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 ...

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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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JoVE Journal

Cancer Research

635 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

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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

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

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

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

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 ...

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 ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance 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. ...

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.