This work is supported by grants from (1) the National Nature Science Foundation (81973877 and 82174408); (2) Shanghai Top Priority Research Center construction project (2022ZZ01009); (3) National Key R&#38;D Program of China (2020YFE0201600); (4) Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation and (5) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).

All studies involving human tissues have been approved by the Institutional Ethics Review Committee of Longhua Hospital, affiliated with Shanghai University of Traditional Chinese Medicine (Shanghai, China) (2013LC52), and written informed consent was obtained from the patients in accordance with the Helsinki Declaration. The IACUC number for this animal study is PZSHUTCM221017013. Four-week-old male CAnN.Cg-Foxn1nu/Crl mice were provided with double lion Irradiated Rodent Diet GB 14924.3 and sterile water, and were housed in IVCs mice cage with five mice per cage, under SPF conditions with a 12-h light/dark cycle. The Table of Materials includes detailed information about all materials, reagents, and instruments used in this protocol.
1. Preparation of human osteosarcoma tissue
NOTE: In this study, the human osteosarcoma tissue was resected12 from the femur lesion of a 15-year-old osteosarcoma patient before chemotherapy.
<ol>
	<li>Immediately store the freshly resected osteosarcoma tissue in a tissue-protective solution to maximize the preservation of osteosarcoma cell activation after rinsing with sterile physiological saline. 
	NOTE: Freshly resected osteosarcoma tissues must be transplanted into mice as soon as possible. They can be stored in the tissue protective solution for a maximum of 24 h before transplantation. Osteosarcoma tissues used for modeling must be from patients who have not received chemotherapy. The activity of tumor cells from patients who received chemotherapy will be poor, leading to the failure of model establishment and loss of high fidelity.</li>
	<li>Transfer the freshly resected osteosarcoma tissues stored in the tissue protective solution to the laboratory as soon as possible.</li>
	<li>Prepare experimental instruments and materials for establishing the PDX model: scalpels (Figure 1A); ophthalmic tweezers (Figure 1B,C); ophthalmic scissor (Figure 1D); suture needle (Figure 1E); suture line (Figure 1F); straight needle holder (Figure 1G); marking pen (Figure 1H). 
	NOTE: Ensure that all surgical instruments are sterilized&#160;by autoclaving before use.</li>
	<li>Rinse the osteosarcoma tissues twice with pre-cooled sterile physiological saline in a sterile hood.</li>
	<li>Remove areas of the osteosarcoma tissue with bleeding and necrosis.</li>
	<li>Cut the cleaned osteosarcoma tissue into 3 mm3 pieces with a scalpel in a culture dish containing pre-cooled sterile physiological saline and keep it on ice.</li>
</ol>
2. Establishment of PDX models by osteosarcoma tissue transplantation at mouse flank region
<ol>
	<li>Administer preoperative analgesia of meloxicam (5 mg/kg/24 h) by subcutaneous injection in mice and place the nude mice on sterile surgical drapes. Provide thermal support throughout the procedure.</li>
	<li>Induce anesthesia in mice by exposing them to 3% isoflurane and 97% oxygen. After the mice are completely anesthetized, transfer them to the nose cone and maintain anesthesia with 1.5% isoflurane and 98.5% oxygen.</li>
	<li>Pinch the toes to ensure the mice are completely anesthetized, and wait longer if there are still spasms or convulsive reactions. 
	NOTE: All procedures must be carried out using sterile equipment in a sterile hood. Avoid dryness of mice during anesthesia by applying ophthalmic ointment to their eyes.</li>
	<li>Secure the mouse in a lateral decubitus position (Figure 2A). Disinfect one side of the mouse flank region for osteosarcoma tissue transplantation 3 times with alternating rounds of povidone iodine and 70% ethanol disinfectant cotton balls. 
	NOTE: Surgical disinfection is performed in a circular pattern, beginning in the center and spiraling outward.</li>
	<li>Mark the surgical incision site on the skin with a marker pen.</li>
	<li>Make a 5 mm incision from the skin to the subcutaneous tissues with a scalpel (Figure 2B).</li>
	<li>Lift the upper side skin of the incision margin with ophthalmic tweezers using the left hand, and perform blunt dissection upward under the dermis of the mice with a straight needle holder using the right hand (Figure 2C).</li>
	<li>Hold the upper side skin of the incision with the ophthalmic tweezers using the left hand, and place the osteosarcoma tissue under the skin approximately 5 mm above the incision margin with ophthalmic tweezers using the right hand to transplant the osteosarcoma tissue (Figure 2C). 
	NOTE: Ensure the osteosarcoma tissue is transplanted just below the dermis of the skin. Blunt dissection is performed with a straight and blunt dissection forceps, such as a straight needle holder, to avoid penetrating the mouse thoracic cavity while finding the dermis layer of the mouse skin.</li>
	<li>Suture the incision using absorbable suture with 2-3 stitches for a 5 mm incision (Figure 2D).</li>
	<li>Return the mice to clean cages and monitor them until they completely recover from anesthesia. 
	NOTE: PDX established by transplanting the human osteosarcoma tissue is designated as passage 0 (P0), PDX established by transplanting the PDX tissue at P0 is designated as passage 1 (P1), followed by P2 and P3.</li>
</ol>
3. Collection of PDX tumor tissues
<ol>
	<li>Measure the tumor volume once a week. When the tumor size reaches 1500 mm3, euthanize the mice using the cervical dislocation method after CO2 inhalation. Humane endpoints include tumor ulceration or issues with front limb mobility. 
	NOTE: Measure the long diameter (a) and short diameter (b) of the tumor using a caliper. Calculate the tumor volume (V) using the formula: V = 1/2 &#215; a &#215; b2.</li>
	<li>Position the mouse laterally to expose the tumor, and disinfect the skin at the tumor site with an alcohol-soaked cotton ball.</li>
	<li>Use ophthalmic scissors to separate the entire tumor. Weigh the tumor mass using an electronic scale.</li>
</ol>
4. Pathological examination of primary clinical and PDX tumor tissues
<ol>
	<li>Fix the tumor tissues13 in a 50 mL tube containing 30 mL of 10% neutral buffered formalin solution for 24 h. Rinse the tumor tissues thoroughly with flowing water to remove the fixative.</li>
	<li>Dehydrate and embed the tumor tissues in paraffin14.</li>
	<li>Slice the tumor tissues and conduct the routine histological examination15.</li>
</ol>

