This work was supported by grant 1R01CA152303-01.

Ethics Statement: Patient-derived colorectal adenocarcinoma tumor specimens were obtained from consenting patients at the University of Colorado Hospital in accordance with a protocol approved by the Colorado Multiple Institutional Review Board (08-0439). All animal work was performed under animal protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC, Protocol # 51412(06)1E and 96813(04)1E).
1. Receiving and Preparing Patient Blood
<ol>
	<li>Collect 1 - 2 ml of blood in a blood/cell separation tube containing sodium citrate (tube phases included are plasma, lymphocyte and monocyte band, density gradient fluid, gel barrier, and erythrocytes and neutrophils). Caution, follow Blood Borne Pathogen guidelines with blood or tissue. 
	Note: PBMCs and plasma could be used for future studies that may include: comparing germline genetic variation with tumor mutations, isolating circulating tumor cells, evaluating cell free DNA (cfDNA), examining proteins and microRNAs, etc.</li>
	<li>Centrifuge the blood/cell separation tube at 2,500 x g for 15 min with no brake at RT.</li>
	<li>Collect peripheral blood mononuclear cells (PBMCs in lymphocyte and monocyte band of tube) and put into a 1.5 ml microcentrifuge tube (clear, autoclavable, DNase, RNase, and pyrogen free), and fill to the top of the tube with sterile Phosphate-Buffered Saline (PBS).
	<ol>
		<li>Centrifuge at 2,300 x g for 3 min at RT to form a pellet of PBMCs at the bottom of the tube. Next, remove the supernatant carefully. Add 1 ml of sterile PBS to wash the pellet and centrifuge at 3,000 x g for 30 sec and then remove supernatant.</li>
	</ol>
	</li>
	<li>Use a pipette to collect plasma from the blood/cell separation tube (in plasma phase of tube) in a 1.5 ml sterile cryogenic vial (DNase and RNase free, no human DNA, endotoxin free) and put the plasma and pellet of PMBC&#39;s into a -80 &#176;C freezer for storage.</li>
</ol>
2. Receiving and Preparing Patient Tumor Sample
<ol>
	<li>Prepare either RPMI or DMEM media with 1:100 (10 %) of Penicillin-Streptomycin and Non-essential Amino Acids, 1:1000 (1%) Plasmocin, and 10 % inactivated Fetal Bovine Serum (complete media) and add 20 - 25 ml to a sterile collection cup for the tumor specimen.</li>
	<li>Retrieve tumor sample in a sterile cup containing complete media on ice or keep at 4 &#176;C. 
	Note: The tumor sample is a piece of excess patient tumor tissue that is removed by a surgeon, processed by a pathologist, and placed into a sterile collection cup with complete media. Caution, follow Blood Borne Pathogen guidelines with blood or tissue. Also, ideally to inject five mice, the tumor should be approximately 1 cm&#179;.</li>
	<li>Bring the tumor sample to the animal facility for injecting into immunocompromised mice. 
	Note: It is best to process the tumor sample as soon as possible.
	<ol>
		<li>In a laminar flow hood, place the tumor sample into a sterile plastic cutting dish from the tumor cup. Use autoclaved-small scissors and forceps to cut into 10 - 12 approximately 3 x 3 x 3 mm pieces and place into an autoclaved 1.5 ml microcentrifuge tube filled with 300 &#181;l of gelatinous protein mixture solution. Keep tube on ice.</li>
		<li>As a priority inject tumor into mice, but if there is any tumor left over, collect flash frozen vials (FF), formalin fixed paraffin embedded (FFPE) cups, and a viable tumor vial.</li>
		<li>For FF, collect small pieces of tumor tissues from a sterile cryogenic vial for further analysis such as protein, RNA, or DNA isolation. Place FF immediately into a liquid nitrogen dewar and store long-term in a -80 &#176;C freezer.</li>
		<li>For FFPE, if there is enough tumor, place 3 small pieces into a 10 ml 10 % formalin cup and process into paraffin embedded blocks once tumor has been in formalin for at least 24 hr. 
		Note: 24 hr is based off our Histology Core suggestions 11.</li>
		<li>Lastly, place the remaining tumor into a cryogenic tube. Make viable media by adding 10 % Dimethyl Sulfoxide (DMSO) to complete media. Next, add 1 ml to a cryogenic tube and store on ice. Note: Do not keep on ice for long periods of time.
		<ol>
			<li>Cut any leftover tumor into small pieces that ideally will be enough for 10 tumors to be injected into 5 mice in the future. Then, place the viable tumor tube on ice until it can be placed into an Isopropyl Alcohol freezing container (slowly freezes the tumor 1&#176;C at a time) and put into -80&#160;&#176;C freezer to ensure that the tumors do not die during the freezing process. 
