This study was funded in part by grants from the R01(CA258113), SPORE (CA261608), and P01 (CA114046). Figure 1 was prepared in BioRender.com.

Human melanoma tissues were obtained from patients receiving treatment at the University of Pennsylvania using the tissue collection protocol (UPCC08607) that is approved by the Institutional Review Board of the University of Pennsylvania. All patients have signed informed consent. Following resection of the human melanoma tissues, the tumor tissue is placed in Dulbecco&#39;s Modified Eagle Medium (DMEM) and kept at 4 &#176;C until processing (within 6 h). Melanoma patient-derived organoids (MPDOs) were derived from a fresh tumor as illustrated in Figure 1.
1. Isolation of cells from human melanoma tissue
<ol>
	<li>Preparations before the start of the procedure 
	NOTE: Two culture methods (Matrigel and collagen gel) can be used to culture dissociated melanoma tissue:
	<ol>
		<li>For the Matrigel method: Prepare a coating solution of Growth Factor Reduced (GFR) Basement Membrane Matrix by diluting GFR Basement Membrane Matrix with organoid culture medium (see Table 1) at a 1:2 ratio. Add approximately 115 &#181;L of the thawed solution to each of several wells of a 48-well plate and leave it to solidify for 30 min at 37 &#176;C in a humidified incubator.</li>
		<li>For the collagen gel method: Add 1 mL of Collagen gel (see Table 1) to a 30 mm inner membrane insert. Allow the gel to solidify for 30 min at 37 &#176;C in a humidified incubator.</li>
	</ol>
	</li>
	<li>Prepare the L-WRN conditioned medium15. L-WRN conditioned medium is produced from an L-WRN cell line engineered to secrete Wnt3a, R-spondin 3, and Noggin into a single conditioned medium that is widely used for cell culture.
	<ol>
		<li>Thaw a cryotube of L-WRN cells in the 37 &#176;C water bath. Add 5 mL of L-cell medium (see Table 1) and the L-WRN cell suspension to a 15 mL conical tube, and centrifuge at 120 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant, ensuring that ~500 &#181;L is left at the bottom to resuspend the pellet of cells.</li>
		<li>Transfer the cell pellet in the supernatant into 30 mL of L-cell medium on ice and add 150 &#181;L of hygromycin (500 &#181;g/mL) and 300 &#181;L of G418 (500 &#181;g/mL). Seed the cells at a density of 3 &#215; 106 on a T-150 flask and incubate at 37 &#176;C in a humidified incubator with 5% CO2 until ~90% confluent.</li>
		<li>Once the cells are ~90% confluent, split cells into five T-150 flasks by washing with 20 mL of phosphate-buffered saline (PBS), aspirating, and digesting with 3 mL of trypsin substitute. After 5 min, neutralize with 9 mL of L-cell medium followed by centrifugation for 5 min at 120 &#215; g at 4 &#176;C . When ~100% confluent, split the five T-150 flasks into 10 T-175 3-layer flasks.</li>
		<li>Once confluent, wash cells with 2 x 50 mL of PBS and then with 25 mL of washing medium (see Table 1).</li>
		<li>Add 75 mL of primary culture medium to each T-175 3-layer flask and incubate for 3 days at 37 &#176;C in a humidified incubator with 5% CO2 (see Table 1).</li>
		<li>After 3 days, harvest the conditioned medium into a 50 mL conical and spin at 1,100 &#215; g for 5 min at 4 &#176;C. Collect the supernatant (conditioned medium), ensuring that 5 mL is left behind.</li>
		<li>Add fresh 75 mL of primary cell culture medium to each T-175 3-layer flask and incubate for an additional 3 days to collect additional conditioned medium.</li>
		<li>To the collected conditioned medium, add an equal volume of primary cell culture medium (50% volume) and vacuum filter the contents using a 0.22 &#181;m filter. Store aliquots into 50 mL tubes and store at -20 &#176;C for long-term usage or 4 &#176;C for up to 2-3 weeks. Use this medium to supplement the organoid culture media (see Table 1). 
		NOTE: Thaw aliquots at 4 &#176;C overnight prior to use.</li>
	</ol>
	</li>
	<li>Harvesting the cellular mixture from fresh melanoma tissue