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

<ol>
	<li>Saraf, A. J., Fenger, J. M., Roberts, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma:+accelerating+progress+makes+for+a+hopeful+future.">Osteosarcoma: accelerating progress makes for a hopeful future.</a> Front Oncol. 8, 4 (2018).</li><li>Ligon, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+immune+exclusion+in+metastatic+osteosarcoma+are+associated+with+inferior+patient+outcomes.">Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes.</a> J Immunother Cancer. 9 (5), e001772 (2021).</li><li>Miwa, 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=Current+and+emerging+targets+in+immunotherapy+for+osteosarcoma.">Current and emerging targets in immunotherapy for osteosarcoma.</a> J Oncol. 2019, 7035045 (2019).</li><li>Eaton, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma.">Osteosarcoma.</a> Pediatr Blood Cancer. 68 (2), e28352 (2021).</li><li>Ritter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma.">Osteosarcoma.</a> Ann Oncol. 21, 320-325 (2010).</li><li>Lowery, C. 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=Broad+spectrum+activity+of+the+checkpoint+kinase+1+inhibitor+prexasertib+as+a+single+agent+or+chemo+potentiator+across+a+range+of+preclinical+pediatric+tumor+models.">Broad spectrum activity of the checkpoint kinase 1 inhibitor prexasertib as a single agent or chemo potentiator across a range of preclinical pediatric tumor models.</a> Clin Cancer Res. 25 (7), 2278-2289 (2019).</li><li>Hingorani, P., 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=Trastuzumab+deruxtecan,+antibody-drug+conjugate+targeting+Her2,+is+effective+in+pediatric+malignancies:+a+report+by+the+pediatric+preclinical+testing+consortium.">Trastuzumab deruxtecan, antibody-drug conjugate targeting Her2, is effective in pediatric malignancies: a report by the pediatric preclinical testing consortium.</a> Mol Cancer Ther. 21 (8), 1318-1325 (2022).</li><li>Brennecke, P., 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=CXCR4+antibody+treatment+suppresses+metastatic+spread+to+the+lung+of+intratibial+human+osteosarcoma+xenografts+in+mice.">CXCR4 antibody treatment suppresses metastatic spread to the lung of intratibial human osteosarcoma xenografts in mice.</a> Clin Exp Metastasis. 31 (3), 339-349 (2014).</li><li>Pan, B., Wei, X., Xu, X. <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+models+in+hepatopancreatobiliary+cancer.">Patient-derived xenograft models in hepatopancreatobiliary cancer.</a> Cancer Cell Int. 22 (1), 41 (2022).</li><li>Tentler, J. 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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=A+practical+protocol+to+prepare+paraffin-embedded+whole+tick+histology+sections.">A practical protocol to prepare paraffin-embedded whole tick histology sections.</a> Ticks Tick Borne Dis. 14 (4), 102162 (2023).</li><li>Chang, 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=Matrine+inhibits+prostate+cancer+via+activation+of+the+unfolded+protein+response/endoplasmic+reticulum+stress+signaling+and+reversal+of+epithelial+to+mesenchymal+transition.">Matrine inhibits prostate cancer via activation of the unfolded protein response/endoplasmic reticulum stress signaling and reversal of epithelial to mesenchymal transition.</a> Mol Med Rep. 18 (1), 945-957 (2018).</li><li>de Alava, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ewing+sarcoma,+an+update+on+molecular+pathology+with+therapeutic+implications.">Ewing sarcoma, an update on molecular pathology with therapeutic implications.</a> Surg Pathol Clin. 10 (3), 575-585 (2017).</li><li>Hidalgo, 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+xenograft+models:+An+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: An emerging platform for translational cancer research.</a> Cancer Discov. 4 (9), 998-1013 (2014).</li><li>Eoh, K. 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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=Clinical+utility+of+patient-derived+xenografts+to+determine+biomarkers+of+prognosis+and+map+resistance+pathways+in+EGFR-mutant+lung+adenocarcinoma.">Clinical utility of patient-derived xenografts to determine biomarkers of prognosis and map resistance pathways in EGFR-mutant lung adenocarcinoma.</a> J Clin Oncol. 33 (22), 2472-2480 (2015).</li><li>Kresse, S. H., Meza-Zepeda, L. A., Machado, I., Llombart-Bosch, A., Myklebost, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+xenograft+models+of+human+sarcoma+show+nonrandom+loss+of+aberrations.">Preclinical xenograft models of human sarcoma show nonrandom loss of aberrations.</a> Cancer. 118 (2), 558-570 (2012).</li><li>Dobrolecki, L. 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=Patient-derived+xenograft+(PDX)+models+in+basic+and+translational+breast+cancer+research.">Patient-derived xenograft (PDX) models in basic and translational breast cancer research.</a> Cancer Metastasis Rev. 35 (4), 547-573 (2016).</li><li>Abdolahi, 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=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>Deng, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+whole-genome+sequencing+identifies+recurrent+genomic+alterations+in+commonly+used+breast+cancer+cell+lines+and+patient-derived+xenograft+models.">Deep whole-genome sequencing identifies recurrent genomic alterations in commonly used breast cancer cell lines and patient-derived xenograft models.</a> Breast Cancer Res. 24 (1), 63 (2022).</li><li>Chen, 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=Generation+and+characterization+of+patient-derived+xenografts+from+patients+with+osteosarcoma.">Generation and characterization of patient-derived xenografts from patients with osteosarcoma.</a> Tissue Cell. 79, 101911 (2022).</li><li>Fortuna-Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+association+between+successful+engraftment+of+osteosarcoma+patient-derived+xenografts+and+clinicopathological+findings.">An association between successful engraftment of osteosarcoma patient-derived xenografts and clinicopathological findings.</a> Histol Histopathol. 35 (11), 1295-1307 (2020).</li><li>Beck, 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=Canine+and+murine+models+of+osteosarcoma.">Canine and murine models of osteosarcoma.</a> Vet Pathol. 59 (3), 399-414 (2022).</li></ol>

The authors declare no competing financial interests.

The PDX models can simulate the characteristics of human cancers and retain more similarity with the primary tumor, including genetic and genomic alterations, histology, heterogeneity, and gene expression profile16,17,18,19. Therefore, they preserve the molecular phenotypes and genotypes of cancer patients, providing innovative approaches for studying biology and evaluating potential therapeuti...

