This work was supported by the Sichuan Province Science and Technology Support Program (Grant Nos. 2019JDRC0019 and 2021ZYD0097), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (Grant No. ZYJC18026), the 1.3.5 project for disciplines of excellence-Clinical Research Incubation Project, West China Hospital, Sichuan University (Grant No. 2020HXFH023), the Fundamental Research Funds for the Central Universities (SCU2022D025), the International Cooperation Project of Chengdu Science and Technology Bureau (Grant No. 2022-GH02-00023-HZ), the Innovation Spark Project of Sichuan University (Grant No. 2019SCUH0015), and the Talent Training Fund for Medical-engineering Integration of West China Hospital - University of Electronic Science and Technology (Grant No. HXDZ22012).

All the animal experiments were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and protocols approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University. NOD-SCID immunodeficient mice aged 4-6 weeks old (of both sexes) and female Balb/c nude mice aged 4-6 weeks old were used for the present study. The animals were obtained from a commercial source (see Table of Materials). The ethics committee of West China Hospital authorized the study with human subjects (protocol number 2020353). Each patient provided written informed consent.
1. Experimental preparation
<ol>
	<li>Arrange disposable blades, sterilized scissors and tweezers, and other instruments required for tumor transplantation, place them on the ultra-clean workbench, and irradiate them with ultraviolet light in advance.</li>
	<li>Prepare sterile saline and Petri dishes for use during the test.</li>
</ol>
2. Acquisition and transport of fresh tumor tissue
<ol>
	<li>Obtain fresh tumor samples (usually larger than 5 mm x 5 mm in size) from the operating room, and place them in a 15 mL or 50 mL centrifuge tube containing sterile HTK solution (see Table of Materials) or saline. Label the centrifuge tubes. 
	NOTE: Fresh tumor samples were obtained by surgical removal or puncture from patients with ATC or HNSCC.</li>
	<li>Put the centrifuge tubes in an ice box prepared in advance. 
	NOTE: During this time, the transplant operator must prepare the necessary items for transplantation (see Table of Materials).</li>
	<li>Ensure that the time between sample collection and transportation to the laboratory for PDX construction does not exceed 2 h. During transport, surround the tubes containing the tissues with an ice-water mixture or ice packs to preserve the tissue activity.</li>
</ol>
3. Tumor transplantation
<ol>
	<li>Once the tumor tissues arrive at the laboratory, record and renumber them. 
	NOTE: For the present study, the patient information was kept strictly confidential. The remaining steps of the procedure were performed in a biosafety level 2 (BSL-2) laboratory. When entering the laboratory, wearing a smock over the work clothes or protective clothing, a hat, and a mask is recommended. The treatment of tumor tissue is conducted in a biosafety cabinet.</li>
	<li>Disinfect the centrifuge tubes containing the tumor tissues with 75% alcohol, and place them on the operating table. Transfer the tumor tissues to 6 cm Petri dishes filled with saline using sterilized ophthalmic forceps. Next, cut them into small pieces of about 2 mm x 2 mm and 3 mm x 3 mm using a blade.</li>
	<li>Transfer the pieces of tumor tissues into a 6 cm Petri dish containing the appropriate amount of saline, wrap the dish with the sealing film, place it in an ice box, and carry it into the specific pathogen-free (SPF) animal room along with the necessary instruments (a pair of scissors, forceps, and inoculation needles).</li>
	<li>Prepare the animal following the steps below.
	<ol>
		<li>Remove the hair on the right lateral thorax of 4-6 week-old female or male NOD-SCID immunodeficient mice, and disinfect the skin with 75% alcohol. Anesthetize the mice by an intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine (see Table of Materials), and smear their eyes with vet ointment to prevent dryness. Confirm anesthesia depth via loss of pedal reflex.</li>
		<li>Make a 2 mm incision with scissors through the skin at the middle of the right lateral thorax of mice.</li>
	</ol>
	</li>
	<li>Take a tumor piece from the Petri dish, and place it into the 2.4 mm x 2.0 mm trocar needle (see Table of Materials) with forceps.</li>