			Note: For long-term storage the tumors are removed (after 2 - 3 days) and placed into a large liquid nitrogen dewar. Please note that viable tubes should not be allowed to thaw unless it is being taken out to inject into mice.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Injection of Patient Derived Tumor Xenografts
<ol>
	<li>Use five 6 - 8 week old male and/or female athymic nude mice (T-cell deficient) for each separate patient derived tumor. Inject a total of 10 tumors (2 injections per mouse) subcutaneously (SQ). 
	Note: The original human tumor is designated as F0 and then once injected into mice the next generation is F1. Also,use autoclaved forceps, scissors, and trocars for each unique explant.</li>
	<li>Load pieces of tumor from the gelatinous protein mixture solution into autoclaved 12 G trocars and ensure that the tumor is completely pushed into the trocar.
	<ol>
		<li>In a sterile laminar flow hood, place 5 mice into an anesthesia box that is connected to an Isoflurane anesthesia machine (started at the initial rate of 5% Isoflurane, 3 - 4% oxygen, and 2 - 3% Isoflurane for maintenance of anesthesia) and charcoal filter (absorbs excess gas). 
		Note: An experienced technician should be able to perform the entire procedure on a cage of 5 mice within approximately 5 min total (roughly 30 sec per mouse). Therefore additional thermal support and eye lubrication is not generally used. For less experienced individuals or during training, it is highly recommended that individuals either perform the procedure on less animals at one time and/or use a warming pad/eye lubrication during procedure if animals will be anesthetized longer than 5 min. Procedure is based upon IACUC standards at our university.</li>
	</ol>
	</li>
	<li>Pinch a mouse toe to ensure the mouse is no longer responsive, then take the mouse out of the Isoflurane box. Place the mouse onto a clean field to inject tumors on the middle dorsal neck region and sliding the trocar down subcutaneously until the flank region is reached with autoclaved 12 G trocars.
	<ol>
		<li>Deliver one tumor subcutaneously to each side of the mouse&#39;s flank. Pinch the tumor when the trocar is pulled out, ensuring that the tumor stays in the desired flank region. The gelatinous protein mixture hardens with the mouse&#8217;s body temperature and encapsulates the tumor for approximately 1 week to help the tumor grow and secure it to the SQ connective tissue. The lesion made by the trocar is no larger than 4 mm and there is no need to close the skin with staples. 
		Note: Our veterinarians determined no closure is needed based on very small size of the lesion, quick healing time (middle dorsal neck region reduces any skin tension or grooming of lesion), no infections noted, and close monitoring of the animals during this time period.</li>
	</ol>
	</li>
	<li>Before the mouse is fully awake inject Meloxicam 2 mg/kg SQ (pain medication) away from the site of tumor injection. Next, place the mouse into the cage and monitor until the mouse is awake and moving. 
	Note: Do not leave recovering animals unattended until fully awake. Meloxicam dosage is based upon IACUC standards at our university.
	<ol>
		<li>Next, repeat the same procedure with the 4 remaining mice and monitor their breathing. 
		Note: Please note this is a very quick procedure and mice wake up very soon after Meloxicam injection, but adjust Isoflurane as needed. Each time a mouse is taken out, turn down the Isoflurane. The Meloxicam will last 24 hr and the lesions from the trocars will fully heal in 1 week.</li>
	</ol>
	</li>
</ol>
4. Maintenance of Patient Derived Tumor Xenograft Bank
<ol>
	<li>Monitor growth and health of mice at least once per week. Use a spreadsheet (or other tracking system) to track tumor sizes, tumor generation, date of tumor injection, and health of mice. 
	Note: If mice have lost 15% of their original body weight, low body condition scores (&#8805; 2), have tumor ulcerations, tumors reaching 2,000 mm3 or total tumors 3,000 mm3, or are sickly (hunched, cold, lethargy, etc.) in any way, mice are euthanized via CO&#8322; or anesthetized followed by cervical dislocation as a secondary method. Euthanize via anesthesia and cervical dislocation if collecting tumor, otherwise mice are euthanized via CO&#8322; and cervical dislocation.</li>