Osteosarcoma is a primary bone malignancy derived from interosseous lobe cells and is most common in adolescents as well as children. It often occurs in the epiphysis of the long diaphysis and is characterized by high malignancy, early metastasis, and poor prognosis1,2. Lung metastasis is the main cause of death in osteosarcoma patients. The 5-year survival rate of patients with non-metastatic osteosarcoma is 65%-70%3. However, over the last 40 years, the 5-year survival rate (only 20%) of patients with metastatic osteosarcoma has not significantly improved, and 25% of osteosarcoma patients have metastases at the time of diagnosis4. Currently, the first-line drugs for osteosarcoma treatment have reached a consensus, but there are still significant differences in chemotherapy regimen and treatment time5. It is important to perform preclinical experiments based on appropriate animal models to obtain more effective chemotherapy regimens.
Currently, models commonly used for osteosarcoma preclinical experiments include cell line-based in vitro cell culture and in vivo cell-derived xenografts (CDX), as well as patient-derived xenografts (PDX)6,7.
The cell lines are convenient for culturing and for use in in vitro studies, or for transplantation into immunodeficient mice to establish CDX models8. However, cell lines cultured in vitro may not accurately reflect the heterogeneity of malignancies and the individual characteristics of patients due to potential mutations that occur to adapt to the in vitro culture environment during repeated passages. Additionally, they lack the microenvironment and immune system necessary for tumor growth and development in vivo. While CDX models offer some advantages over in vitro cell culture, they still may not fully reflect the individual characteristics of osteosarcoma patients, although tumor tissues obtained from CDX models have limited intratumoral heterogeneity and immune system representation compared to cell lines cultured in vitro9. Therefore, establishing a preclinical model with high fidelity is crucial.
PDX models involve the immediate transplantation of freshly resected human cancer tissues into immunodeficient mice. This method allows for the faithful preservation of genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient, even after successive passages in mice. Furthermore, PDX models are known to accurately predict later clinical outcomes10, making them valuable tools for creating individualized treatments and advancing precision medicine research11.
This work describes the procedure for establishing a PDX model in immunodeficient mice by transplanting human osteosarcoma tissue. Such models serve as platforms for conducting preclinical experiments for osteosarcoma.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% formalin neutral solution</td>
			<td>Wuhan Saiweier Biotechnology Co., Ltd</td>
			<td>G1101-500ml</td>
			<td>Fix the tissues</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Japan Hiryama Company</td>
			<td>HVE-50</td>
			<td>Sterilization surgical instruments</td>
		</tr>
		<tr>
			<td>CAnN.Cg-Foxn1nu/Crl</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd.</td>
			<td>/</td>
			<td>Animal</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Yantai Green Forest Tools Co., Ltd.</td>
			<td>034180A</td>
			<td>Measure the tumor volume</td>
		</tr>
		<tr>
			<td>Dish (60mm)</td>
			<td>Shanghai NianYue Biotechnology Co., Ltd</td>
			<td>430166, Corning</td>
			<td>Sample placment during transplantation</td>
		</tr>
		<tr>
			<td>Disinfectant cotton balls</td>
			<td>Shanghai Honglong Industrial Co., Ltd.</td>
			<td>20230627</td>
			<td>Disinfect the skin of mice</td>
