This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.
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.
Cancer is characterized by excess proliferation of cells that can evade apoptosis and also metastasize to distant sites1. Breast cancer is one of the most common forms among females in the US, with an estimated 266,000 new cases and 40,000 deaths in 20182. A particularly aggressive and difficult to treat subtype is triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor (HER2). Radiation therapy is commonly used in breast cancer to eliminate residual tumor cells following lumpectomy, but over 13% of TNBC patients still experience recurrence at the primary tumor site3.
It is known that radiation therapy is effective in mitigating metastasis and recurrence because the combination of lumpectomy and radiation results in the same long-term survival as mastectomy4. However, it has recently been shown that radiation treatment is associated with local recurrence to the primary tumor site in immunocompromised settings5,6. Also, it is well known that radiation changes the extracellular matrix (ECM) of normal tissue by inducing fibrosis7. Therefore, it is important to understand the role of radiation-induced ECM changes in dictating tumor cell behavior.
Decellularized tissues have been used as in vitro models to study disease8,9. These decellularized tissues preserve ECM composition and recapitulate the complex in vivo ECM. This decellularized tissue ECM can be further processed and digested to form reconstituted ECM hydrogels that can be used to study cell growth and function10,11. For example, injectable hydrogels derived from decellularized human lipoaspirate and from myocardial tissue served as non-invasive methods of tissue engineering, and a hydrogel derived from porcine lung tissue was utilized as an in vitro method of testing mesenchymal stem cell attachment and viability12,13,14. The effect of normal tissue radiation damage on ECM properties, however, has not been investigated.
Hydrogels derived from ECM have the greatest potential for in vitro study of in vivo phenomena. Several other materials have been studied, including collagen, fibrin, and matrigel, but it is difficult to synthetically recapitulate the composition of the ECM13. An advantage of using ECM-derived hydrogels is that the ECM contains the necessary proteins and growth factors for a particular tissue14,15. Irradiation of normal tissue during lumpectomy causes significant changes to the ECM, and ECM-derived hydrogels can be used to study this effect in vitro. This method could lead to more complex and more accurate in vitro models of disease.
In this study, we subjected murine mammary fat pads (MFPs) to radiation ex vivo. The MFPs were decellularized and made into pre-gel solution. Hydrogels were formed with embedded 4T1 cells, a murine TNBC cell line. The rheological properties of the hydrogel material were examined, and tumor cell dynamics were evaluated within the hydrogels. Hydrogels fabricated from irradiated MFPs enhanced tumor cell proliferation. Future studies will incorporate other cell types to study cell-cell interactions in the context of cancer recurrence following therapy.
Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparation and ex vivo irradiation of MFPs
2. Decellularization (adapted from references12,16,17)
NOTE: This procedure was adapted from previously published methods focused on adipose decellularization, which included the sodium deoxycholate ionic detergent rather than sodium dodecyl sulfate to remove DNA efficiently12,16,17.
3. Lyophilization
4. Milling
5. Hydrogel formation
6. Encapsulating cells in hydrogels
7. H & E staining
8. 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining
9. Rheology
10. Phalloidin conjugate staining of F-actin
11. Viability assay
MFPs were decellularized following irradiation using the procedure shown in Figure 1A. MFPs pre-decellularization (Figure 1B) and post-decellularization (Figure 1C) are shown. Decellularization was confirmed using hematoxylin and eosin (H & E) staining, and 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining was used to evaluate lipid content (Figure 2). Rheological properties of the ECM hydrogels were also assessed at 37 °C (Figure 3). The storage modulus was higher than the loss modulus for all conditions, demonstrating stable hydrogel formation.
GFP- and luciferase-labeled 4T1 mammary carcinoma cells were encapsulated in the hydrogels. Cell proliferation was examined by fluorescence microscopy, bioluminescence measurements, and viability staining 48 h after encapsulation (Figure 4). Irradiated hydrogels showed an increasing trend in tumor cell proliferation. Phalloidin conjugate was used to visualize F-actin in the encapsulated cells (Figure 5). This technique can be used to examine cell morphology and cytoskeletal properties.