	<li>When a tumor is approximately 1,500-2,000 mm3 anesthetize the mice as described above and then perform cervical dislocation to euthanize.
	<ol>
		<li>Check that the mouse has no heartbeat. Excise the SQ tumor with autoclaved scissors and forceps. 
		NOTE: Allow tumors to grow for up to 1 year and if no tumor is seen then euthanize the mice via CO2, followed by cervical dislocation as a secondary method.</li>
		<li>Passage the best growing tumor into the next generation (aka a new set of 5 mice). Use instructions above to collect 10 - 12 tumors for passing and then collect the leftover tumor as also described above. 
		Note: Collecting numerous viable tubes is very important in the early generations (F1 - F8); therefore collect several viable tubes, FF tubes, and 1 FFPE per generation.</li>
		<li>Keep the remaining mice until a new generation of mice has tumor growth of approximately 300 mm3. When remaining mice have tumors that are large, continue to collect as described above.</li>
	</ol>
	</li>
	<li>Continue to passage tumors and collect at each stage until F15. At this point, take a viable tube of the earliest generation possible out of liquid nitrogen and let thaw on ice and follow tumor-injecting procedures described above. 
	Note: Tumors that grow from viable tubes take longer to grow in mice compared to passing from generation to generation, so keep that in mind when planning. If a particular PDTX model is no longer needed at any passage, euthanize and collect tumor, to ensure numerous viable tubes are collected for future use.</li>
</ol>
5. Developmental Therapeutics with Patient Derived Tumor Xenografts
Note: Most tumors at F3 generation have good growth kinetics (grow faster and more consistent), therefore, proceed to PDTX drug efficacy studies.
<ol>
	<li>When the desired tumor is very large (1,500 - 2,000 mm3), follow procedures above to collect tumor to expand into desired amount of mice.
	<ol>
		<li>Depending on the hypothesis being tested determine the number of tumors needed per treatment group. 
		Note: 1 ml of gelatinous protein mixture solution can approximately fit 30 small tumor pieces (depending on tumor morphology) for injecting into mice.</li>
	</ol>
	</li>
	<li>Check mice with tumors weekly and when most tumors are visibly small (approximately between 50 - 300 mm&#179;) measure the tumors with calipers to determine the tumor volume. Tumor volume = [width&#178; (smallest measurement) x length (largest measurement)] x 0.52. 
	Note: The gelatinous protein mixture solution surrounds the injected tumor pieces for one week, then after one-week tumor growth will be accurate.</li>
	<li>Use tumor volumes between 50 - 300 mm&#179; and then average the left and right tumors. Then randomize into treatment groups with 10 tumors per group and a group average within a few numbers of each other. Next, sort the mice into groups and begin the desired treatment study.</li>
	<li>Dose the mice with drug (schedule dependent on drug), weigh and measure (tumor) twice per week. At the end of study, euthanize mice via anesthesia and cervical dislocation and collect tumor for future pharmacodynamic analysis in the lab. 
	Note: The study lasts for 30 days depending on vehicle tumor size and health of mice.</li>
</ol>
6. Organization of a PDTX bank
<ol>
	<li>Keep a well-documented form to prevent repeating of research and appropriate use of animals. 
	NOTE: This is key to a successful PDTX bank.
	<ol>
		<li>Use spreadsheets to keep track of a tumor from the human patient in the clinic, to mice in the PDTX bank, treatments, data, and what is collected. Note: this includes freezer, liquid nitrogen dewars, and FFPE storage as well.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The PDTX drug discovery platform offers an improved model to the shortcomings of other preclinical models that are unreliable in predicting clinical activity of novel compounds. Importantly, tumors in this model are biologically stable, retain metastatic potential, and exhibit similar drug responsiveness from generation to generation. In this model, patient derived tumors are injected into athymic nude mice, passaged, and subsequently used in therapeutic evaluation. There are several critical steps for a successful PDTX ...

