Poster S11 - Characterization of Oncofetal Fibronectin in Tumor Derived Matrices

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jessica Longstreth

Research Assistant
Temple University
Cherry Hill, New Jersey, United States

Co-Author(s)

Ghazal Bashiri

Research Assistant
Temple University, United States

Primary Investigator(s)

KW

Karin Wang, PhD

Assistant Professor
Temple University, United States

Introduction: Despite promising advances in treating even aggressive forms of breast cancer (1), it remains the most diagnosed cancer worldwide and (2) and metastatic tumors often develop resistance to current therapies or have higher rates of recurrence (1). The surrounding extracellular matrix (ECM) and cancer-associated cells, which make up the tumor microenvironment (TME), contribute to the metastatic potential of the tumor (3–6); however, investigation of how TME structure and composition affects tumor behavior is still unknown. Decellularized ECMs produced by cancer associated fibroblasts (CAFs) offer a promising in vitro platform for understanding how TME characteristics such as matrix composition and microarchitecture affect tumor cell behavior. Unlike animal or whole-tissue derived matrices, cell derived matrices can be tuned to study the effects of specific variables, such as fibroblast type, cancer type, and even patient demographic (7). For instance, oncofetal fibronectin (oFN), which is formed when a glycotransferase adds a GalNAc unit at a specific threonine residue of the type-III connecting segment of FN, has increased expression in multi-drug resistant cancer tissues (8). oFN contributes to epithelial-mesenchymal transition, a hallmark of cancer progression (9,10). But exploration into how oFN affects ECM structure-function and the specific mechanisms by which it aids in cancer progression is largely unknown (8). Here, we use MDA-MB-231 (a metastatic breast cancer cell line) conditioned human dermal fibroblasts (HDFas) as a model of cancer-associated fibroblasts to produce decellularized matrices containing oFN and identify distinct matrix characteristics that may facilitate cancer progression.

Materials and

Methods: MDA-MB-231 (ATCC #HTB-26) cells cultured in Dulbecco’s Eagle Modified Medium (DMEM) (Gibco #11995073) (10% fetal bovine serum [FBS] [Gibco #25200072] and 1% penicillin-streptomycin [P/S] [Gibco #15140122]) were used to collect metastatic tumor conditioned media (mTCM). 24 hours prior to collecting mTCM, fresh low serum (1%) media was provided. DMEM media without cells underwent the same collection process to act as a control treatment (hereon referred to as INC media).

HDFa (ATCC # PCS-201-012) cells were then preconditioned with either mTCM or INC media for 3 days prior to seeding 500,000 cells per gelatin coated glass-bottom 35 mm petri dishes. Cells were fed every 48 hours with their appropriate media supplemented with 50 μg/mL L-ascorbic acid (FisherScientific #AC401471000). After 7 and 10 days, some samples were fixed in 4% paraformaldehyde (PFA) solution (FisherScientific #AAJ61899AK) while other parallel samples were decellularized as described by the Cukierman group (11) and fixed in 4% PFA solution.

Dishes with cells were stained with DAPI (1:5000) (LifeTechnologies #D1306), and Alexa Fluor 568 Phalloidin (1:250), collagen I monoclonal antibody (1:250) (Life Technologies #MA126771) and polyclonal fibronectin (FN) antibody (1:200) (LifeTechnologies #PA529578). Decellularized plates were not stained with phalloidin. Additional decellularized samples were immunostained for oFN (1:500) (Creative Biolabs #AGM-190YJ) against total FN.

Four regions of interest were imaged per condition using an Olympus H2P confocal microscope with a 20x objective. Images were analyzed using Color Pixel Counter and OrientationJ Dominant Direction Fiji plugins. Statistical analysis was performed in GraphPad Prism.

Results, Conclusions, and Discussions: Matrices were decellularized and immunostained to highlight collagen I and FN (Fig. 1A-B). There was no significant difference in FN area between the INC and mTCM groups. (Fig. 1C). The 7 day mTCM group had significantly more area covered by collagen I than the INC group (p = 0.0032) (Fig. 1D). This matches previous studies in which tumor conditioned matrices contain more collagen (3). Increased fibrillar collagen I is correlated with lower survival rates and contributes to metastatic signaling (12,13). Although a difference at 10 days was not observed, this data set is limited and further biological replicates are required.
Notably, oFN was found in the INC group and mTCM group at both timepoints (Fig. 2A-B). There was also more oFN than total FN in most of the samples (Fig. 2C). This was unexpected, as oFN is absent in normal adult tissue (14). This suggests that the fibroblasts are in a myofibroblast state, even in the control group. To provide a better control group, it may be useful use MCF-10A conditioned media, as this cell line is often used as a model for normal human mammary epithelial cells (15). Additionally, human mammary fibroblasts (ScienCell #7063) will be used in future experiments as they are more relevant to the breast cancer model. After 7 days, the INC group had more total FN and oFN colocalization than the mTCM group (p = 0.0001) (Fig 2D), suggesting that the oFN matrix may have functions beyond that of the overall FN matrix – an occurrence seen with other FN variants (16). Coherency (i.e., the fraction of identified fibers sharing the same orientation) of the oFN matrix was higher than the total FN in the 7 day mTCM group (p = 0.0031) (Fig. 2E). This implies the oFN fibers are more aligned than typical FN fibers which may contribute to a metastatic TME by promoting directional migration (17).

The utility of fibroblast-derived matrices goes beyond identifying differences in matrix composition and architecture. In the future, cancer organoids can be seeded onto matrices to observe the effect of specific characteristics on cell-integrin signaling and migration.

Acknowledgements (Optional): References
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