	<ol>
		<li>Wash the freshly resected melanoma tissue in a 10 mL Petri dish containing washing medium. After the first rinse, use sterile tweezers to transfer the tumor into a second Petri dish for further washing and repeat this process 3x on ice to preserve tumor integrity and viability. Throughout these rinsing cycles, use sterile scissors and a blade to remove excess connective tissue, fat, and residual blood. After the three washes, place the tumor in an empty Petri dish and use sterile blades to finely mince it. 
		NOTE: After mincing, the tumor should exhibit a gel-like consistency. The finer the mincing of the tumor, the higher the cell yield will be. This is because larger clumps of the tumor will be more challenging to digest and may not pass through the 70 &#181;m Nylon cell strainer in the subsequent step.</li>
		<li>Once the tumor tissue has been thoroughly washed and minced, transfer it into a 50 mL conical tube containing 10 mL of the prepared digestion medium (see Table 1). Place the conical in a water bath at 37 &#176;C, ensuring the contents are vortexed every 5 min.</li>
		<li>After 25 min, add 30 mL of the ADMEM/F12 medium containing 10% FBS to stop digestion. 
		NOTE: These specified quantities are intended for tumors weighing no more than 0.2 g.</li>
		<li>Filter the digested cell suspension through a 70 &#181;m Nylon cell strainer, then rinse the strainer with ADMEM/F12 (1x) Reduced Serum Medium (1:1) containing 10% FBS. Following the washing step, use a pipette to retrieve any remaining media on the bottom of the filter to ensure that the highest possible number of cells is retrieved. Then, centrifuge at 300 &#215; g for 7 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in organoid culture medium (see Table 1).</li>
		<li>Culture the melanoma single-cell suspension in either Matrigel or collagen culture systems:
		<ol>
			<li>Matrigel: Count the single-cell suspension to ensure that approximately 2 &#215; 105 cells are seeded in the prewarmed Matrigel-coated wells supplemented with 200 &#181;L of organoid culture medium. 
			NOTE: Matrigel-based organoids are used to study pathophysiology, screen drug efficacy, and test drug toxicity16.</li>
			<li>Collagen gel: Count the single-cell suspension to ensure that approximately 106 cells are mixed with 1 mL of collagen gel. Add the mixture to the pre-solidified collagen gel membrane inserts and place these in a 6-well plate. Add enough organoid culture medium to submerge the inserts containing the cells and collagen gel. 
			NOTE: Collagen gel organoids are used to measure T cell migration and study tissue development16.</li>
		</ol>
		</li>
		<li>Allow the organoids to grow while ensuring that fresh medium is added every 3 days.</li>
		<li>Passage the organoids once they reach 150-200 &#181;m in size or cryopreserve them until further usage.
		<ol>
			<li>For passage of organoids, transfer the organoids into a 15 mL conical and wash with 1x DPSB. Centrifuge the mixture at 300 &#215; g for 7 min at 4 &#176;C. Remove the supernatant and add trypsin substitute (50-100 &#181;L per organoid) for 10 min in a 37 &#176;C water bath, ensuring that contents are vortexed every 3 min.</li>
			<li>Stop the digestion using ADMEM/F12 (1x) Reduced Serum Medium (1:1) medium containing 10% FBS. Centrifuge the mixture at 300 &#215; g for 7 min and resuspend in organoid culture medium.</li>
			<li>Seed the single-cell suspension into a new Matrigel/Collagen-coated well at the same initial seeding density (2 &#215; 105 cells in 200 &#181;L of organoid culture media for Matrigel-coated wells in a 48 well plate or 106 cells in 1 mL of organoid culture media for collagen coated 30 mm inner membrane insert).</li>
			<li>For cryopreservation, digest MPDOs using the trypsin substitute into single-cell suspensions followed by resuspension of 106 cells into 1 mL of freezing medium (see Table 1).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

