Name: an enrichment method for small extracellular vesicles derived from liver cancer tissue
Uploaded: 2023-02-03T12:50:00
Description: Watch this Scientific Journal Video about An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue at JoVE.com

The authors thank the First Affiliated Hospital of Gannan Medical University for supporting this work.&#160;This work was supported by the National Natural Science Foundation of China (grant numbers 82260422).

Human liver cancer tissue was collected from patients diagnosed with hepatic malignancy at the First Affiliated Hospital of Gannan Medical University. All patients signed an informed consent form, and the collection of human tissue samples was approved by the ethics committee of the First Affiliated Hospital of Gannan Medical University. See the Table of Materials for details related to all materials, equipment, and software used in this protocol.
1. Preparation...

<ol>
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C., 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=Mitochondrial+protein+enriched+extracellular+vesicles+discovered+in+human+melanoma+tissues+can+be+detected+in+patient+plasma.">Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma.</a> Journal of Extracellular Vesicles. 8 (1), 1635420 (2019).</li><li>Muralidharan-Chari, V., 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=ARF6-regulated+shedding+of+tumor+cell-derived+plasma+membrane+microvesicles.">ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles.</a> Current Biology. 19 (22), 1875-1885 (2009).</li><li>Matejovi&#269;, A., Wakao, S., Kitada, M., Kushida, Y., Dezawa, M. <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+separation+methods+for+tissue-derived+extracellular+vesicles+in+the+liver,+heart,+and+skeletal+muscle.">Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle.</a> FEBS Open Bio. 11 (2), 482-493 (2021).</li><li>Zhou, 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=Brown+adipose+tissue-derived+exosomes+mitigate+the+metabolic+syndrome+in+high+fat+diet+mice.">Brown adipose tissue-derived exosomes mitigate the metabolic syndrome in high fat diet mice.</a> Theranostics. 10 (18), 8197-8210 (2020).</li><li>Chen, J., 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+the+variability+of+small+extracellular+vesicles+derived+from+human+liver+cancer+tissues+and+cultured+from+the+tissue+explants+based+on+a+simple+enrichment+method.">Comparison of the variability of small extracellular vesicles derived from human liver cancer tissues and cultured from the tissue explants based on a simple enrichment method.</a> Stem Cell Reviews and Reports. 18 (3), 1067-1077 (2022).</li><li>Fang, T., 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=Tumor-derived+exosomal+miR-1247-3p+induces+cancer-associated+fibroblast+activation+to+foster+lung+metastasis+of+liver+cancer.">Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer.</a> Nature Communications. 9 (1), 1247-1243 (2018).</li><li>Huang, 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=RNA+sequencing+of+plasma+exosomes+revealed+novel+functional+long+noncoding+RNAs+in+hepatocellular+carcinoma.">RNA sequencing of plasma exosomes revealed novel functional long noncoding RNAs in hepatocellular carcinoma.</a> Cancer Science. 111 (9), 3338-3349 (2020).</li><li>Chen, L., 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=HCC-derived+exosomes+elicit+HCC+progression+and+recurrence+by+epithelial-mesenchymal+transition+through+MAPK/ERK+signalling+pathway.">HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway.</a> Cell Death &amp; Disease. 9 (5), 513 (2018).</li><li>Jingushi, K., 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=Extracellular+vesicles+isolated+from+human+renal+cell+carcinoma+tissues+disrupt+vascular+endothelial+cell+morphology+via+azurocidin.">Extracellular vesicles isolated from human renal cell carcinoma tissues disrupt vascular endothelial cell morphology via azurocidin.</a> International Journal of Cancer. 142 (3), 607-617 (2018).</li><li>Camino, T., 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=Deciphering+adipose+tissue+extracellular+vesicles+protein+cargo+and+its+role+in+obesity.">Deciphering adipose tissue extracellular vesicles protein cargo and its role in obesity.</a> International Journal of Molecular Sciences. 21 (24), 9366 (2020).</li></ol>

This protocol describes a repeatable method for extracting sEVs from liver cancer tissue. High-quality sEVs are obtained by sharp tissue isolation, treatment with digestive enzymes, differential ultracentrifugation, and 0.22 &#181;m filter membrane filtration and purification. For downstream analysis, it is extremely important to ensure the high purity of the sEVs. In the process of differential centrifugation, a layer of white substances (unknown composition) will appear on the surface of the supernatant. As this layer ...

