Name: studying normal tissue radiation effects using extracellular matrix hydrogels
Uploaded: 2019-07-24T13:00:00
Description: Watch this Scientific Journal Video about Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels at JoVE.com

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.

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
<ol>
	<li>Sacrifice athymic Nu/Nu mice (8&#8211;10 weeks) using CO&#173;2 asphyxiation followed by cervical dislocation.</li>
	<li>Clean the skin using 70% ethanol.</li>
	<li>Collect mammary fat pads (MFPs) from sacrificed mice using pre-sterilized scissors and force...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2018.">Cancer statistics, 2018.</a> CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).</li><li>Lowery, A. J., Kell, M. R., Glynn, R. W., Kerin, M. J., Sweeney, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locoregional+recurrence+after+breast+cancer+surgery:+a+systematic+review+by+receptor+phenotype.">Locoregional recurrence after breast cancer surgery: a systematic review by receptor phenotype.</a> Breast Cancer Research and Treatment. 133 (3), 831-841 (2012).</li><li>Miller, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+treatment+and+survivorship+statistics,+2016.">Cancer treatment and survivorship statistics, 2016.</a> CA: A Cancer Journal for Clinicians. 66 (4), 271-289 (2016).</li><li>Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Circulating+Breast+Cancer+Cells+Is+Stimulated+by+Radiotherapy.">Recruitment of Circulating Breast Cancer Cells Is Stimulated by Radiotherapy.</a> Cell Reports. 8 (2), 402-409 (2014).</li><li>Rafat, M., Aguilera, T. A., Vilalta, M., Bronsart, L. L., Soto, L. A., von Eyben, R., Golla, M. A., Ahrari, Y., Melemenidis, S., Afghahi, A., Jenkins, M. J., Kurian, A. W., Horst, K. C., Giaccia, A. J., Graves, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+Promote+Circulating+Tumor+Cell-Mediated+Local+Recurrence+Following+Radiation+Therapy+in+Immunosuppressed+Patients.">Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence Following Radiation Therapy in Immunosuppressed Patients.</a> Cancer Res. 75 (15), 4241-4252 (2018).</li><li>Haubner, F., Ohmann, E., Pohl, F., Strutz, J., Gassner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+after+radiation+therapy:+Review+of+the+literature.">Wound healing after radiation therapy: Review of the literature.</a> Radiation Oncology. 7 (1), 1-9 (2012).</li><li>Beachley, V. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+matrix+arrays+for+high+throughput+screening+and+systems+analysis+of+cell+function.">Tissue matrix arrays for high throughput screening and systems analysis of cell function.</a> Nature Methods. 12 (12), 1197-1204 (2015).</li><li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+metastases+obtained+by+culturing+colorectal+cancer+cells+on+tissue-specific+decellularized+scaffolds.">Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds.</a> Nature Biomedical Engineering. 2 (6), 443-452 (2018).</li><li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogels+from+decellularized+tissues:+Structure+and+function.">Extracellular matrix hydrogels from decellularized tissues: Structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li><li>Hinderer, S., Layland, S. L., Schenke-Layland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+and+ECM-like+materials+&#8212;+Biomaterials+for+applications+in+regenerative+medicine+and+cancer+therapy.">ECM and ECM-like materials &#8212; Biomaterials for applications in regenerative medicine and cancer therapy.</a> Advanced Drug Delivery Reviews. 97, 260-269 (2016).</li><li>Young, D. A., Ibrahim, D. O., Hu, D., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+hydrogel+scaffold+from+decellularized+human+lipoaspirate.">Injectable hydrogel scaffold from decellularized human lipoaspirate.</a> Acta Biomaterialia. 7 (3), 1040-1049 (2011).</li><li>Singelyn, J. M., Christman, K. L., Littlefield, R. B., Schup-Magoffin, P. J., DeQuach, J. A., Seif-Naraghi, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30 (29), 5409-5416 (2009).</li><li>Pouliot, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> Journal of Biomedical Materials Research Part A. 104 (8), 1922-1935 (2016).</li><li>Bonvillain, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nonhuman+Primate+Model+of+Lung+Regeneration:+Detergent-Mediated+Decellularization+and+Initial+In+Vitro+Recellularization+with+Mesenchymal+Stem+Cells.">A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells.</a> Tissue Engineering Part A. 18 (23-24), 2437-2452 (2012).</li><li>Brown, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Three+Methods+for+the+Derivation+of+a+Biologic+Scaffold+Composed+of+Adipose+Tissue+Extracellular+Matrix.">Comparison of Three Methods for the Derivation of a Biologic Scaffold Composed of Adipose Tissue Extracellular Matrix.</a> Tissue Engineering Part C: Methods. 17 (4), 411-421 (2011).</li><li>Link, P. A., Pouliot, R. A., Mikhaiel, N. S., Young, B. M., Heise, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+Hydrogels+from+Pulmonary+Extracellular+Matrix+for+3D+Cell+Culture.">Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture.</a> Journal of Visualized Experiments. (119), 1-9 (2017).</li><li>Massensini, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration-dependent+rheological+properties+of+ECM+hydrogel+for+intracerebral+delivery+to+a+stroke+cavity.">Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity.</a> Acta Biomaterialia. 27, 116-130 (2015).</li><li>Mierke, C. T., Frey, B., Fellner, M., Herrmann, M., Fabry, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+5+1+facilitates+cancer+cell+invasion+through+enhanced+contractile+forces.">Integrin 5 1 facilitates cancer cell invasion through enhanced contractile forces.</a> Journal of Cell Science. 124 (3), 369-383 (2011).</li><li>Ahmadzadeh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+two-way+feedback+between+contractility+and+matrix+realignment+reveals+a+nonlinear+mode+of+cancer+cell+invasion.">Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion.</a> Proceedings of the National Academy of Sciences. 114 (9), E1617-E1626 (2017).</li></ol>

