials indicate further exploration into the role of integrin α2β1 and how collagen organization is impacted with reduced levels of integrin α2β1. Additional trials are underway for the M1 and M2 cell lines in the series to understand how integrin α2β1 impacts cellular migration in different stages of breast cancer progression. We plan to expand our imaging acquisition to include Second Harmonic Generation microscopy, a label-free, collagen specific imaging modality to evaluate any correlation between COL1 structural patterns, a likely critical biomarker of breast cancer progression, and integrin α2β1 expression levels. Lastly, we are in the process of increasing our sampling speed in the migration trials from 12 hours to potentially achieve single cell tracking in the model.

Acknowledgements (Optional): The authors would like to thank Drs. Peter Brooks, Jennifer Caron, Xianghua Han, and Allison M. Cross for their support. This work was funded by the Agilent Research Gifts #4624 and #4845, NIH T32 Training Grant #1T32GM132006-01, Bioscience Association of Maine (BioME) Seed Grant, Northern New England Clinical Oncology Society (NNECOS) Student and Trainee Project Grant, and the University of Maine NIH COBRE Pilot Grant.