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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

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.

Introduction

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....

Protocol

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

  1. Sacrifice athymic Nu/Nu mice (8–10 weeks) using CO­2 asphyxiation followed by cervical dislocation.
  2. Clean the skin using 70% ethanol.
  3. Collect mammary fat pads (MFPs) from sacrificed mice using pre-sterilized scissors and force.......

Representative Results

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 .......

Discussion

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

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
10% Neutral Buffered Formalin, Cube with SpigotVWR16004-128-
2-methylbutaneAlfa Aesar19387-
AR 2000ex RheometerTA Instruments10D4335rheometer
Bovine Serum AlbuminSigma-AldrichA1933-25G-
calcein acetoxymethyl (calcein AM)Molecular Probes, Inc.C1430-
D-Luciferin Firefly, potassium saltBiosynth Chemistry & BiologyL-8820(S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt
DPX Mountant for HistologySigma-Aldrich06522-500ML-
Dulbecco's phosphate-buffered salineGibco14040133-
Eosin-Y with PhloxineRichard-Allan Scientific71304eosin
ethidium homodimerMolecular Probes, Inc.E1169ethidium homodimer-1 (EthD-1)
Fetal Bovine SerumSigma-AldrichF0926-500ML-
Fisher Healthcare Tissue-Plus O.C.T. CompoundFisher Scientific23-730-571cryostat embedding medium
Fluoromount-GSouthernBiotech0100-01aqueous based mounting medium
FreeZone 4.5Labconco7751020lyophilizer
Hoechst 33342 Solution (20 mM)Thermo Scientific62249blue fluorescent dye
Hydrochloric acidSigma-Aldrich258148-500ML-
IVIS Lumina IIIPerkinElmerCLS136334bioluminescence imaging system
Kimtech Science KimwipesKimberly Clarkdelicate task wipes
n-Propanol (Peroxide-Free/Sequencing), Fisher BioReagentsFisher ScientificBP1130-500-
Oil Red OSigma-AldrichO0625-25G1-([4-(Xylylazo)xylyl]azo)-2-naphthol
OPS Diagnostics CryoGrinderOPS Diagnostics, LLCCG-08-02-
PBS (10X), pH 7.4Quality Biological, Inc.119-069-151Phosphate-buffered saline
Penicillin-StreptomycinGibco15140-122-
Pepsin from porcine gastric mucosaSigma-AldrichP6887-5Gpepsin
Peracetic acidSigma-Aldrich77240-100ML-
Phalloidin-iFluor 594 Reagent (ab176757)abcamab176757phalloidin conjugate
Propylene glycolSigma-AldrichW294004-1KG-K-
Richard-Allan Scientific Signature Series Bluing ReagentRichard-Allan Scientific7301Lbluing agent
Richard-Allan Scientific Signature Series Hematoxylin 7211Richard-Allan Scientific7211-
RPMI Medium 1640Gibco11875-093-
Sodium deoxycholate, 98%Frontier ScientificJK559522deoxycholic acid
SucroseSigma-AldrichS5016-
Triton x-100Sigma-AldrichX100-100MLt-Octylphenoxypolyethoxyethanol
Trypsin-EDTA (0.25%), phenol redGibco25200-056-
Whatman qualitative filter paper, Grade 4Whatman1004-110grade 4 qualitative filter paper
Xylenes (Certified ACS), Fisher ChemicalFisher ScientificX5-4-

References

  1. Hanahan, D., Weinberg, R. A. The hallmarks of cancer. Cell. 100 (1), 57-70 (2000).
  2. Siegel, R. L., Miller, K. D., Jemal, A. Cancer statistics, 2018. CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).
  3. Lowery, A. J., Kell, M. R., Glynn, R. W., Ke....

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Extracellular MatrixHydrogelsRadiation EffectsDecellularizationMammary Fat PadBreast CancerRecurrence MechanismsTrypsin EDTAT octylphenoxypolyethoxyethanolDeoxycholic Acid

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