Name: isolation and characterization of tumor-initiating cells from sarcoma patient-derived xenografts
Uploaded: 2019-06-13T12:30:00.000+00:00
Duration: 7 min 18 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts at JoVE.com

This research was supported by NCI-P01-CA087497 (to C.C.-C. and D.H.) and NIH-U 54-0OD020353 (to C.C.-C., D.H., and J.D.-D.), the Agilent Thought Leader Award (to C.C.-C.), and the Martel Foundation (to C.C.-C. and J.D.-D.).

All protocols for mouse experiments discussed here were in accordance with institutional guidelines and approved by the Mount Sinai Medical Center Institutional Human Research Ethics Committee and Animal Care and Use Committee.
1. Processing of the Sarcoma Tissue Sample, and PDX Formation
<ol>
	<li>Under an Institutional Review Board-approved protocol, have pathology service personnel prepare sarcoma samples from surgery specimens and immediately put each sample on ice in a 100 mm Petri dish.</li>
	<li>Place the sample into a 15 mL polystyrene conical tube with 6 mL of cold Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Process the tissue sample immediately.</li>
	<li>(Optional) For osteosarcoma only, follow the following steps, which can be skipped for soft tissue sarcoma.
	<ol>
		<li>Cut the tissue into 20 mm3 pieces using a scalpel. Transfer the tissue pieces to a 15 mL tube containing 3 mL of collagenase solution (RPMI 1640 with 1 mg/mL collagenase).</li>
		<li>Put the tube into a water bath at 37 &#176;C for 30 min.</li>
		<li>Thoroughly vortex the tube and, then, add 3 mL of RPMI 1640 supplemented with 10% FBS to neutralize the collagenase activity.</li>
		<li>Centrifuge for 5 min at 350 x g at room temperature. Remove the supernatant.</li>
	</ol>
	</li>
	<li>Working in a sterile biosafety cabinet, place the tissue sample in a 100 mm Petri dish. Add 500 &#956;L of sterile 1x phosphate-buffered saline (PBS) to the tissue. With a sterile scalpel, mechanically triturate the tissue into small pieces until no visible tissue piece is larger than 0.1 mm.</li>
	<li>Transfer the 500 &#956;L cell suspension to a 35 &#956;m cell strainer and collect the filtered suspension with a 50 mL polystyrene tube. Put this 50 mL polystyrene tube with the cell suspension on ice.</li>
	<li>Add another 500 &#956;L of PBS to the tissue. Triturate for the second time, transfer the suspension through the cell strainer, and collect it into the same 50 mL tube.</li>
	<li>Repeat these steps (steps 1.5&#8211;1.6) until the tissue section is completely dissociated, usually 6x - 8x.</li>
	<li>Pellet the cell suspension by centrifugation at 350 x g for 10 min at room temperature. Discard the supernatant, resuspend the pellet using 5 mL of hemolysis buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA), and incubate the solution for 5 min at room temperature to remove red blood cells.</li>
	<li>Centrifuge for 5 min at 350 x g at room temperature and remove the hemolysis buffer. Wash the pellet with 5 mL of PBS. Centrifuge again for 5 min at 350 x g and remove the supernatant.</li>
	<li>Resuspend the pellet with 1 mL of PBS and count the viable cell number using a hemocytometer or any other alternative method. Dilute the cells to a final concentration of 1 x 107 cells in 200 &#956;L of PBS. Leave the cell suspension on ice.</li>
	<li>Leave the basement membrane matrix on ice to allow it to melt. Add 200 &#956;L of basement membrane matrix to the 200 &#956;L cell suspension. Gently mix and keep on ice.</li>
	<li>Subcutaneously inject the cell suspension:basement membrane matrix (1:1) mixture into two NOD scid gamma (NSG) mice into their flanks. Use 200 &#956;L for each injection.</li>
	<li>Monitor the PDX formation by checking the injection site of the mice 2x a week. Remove the tumor xenograft when it reaches 1 cm in diameter.</li>
</ol>
2. Isolation of Tumor-initiating Cells by FACS from the PDX
<ol>
	<li>Surgically remove the PDX from the mice as described previously12.</li>
	<li>Cut the tumor in half. Fix half of the xenograft with 4% paraformaldehyde overnight. This is for histological analysis. Process the other half of the tissue as described above (steps 1.3&#8211;1.8) to get a tumor cell suspension.</li>
	<li>Resuspend the pellet with PBS and count the viable cell number.</li>
	<li>Dilute the cell suspension in PBS supplemented with 5% FBS to a concentration of 2 x 106 cells/mL. Leave the cell suspension on ice for 30 min. Divide the cell suspension over two tubes. Mark the tubes with isotope control and antibody, respectively. Note the number of cells in each tube.</li>
	<li>Prepare 2x HLA-1-PE antibody by diluting the antibody with PBS supplemented with 5% FBS (1:250). Dilute the negative isotype control antibody with the same condition. Mix the cell suspension from the &#8220;antibody&#8221; tube with diluted antibody (1:1) to make the final antibody dilution 1:500. Mix the cell suspension from the &#8220;isotope control&#8221; tube with isotope control (1:1). Put the cell suspensions on ice for 90 min.</li>
	<li>Centrifuge for 5 min at 350 x g at 4 &#176;C and remove the supernatant. Add 10 mL of PBS to wash the pellet 2x.</li>
	<li>Add 4&#39;,6-diamidino-2-phenylindole (DAPI) to PBS to a final concentration of 10 &#956;g/mL. Add this DAPI solution to the cell pellet to make a cell suspension of 107 cells/mL (using the cell numbers from step 2.4).</li>
	<li>Filter the cell suspension through 35 &#956;m strainer caps into 12 mm x 75 mm polystyrene tubes.</li>
	<li>Use a flow cytometer to sort the HLA-1-negative TIC subpopulation6. Gate viable cells (DAPI-negative) and collect both HLA-1-negative and -positive subpopulations into two 15 mL collection tubes, each containing 4 mL of RPMI 1640 culture medium.</li>
</ol>
3. Characterization of Tumor-initiating Cells
<ol>
	<li>Sarcosphere formation
	<ol>
		<li>Make sarcosphere growth medium, using alpha-MEM cell culture medium. Add supplements to make a final concentration of B-27 supplements (1x), N2 supplements (1x), basic fibroblast growth factor (bFGF) (20 ng/mL), and epidermal growth factor (EGF) (20 ng/mL) and penicillin/streptomycin (100 IU/mL). Filter the medium with a 0.2 &#956;m cell culture filter before use.</li>
		<li>Pellet the sorted HLA-1-negative and -positive subpopulation from step 2.9 and count the cell numbers of each subpopulation.</li>
		<li>Dilute 1.5 x 106 cells into 15 mL of the sarcosphere growth medium to make the cell dilution of 1 x 105 cells/mL.</li>
		<li>Serially dilute the cells with fresh sarcosphere growth medium to make 15 mL of each cell dilution of 104, 103, and 102 cells/mL. Then, prepare four 96-well ultra-low attachment cell culture plates, each for a cell dilution.</li>
		<li>Transfer 100 &#181;L of cell suspension from the 105 cells/mL dilution to each well of the first 96-well ultra-low attachment cell culture plate. This plate has 104 cells in each well.</li>
		<li>Use the other three 96-well ultra-low attachment cell culture plates for the other three cell dilutions: 104, 103, and 102 cells/mL. Transfer 100 &#181;L of cell suspension to each well of the 96-well ultra-low attachment cell culture plates. These three culture plates have 1,000 cells/well, 100 cells/well, and 10 cells/well, respectively.</li>
		<li>Put the plates into a 37 &#176;C, 5% CO2 cell culture incubator.</li>
		<li>Add new bFGF and EGF directly to the cell culture medium (final concentration of 20 ng/mL) every three days without changing the medium to avoid cells lost in the suspension culture.</li>