Figure 1: Experimental workflow. (A) Schematic of hydrogel formation. Digital camera images were taken of MFPs pre- (B) and post-decellularization (C). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Tissue weight (g) | Control (0 Gy) |
Irradiated (20 Gy) |
Initial MFP weight | 0.461 | 0.457 |
MFP weight following histology sample removal | 0.423 | 0.416 |
MFP weight after decellularization | 0.025 | 0.025 |
Decellularized MFP weight after histology sample removal | 0.015 | 0.016 |
Table 1: Tissue weights before and after decellularization. Representative tissue weights for each condition were measured before and after MFP decellularization.
This method of hydrogel formation is largely dependent on the amount of starting tissue. Murine MFPs are small, and the decellularization process results in a significant reduction of material (Table 1). The process can be repeated with more MFPs to increase final yield. Milling is another important step that may lead to loss of material. Others have shown success with a cryogenic mill, but this protocol is based on milling via a handheld mortar and electric drill with a pestle attachment8,17. This has the advantage of lower capital costs and minimizing material loss but may introduce variability in the final product.
A challenge to confirmation of decellularization and de-lipidation is in freezing adipose tissue in cryostat embedding medium. Figure 2A shows H & E staining of an unirradiated MFP embedded and sectioned in paraffin. Distinct nuclei are visible on the edges of adipose cells near junctions with other cells, and adipocyte morphology is well-maintained. Figure 2B,C,E,F show MFPs prepared by embedding and freezing MFPs in cryostat embedding medium and sectioning 5 μm slices. This process was unable to retain adipocyte morphology and shape. However, decellularization was confirmed through the loss of nuclei and other traces of DNA (Figure 2C), and de-lipidation was visualized with the loss of neutral lipid content staining (Figure 2F). Adipocyte morphology was maintained by incubating MFPs in sucrose, embedding and freezing in cryostat embedding medium, and sectioning 30 μm slices (Figure 2D,G). While the visualization of H & E staining was difficult with this method (Figure 2D), 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining confirmed the retention of adipocyte morphology (Figure 2G).
We have developed an in vitro hydrogel model that can mimic the in vivo normal tissue microenvironment and its response to radiation damage. ECM hydrogels have been fabricated in similar studies, but the impact of radiation damage on tumor cell behavior has not been assessed9,12,16,17,18. We expect that irradiation of MFPs will alter ECM remodeling and composition, and these compositional changes will be characterized in future studies. We observed an increasing trend in the proliferation of 4T1 cells within irradiated ECM hydrogels using both bioluminescence imaging and viability staining (Figure 4). In addition, we used phalloidin conjugate to stain F-actin filaments in encapsulated tumor cells and found a qualitative increase in actin alignment and tumor cell elongation in irradiated ECM hydrogels, which suggests an increase in adhesion strength and invasive capacity (Figure 5)19,20. Future experiments will explore changes in focal adhesion dynamics and protease-mediated remodeling for the evaluation of cell migration and invasion.
This method was developed using a murine TNBC cell line, but this method may be used as a platform for evaluating the proliferation and invasiveness of other cell types. Future studies may incorporate immune cells to determine their role in response to radiation as well as other forms of tissue damage (e.g., surgery). Although this study evaluated ECM hydrogels from MFPs irradiated ex vivo, additional studies will explore in vivo radiation of MFPs to evaluate the effect of physiological radiation response and infiltrating immune cells on ECM characteristics. We have established a method to fabricate ECM hydrogels from mouse MFPs to study the effect of normal tissue radiation on tumor cell behavior, and this technique may be extended to human tissue for a more relevant hydrogel model. Overall, examining normal tissue damage through ECM hydrogels may lead to insights into the role of ECM changes following radiation therapy in local recurrence.
