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Cancer Technologies

Cancer Technologies - Poster Session E

Poster S16 - Developing a Platform to Investigate Matrix Remodeling in the Tumor Stroma

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

Introduction:  The extracellular matrix (ECM) is an important, dynamic component of the tumor microenvironment[1]. As the tumor stroma evolves, the ECM is continuously remodeled, leading to spatial heterogeneity in ECM protein deposition and microarchitecture. The initial tumor stroma ECM (early) and the remodeled tumor stroma ECM (mature) could have dramatic differences in how they dysregulate cell signaling in the tumor microenvironment. Fibronectin (FN) is the first ECM protein assembled by cells and acts as a template for collagen assembly[2]. FN regulates various cell functions such as adhesion, differentiation, proliferation, and migration[3]. The extra domain A (EDA) FN is a splice variant of FN expressed in tumor stroma and strongly correlated with tumor growth and metastasis[4]. The EDA is an extra repeating FN domain included between FNIII11 and FNIII12 and its overexpression is often correlated with enhanced ECM remodeling, matrix metalloproteinase (MMP) expression, actin cytoskeleton reorganization, and fibrotic stiff tissue. The impact of tumor stroma evolution on ECM remodeling, including changes in composition, the insertion of the EDA site within FN, and alterations in microarchitecture remains poorly understood. Furthermore, how these changes regulate the behavior of surrounding cells requires further investigation. Therefore, to address these questions, in this study, we developed a tumor stroma screening platform, which enables us to investigate the evolving composition and microarchitecture of the ECM, as well as the presence of EDA-FN isoform, within tumor-conditioned cell-derived matrices.

Materials and

Methods:  Tumor-associated human dermal fibroblast cells (HDFA) (p4) were used as an in-vitro model of cancer-associated stromal cells. Tumor soluble factors (TSF) from an aggressive metastatic breast cancer line (MDA-MB231) were collected, concentrated 10X, and used to differentiate HDFA into cancer-associated fibroblasts (CAFs) (Fig.1). HDFA cells were conditioned in MDA-MB231-derived medium containing TSF (experimental group) or in low-serum medium containing 1% FBS (control group) for 3 days[5]. Next, the preconditioned cells were trypsinized and seeded onto gelatin-coated glass insets within 35-mm Petri dishes. Half of the samples were decellularized at days 7 and 14 simulating the early and mature stages of the remodeled tumor stroma, while the other half of the samples were fixed with cells on days 7 and 14[6]. To confirm the proper decellularization process and the potential differences in the composition of tumor-derived and control matrices, cell-derived ECMs were fixed and immunofluorescently stained for nuclei (DAPI, ThermoFisher), total FN (Polyclonal FN antibody, ThermoFisher), collagen type I (Col I antibody, ThermoFisher), EDA FN (IST-9, Abcam), and F-actin (AlexaFluor 568 Phalloidin, ThermoFisher). Confocal images were acquired with an Olympus IX 81 (Olympus Life Sciences) and 20x objective; fluorescence and microarchitecture were characterized via ImageJ. Color Pixel Counter, an ImageJ/Fiji Plugin, was used to calculate the percentage of area covered by either FN or Col I. JaCoP, an ImageJ/Fiji plugin was used to calculate the colocalization of EDA-FN and total FN. One-way ANOVA, Tukey’s multiple comparisons post hoc, and unpaired t-test analysis were performed in GraphPad Prism 9.

Results, Conclusions, and Discussions: We successfully developed a cell-derived ECM platform simulating early and mature tumor stroma. Fig. 2 illustrates tumor-derived and control matrices before decellularization. Tumor-derived ECM decellularized at day 7 (early tumor-derived ECM), has a high amount of FN (Fig. 3A and B). At this initial stage of ECM assembly, although Col I is present, it does not exhibit an organized, fibrillar structure like FN (Fig 3A and C). This confirms previous studies demonstrating that the initial assembly of the FN matrix is necessary for enhanced deposition and assembly of Col l [2], [7]. Tumor-derived ECM decellularized at day 14 represents the mature tumor-derived ECM with enhanced Col l assembly into more organized fibrils, indicating the dynamic nature of the tumor microenvironment. Furthermore, for both matrices decellularized at days 7 and 14, tumor-derived ECM shows higher assembly and deposition of both FN and Col I compared to control ECM (Fig. 3B and C). This suggests that contrary to the highly dynamic nature of the tumor-derived ECM, control ECM is less remodeled. Fig. 4A shows the presence and colocalization of EDA-FN with total FN in tumor-derived ECM decellularized at day 7. The colocalization values for Pearson's correlation coefficients demonstrated that in tumor-derived matrices, EDA-FN is less colocalized with total FN compared to control matrices (Fig. 4B). The differential colocalization of EDA-FN and total FN between tumor-derived and control ECM can be contextualized by the extensive remodeling and microarchitectural changes in tumor stroma that can affect how ECM proteins are deposited, assembled and spatially organized. Our platform successfully mimics the ECM remodeling during tumor progression as it captures the changes in the spatial organization of EDA-FN between tumor and control-derived matrices. Moreover, dysregulated microarchitecture of the ECM in tumor stroma can directly affect cell behaviors such as adhesion, differentiation, and proliferation. Therefore, the platform that we developed, holds significant importance as it enables us for future studies of how evolving tumor-derived ECM microarchitecture dysregulates surrounding cell activities to promote tumor progression.

Acknowledgements (Optional):  

References:
[1] Z. Yuan et al., ‘Extracellular matrix remodeling in tumor progression and immune escape: from mechanisms to treatments’, Mol Cancer, vol. 22, no. 1, pp. 1–42, 2023, doi: 10.1186/s12943-023-01744-8.
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[6] J. Franco-Barraza, D. A. Beacham, M. D. Amatangelo, and E. Cukierman, ‘Preparation of extracellular matrices produced by cultured and primary fibroblasts’, Curr Protoc Cell Biol, vol. 2016, no. June, pp. 10.9.1-10.9.34., 2016, doi: 10.1002/cpcb.2.
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