<ol>
	<li>Mirabelli, P., Coppola, L., Salvatore, 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+Cell+Lines+Are+Useful+Model+Systems+for+Medical+Research.">Cancer Cell Lines Are Useful Model Systems for Medical Research.</a> Cancers (Basel). 11 (8), 1098 (2019).</li><li>Shannon, A. E., Boos, C. E., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culturing+multicellular+tumor+models:+Modeling+the+tumor+microenvironment+and+analysis+techniques.">Co-culturing multicellular tumor models: Modeling the tumor microenvironment and analysis techniques.</a> Proteomics. 21 (9), e2000103 (2021).</li><li>Jubelin, C., Mu&#241;oz-Garcia, J., Griscom, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+culture+models+in+oncology+research.">Three-dimensional in vitro culture models in oncology research.</a> Cell Biosci. 12, 155 (2022).</li><li>Jin, J., Yoshimura, K., Sewastjanow-Silva, M., Song, S., Ajani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+prospects+of+patient-derived+xenografts+for+cancer+research.">Challenges and prospects of patient-derived xenografts for cancer research.</a> Cancers (Basel). 15 (17), 4352 (2023).</li><li>Nabel, C. S., Heiden, M. G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenografts+to+study+cancer+metabolism:+when+does+X+mark+the+spot).">Patient-derived xenografts to study cancer metabolism: when does X mark the spot).</a> Cancer Res. 81 (17), 4399 (2021).</li><li>Choi, Y., 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=Studying+cancer+immunotherapy+using+patient-derived+xenografts+(PDXs)+in+humanized+mice.">Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.</a> Exp Mol Med. 50 (8), 99 (2018).</li><li>Woo, X. Y., 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=Conservation+of+copy+number+profiles+during+engraftment+and+passaging+of+patient-derived+cancer+xenografts.">Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.</a> Nat Genet. 53 (1), 86 (2021).</li><li>Somasundaram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-infiltrating+mast+cells+are+associated+with+resistance+to+anti-PD-1+therapy.">Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.</a> Nat Commun. 12 (1), 346 (2021).</li><li>Abdolahi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+(PDX)+models,+applications+and+challenges+in+cancer+research.">Patient-derived xenograft (PDX) models, applications and challenges in cancer research.</a> J Transl Med. 20 (1), 206 (2022).</li><li>Yang, S., 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=Organoids:+The+current+status+and+biomedical+applications.">Organoids: The current status and biomedical applications.</a> Med Comm. 4 (3), e274 (2023).</li><li>Minoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+cancer+organoids+as+a+functional+system+to+model+different+disease+stages+and+therapy+response.">Bladder cancer organoids as a functional system to model different disease stages and therapy response.</a> Nat Commun. 14, 2214 (2023).</li><li>Ou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+melanoma+organoid+models+facilitate+the+assessment+of+immunotherapies.">Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.</a> eBioMedicine. 92, 104614 (2023).</li><li>Gr&#246;nholm, M., 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=Patient-derived+organoids+for+precision+cancer+immunotherapy.">Patient-derived organoids for precision cancer immunotherapy.</a> Cancer Res. 81 (12), 3149-3155 (2021).</li><li>Ou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+platforms+to+study+therapeutic+efficacy+of+human+&#947;&#948;+T+cells.">Preclinical platforms to study therapeutic efficacy of human &#947;&#948; T cells.</a> Clin Transl Med. 12 (6), e814 (2022).</li><li>Murine L-Wrn conditioned medium protocol. The University of Texas MD Anderson Cancer Center Available from: <a target="_blank" href="https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf">https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf</a> (2024)</li><li>Jee, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+collagen-based+3D+matrix+for+gastrointestinal+tract-derived+organoid+culture.">Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture.</a> Stem Cells Int. 13 (2019), 8472712 (2019).</li><li>Pipkin, M. E., Rao, A., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+control+of+the+perforin+locus.">The transcriptional control of the perforin locus.</a> Immunol Rev. 235 (1), 55 (2010).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tim-3+suppresses+the+killing+effect+of+V&#947;9V&#948;2+cells+on+colon+cancer+cells+by+reducing+perforin+and+granzyme+B+expression.">Tim-3 suppresses the killing effect of V&#947;9V&#948;2 cells on colon cancer cells by reducing perforin and granzyme B expression.</a> Exp Cell Res. 386 (1), 111719 (2020).</li><li>Li, L. T., Jiang, G., Chen, Q., Zheng, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki67+is+a+promising+molecular+target+in+the+diagnosis+of+cancer+(Review).">Ki67 is a promising molecular target in the diagnosis of cancer (Review).</a> Mol Med Rep. 11 (3), 1566-1572 (2015).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Li, H. Y., Ren, K., Wang, C., Bu, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+organoid+research+progress+and+potential+applications.">Skin organoid research progress and potential applications.</a> International Journal of Dermatology and Venereology. 5 (2), 101-106 (2022).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+mucosal+melanoma+organoid+platform+for+modeling+tumor+heterogeneity+and+exploring+immunotherapy+combination+options.">A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options.</a> Sci Adv. 9 (43), (2023).</li><li>Vlachogiannis, G., 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=Patient-derived+organoids+model+treatment+response+of+metastatic+gastrointestinal+cancers.">Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.</a> Science. 359 (6378), 920-926 (2018).</li><li>Ganesh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rectal+cancer+organoid+platform+to+study+individual+responses+to+chemoradiation.">A rectal cancer organoid platform to study individual responses to chemoradiation.</a> Nat Med. 25 (10), 1607-1614 (2019).</li><li>Vilgelm, A. E., 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=Fine-needle+aspiration-based+patient-derived+cancer+organoids.">Fine-needle aspiration-based patient-derived cancer organoids.</a> iScience. 23 (8), 101408 (2020).</li></ol>