	<li>Hold the mouse, tighten the skin at the puncture site, use the trocar containing the tumor pieces to insert the tumor through the initial 2 mm skin incision, move to the back of the shoulder, and push the trocar core.</li>
	<li>Ensure that the tumor piece is pushed out and is left in the transitional sinus formed by the trocar puncture, and then pull out the trocar.</li>
	<li>If the tumor moves with the needle when it is withdrawn, use the trocar to reset it and suture the incision. 
	NOTE: In this study, each mouse was inoculated at the dorsal fore and hind limbs. One to three mice were inoculated per tumor sample from each patient based on the tumor size.</li>
</ol>
4. Tumor tissue preservation, fixation, and protein freezing
NOTE: The remaining tumor tissues were used for seed preservation, fixation, and DNA/RNA/protein freezing, respectively.
<ol>
	<li>Remove the saline from the tumor surface with a sterile gauze before placing it in the cryopreservation tube to ensure that the tumor surface is not excessively wet.</li>
	<li>Put four to six pieces of 2 mm x 2 mm tumor tissue in a 2 mL cell cryopreservation tube, add 1 mL of cryopreservation solution composed of 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) into the tube, put the tube in a gradient cooling box, freeze it at &#8722;80 &#176;C overnight, and finally, transfer it to liquid nitrogen.</li>
	<li>Place the 3 mm x 3 mm tumor tissue blocks in 10% buffered formalin for tissue fixation for pathological examination.</li>
	<li>Put the 3 mm x 3 mm tissue block into a 2 mL cell cryopreservation tube, quickly freeze it in liquid nitrogen, and then transfer to a &#8722;80 &#176;C refrigerator for DNA/RNA and protein extraction.</li>
	<li>Collect the clinical information of the patients, such as the smoking history, tumor size, differentiation, pathological subtype, cancer grade, cancer stage, distant metastasis, origin, medical history, immunohistochemistry, human papillomavirus (HPV) infection in HNSCC patients, and treatment medication.</li>
</ol>
5. Passaging, cryopreservation, and resuscitation of PDX model tumors
<ol>
	<li>Measure the length and width of the subcutaneous tumors in mice by using vernier calipers once a week, and calculate the tumor volume according to the formula: tumor volume = 0.5 &#215; length &#215; width2. Draw the tumor growth curve.</li>
	<li>When the PDX tumor reaches 2,000 mm3, passage it to the next generation of mice, and perform tumor re-transplantation. Perform the preparation of the instruments following step 4.</li>
	<li>Euthanize the mice by cervical dislocation after anesthetizing with 80 mg/kg ketamine.</li>
	<li>Disinfect the skin with 75% alcohol. Then, cut the skin surrounding the tumor using scissors, then remove the tumor with forceps, and place it in a Petri dish.&#160;</li>
	<li>Perform the tumor transplantation procedure following step 3.</li>
	<li>Perform the preservation and cryopreservation of the PDX model tumors following step 4.</li>
	<li>For the resuscitation of the tumor tissue, follow the principle of slow freezing and quick dissolving. After taking out the cryovials from liquid nitrogen, quickly place them in a water bath at 37 &#176;C for rapid dissolving.</li>
	<li>Gently shake the cryovials in the water bath to accelerate the thawing process.</li>
	<li>Thaw, transfer the tumor pieces to the prepared normal saline for washing, and then inoculate the next generation of mice. For the specific operation, please see the tissue transplantation procedure in step 3.</li>
</ol>
6. Determining the therapeutic efficacy of lenvatinib and cisplatin in the ATC PDX model
NOTE: The ATC PDX model was used to test the therapeutic effect of the tyrosine kinase inhibitor lenvatinib and the chemotherapeutic drug cisplatin25,26,27.
<ol>
	<li>Select the P5 generation tumor tissue of an ATC PDX model (THY-017), cut into 2-4 mm3 tissue pieces, and inoculate subcutaneously (step 3) to the right back of ten 4-6 week female Balb/c nude mice.</li>
	<li>Select 15 mice with tumor volumes between 50-150 mm3, and divide them into three groups.</li>
	<li>Administer lenvatinib (10 mg/kg) intragastrically to one group once daily for 15 days, administer cisplatin (3 mg/kg) intraperitoneally to one group every 3 days for a total of six doses, and administer the control group with the same volume of normal saline.</li>
	<li>Measure the body weight and tumor volume of the mice twice per week.</li>
	<li>At the end of the test, euthanize the mice (step 5.3), and weigh the tumors.</li>
</ol>

No potential conflicts of interest are disclosed.

