We thank the members of the Mayo Clinic Center for Individualized Medicine (CIM) Dr. Lin Yang and Faye R. Harris, MS, for the help in conducting experiments. This work was supported by Mr. and Mrs. Neil E. Eckles&#8217; Gift to the Mayo Clinic Center for Individualized Medicine (CIM).

Fresh tissues from consenting patients with ovarian cancer were collected at the time of debulking surgery according to a protocol approved by Mayo Clinic Institutional Review Board (IRB). All animal procedures and treatments used in this protocol were approved by Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and followed animal care guidelines.
1. Mate pair sequencing and analyses
NOTE: Either fresh or flash frozen tissue must be used for mate pair (MPseq) sequencing. Paraffin embedded material is not suitable because it contains fragmented DNA.
<ol>
	<li>Isolate DNA from frozen tumor tissue. Use original human specimen obtained from surgical material or biopsy36.</li>
	<li>Use 1000 ng of DNA to make MPseq libraries and sequence as 2 samples per lane on the next generation sequencer (see Table of Materials)36.</li>
	<li>Analyze data using a set of algorithms to detect large chromosomal aberrations (deletions, insertions, amplifications, inversions and translocations) as described earlier36,37.</li>
</ol>
2. Selection of therapeutic targets
<ol>
	<li>Use the open access Panda tool (Pathway and Annotation) or an analogous tool to identify targetable alterations (http://bioinformaticstools.mayo.edu/Panda).
	<ol>
		<li>Make a list of genes that are identified by MSeq as altered, as a simple tab-delimited file, using standard accepted gene symbols. 
		NOTE: The included example features analysis of amplifications and gains.</li>
		<li>Add a &#8220;#&#8221; sign to the header line of the list to ensure that the table header is transferred to the pathway level view of the software.</li>
		<li>Upload the file by clicking the Upload Annotation Set navigation tab.</li>
		<li>Assign a single icon from the menu to represent the underlying data by clicking on the icon of choice and then clicking the Finalize tab.</li>
		<li>After the annotation files are uploaded, identify a column that displays the number of annotated genes per pathway. This is the last column on the right.</li>
		<li>Use Pathway Filter on the upper left of the main window to show pathways containing genes of interest.</li>
		<li>Identify pathways that have more annotated genes than is expected by chance. Use the function located under the Enrichment tab.</li>
		<li>Select a database to display potentially druggable genes from Preset Annotation by checking an appropriate icon on the left of the main window (e.g., DGIdb, PharmGKB).</li>
		<li>Select pathway for visualization by clicking on its name displayed in the Pathway Viewer page. 
		NOTE: Icons representing each annotation set are shown next to the associated gene. Click on the gene of interest to open the corresponding GeneCards webpage.</li>
		<li>Select the pathways that showed the annotated genes of interest (i.e., altered in a given tumor) and &#8220;hits&#8221; for potential drugs for further analysis.</li>
	</ol>
	</li>
	<li>Use a database containing drugs approved for clinical application (https://clinicaltrials.gov/) to cross-reference the identified targets.</li>
	<li>Prioritize targetable alterations for further testing in PDX models by performing a literature review (e.g., PubMed) to confirm relevance to the biology of a particular type of tumor.</li>
</ol>
3. Validation of genomic rearrangements by PCR and Sanger sequencing
<ol>
	<li>Design primers using sequencing reads obtained from MPseq data.
	<ol>
		<li>Select a junction of interest for the validation (i.e., potential therapeutic target) based on MPseq analyses.</li>
		<li>Design primers directionally such that the amplicon contains the junction. Design 2 primers on each side of the junction, for a total of 4, to increase the chances of amplifying the junction. 
		NOTE: Name primers according to the case and chromosome location.</li>
		<li>Use standard PCR parameters for the primer design and a software of choice. Select melting temperature (60-62 &#730;C) and the GC content (40-60%). Make sure that the primer sequence does not form primer dimers, palindromes or hairpin loops.</li>
		<li>Confirm that the primer sequence lacks homology to other areas of the human genome by checking it using BLAT (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start).</li>
	</ol>
	</li>
	<li>Run a PCR to amplify the junction of interest.