The authors have no conflicts of interest to declare.

The emergence of PDOs has addressed multiple limitations posed by other previously established cancer research methods while introducing transformative potential applications in the field. This organoid technology was initially proposed in 2009 by Hans Clevers and colleagues, who were able to successfully establish an intestinal organoid culture system by culturing Lgr5+ stem cells derived from the intestine of mice in a 3D matrix gel containing R-sponsin, EGF, and Noggin factors20. A f...

Conventional 2-D cell culture models are essential tools in cancer research for studying cancer progression and therapy1. These models not only allow for controlled experimental conditions to investigate the molecular and cellular mechanisms underlying cancer development and progression but also provide cost-effective and relatively rapid experimental results. Their usage, however, is restricted due to the limited cellular diversity in the tumor microenvironment (TME), which cannot be recapitulated in a coplanar nature of mono-culture models2. Additionally, cell culture models offer an oversimplified environment compared to the human body3. Thus, to better understand cancer progression and develop effective treatments, researchers have turned to innovative models that aim to recapitulate the TME in nature closely. Consequently, maintaining fidelity to native tumors is paramount in these models. Given the intricate nature of cancer, any significant deviation from the original tumor could introduce unforeseen variables that might confound meaningful conclusions about tumor behavior. 
While patient-derived xenografts (PDX) have emerged as a model system that allows for monitoring of tumor progression in vivo, some limitations question how well the developed tumor recapitulates human cancer biology4. For instance, melanoma TME contains tumor-infiltrating immune cells and stromal cells. When passaged as a xenograft, the non-cancer cells from the patient will be gradually lost, which can select adaptations that differ from those in patient tumors. While these limitations can be mitigated by performing limited passages of the PDX in vivo, the differences between the species can still pose a risk of genetic changes to the PDX that deviate from that of the native tumor5. Moreover, PDX models are typically established in immunocompromised mice to prevent rejection of human tissues. This limits the ability to study the role of the host immune system in cancer, especially in immunotherapeutic studies6. We and others have engrafted PDXs in "humanized" mice7,8. However, establishing PDX models in humanized mice is time-consuming and expensive. It can take several months to years to develop a single PDX model from a patient's tumor specimen, thus limiting the speed and scale of research. Despite these limitations, PDX models have been shown to accurately represent the biology of the TME, thus having been extensively used for the development of cancer therapeutics, personalized medicine, and immunotherapy, among others9. 
An alternative 3D culture technique that has emerged is the patient-derived organoid (PDO), where a freshly resected patient tumor is cultured to create models that maintain phenotypic heterogeneity, histoarchitecture, and intercellular communications10. Its ability to closely resemble native tumors has been previously demonstrated where cultured organoids from non-muscle invasive (NMIBC) and muscle-invasive bladder cancer (MIBC) successfully recapitulated key aspects of the parental tumors11. Due to their potential and advantages over other models, PDOs offer a wide variety of tailored applications that other models lack, including but not limited to the study and development of immune checkpoint inhibitors along with cellular and targeted therapies. For example, we have previously used melanoma patient-derived organoids (MPDOs) to study the ability of tumor-infiltrating lymphocytes (TILs) to be expanded by IL-2 and anti-PD1 antibodies (&#945;PD-1). Results showed that the TILs expanded by IL-2 and &#945;PD-1 not only had a higher number but could also successfully infiltrate MPDOs and kill melanoma cells with higher efficiency than TILs expanded using other methods12. Other groups have shown similar results where the usage of anti-PD-1 and/or anti-PD-L1 leads to TILs remaining functional in PDOs, which subsequently leads to tumor killing13. Additionally, our group has also used MPDOs to assess &#947;&#948; T-cell infiltration and killing ability14. PDOs' applicability spans a wide range of areas, including TIL expansion, cytotoxicity studies, and small molecule screening. All these applications highlight the potential and usability that this model confers. As such, we describe the detailed protocol for the culture of MPDOs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mL Red Cap Internal Threaded Polypropylene Cryogenic Vial</td>