This study has successfully established the subcutaneous PDX models of ATC and HNSCC. There are many aspects to pay attention to during the process of PDX model construction. When the tumor tissue is separated from the patient, it should be put into the ice box and sent to the laboratory for inoculation as soon as possible. After the tumor arrives at the laboratory, the operator must pay attention to maintaining a sterile field and practice aseptic procedures. For needle biopsy samples, because the tumor tissue is particularly small, inoculation after mixing the sample with the matrix gel would be more conducive to establishing the model. The primary tumor tissue should also be preserved, fixed, and frozen as much as possible for future research. During inoculation, the air in the trocar needs to be expelled as much as possible after the tumor pieces have been put into the trocar before use. After tumor inoculation, the tumor growth should be observed in mice for 1-4 months, and mice without tumor growth for more than 6 months can be euthanized29.
Immunodeficient mice are generally chosen as the host for PDX model construction29,30. From the P0 generation to the P2 generation, non-obese diabetic-severely compromised immune deficient (NOD-SCID) mice or NOD Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are generally used. In the P3 generation and beyond, the samples are considered to be stably passed, so nude mice can usually also serve as the host, and tumors can also grow normally. Additionally, the total operation time, tumor isolation time, disease-free survival, and overall survival rate of the patients, the tumor malignant degree, and the histologic subtype were all associated with PDX model tumorigenicity31,32,33,34. The transplantation site also has an impact on the success rate of PDX modeling, and studies have shown that renal capsule and orthotropic transplantation have a high tumorigenic rate33,35. In addition, the use of Matrigel may also improve the tumorigenic rate36,37. It has been reported that human papillomavirus (HPV) infection affects the success rate of transplantation in HNSCC tumors; HPV-negative tumors have a superior take rate compared to HPV-positive tumors38,39. This study did not reach the same conclusion, probably due to the small sample numbers and incomplete information on HPV infection.
Different from the orthotopic and renal capsule transplantation models, the subcutaneous model is more convenient for observing the growth of tumors and is also conducive to operation40,41,42. Based on the tumor growth data of the ATC and HNSCC PDX model, we found that the growth rates of tumors from different patients were inconsistent, reflecting inter-tumor heterogeneity. The tumor growth rate of the P0 generation from most PDX models was relatively slower than for the latter passages, which was likely due to the adaption of the mouse microenvironment. Notably, the growth rate from some patient-derived tumors increased in different passages after the P1 generation, consistent with the shortened passage interval reported by Pearson et al.43. Histopathological examination demonstrated that the PDX tumors retained the morphological characteristics of the primary tumors. The correlation between the PDX model and the clinical ATC patients was also reflected in the results of the in vivo pharmacodynamic tests, which demonstrated that Lenvartinib exhibited a good anti-tumor effect, consistent with clinical reports25,26,27.
However, the PDX model also has certain disadvantages. For example, the tumor formation time is relatively long, which is unsuitable for patients with advanced or aggressive tumors. In addition, the time and monetary costs of high-throughput drug screening are too high44. Indeed, combining the PDX model with tumor organoids and establishing a patient-derived organoid (PDO) model corresponding to the PDX model would compensate for this deficiency44,45,46. Orthotopic transplantation models can be used to study the pathogenesis and metastatic mechanisms of tumors40,41,47. The lack of a functional immune system is another disadvantage of the PDX model, so increasing numbers of experiments are using humanized mice to construct the PDX model for tumor immunology research48,49,50.