		<li>Using a light microscope, monitor the sarcosphere formation every day for three weeks, as shown in Figure 2A.</li>
		<li>After three weeks, count the number of sarcosphere-positive wells and sarcosphere-negative wells of each cell dilution for both HLA-1-negative and HLA-1-positive cells.</li>
		<li>Calculate the sphere-forming cell frequency based on a Poisson probability distribution13. Compare the HLA-1-negative TICs with the HLA-1-positive bulk cells.</li>
	</ol>
	</li>
	<li>Serial-dilution tumor-formation assay
	<ol>
		<li>Count the HLA-1-negative and -positive subpopulations from step 2.9.</li>
		<li>Make serial dilutions of the cells with PBS to concentrations of 106, 105, 104, and 103 cells/mL. Use 1 mL of each dilution for the tumor formation in 10 mice.</li>
		<li>Add 1 mL of basement membrane matrix to the 1 mL cell suspension of each dilution (1:1). Keep the 2 mL of each mixture on ice.</li>
		<li>Subcutaneously inject 200 &#181;L of the cell:basement membrane matrix mixture into the flanks of NGS mice, using HLA-1-negative cells for one flank and HLA-1-positive cells for the other flank of the same mouse. For each dilution, use 10 mice. Use 25G syringes with a needle for the injections.</li>
		<li>Monitor the tumor formation in the mice for four to eight weeks, depending on the rate of tumor growth.</li>
		<li>Calculate the tumor-initiating cell frequency by the percentage of tumor formation at different input cell numbers. Compare the HLA-1-negative TICs with the HLA-1-positive bulk cells.</li>
	</ol>
	</li>
	<li>Induced differentiation along mesenchymal pathways
	<ol>
		<li>Use the sarcospheres formed during previous steps (step 3.1.9). Transfer the sarcospheres to a new 6-well plate. Culture the sarcosphere with 2.5 mL of alpha-MEM supplemented with 10% FBS to let the cells attach to the culture plate surface.</li>
		<li>After an attachment for two days, switch the culture medium from alpha-MEM supplemented with 10% FBS to the 1:1 mixture of alpha-MEM supplemented with 10% FBS and human mesenchymal stem cell (hMSC) growth medium.</li>
		<li>After two days, switch to complete hMSC growth medium.</li>
		<li>When the cells reach 90% confluency, aspirate the hMSC medium and add differentiation medium. For osteogenic differentiation, add osteogenic differentiation medium (hMSC growth medium supplemented with 10 nM dexamethasone, 5 mM &#946;-glycerophosphate, 50 &#181;g/mL L-ascorbic acid, and 10 mM lithium chloride). For lipogenic differentiation, add adipocyte differentiation medium (hMSC growth medium supplemented with 0.5 &#181;M dexamethasone, 0.5 &#181;M isobutylmethylxanthine, and 50 &#181;M indomethacin).</li>
		<li>Change the differentiation medium every three days.</li>
		<li>After three to four weeks, stop the differentiation and wash the cells with PBS. Then, aspirate the PBS and add 2 mL of 10% formalin to the cells for fixation. Let the cells sit for 45 min at room temperature. Wash them with deionized water. The cells are now ready for Alizarin Red S staining (step 3.3.6.1) or Oil Red O staining (step 3.3.6.2).
		<ol>
			<li>To detect osteogenic differentiation, perform Alizarin Red S staining. Aspirate water and add 2 mL of Alizarin Red S working solution (2% Alizarin Red S, pH 6.0) to the cells, and let them sit for 5 min for staining. Wash the cells with deionized water and observe the reaction microscopically.</li>
			<li>To detect adipogenic differentiation, perform Oil Red O staining.
			<ol>
				<li>Make an Oil Red O solution. Prepare a stock solution by adding 300 mg of Oil Red O powder to 100 mL of isopropanol.</li>
				<li>Within 2 h before using, mix three parts (30 mL) of Oil Red O stock solution with two parts (20 mL) of deionized water. Allow the mixture to sit for 10 min at room temperature.</li>
				<li>Filter the working solution using before use.</li>
				<li>Remove the water from the cells prepared according to step 3.3.6. Add 2 mL of 60% isopropanol to cover the cell monolayer and the cells sit for 2 min.</li>
				<li>Remove the isopropanol and add 2 mL of Oil Red O working solution. Allow the cells to sit for 5 min at room temperature.</li>
				<li>Rinse the cells with deionized water and observe the reaction under a light microscope.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Medema, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+the+challenges+ahead.">Cancer stem cells: the challenges ahead.</a> Nature Cell Biology. 15 (4), 338-344 (2013).</li><li>Jordan, C., Guzman, M., Noble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells.">Cancer stem cells.</a> The New England Journal of Medicine. 355 (12), 1253-1261 (2006).</li><li>Jordan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+controversial+or+just+misunderstood?.">Cancer stem cells: controversial or just misunderstood?.</a> Cell Stem Cell. 4 (3), 203-205 (2009).</li><li>Bapat, S., Mali, A., Koppikar, C., Kurrey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor-like+cells+contribute+to+the+aggressive+behavior+of+human+epithelial+ovarian+cancer.">Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer.</a> Cancer Research. 65 (8), 3025-3029 (2005).</li><li>Charafe-Jauffret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature.">Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.</a> Cancer Research. 69 (4), 1302-1313 (2009).</li><li>Domingo-Domenech, 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=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer Cell. 22 (3), 373-388 (2012).</li><li>Reya, T., Morrison, S., Clarke, M., Weissman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+cancer,+and+cancer+stem+cells.">Stem cells, cancer, and cancer stem cells.</a> Nature. 414 (6859), 105-111 (2001).</li><li>Yi, 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=Abnormal+DNA+methylation+of+CD133+in+colorectal+and+glioblastoma+tumors.">Abnormal DNA methylation of CD133 in colorectal and glioblastoma tumors.</a> Cancer Research. 68 (19), 8094-8103 (2008).</li><li>Ginestier, 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=ALDH1+is+a+marker+of+normal+and+malignant+human+mammary+stem+cells+and+a+predictor+of+poor+clinical+outcome.">ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.</a> Cell Stem Cell. 1 (5), 555-567 (2007).</li><li>Garrido, F., 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=Natural+history+of+HLA+expression+during+tumour+development.">Natural history of HLA expression during tumour development.</a> Immunology Today. 14 (10), 491-499 (1993).</li><li>Chang, C. C., Campoli, M., Ferrone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+and+nonclassical+HLA+class+I+antigen+and+NK+cell-activating+ligand+changes+in+malignant+cells:+current+challenges+and+future+directions.">Classical and nonclassical HLA class I antigen and NK cell-activating ligand changes in malignant cells: current challenges and future directions.</a> Advances in Cancer Research. 93, 189-234 (2005).</li><li>Han, 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=Targeting+sarcoma+tumor-initiating+cells+through+differentiation+therapy.">Targeting sarcoma tumor-initiating cells through differentiation therapy.</a> Stem Cell Research. 21, 117-123 (2017).</li><li>Hu, Y., Smyth, G. 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The authors have nothing to disclose.

