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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 a....
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
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 .......
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 attachment
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.
....Name | Company | Catalog Number | Comments |
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 | - |
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