		</tr>
		<tr>
			<td>Disposable sterile gloves</td>
			<td>Guilin Hengbao Health Protection Co., Ltd.</td>
			<td>YT21131</td>
			<td>Sterile operation</td>
		</tr>
		<tr>
			<td>Double lion Irradiated Rodent Diet</td>
			<td>Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.</td>
			<td>GB 14924.3</td>
			<td>Animal feed</td>
		</tr>
		<tr>
			<td>Electronic scale</td>
			<td>Shanghai NianYue Biotechnology Co., Ltd</td>
			<td>1-2000</td>
			<td>Weigh the weight of the tumor</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Shanghai Gengyun Biotechnology Co., Ltd</td>
			<td>E4009-25G</td>
			<td>Hematoxylin eosin stain</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Shanghai Gengyun Biotechnology Co., Ltd</td>
			<td>H3136-25G</td>
			<td>Hematoxylin eosin stain</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>VETEASY</td>
			<td>Mouse anesthesia&#160;</td>
		</tr>
		<tr>
			<td>IVCs mice cage</td>
			<td>Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.</td>
			<td>HH-MMB-2</td>
			<td>Animal barrier</td>
		</tr>
		<tr>
			<td>Mark pen</td>
			<td>Zebra Trading (Shenzhen) Co., Ltd.</td>
			<td>YYST5</td>
			<td>Mark the surgical incision</td>
		</tr>
		<tr>
			<td>Olympus Optical microscope</td>
			<td>Japanese Olympus Company</td>
			<td>BH20</td>
			<td>Scan tissue slices</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Shanghai Gengyun Biotechnology Co., Ltd</td>
			<td>SOICOEYEGRL</td>
			<td>Avoid dry eyes of mice during anesthesia</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai NianYue Biotechnology Co., Ltd</td>
			<td>Y00030 JZ</td>
			<td>Cut the skin</td>
		</tr>
		<tr>
			<td>Ophthalmic tweezers</td>
			<td>Shanghai NianYue Biotechnology Co., Ltd</td>
			<td>BS-ZER-S-100 Biosharp</td>
			<td>Hold osteosarcoma tissues during transplantation</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Jiangsu Shitai Experimental Equipment Co., Ltd.</td>
			<td>80200-0015</td>
			<td>Buried osteosarcoma tissue</td>
		</tr>
		<tr>
			<td>Paraffin slicing machine</td>
			<td>Lyca Microsystem (Shanghai) Trading Co., Ltd.</td>
			<td>RM2235</td>
			<td>Osteosarcoma tissue section</td>
		</tr>
		<tr>
			<td>physiological saline</td>
			<td>Guangzhou Jinsheng Biotechnology Co., Ltd.</td>
			<td>605-004057</td>
			<td>Rinse and temporary storage of osteosarcoma tissue</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Surgical Instrument Factory of Shanghai Medical Devices (Group) Co., Ltd.</td>
			<td>J11010-10# JZ</td>
			<td>Separation of osteosarcoma tissue and making surgical incisions</td>
		</tr>
		<tr>
			<td>Sterile hood</td>
			<td>Thermo Fisher Technology (China) Co., Ltd.</td>
			<td>ECO0.9</td>
			<td>Surgical operation table</td>
		</tr>
		<tr>
			<td>sterile surgical drapes</td>
			<td>Henan Huayu Medical Equipment Co., Ltd.</td>
			<td>20160090</td>
			<td>Provide sterile surgery area</td>
		</tr>
		<tr>
			<td>Straight needle holder</td>
			<td>Shanghai Gengyun Biotechnology Co., Ltd</td>
			<td>J31050 JZ</td>
			<td>Suture the wound</td>
		</tr>
		<tr>
			<td>Suture line</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd</td>
			<td>F3124</td>
			<td>Suture the wound</td>
		</tr>
		<tr>
			<td>Suture needle</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd</td>
			<td>F3124</td>
			<td>Suture the wound</td>
		</tr>
		<tr>
			<td>Tissue protective solution</td>
			<td>Nanjing Shenghang Biotechnology Co., LTD</td>
			<td>BC-CFM-03</td>
			<td>Maintain the activity of tissue cells</td>
		</tr>
		<tr>
			<td>Tube (50 mL)</td>
			<td>Shanghai Baisai, Biotechnology Co., Ltd.</td>
			<td>BLD-BL2002500</td>
			<td>Install formalin fixation solution</td>
		</tr>
	</tbody>
</table>