Small extracellular vesicles (sEVs) are approximately 30 nm to 150 nm in diameter and are secreted by various cells1. They can communicate with tissue cells and regulate the local or distant microenvironment by transporting important biological molecules such as lipids, proteins, DNA, and RNA to various organs, tissues, cells, and intracellular parts. Thus, they can also change the behavior of recipient cells2,3. The isolation and purification of specific sEVs is an essential prerequisite to studying their biological behavior during the development and course of disease. Differential ultracentrifugation-regarded as the gold standard-is commonly used to separate sEVs from the tissues in which they normally reside4. Tissue debris, cell debris, large vesicles, and apoptotic bodies can be removed by this technique, leaving only the sEVs.
Collagenase D and DNase I have been shown not to affect the molecular characteristics of cells or vesicles, with the properties of both enzymes contributing to the release of vesicles in the extracellular matrix 5. These enzymes have been used to extract sEVs from human metastatic melanoma tissue, colon cancer tissue, and colonic mucosal tissue5,6,7. However, the concentration and digestion time of collagenase D and DNase I in these methods differ, leading to inconsistent conclusions. To avoid the coprecipitation of other subtypes of sEVs, researchers have removed larger extracellular vesicles (0.1 &#181;m or 0.2 &#181;m in diameter) by filtration and/or differential centrifugation8. Depending on the source tissue, different methods of isolation and purification may be required9,10.
Using the traditional differential ultracentrifugation method to extract sEVs from liver tissue results in a layer of white matter on the surface of the supernatant, without any way of determining its properties. In a previous study11, this layer of white matter was found to affect the purity of the sEVs. Although the particle number and protein concentration of samples isolated by the traditional method were higher than those of the current method, the coefficient of variation was large, possibly because many pollutants can lead to poor repeatability of the results. That is, using detergent (i.e., detecting the solubility of particles in 1% Triton X-100), we found that the purity of sEVs obtained by this method was greater. Hence, we use this method to isolate and purify sEVs derived from colorectal cancer tissue for proteomic research.
At present, the research on sEVs in liver cancer is focused mainly on serum, plasma, and the cell culture&#39;s supernatant12,13,14. However, sEVs derived from liver cancer tissue can more accurately reflect the physiologic pathology and the surrounding microenvironment of liver cancer and effectively avoid the degradation and pollution of other EVs15,16. With the use of differential ultracentrifugation, this method can enrich the yield and obtain high-quality sEVs, providing an important basis for the further study of liver cancer. This method enables the liver cancer tissue to be separated by sharp separation and to be dissociated by collagenase D and DNase I. Then, the cellular debris, large vesicles, and apoptotic bodies are further removed by filtration and differential ultracentrifugation. Finally, the sEVs are isolated and purified for later studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Membrane Filter Unit</td>
			<td>Millex</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Sterile syringe</td>
			<td>Hubei Xianming Medical Instrument Company</td>
			<td>YL01329</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400-2</td>
			<td></td>
		</tr>
		<tr>
			<td>4.7 mL Centrifuge Tube</td>
			<td>Beckman Coulter</td>