The PDX model is an animal model in which human tumor tissue is transplanted into immunodeficient mice and grows in the environment provided by the mice1. Traditional tumor cell line models suffer from several disadvantages, such as the lack of heterogeneity, the inability to retain the tumor microenvironment, the vulnerability to genetic variations during repeated in vitro passages, and the poor clinical application2,3. The main drawbacks of genetically engineered animal models are the potential loss of the genomic features of human tumors, the introduction of new unknown mutations, and the difficulty in identifying the degree of homology between mouse tumors and human tumors4. In addition, the preparation of genetically engineered animal models is expensive, time-consuming, and relatively inefficient4.
The PDX model has many advantages over other tumor models in terms of reflecting tumor heterogeneity. From the perspective of histopathology, although the mouse counterpart replaces the human stroma over time, the PDX model preserves the morphological structure of the primary tumor well. In addition, the PDX model conserves the metabolomic identity of the primary tumor for at least four generations and better reflects the complex inter-relationships between tumor cells and their microenvironment, making it unique in simulating the growth, metastasis, angiogenesis, and immunosuppression of human tumor tissue5,6,7. At the cellular and molecular levels, the PDX model accurately reflects the inter- and intra-tumor heterogeneity of human tumors, as well as the phenotypic and molecular characteristics of original cancer, including gene expression patterns, mutation status, copy number, and DNA methylation and proteomics8,9. PDX models with different passages have the same sensitivity to drug therapy, indicating that the gene expression of PDX models is highly stable10,11. Studies have shown an excellent correlation between the response of the PDX model to a drug and the clinical responses of patients to that drug12,13. Therefore, the PDX model has emerged as a powerful preclinical and translational research model, particularly for drug screening and clinical prognosis prediction.
Thyroid cancer is a common malignant tumor of the endocrine system and is a human malignancy that has shown a rapid increase in incidence in recent years14. Anaplastic thyroid carcinoma (ATC) is the most malignant thyroid cancer, with a median patient survival of only 4.8 months15. Although only a minority of thyroid cancer patients are diagnosed with ATC each year in China, the mortality rate is close to 100%16,17,18. ATC usually grows rapidly and invades the adjacent tissues of the neck as well as the cervical lymph nodes, and about half of the patients have distant metastases19,20. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and one of the leading causes of cancer deaths, with an estimated 600,000 people suffering from HNSCC each year21,22,23. HNSCC includes a large number of tumors, including those in the nose, sinuses, mouth, tonsils, pharynx, and larynx24. ATC and HNSCC are two of the main head and neck malignancies. In order to facilitate the development of novel therapeutic agents and personalized treatments, it is necessary to develop robust and advanced preclinical animal models such as PDX models of ATC and HNSCC.
This article introduces detailed methods for establishing the subcutaneous PDX model of ATC and HNSCC, analyzes the key factors affecting the tumor take rate in model construction, and compares the histopathological characteristics between the PDX model and the primary tumor. Meanwhile, in this work, in vivo pharmacodynamic tests were performed using the successfully constructed PDX models in order to validate their clinical relevance.

<table><tbody><tr><td>2.4 mm x 2.0 mm trocar</td><td>Shenzhen Huayang Biotechnology Co., Ltd</td><td>18-9065</td><td></td></tr><tr><td>Balb/c nude mice</td><td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td><td>401</td><td></td></tr><tr><td>Biosafety cabinet</td><td>Suzhou Antai</td><td>BSC-1300IIA2</td><td></td></tr><tr><td>Blade</td><td>Shenzhen Huayang Biotechnology Co., Ltd</td><td>18-0823</td><td></td></tr><tr><td>Centrifuge tube&amp;nbsp;</td><td>Corning</td><td>430791/430829</td><td></td></tr><tr><td>Cryopreservation tube</td><td>Chengdu Dianrui Experimental Instrument Co., Ltd</td><td>/</td><td></td></tr><tr><td>Custodiol HTK-Solution</td><td>Custodiol</td><td>2103417</td><td></td></tr><tr><td>Dimethyl sulfoxide(DMSO)</td><td>SIGMA-ALORICH</td><td>D5879-500mL</td><td></td></tr><tr><td>Electronic balance</td><td>METTLER</td><td>ME104</td><td></td></tr><tr><td>Electronic digital caliper</td><td>Chengdu Chengliang Tool Group Co., Ltd</td><td>0-220</td><td></td></tr><tr><td>fetal bovine serum(FBS)</td><td>VivaCell</td><td>C04001-500</td><td></td></tr><tr><td>IBM SPSS Statistics 26</td><td>IBM</td><td></td><td></td></tr><tr><td>Ketamine</td><td>Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd&amp;nbsp;</td><td>100761663</td><td></td></tr><tr><td>Lenvatinib</td><td>ApexBio</td><td>A2174</td><td></td></tr><tr><td>NOD-SCID immunodeficient mice</td><td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td><td>406</td><td></td></tr><tr><td>Pen-Strep Solution</td><td>Biological Industries</td><td>03-03101BCS</td><td></td></tr><tr><td>Petri dish</td><td>WHB</td><td>WHB-60/WHB-100</td><td></td></tr><tr><td>Saline&amp;nbsp;</td><td>Sichuan Kelun</td><td>W220051705</td><td></td></tr><tr><td>Scissor</td><td>Shenzhen Huayang Biotechnology Co., Ltd</td><td>18-0110</td><td></td></tr><tr><td>Tweezer</td><td>Shenzhen Huayang Biotechnology Co., Ltd</td><td>18-1241</td><td></td></tr><tr><td>Vet ointment</td><td>Pfizer Inc.</td><td>P10015353</td><td></td></tr><tr><td>Xylazine</td><td>Dunhua Shengda Animal Medicine Co., Ltd</td><td>070031777</td><td></td></tr></tbody></table>

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.