There are several critical steps which limit the success of this protocol to isolate and characterize tumor-initiating cells from human sarcoma PDXs. Human sarcoma includes many different subtypes. We observed that PDX formation is highly dependent on sarcoma subtypes. Clinical aggressive sarcomas with a histologically undifferentiated phenotype (e.g.,&#160;pleomorphic undifferentiated sarcomas [success rate 100%, n = 2], dedifferentiated liposarcomas [success rate 100%, n = 2], and synovial sarcomas [success rate 100%, n = 3]) have a high success rate of PDX formation. Meanwhile, sarcomas with differentiated phenotypes (e.g., well-differentiated liposarcomas [success rate 0%, n = 3]) show lower PDX formation rates. It is possible that tumor-initiating cells are present in more malignant subtypes at a higher percentage than in less malignant, differentiated subtypes. In addition, we recommend finishing the tumor-initiating cell isolation procedure within one day without any stop to minimize any loss of cell viability.
We have identified that HLA-1-negative cells exist widely in human sarcomas. But the percentage of HLA-1-negative cells can vary among samples from different patients. By this method, we have successfully isolated tumor-initiating cells from samples that have HLA-1-negative cells ranging from less than 0.5% to more than 30%12. It is important to characterize the HLA-1-negative cells functionally by sphere formation and tumor formation assays to confirm the tumor-initiating cell identity of isolated HLA-1-negative cells.
However, the protocol presented here also has limitations. Previous data showed that HLA-1 expression is epigenetically regulated, which is consistent with the observation of HLA-1 expression&#39;s cellular heterogeneity within the same tumor12. HLA-1 genomic mutations were detected in sarcomas and other cancer types. Mutations in HLA-1 genes may lead to the complete loss of HLA-1 on the cell surface in the whole tumor or to expressing nonfunctional mutated HLA-1. In either case, HLA-1 negativity cannot be used to identify TICs within the tumor.
Using HLA-1 as a negative marker, we have successfully isolated TICs from a variety of human sarcoma subtypes and validated our results by functional analysis. Thus, we were able to perform molecular studies including gene expression analysis on the TICs, in order to develop specific treatment targeting these TICs.