Colorectal cancer (CRC) is a significant contributor to cancer deaths in the United States. In 2015, there were an estimated 132,700 new cases of CRC with 49,700 deaths 1. Although the prognosis in patients with localized disease is excellent, patients with advanced disease have poor outcomes, making this a major priority in the development of novel therapies. Despite standard of care chemotherapeutic regimens and newer biologics that are deployed against this disease, there has been only an incremental increase in overall survival. Accordingly, there is a significant effort in understanding the driver pathways involved in facilitating tumor growth in this disease. The Cancer Genome Atlas Network has recently identified numerous main pathways that are implicated in CRC dysregulation and include: WNT, phosphoinositide 3-kinase (PI3K), RAS, transforming growth factor-&#946; (TGF- &#946;) and TP53 2. Together, with investigations describing other pathways that potentiate growth in CRC have ignited the development of newer therapies aimed at significantly improving the survival in this patient population 3-5. Utilizing preclinical models in oncology drug development have been essential in this process in predicting the clinical activity of these novel compounds.
Various preclinical models have been utilized in the drug development process. Considering that preclinical transgenic animal models and immortalized cell lines have been unsuccessful in determining the clinical activity of novel oncology therapies, largely due to their inability to reflect the complexity of human tumors, patient-derived tumor xenograft (PDTX) models have been established. The greatest advantage of this model is that tumor heterogeneity remains intact and closely reflects the molecular characteristics and clonality of the originating patient tumor 6-9. PDTX models provide an excellent in vivo preclinical platform to study novel agents, drug resistance pathways, combinational strategies, and cancer stem cell biology 10.
A general overview of the PDTX process is illustrated in Figure 1. It begins in the clinic, consenting patients to allow some of their excess tumor tissue to be used for this research. Next, at surgery, a piece of the tumor is grossed by a pathologist and put into media to be transported to research personnel. Immediately after this, a section of the tumor is cut into small pieces and transplanted into immunodeficient mice subcutaneously. Once the tumor grows, it is passaged into different generations of mice in order to maintain the tumor10. Typically, after the F3 generation the tumor can be expanded into a treatment study where novel compounds and/or combinational therapies are evaluated. Utilizing Next Gen Seq (Exome Seq, RNA Seq and SNP array) potential predictive biomarkers are discovered that assist in the selection of patients that may derive benefit from a particular treatment.
The overarching goals of using PDTX models are to: 1) evaluate the efficacy of novel therapies as single agent or in combination and 2) identify predictive biomarkers of sensitivity or resistance prior to clinical investigation. In this manuscript, we provide the methodology in the initiation and maintenance of a CRC PDTX bank and provide the advantages and limitations of this model in drug development discovery.
<img alt="Figure 1" src="/files/ftp_upload/54393/54393fig1.jpg" /> 
Figure 1. Overview of the CRC PDTX Model Protocol. A patient derived tumor is received from surgery and immediately injected into athymic nude mice subcutaneously. Once the tumor grows it is expanded into subsequent generations and eventually expanded for treatment studies. Treatment responses are assessed and predictive biomarkers are identified that may aid in patient selection. <a href="https://www.jove.com/files/ftp_upload/54393/54393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

Similarities of Common Mutations in the CRC PDTX Models and the TCGA
We investigated whether the percentage of common mutations (KRAS, NRAS, BRAF, PIK3CA, APC, CTNNB1 and TP53) in the CRC PDTX bank were representative to the mutation frequency seen in the CRC patient population. As shown in Figure 2A (TCGA) and B (CRC PDTX bank), the frequency of mutations in these gene...

Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

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

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 ...

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Cancer Research

13.6K Views.  University of Colorado Denver Anschutz Medical Campus. 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.

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

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

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

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

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

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

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 ...

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 ...

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

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

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

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

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

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

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

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

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

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

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

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

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

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

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.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...