A total of 18 thyroid cancer specimens were transplanted, and five PDX models of thyroid cancer were successfully constructed (27.8% tumor take rate), including four cases of undifferentiated thyroid cancers and one case of anaplastic thyroid cancer. The correlation between the success rate of model construction and the age, gender, tumor diameter, tumor grade, and differentiation were analyzed. Although the model success rate of grade 4 tumor samples was higher than for samples with lower grades, and the success rate of undifferentiated tumor samples was also higher than that of highly differentiated samples, the correlation analysis results showed that these factors were not associated with the success rate of the PDX model (Table 1). Seventeen HNSCC samples were inoculated, and four PDX models of HNSCCC were successfully constructed. The correlation analysis between the tumor take rate in the model construction and the clinical parameters of the tumor samples demonstrated that the degree of differentiation was associated with model success rate, while age, gender, smoking history, tumor diameter, cancer grade, metastasis, and HPV infection did not affect the tumor take rate (Table 2).
The tumor growth curves for each PDX model were plotted to better understand the growth rates of the PDX models from different patients (Figure 1, Figure 2,&#160;and Table 3). The average tumorigenic cycles (time from inoculation to a tumor size of 1,000 mm3) of THY-004 from generations P0 to P5 were 68 days, 87 days, 29 days, 34 days, 28 days, and 26 days, respectively. The average tumorigenic cycles of THY-012 from generations P0 to P5 were 119 days, 61 days, 66 days, 55 days, 87 days, and 116 days, respectively. The average tumorigenic cycles of THY-017 from generations P0 to P5 were 27 days, 17 days, 30 days, 13 days, 22 days, and 15 days, respectively. The average tumorigenic cycles of THY-018 from generations P0 to P3 were 134 days, 70 days, 48 days, and 48 days, respectively. The average tumorigenic cycles of THY-021 from generations P0 to P3 were 53 days, 66 days, 35 days, and 49 days, respectively. The average tumorigenic cycles of OTO-017 from generations P0 to P4 were 118 days, 86 days, 67 days, 129 days, and 88 days, respectively. The average tumorigenic cycles of OTO-022 from generations P0 to P5 were 155 days, 55 days, 32 days, 37 days, 27 days, and 46 days, respectively. The average tumorigenic cycles of OTO-030 from generations P0 to P2 were 133 days, 93 days, and 104 days, respectively. The average tumorigenic cycles of OTO-031 from generations P0 to P5 were 144 days, 58 days, 33 days, 34 days, 52 days, and 50 days, respectively. The ATC samples were stably passed to the P3 generation and later, while two cases of HNSCC samples failed to form tumors after passing to the P1 generation. The growth rates of some samples were relatively slow in the P0 generation, but their growth rates were accelerated after passing to the P1 and later generations. The histopathological characteristics of the patient tumors with those of different generations of PDX models were compared. The results showed that PDX tumors and patient-derived primary tumors were morphologically almost similar (Figure 3), with slight differences that may be due to the heterogeneity in the sampling area between patients and different generations of PDX.
The anti-tumor efficacy of lenvatinib (a multi-target tyrosine kinase inhibitor approved for the treatment of advanced thyroid cancer28) was evaluated in the PDX model of ATC. As shown in Figure 4A, lenvatinib treatment significantly inhibited the tumor growth in the ATC PDX model compared to the normal saline control group (P &#60; 0.05). At the end of the experiment, the tumor tissue was excised and weighed to determine the tumor weight. Compared with the control group, the tumor weight of the lenvatinib treatment group was lower, although a statistical difference was not achieved (Figure 4B). In addition, no obvious changes in general status and body weight were observed in the mice treated with lenvatinib (Figure 4C). Due to the excessive frequency of cisplatin administration during the experiments, the mice showed significant toxicity, manifested by weight loss and even death. The anti-tumor efficacy of cisplatin is shown in Supplementary Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64623/64623fig01.jpg" /> 
Figure 1: Tumor growth curve of ATC PDX models from different patients. Each color represents the specified generation, and each curve represents a single tumor. One to three mice were inoculated at the passage 0 (P0) generation, and five mice were inoculated in the subsequent passages (P1-P5). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64623/64623fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64623/64623fig02.jpg" /> 