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<sup class="xref...

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.

<table>
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin, Cube with Spigot</td>
			<td>VWR</td>
			<td>16004-128</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Alfa Aesar</td>
			<td>19387</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AR 2000ex Rheometer</td>
			<td>TA Instruments</td>
			<td>10D4335</td>
			<td>rheometer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A1933-25G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>calcein acetoxymethyl (calcein AM)</td>
			<td>Molecular Probes, Inc.</td>
			<td>C1430</td>
			<td>-</td>
		</tr>
		<tr>
			<td>D-Luciferin Firefly, potassium salt</td>
			<td>Biosynth Chemistry &#38; Biology</td>
			<td>L-8820</td>
			<td>(S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt</td>
		</tr>
		<tr>
			<td>DPX Mountant for Histology</td>
			<td>Sigma-Aldrich</td>
			<td>06522-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eosin-Y with Phloxine</td>
			<td>Richard-Allan Scientific</td>
			<td>71304</td>
			<td>eosin</td>
		</tr>
		<tr>
			<td>ethidium homodimer</td>
			<td>Molecular Probes, Inc.</td>
			<td>E1169</td>
			<td>ethidium homodimer-1 (EthD-1)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F0926-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fisher Healthcare Tissue-Plus O.C.T. Compound</td>
			<td>Fisher Scientific</td>
			<td>23-730-571</td>
			<td>cryostat embedding medium</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>aqueous based mounting medium</td>
		</tr>
		<tr>
			<td>FreeZone 4.5</td>
			<td>Labconco</td>
			<td>7751020</td>
			<td>lyophilizer</td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution (20 mM)</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td>blue fluorescent dye</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IVIS Lumina III</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td>bioluminescence imaging system</td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes</td>
			<td>Kimberly Clark</td>
			<td></td>
			<td>delicate task wipes</td>
		</tr>
		<tr>
			<td>n-Propanol (Peroxide-Free/Sequencing), Fisher BioReagents</td>
			<td>Fisher Scientific</td>
			<td>BP1130-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>O0625-25G</td>
			<td>1-([4-(Xylylazo)xylyl]azo)-2-naphthol</td>
		</tr>
		<tr>
			<td>OPS Diagnostics CryoGrinder</td>
			<td>OPS Diagnostics, LLC</td>
			<td>CG-08-02</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>Quality Biological, Inc.</td>
			<td>119-069-151</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pepsin from porcine gastric mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>pepsin</td>
		</tr>
		<tr>
			<td>Peracetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>77240-100ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 594 Reagent (ab176757)</td>
			<td>abcam</td>
			<td>ab176757</td>
			<td>phalloidin conjugate</td>
		</tr>
		<tr>
			<td>Propylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>W294004-1KG-K</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific Signature Series Bluing Reagent</td>
			<td>Richard-Allan Scientific</td>
			<td>7301L</td>
			<td>bluing agent</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific Signature Series Hematoxylin 7211</td>
			<td>Richard-Allan Scientific</td>
			<td>7211</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate, 98%</td>
			<td>Frontier Scientific</td>
			<td>JK559522</td>
			<td>deoxycholic acid</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td>t-Octylphenoxypolyethoxyethanol</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper, Grade 4</td>
			<td>Whatman</td>
			<td>1004-110</td>
			<td>grade 4 qualitative filter paper</td>
		</tr>
		<tr>
			<td>Xylenes (Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>X5-4</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