establishment of patient-derived xenograft mouse model with human osteosarcoma tissues

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

This protocol describes the detailed procedure for establishing a PDX mouse model, preserving the morphological features of human osteosarcoma after immediate transplantation of freshly resected human osteosarcoma tissue and successive passages in mice. Here, a PDX mouse model was successfully established using human osteosarcoma tissue.
Figure 3A shows a representative mouse of PDX at P0, two months after the transplantation of femoral osteosarcoma tissue from a ...

Watch this Scientific Journal Video about Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study at JoVE.com

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

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

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

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

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Research

JoVE Journal

Cancer Research

613 Views.  Shanghai University of Traditional Chinese Medicine. The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

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

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

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

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

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

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.

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 ...

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 ...

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. ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Medicine

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine.&#160;Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by ...

Patient-derived Xenograft Modeling: A Technique to Generate Melanoma Mouse Models

Source: Min Xiao et al. <a href="https://www.jove.com/v/59508"> A Melanoma Patient-Derived Xenograft Model</a>. J. Vis. Exp. (2018)
In this video, human-derived melanoma cells are implanted subcutaneously into the flank region of an immunocompromised mouse. The PDX model allows preclinical investigation of melanoma cells that better recapitulate the tumor heterogeneity and melanoma aggressiveness observed in in vivo conditions.