Figure 2: Tumor growth curve of the HNSCC PDX models from different patients. Each color represents the specified generation, and each curve represents a single tumor. One to three mice were inoculated at the P0 generation, and five mice were inoculated at P1 and later generations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64623/64623fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64623/64623fig03.jpg" /> 
Figure 3: Histopathological study. Histopathological comparison between the patient primary tumors and corresponding PDXs (passage 1 and passage 3) of ATC (THY-012, THY-017) and HNSCC (OTO-017) (hematoxylin-eosin staining, 100x). The pathological subtypes of THY-012 and THY-017 were anaplastic thyroid carcinoma, and the pathological subtype of OTO-017 was squamous cell carcinoma. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64623/64623fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64623/64623fig04.jpg" /> 
Figure 4: The therapeutic efficacy of lenvatinib in the ATC PDX model. Changes in the (A) tumor volume, (B) tumor weight, and (C) body weight of ATC PDX-bearing mice after treatment with lenvatinib (10 mg/kg). Statistical analyses were performed using T-tests to compare levatinib with control. *P &#60; 0.05&#160;versus control was considered to be statistically significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64623/64623fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td>Class</td>
			<td>Tumor take rate (%)</td>
			<td>P</td>
		</tr>
		<tr>
			<td>Age (years)</td>
			<td>&#60;60</td>
			<td>16.67 (1/6)</td>
			<td>0.615</td>
		</tr>
		<tr>
			<td></td>
			<td>&#8805;60</td>
			<td>33.33 (4/12)</td>
			<td></td>
		</tr>
		<tr>
			<td>Gender</td>
			<td>Male</td>
			<td>16.67 (1/6)</td>
			<td>0.615</td>
		</tr>
		<tr>
			<td></td>
			<td>Female</td>
			<td>33.33 (4/12)</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor diameter</td>
			<td>&#60;6cm</td>
			<td>37.50 (3/8)</td>
			<td>0.608</td>
		</tr>
		<tr>
			<td></td>
			<td>&#8805;6cm</td>
			<td>20.00 (2/10)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathologic TNM stage</td>
			<td>I</td>
			<td>0.00 (0/1)</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>&#1064;</td>
			<td>0.00 (0/1)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td><img alt="Equation 2" src="/files/ftp_upload/64623/64623eq02.jpg" style="margin: auto;" /></td>
			<td>31.25 (5/16)</td>
			<td></td>
		</tr>
		<tr>
			<td>Differentiation</td>
			<td>High</td>
			<td>0.00 (0/7)</td>
			<td>0.059</td>
		</tr>
		<tr>
			<td></td>
			<td>Poor</td>
			<td>25.00 (1/4)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Undifferentiated</td>
			<td>57.14 (4/7)</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Correlation between the ATC tumor take rate and the clinical characteristics of the patients.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td>Class</td>
			<td>Tumor take rate (%)</td>
			<td>P</td>
		</tr>
		<tr>
			<td>HPV</td>
			<td>Negative</td>
			<td>33.33 (2/6)</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>Unknown or positive</td>
			<td>36.36 (4/11)</td>
			<td></td>
		</tr>
		<tr>
			<td>Age (years)</td>
			<td>&#60;60</td>
			<td>33.33 (3/9)</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>&#8805;60</td>
			<td>37.50 (3/8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Gender</td>
			<td>Male</td>
			<td>50.00 (5/10)</td>
			<td>0.304</td>
		</tr>
		<tr>
			<td></td>
			<td>Female</td>
			<td>14.29 (1/7)</td>
			<td></td>
		</tr>
		<tr>
			<td>Smoking status</td>
			<td>Ever</td>
			<td>44.44 (4/9)</td>
			<td>0.62</td>
		</tr>
		<tr>
			<td></td>
			<td>Never</td>
			<td>25.00 (2/8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Tumor diameter</td>
			<td>&#60;3cm</td>
			<td>40.00 (4/10)</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>&#8805;3cm</td>
			<td>28.57 (2/7)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathologic TNM stage</td>
			<td>I</td>
			<td>75.00 (3/4)</td>
			<td>0.423</td>
		</tr>
		<tr>
			<td></td>
			<td><img alt="Equation 1" src="/files/ftp_upload/64623/64623eq01.jpg" style="margin: auto;" /></td>
			<td>25.00 (2/8)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>&#1064;</td>
			<td>0.00 (0/1)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td><img alt="Equation 2" src="/files/ftp_upload/64623/64623eq02.jpg" style="margin: auto;" /></td>
			<td>33.33 (1/3)</td>
			<td></td>
		</tr>
		<tr>
			<td>Distant metastasis</td>
			<td>Y</td>
			<td>28.57 (2/7)</td>
			<td>0.633</td>
		</tr>
		<tr>
			<td></td>
			<td>N</td>
			<td>44.44 (4/9)</td>
			<td></td>
		</tr>
		<tr>