studying normal tissue radiation effects using extracellular matrix hydrogels

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.

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 &#38; 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 ...

Watch this Scientific Journal Video about Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels at JoVE.com

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

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 ...

This protocol shows that radiation influences extracellular matrix properties of adipose tissue in the murine mammary fat pad. The addition of radiation in a decellularization model has not been done before. This technique uses several washing steps that each serve a unique chemical purpose in the decellularization process.Specificity of this technique allows for gentler chemical washes, to better preserve tissue properties. Breast cancer relapse occurs following therapy, especially in triple negative cases. Evaluating how the irradiated extracellular matrix alters tumor cell behavior will lead to important discoveries about recurrence mechanisms.After irradiating samples, using a cesium source, transfer the irradiated MFP's and complete RPMI media into a biosafety cabinet. Fill six centimeter or 10 centimeter dishes with enough media to submerge the MFP's. Incubate at 37 degrees Celsius, with 5%carbon dioxide, for two days.First, place the MFP's in six centimeter dishes with five milliliters of Trypsin-EDTA solution. Spray and wipe the dishes with 70%ethanol and incubate at 37 degrees Celsius for one hour. Use 0.7 milliliter strainers to wash the MFP's with deionized water, by pouring water over the tissue three times.Use forceps to manually massage the tissue in between washes. Next, briefly dry the tissue on a delicate task wipe and weigh it. Place the dried tissues in a pre-autoclaved beaker, containing an appropriate sized stir bar and cover the tissues with 60 milliliters of 3%t-octylphenoxypolyethoxyethanol per gram of tissue.Stir for one hour at room temperature. Then dump the beaker's contents into a strainer. Rinse the beaker with deionized water and pour this onto the tissues.Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses. After this, briefly dry the tissue on a delicate task wipe and weigh. Place the tissues and the stir bars back into the same beakers and cover them with 60 milliliters of 4%deoxycholic acid per gram of tissue.Stir at room temperature for one hour. Next, dump the beaker's contents into a mesh strainer, rinse the beaker with deionized water and pour this onto the tissues. Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses.Briefly dry the rinsed tissue on a delicate task wipe and weigh it. Place the dried tissues into the same beaker, along with fresh deionized water, supplemented with 1%penicillin-streptomycin. Cover the beaker tightly with paraffin film and leave at 4 degrees Celsius overnight.The next day drain the beaker contents into a strainer, briefly dry the tissue on a delicate task wipe and weigh it. Then, place the MFP's in the same beaker with an appropriate sized stir bar. Cover the tissue with 60 milliliters of a solution containing 4%ethanol and 0.1%peracetic acid per gram of tissue.Stir at room temperature for two hours. Dump the beaker's contents into a 0.7 millimeter strainer. Use forceps to manually massage the tissue and place the contents back into the beaker.Wash the tissue by covering it with 60 milliliters of 1X PBS per gram of tissue. Stir at room temperature for 15 minutes. Repeat this entire washing process one more time.Next, dump the beaker's contents into a 0.7 millimeter strainer. Using forceps, manually massage the tissue and place the contents back into the beaker. Wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.Stir at room temperature for 15 minutes. Repeat this entire washing process one more time. Briefly dry the washed tissue on a delicate task wipe and weigh it.Dump the tissue and contents into a strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and cover the tissues with 60 milliliters of 100%n-propanol per gram of tissue. Stir at room temperature for one hour.Then, briefly dry the tissue on a delicate task wipe and weigh it. Dump the tissue and contents into a 0.7 millimeter strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.Stir