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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELDA:+Extreme+limiting+dilution+analysis+for+comparing+depleted+and+enriched+populations+in+stem+cell+and+other+assays.">ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.</a> Journal of Immunology. 347, 70-78 (2009).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:300px;">0.2 &#956;m cell culture filter</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:119px;">450-0020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">100 mm Petri dish</td>
			<td style="width:123px;">Falcon</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">15 mL conical tube</td>
			<td style="width:123px;">Falcon</td>
			<td style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">35 &#956;m cell strainer</td>
			<td style="width:123px;">Falcon</td>
			<td style="width:119px;">352340</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">50 mL polystyrene tube</td>
			<td style="width:123px;">Falcon</td>
			<td style="width:119px;">352070</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">96-well ultra-low attachment cell culture plate</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">7007</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Alizarin Red S&#160;</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5533</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">B-27</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">08-0085SA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">bFGF</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">PHG0021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">dexamethasone</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">EGF</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">PHG0311</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">HLA-1-PE antibody&#160;</td>
			<td style="width:123px;">Abcam</td>
			<td>ab43545</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">hMSC growth medium</td>
			<td style="width:123px;">ATCC</td>
			<td style="width:119px;">PCS-500-030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">indomethacin</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7378</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">isobutylmethylxanthine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5879</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">isopropanol</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">W292907</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:300px;">Isotype Control Antibody</td>
			<td style="width:123px;">Abcam</td>
			<td style="width:119px;">ab103534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">L-ascorbic acid&#160;</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5960</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">lithium chloride&#160;</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">62476</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Matrigel basement membrane matrix&#160;</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">354230</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:300px;">MEM Alpha</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">12571-063</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">N2</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">17502-048</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">NSG mice</td>
			<td style="width:123px;">The Jackson Lab</td>
			<td style="width:119px;">005557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Oil Red O&#160;</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">O0625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">PBS</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">21-040-CM</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">penicillin/streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">RPMI 1640&#160;</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:300px;">Syringe with needle</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">&#946;-glycerophosphate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/57011/57011fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.8K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

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.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

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

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...