Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Abstract

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

Figure 1:. Experimental workflow.

(A) Schematic of hydrogel formation. Digital camera images were taken of MFPs pre- (B) and post-decellularization (C).

Figure 2:. Confirmation of decellularization and de-lipidation in MFPs.

Hematoxylin and eosin staining (H & E) of unirradiated MFPs embedded in paraffin and sectioned at 5 μm (A) was compared to MFPs frozen in cryostat embedding medium (5 μm sections) before (B) and after decellularization (C), incubated with sucrose prior to freezing in cryostat embedding medium and sectioned at 30 μm (D). 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining was done to visualize lipid retention in MFPs frozen in cryostat embedding medium (5 μm sections) before (E) and after decellularization (F) and incubated with sucrose prior to freezing in cryostat embedding medium and sectioned at 30 μm sections (G). Scale bars represent 50 μm. Decell = decellularization.

Figure 3:. Confirmation of hydrogel formation.

Rheology was used to determine the storage and loss modulus of control (A) and irradiated (B) pre-gel solution made from MFPs at 37 °C and 0.5% strain. Error bars show standard deviation.

Figure 4:. Tumor cell proliferation in irradiated ECM hydrogels.

4T1 cell proliferation 48 h after inoculation is shown with pre-gel derived from control (A) and irradiated (B) MFPs. (C) Bioluminescence signal from 4T1 cells embedded within control and irradiated hydrogels. Calcein AM stained live cells and ethidium homodimer stained dead cells were evaluated in control (D) and irradiated (E) hydrogels, and the live/dead ratio was quantified (F). Scale bars represent 200 μm. Error bars show standard error.

Figure 5:. Cytoskeletal properties in ECM hydrogels.

Cells within (A) control and (B) irradiated ECM hydrogels are stained with phalloidin conjugate to visualize F-actin (red) and blue fluorescent dye to visualize nuclei (blue) in irradiated MFPs. Scale bar represents 100 μm.