at room temperature for 15 minutes. Repeat this wash process three times. After this, briefly dry the tissue on a delicate task wipe and weigh it.Transfer the tissue to a labeled 15 milliliter tube and freeze at negative 80 degrees Celsius overnight. First, fill a shallow container with liquid nitrogen. Remove the samples from the freezer and weigh each lyophilized MFP.Place one sample in the mortar, use a cryogenic glove to hold the mortar in the liquid nitrogen. Then, use a pestle, attached to a hand-held drill, to mill the sample. Mill in one minute intervals to check the progress and remove the gloved hand from the liquid nitrogen.Repeat this milling process for all samples, making sure to spray and wipe the mortar and pestle with ethanol between each sample. Store the powdered samples in 15 milliliter tubes at negative 80 degrees Celsius until ready to use. To begin, remove the samples from the freezer and thaw at room temperature.While the samples are thawing, mix pepsin into hydrochloric acid to form a Pepsin-HCL solution. Next, add sample powder and the Pepsin-HCL solution to a 15 milliliter tube, add a small stir bar and stir for 48 hours. After this, place the tubes on ice for five minutes.Add 10X PBS to each sample, such that the final solution has a concentration of 1X PBS. Then, add 10%volume/volume, 0.1 molar sodium hydroxide to each solution, to reach pH 7.4, using pH paper to test each solution individually. Using a pH 7.4 gel solution, resuspend the pelleted GFP and luciferase labeled 4T1 cells to a concentration of either 500, 000 or 1, 000, 000 cells per milliliter of gel solution.Add 16 microliters of gel cell solution to each well of a 16-well chamber slide and incubate at 37 degrees Celsius for 30 minutes. After this, add 100 microliters of complete RPMI media to each well. Continue to incubate at 37 degrees Celsius for 48 hours.Then, use a fluorescence microscope to observe the cells at an excitation wavelength of 519 nanometers and an emission wavelength of 618 nanometers. In this study, normal tissue radiation effects are studied, using extracellular matrix hydrogels. Hematoxylin and eosin staining is used to confirm decellularization, through the loss of nuclei and other traces of DNA.While Oil Red O staining is used to evaluate lipid content and confirm the retention of a dipasite morphology. The rheological properties of the ECM hydrogels are assessed at 37 degrees Celsius. The storage modulus is higher than the loss modulus for all conditions, demonstrating stable hydrogel formation.GFP and luciferase labeled 4T1 mammary carcinoma cells are then encapsulated in the hydrogels. Cell proliferation is examined by fluorescence microscopy, by illuminescence measurements and viability staining, 48 hours after encapsulation. Irradiated hydrogels show an increasing trend in tumor cell proliferation.Phalloidin conjugate is used to visualize F-Actin in the encapsulated cells. Remember to cool the mortar before placing the tissue inside the mill, a warm mortar may lead to incomplete milling. Use caution when handling n-propanal and pepsin, only open n-propanal and other decellularizationary agents under a chemical hood.If possible, weigh the pepsin under a chemical hood, as there is danger of inhalation. ECM composition changes after irradiation and decellularization can be evaluated using mass spectrometry and Raman spectroscopy. In addition, the fiber structure of the ECM hydrogels can be analyzed by scanning electron microscopy.This method has the potential to be expanded to examine the effects of radiation on, not only tumor cells but also immune cells, as well as tissues that experience radiation damage, as a result of therapy. This decellularization technique will allow researchers to evaluate the extracellular matrix properties of the tissue following radiation, which is an important treatment option in most cancers.

studying-normal-tissue-radiation-effects-using-extracellular-matrix

Research

JoVE Journal

Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Cell Cycle-specific Measurement of &amp;#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

Studying TGF-&amp;#946; Signaling and TGF-&amp;#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (&#60;1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (&#62;500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...