The authors have nothing to disclose.
The authors thank Dr. Laura L. Bronsart for providing the GFP- and luciferase-4T1 cells, Dr. Edward L. LaGory for advice on 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining, Dr. Craig L. Duvall for IVIS and lyophilizer use, and Dr. Scott A. Guelcher for rheometer use. This research was financially supported by NIH grant #R00CA201304.
10% Neutral Buffered Formalin, Cube with Spigot | VWR | 16004-128 | – |
2-methylbutane | Alfa Aesar | 19387 | – |
AR 2000ex Rheometer | TA Instruments | 10D4335 | rheometer |
Bovine Serum Albumin | Sigma-Aldrich | A1933-25G | – |
calcein acetoxymethyl (calcein AM) | Molecular Probes, Inc. | C1430 | – |
D-Luciferin Firefly, potassium salt | Biosynth Chemistry & Biology | L-8820 | (S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt |
DPX Mountant for Histology | Sigma-Aldrich | 06522-500ML | – |
Dulbecco's phosphate-buffered saline | Gibco | 14040133 | – |
Eosin-Y with Phloxine | Richard-Allan Scientific | 71304 | eosin |
ethidium homodimer | Molecular Probes, Inc. | E1169 | ethidium homodimer-1 (EthD-1) |
Fetal Bovine Serum | Sigma-Aldrich | F0926-500ML | – |
Fisher Healthcare Tissue-Plus O.C.T. Compound | Fisher Scientific | 23-730-571 | cryostat embedding medium |
Fluoromount-G | SouthernBiotech | 0100-01 | aqueous based mounting medium |
FreeZone 4.5 | Labconco | 7751020 | lyophilizer |
Hoechst 33342 Solution (20 mM) | Thermo Scientific | 62249 | blue fluorescent dye |
Hydrochloric acid | Sigma-Aldrich | 258148-500ML | – |
IVIS Lumina III | PerkinElmer | CLS136334 | bioluminescence imaging system |
Kimtech Science Kimwipes | Kimberly Clark | delicate task wipes | |
n-Propanol (Peroxide-Free/Sequencing), Fisher BioReagents | Fisher Scientific | BP1130-500 | – |
Oil Red O | Sigma-Aldrich | O0625-25G | 1-([4-(Xylylazo)xylyl]azo)-2-naphthol |
OPS Diagnostics CryoGrinder | OPS Diagnostics, LLC | CG-08-02 | – |
PBS (10X), pH 7.4 | Quality Biological, Inc. | 119-069-151 | Phosphate-buffered saline |
Penicillin-Streptomycin | Gibco | 15140-122 | – |
Pepsin from porcine gastric mucosa | Sigma-Aldrich | P6887-5G | pepsin |
Peracetic acid | Sigma-Aldrich | 77240-100ML | – |
Phalloidin-iFluor 594 Reagent (ab176757) | abcam | ab176757 | phalloidin conjugate |
Propylene glycol | Sigma-Aldrich | W294004-1KG-K | – |
Richard-Allan Scientific Signature Series Bluing Reagent | Richard-Allan Scientific | 7301L | bluing agent |
Richard-Allan Scientific Signature Series Hematoxylin 7211 | Richard-Allan Scientific | 7211 | – |
RPMI Medium 1640 | Gibco | 11875-093 | – |
Sodium deoxycholate, 98% | Frontier Scientific | JK559522 | deoxycholic acid |
Sucrose | Sigma-Aldrich | S5016 | – |
Triton x-100 | Sigma-Aldrich | X100-100ML | t-Octylphenoxypolyethoxyethanol |
Trypsin-EDTA (0.25%), phenol red | Gibco | 25200-056 | – |
Whatman qualitative filter paper, Grade 4 | Whatman | 1004-110 | grade 4 qualitative filter paper |
Xylenes (Certified ACS), Fisher Chemical | Fisher Scientific | X5-4 | – |