Intratumoral cellular heterogeneity of human cancers has been supported by increasing evidence during the past decade1. Similar to normal tissue, cancer tissue consists of a small subpopulation of TICs (also called cancer stem cells), which exhibit tumor-forming ability; meanwhile, the bulk of cancer cells exhibit differentiated phenotypes2. These TICs show stem cell-like properties, including the expression of a stem cell marker and the ability of both self-renewal and asymmetric cell division, and thus, can initiate the formation of a cellular heterogeneous tumor3. Recent studies have revealed that TICs are not only responsible for tumor initiation but are also associated with tumor aggressiveness4, metastasis5, and drug resistance6. Therefore, it is important to understand the biology of TICs and, thus, develop a specific treatment strategy targeting these TICs.
FACS-based methods have been used to identify TICs using TICs markers, including CD133, CD24, and CD441. Most of these markers are also expressed in normal stem cells7. However, none of these markers solely mark TICs. The roles these molecules play in the malignancy of TICs are still not clear. For example, CD133 can be frequently inactivated by DNA methylation, and thus, this intertumoral heterogeneity may render the accuracy of these markers8. ALDH1 is a marker that also functions to maintain the stemness of TICs9. It seems to be more effective in identifying breast cancer TICs but is still questionable in other tumor types9. Some signaling pathways play important roles in stem cell biology, including Wnt (wingless-related integration site), TGF-&#946; (transforming growth factor beta), and Hedgehog1. But it is difficult to prove that these pathways are TIC-specific and to use the activity of these pathways to isolate TICs from primary tumors. Thus, a reliable novel TIC marker is urgently needed.
Human MHC class I, also called HLA-1, is a cell surface protein expressed in almost all nucleated cells10. HLA-1 functions as an antigen presenting a molecule that is specifically recognized by CD8 T cells10. The cytotoxic effect of CD8 T cells can be activated when cancer cells present a tumor antigen by HLA-1. Therefore, lacking HLA-1 on the cell surface of the cancer cells can lead to an immune escape from the cytotoxic CD8 T cells. The downregulation of HLA-1 has been described in different types of human cancer and is correlated with poor prognosis, metastasis, and drug resistance11. We have shown that the loss of HLA-1 expression on the cell surface can be used to identify TICs in sarcomas, as well as in prostate cancer6,12.
Here, we describe a detailed protocol to isolate TICs from human sarcoma PDXs by FACS, using HLA-1 as a negative marker, and to further validate and characterize these HLA-1-negative TICs.