			<td>Corning</td>
			<td>431420</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>105 mm Polystyrene Forceps Sterile</td>
			<td>Caplugs Evergreen&#160;</td>
			<td>222-1121-B1I</td>
			<td></td>
		</tr>
		<tr>
			<td>150 cm2 Cell Culture Flask</td>
			<td>Corning</td>
			<td>CLS430825</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12 (1x) Reduced Serum Medium (1:1)</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-human Perforin Antibody</td>
			<td>BioLegend</td>
			<td>308120</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-human Ki-67 Antibody</td>
			<td>BioLegend</td>
			<td>350522</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 anti-human CD366 (Tim-3) Antibody</td>
			<td>BioLegend</td>
			<td>345028</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 711 anti-human CD45 Antibody</td>
			<td>BioLegend</td>
			<td>304050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts 0.4 &#181;m, 30 mm Diameter</td>
			<td>Millipore Sigma</td>
			<td>PIHP03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Staining Buffer</td>
			<td>BioLegend</td>
			<td>420201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning&#160;</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning&#160;</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type IV, powder</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Plate, PS, 48 wells, TC treated with lid, sterile</td>
			<td>Max Scientific&#160;</td>
			<td>07-6048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - high glucose 4.5 mg/mL</td>
			<td>Corning</td>
			<td>MT10-0130CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I - Grade II</td>
			<td>Millipore Sigma</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1%&#160;</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, FBS</td>
			<td>Corning</td>
			<td>MT35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Annexin V Apoptosis Detection Kit I</td>
			<td>BD Biosciences&#160;</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris</td>
			<td>1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td>
			<td>ThermoFisher</td>
			<td>10131035</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>ThermoFisher</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B (50 mg/mL)</td>
			<td>ThermoFisher</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN cell</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Phenol Free &#38; Growth Fact. Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex PVDF syringe filter, 0.22 &#956;M</td>
			<td>Millipore Sigma</td>
			<td>SLGVR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc TripleFlask Treated Cell Culture Flask</td>
			<td>ThermoFisher</td>
			<td>132867</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine5 anti-human CD8a Antibody</td>
			<td>BioLegend</td>
			<td>301010</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Cy 7 Mouse anti-Human CD279 (PD-1)</td>
			<td>BD Biosciences&#160;</td>
			<td>561272</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-acidic</td>
			<td>Peprotech</td>
			<td>100-17A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate Solution (NaHCO3) (7.5%)</td>
			<td>Quality Biological&#160;</td>
			<td>118-085-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Surgical Blades no. 22</td>
			<td>Integra</td>
			<td>4-322</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express&#160;</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td></td>
		</tr>
	</tbody>
</table>

unveiling therapeutic opportunities with melanoma patient-derived organoid models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

The shape and size of the MPDOs were monitored over time to study their growth dynamics. As seen in Figure 2, during the initial growth period, the organoid size increased substantially throughout the 7 days depicted. Following the observation of MPDO growth dynamics, attention turned to evaluating the immune cell composition to verify the faithful recapitulation of organoids to the original tumors. This assessment, primarily focusing on &#945;&#946; T cell abundance, provided insights into ...

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

unveiling-therapeutic-opportunities-with-melanoma-patient-derived

Research

JoVE Journal

Cancer Research

1.1K Views.  University of Pennsylvania. Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches. 

Video: Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

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 ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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. ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

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 ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.