			<td>361621</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell cuture plate</td>
			<td>LABSELECT</td>
			<td>11110</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Beaker</td>
			<td>Tianjin Kangyiheng Experimental Instrument Sales Company</td>
			<td>CF2100800</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell strainer</td>
			<td>Biosharp</td>
			<td>BS-70-XBS</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell culture dish</td>
			<td>CELL TER</td>
			<td>CS016-0128</td>
			<td></td>
		</tr>
		<tr>
			<td>600 &#181;L Centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT060C</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification kit</td>
			<td>Thermo Fisher</td>
			<td>RJ240544</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Optima-Max-TL</td>
			<td>Beckman&#160;</td>
			<td>A95761</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Mini trans-blot</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Mini-Protean</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>CD63 Antibody</td>
			<td>Abcam</td>
			<td>ab134045</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9 Antibody</td>
			<td>Abcam</td>
			<td>ab263019</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R</td>
			<td>Eppendorf</td>
			<td>5428HQ527333</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning Solution</td>
			<td>NanoFCM</td>
			<td>C1801</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper net</td>
			<td>Henan Zhongjingkeyi Technology Company</td>
			<td>DJZCM-15-N1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Thermostat</td>
			<td>Hangzhou allsheng instruments company</td>
			<td>AS-01030-00</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Anti Human CD9 Antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1086C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Solarbio</td>
			<td>G8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat horseradish peroxidase (HRP)-coupled secondary anti-mouse antibody&#160;</td>
			<td>Proteintech</td>
			<td>SA00001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat horseradish peroxidase (HRP)-coupled secondary anti-rabbit antibody&#160;</td>
			<td>Proteintech</td>
			<td>SA00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Shanghai Zhenxing Chemical Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle flow cytometer</td>
			<td>NanoFCM INC</td>
			<td>FNAN30E20112368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors(PhosSTOP)</td>
			<td>Roche</td>
			<td>4906845001</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline(PBS)</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene Difluoride Membrane</td>
			<td>Solarbio</td>
			<td>ISEQ00010</td>
			<td></td>
		</tr>
		<tr>
			<td>QC Beads</td>
			<td>NanoFCM</td>
			<td>QS2502</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 basic medium</td>
			<td>Biological Industries&#160;</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Guangzhou Kehua Trading Company</td>
			<td>NN-0623-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Nanospheres</td>
			<td>NanoFCM</td>
			<td>S16M-Exo</td>
			<td></td>
		</tr>
		<tr>
			<td>Transference Decoloring Shaker TS-8</td>
			<td>Kylin-Bell</td>
			<td>E0018</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Thermo Scientific</td>
			<td>Talos L120C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td></td>
		</tr>
		<tr>
			<td>TSG101 Antibody</td>
			<td>Proteintech</td>
			<td>28283-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Guangzhou Lige Technology Company</td>
			<td>LG01-105-4X</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enrichment method for small extracellular vesicles derived from liver cancer tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