<table><tbody><tr><td>0.2 &amp;mu;m cell culture filter</td><td>ThermoFisher</td><td>450-0020</td><td></td></tr><tr><td>100 mm Petri dish</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>15 mL conical tube</td><td>Falcon</td><td>352196</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 mL polystyrene tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>96-well ultra-low attachment cell culture plate</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>08-0085SA</td><td></td></tr><tr><td>bFGF</td><td>Invitrogen</td><td>PHG0021</td><td></td></tr><tr><td>dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>PHG0311</td><td></td></tr><tr><td>HLA-1-PE antibody</td><td>Abcam</td><td>ab43545</td><td></td></tr><tr><td>hMSC growth medium</td><td>ATCC</td><td>PCS-500-030</td><td></td></tr><tr><td>indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td></td></tr><tr><td>isobutylmethylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>isopropanol</td><td>Sigma-Aldrich</td><td>W292907</td><td></td></tr><tr><td>Isotype Control Antibody</td><td>Abcam</td><td>ab103534</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma-Aldrich</td><td>A5960</td><td></td></tr><tr><td>lithium chloride</td><td>Sigma-Aldrich</td><td>62476</td><td></td></tr><tr><td>Matrigel basement membrane matrix</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>MEM Alpha</td><td>Gibco</td><td>12571-063</td><td></td></tr><tr><td>N2</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>NSG mice</td><td>The Jackson Lab</td><td>005557</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Syringe with needle</td><td>BD</td><td>309626</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma-Aldrich</td><td>G9422</td><td></td></tr></tbody></table>

isolation and characterization of tumor-initiating cells from sarcoma patient-derived xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