			<td>Differentiation</td>
			<td>High</td>
			<td>12.50 (1/8)</td>
			<td>0.036*</td>
		</tr>
		<tr>
			<td></td>
			<td>Moderate to high</td>
			<td>100.00 (2/2)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Moderate</td>
			<td>0.00 (0/2)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Moderate to poor</td>
			<td>66.67 (2/3)</td>
			<td></td>
		</tr>
		<tr>
			<td>* P &#60; 0.05</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Correlation between the HNSCC tumor take rate and the clinical characteristics of the patients. *P &#60; 0.05.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample name</td>
			<td>Generation P to P0</td>
			<td>Generation P0 to P1</td>
			<td>Generation P1 to P2</td>
			<td>Generation P2 to P3</td>
			<td>Generation P3 to P4</td>
			<td>Generation P4 to P5</td>
		</tr>
		<tr>
			<td>THY-004</td>
			<td>68</td>
			<td>87</td>
			<td>29</td>
			<td>34</td>
			<td>28</td>
			<td>26</td>
		</tr>
		<tr>
			<td>THY-012</td>
			<td>119</td>
			<td>61</td>
			<td>66</td>
			<td>55</td>
			<td>87</td>
			<td>116</td>
		</tr>
		<tr>
			<td>THY-017</td>
			<td>27</td>
			<td>17</td>
			<td>30</td>
			<td>13</td>
			<td>22</td>
			<td>15</td>
		</tr>
		<tr>
			<td>THY-018</td>
			<td>134</td>
			<td>70</td>
			<td>48</td>
			<td>48</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>THY-021</td>
			<td>53</td>
			<td>66</td>
			<td>35</td>
			<td>49</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>OTO-017</td>
			<td>118</td>
			<td>86</td>
			<td>67</td>
			<td>129</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>OTO-022</td>
			<td>155</td>
			<td>55</td>
			<td>32</td>
			<td>37</td>
			<td>27</td>
			<td>46</td>
		</tr>
		<tr>
			<td>OTO-030</td>
			<td>133</td>
			<td>93</td>
			<td>104</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>OTO-031</td>
			<td>144</td>
			<td>58</td>
			<td>33</td>
			<td>34</td>
			<td>52</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 3:&#160;The average tumorigenic cycle (time from inoculation to a tumor size of 1,000 mm3) of the ATC and HNSCC models.
Supplementary Figure 1: The therapeutic efficacy of cisplatin in the ATC PDX model. Changes in the (A) tumor volume, (B) tumor weight, and (C) body weight of ATC PDX-bearing mice after treatment with cisplatin (3 mg/kg). Statistical analyses were performed using T-tests to compare cisplatin with the control. *P &#60; 0.05 versus control was considered to be statistically significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64623/Supplementary Figure 1.png" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma at JoVE.com

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

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.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

establishment-characterization-patient-derived-xenograft-models

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.7K Views.  Sichuan University. Dit protocol helpt bij het opzetten van ATC- en HNSCC PDX-modellen die tumorheterogeniteit, moleculaire diversiteit beter weerspiegelen en kliniekresultaten voorspellen. De techniek is eenvoudig te bedienen, heeft een hoog slagingspercentage en kan ook geschikt zijn voor andere PDX-modellen. Deze techniek kan worden uitgebreid naar anti-kanker drug gevoeligheid screening om de gepersonaliseerde behandeling van kankerpatiënt te begeleiden.Een persoon die deze techniek nog nooit heeft u...

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

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 ...

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

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

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

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

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

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

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

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

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Medicine

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 ...

Spontaneous Murine Model of Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the ...

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

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

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

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

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

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

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of Anticancer Drugs

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

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

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