sEVs from human liver cancer tissues have played a crucial role in the diagnosis, treatment, and prognosis of patients with liver cancer. This method used common laboratory instruments to isolate and purify sEVs derived from liver cancer tissues; this may provide methodologic support for the study of sEVs. Figure 2 illustrates the general process of enriching sEVs from liver cancer tissues. sEVs in the intercellular spaces of tissues are fully released through tissue cutting and enzymatic hy...

Watch this Scientific Journal Video about An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue at JoVE.com

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

This method allows for the isolation of liver cancer tissue-derived small extracellular vesicles with a purity higher than that achieved by classic, differential ultracentrifugation. This technology is simple, convenient, and easy to operate, which can provide methodologic support for the study of small extracellular vesicles. To begin, remove the tissue sample from the 80 degrees Celsius freezer.Place approximately 400 milligrams of the tissue into a 10-centimeter cell culture dish on the ice box, and finely mince the tissue with a scalpel. Transfer the tissue to a six-well plate with 1.5 milliliters of the digestive solution. Then, place the plate on the transference decoloring shaker, and incubate at 37 degrees Celsius for 20 minutes for complete dissociation of the liver cancer tissue and release of the small extracellular vesicles, or sEVs.After incubation, place the six-well plate on the ice box. Add 80 microliters of phosphatase inhibitor and 200 microliters of complete protease inhibitor solution to stop the digestion. Then, transfer the digestive solution to a 70-micrometer cell strainer placed on a two-milliliter centrifuge tube, and filter it slowly to remove any large tissue debris.Centrifuge the filtrate at 500 g for 10 minutes to remove the small tissue debris, and collect the supernatant in a new two-milliliter centrifuge tube. Centrifuge the supernatant at 3, 000 g for 20 minutes to remove the cell debris. Carefully transfer the supernatant to a new centrifuge tube until the tip of the pipette is about to touch the white debris at the bottom.After repeating centrifugation of the remaining liquid, pool the supernatant into the tube without disturbing the debris. Centrifuge the collected supernatant at 12, 000 g for 20 minutes, and transfer 900 microliters of the supernatant to a 4.7-millimeter ultracentrifuge tube. Repeat centrifugation of the remaining liquid, and then collect and mix both supernatants into the same ultracentrifuge tube.Fill the tube completely with PBS. Centrifuge the supernatant at 100, 000 g for 60 minutes. After discarding the supernatant, resuspend the pellet by filling the ultracentrifuge tube with PBS.After repeating the centrifugation at 100, 000 g for 60 minutes, resuspend the pellet with 50 microliters of PBS. Aspirate the sEV suspension using a one-milliliter sterile syringe, and filter it through a 0.22-micrometer membrane filter into a 600-microliter centrifuge tube. Store the filtrate at 80 degrees Celsius for further analysis.Use a nanoparticle flow cytometer to determine the size distribution and purity of the sEVs. Check the volume of the cleaning solution. Then, note the difference between the levels of the sheath and the waste liquid.After 20 seconds of turning on the main power supply of the instrument, turn on the computer. Wait for the beeps indicating that the instrument is properly connected to run the software. Click on Start Up to turn on the camera, laser, and air pump.Next, click on Sheath Flow, and select Start Up.Place ultrapure water on the loading platform. After four minutes, click on Sample and Boosting, and then select Unload in Sample. After 30 seconds, place the blank tube on the loading platform.Click on Sample, select Boosting, and then select Unload in Sample. Then, place the tube containing the ultrapure water on the loading platform. Simultaneously, click on Sample and Boosting, followed by Sheath Flow and Purge.Click on Manual Operation, and place the particle concentration tube on the loading platform. Then, click on Sample, select Boosting for one minute, and simultaneously select 250 nanometer Standard Fluorescent Silica Nanospheres quality control in Sample Information. Select Sampling from Sample, and turn the detector on by clicking on SPCM.Then, click on Auto Sampling, and enter 1.0 into Sampling Set to fix the pressure at one kilopascal. Click on the toolbar, and select Large Signal. Adjust the horizontal position of the laser, and set the laser to two micrometers.Then, continuously click on L and R to ensure the signal is strong and uniform. Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in Nfa File, and click on Sample to select Unload.Place the cleaning solution tube on the loading platform. Click on Sample, select Boosting, and after one minute click on Sample and Unload. Use ultrapure water to remove the residual cleaning solution from the capillary tip.Place the particle size standard tube on the loading platform, and click on Sample Boosting for one minute. Select 68 to 155 S16M-Exo quality control in Sample Information, and click on Sample, then Sampling. Next, click on the toolbar, and select Small Signal.Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in an Nfa file, and click on Sample to select Unload. After removing the residues with the cleaning solution, place the PBS tube on the loading platform, and perform the steps demonstrated for the particle size standard tube.Next, clean the capillary tip as demonstrated earlier, and load the sEV sample to analyze the data described earlier for the particle size standard tube. The purified sEVs were examined under a transmission electron microscope, which revealed the presence of sEVs with a cup-shaped morphology. Flow cytometry for the sEVs was carried out.And the particle size ranged from 40 to 200 nanometers, and the average particle size was 89 nanometers. The purity of the enriched sEVs was found to be 68.32%The protein markers of sEVs such as CD63, CD9, and TSG101 were detected by western blot. GM130 was used as the negative control marker of sEVs, which was found in tissue cells but not in the sEVs.It is crucial to perform the steps of enzymatic digestion, differential ultracentrifugation, and membrane filtration with utmost care to ensure the purity of small extracellular vesicles.

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Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

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Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

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 ...

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.

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 ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.

This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &amp;#947;&amp;#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...