A human sarcoma PDX was generated and stained. Intratumoral heterogeneity was shown by immunohistochemistry using HLA-1 antibody. The xenograft consisted of two distinct subpopulations, namely HLA-1 positive and negative (Figure 1A)12. The sarcoma PDX showed histological similarities with the parental primary tumor (Figure 1A). Sarcoma PDX TICs were isolated by FACS. Using a double sorting method, the HLA-1-negative cells were highly enriched from the parental cell population (Figure 1B)12.
Genes expressed in stem cells (e.g.,&#160;Oct4, Nanog, and Myc) were found to be highly expressed in isolated HLA-1-negative cells when compared to their HLA-1-positive counterpart (Figure 1C). Sox-9, a developmental gene reported to play important roles in other cancer stem cells, such as in breast carcinoma, were specifically expressed in HLA-1-negative cells (Figure 1D).
To validate the isolated HLA-1-negative subpopulation, a sarcosphere formation assay was performed to examine the self-renewal ability of the cells. HLA-1-negative cells were able to form spheres with an initial input of as little as 10 cells (Figure 1E)12. In order to examine the tumor-forming ability, a serial-dilution tumor-formation assay was performed. The same numbers of HLA-1-negative and -positive cells were injected subcutaneously into each flank of the same mouse. HLA-1-negative cells showed a significantly higher tumor formation ability (Figure 1F)12, while xenografts formed by both HLA-1-negative and -positive subpopulations were cellular heterogeneous tumors (Figure 1H).
We performed a gene expression analysis of the isolated HLA-1-negative TICs12. Genes associated with normal mesenchymal cell differentiation were elevated in TICs12. Thus, we also tested whether HLA-1 TICs can be induced to terminal differentiation and result in a decreased tumor formation ability. The results showed that HLA-1-negative cells can be induced to differentiate along both lipogenic and osteogenic pathways and show strong Oil Red O and Alizarin Red S staining (Figure 1G). In contrast, HLA-1-positive cells do not differentiate under the same conditions. Thus, these results indicate a promising differentiated therapy strategy that may be used to target sarcoma TICs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57011/57011fig1.jpg" /> 
Figure 1: Isolation of HLA-1-negative cells from intratumoral heterogeneous sarcoma PDXs. (A) HLA-1-negative cells (arrows) were found in different subtypes of human sarcomas by immunohistochemistry (IHC). (a and b) Clear cell sarcoma. (c and d) Pleomorphic liposarcoma. (e and f) Leiomyosarcoma. (g and h) Malignant peripheral nerve sheath tumor. (i and j) Liposarcoma, not otherwise specified. (k and l) Dedifferentiated liposarcoma. Scale bar = 100 &#181;m. (B) Sarcoma PDXs were histologically similar to the parental tumor (hematoxylin and eosin [H&#38;E] stain) and showed cellular heterogeneity in the HLA-1 expression by IHC. Here are shown representative pictures of sarcoma PDXs, including a clear cell sarcoma (CCS), a dedifferentiated chondrosarcoma (DCS), and a dedifferentiated liposarcoma (DDL). Scale bar = 100 &#181;m. (C) The subpopulation of HLA-1-negative cells was isolated by flow cytometry with a double-sort method. From top to bottom: first sort, second sort, and purity check. Isolated HLA-1-negative and HLA-1-positive cells were subjected to a subsequent functional analysis, including a tumor formation assay. The results from this figure are from a previous publication12. <a href="https://www.jove.com/files/ftp_upload/57011/57011fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57011/57011fig2.jpg" /> 
Figure 2: Characterization of HLA-1-negative TICs by functional assays. (A) A sphere formation assay showed that as few as 10 HLA-1-negative cells were able to form sarcoma spheres. Left: representative pictures of sarcoma spheres. Right: sphere-forming frequency; mean &#177; SD. Scale bar = 100 &#181;m. (B) HLA-1-negative cells isolated from the sarcoma PDX were highly tumorigenic. Here shown are representative pictures of the tumor formed by HLA-1-negative and -positive cells from DDL. A thousand cells of HLA-1-negative and HLA-1-positive DDL cells were injected in separate flanks of the same mouse. (C) The mRNA levels of stem cell genes Oct4, Nanog, and Myc were expressed at higher levels in HLA-1-negative cells compared to HLA-1-positive cells. The data represent the mean &#177; SD (n = 5). (D) Strong positive staining of Oil Red O and Alizarin Red S shows a terminal differentiation along lipogenic and osteogenic pathways that are induced from sarcoma TICs. (E) HLA-1 immunostaining of PDXs formed by HLA-1-positive (left) and -negative (right) subpopulations. Scale bar = 100 &#181;m. The results from this figure are from a previous publication12. <a href="https://www.jove.com/files/ftp_upload/57011/57011fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

isolation-characterization-tumor-initiating-cells-from-sarcoma

Research

JoVE Journal

Cancer Research

6.9K Views.  Icahn School of Medicine at Mount Sinai. We ontdekten dat HLA-I epigenetisch naar beneden is gereguleerd in een subpopulatie van kankercellen die stamceleigenschappen en tumorinitierende capaciteit vertonen. HLA-I downregulation is direct gerelateerd aan immuun ontsnappen, het verstrekken van overlevingsvoordelen voor kanker stamcellen, waardoor HLA-I negativiteit een nauwkeurige functionele marker voor deze cellen. Het demonstreren van de procedure zal Sudeh Izadmehr zijn, een postdoc uit ons laboratorium.Bij aankoop van het...

Video: Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetics

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Biology

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

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