	<ol>
		<li>Dilute primers with water to 10 mM and combine 10 mL of each primer so that each forward primer is paired with each reverse primer into a primer mix. 
		NOTE: Sequences for the primers for the selected example are shown in Table 1.</li>
		<li>Label 0.2 mL strip tubes as: C1, T1, C2, T2, C3, T3, C4 and T4 where C=control human genomic DNA (commercial), T=Tumor DNA, isolated from patient or PDX tumor, and the number indicates the primer mix.</li>
		<li>Add 1 mL of each primer mix into its labeled tubes.</li>
		<li>Prepare the Taq mastermix by combining reagents listed in Table 2, leaving the enzyme in the freezer until needed, adding it to the mix at the very end.</li>
		<li>Make 2 master mixes, one for each template DNA, control human DNA and tumor DNA. Add 24 mL of each Taq master mix to its respective strip tube. The total reaction volume is 25 mL. Vortex very briefly and then spin the strip tube down.</li>
		<li>Run a PCR in a thermocycler. Use the parameters shown in Table 3. Adjust the annealing temperature so that it is at least 1 &#176;C colder than the melting temperature of the primers.</li>
		<li>Store the completed PCR product at -20 &#176;C (long term) or refrigerated at 4 &#176;C (short term) until needed.</li>
		<li>Perform electrophoresis at 1-5 V/cm to visualize PCR product using a 1.5% agarose gel. Leave 2 mL aliquot of the product to be used for Sanger sequencing.</li>
	</ol>
	</li>
	<li>Perform Sanger sequencing to confirm the junction and identify the exact breakpoint38.
	<ol>
		<li>Use the PCR product if the PCR generated a single product (band). Alternatively, cut the band out of gel, purify and submit for Sanger sequencing along with the primer used for amplification. 
		NOTE: The tumors for which genomic analyses were performed then are used for implantation in mice.</li>
	</ol>
	</li>
</ol>
4. Tumor engraftment and maintenance
<ol>
	<li>Set up preparations to engraft tumors into PDX mouse models. Select for engraftment tumors for which genomic analyses will be or has been performed.
	<ol>
		<li>Make sure that a supportive infrastructure is in place at the time of the start of the development of any PDX models, including dedicated laboratory and animal facilities, skilled technical staff and detailed standard operating procedures.</li>
		<li>Ensure the quick transportation and processing of specimens as speed is crucial for cell viability and successful engraftment.</li>
		<li>Use a sterile environment to reduce bacterial and fungal contamination for processing and engrafting specimens.</li>
		<li>Handle human specimens with caution, in accordance with institutional policies regarding potentially biohazardous materials, as they may harbor blood-borne pathogens.</li>
		<li>Prepare the tissue (0.5-0.7 cm3 in size) for engraftment by putting the surgical specimen into a pre-chilled 50 mL tube with 20 mL of tissue culture media. 
		NOTE: The tumor tissue can be fresh or recovered from previously cryopreserved material5.</li>
		<li>Confirm the tumor content in the specimen by consulting a pathologist.</li>
		<li>Place the tumor tissue in a dish containing 10-15 mL of cold PBS, or tissue culture media such as RPMI 1640 or DMEM containing antibiotics (1% penicillin and streptomycin).</li>
		<li>Identify and isolate viable tumor material from adjacent normal and necrotic tissue with the help from a pathologist. Use sterile forceps and scalpel to remove necrotic material pointed out by a pathologist. 
		NOTE: The tumor can be implanted either intraperitoneally or subcutaneously into the mice. Follow step 4.2 to perform an intraperitoneal implantation or skip to step 4.3 to perform a subcutaneous engraftment. In this study, targeted therapies selected based on genomic analyses were tested in a series of PDX models for high grade serous ovarian cancer with intraperitoneal implantation.</li>
	</ol>
	</li>
	<li>Prepare the tissue for intraperitoneal (IP) implantation mincing the tissue with sterile forceps and a scalpel on ice to make pieces approximately 1-1.5 mm3 in size and mixing with cold culture medium. Inject 0.3&#8211;0.5 mL of tumor slurry using a 16-17 gauge needle. 
	NOTE: All surgical procedures are performed by using aseptic techniques. Sterile gloves, sterile instruments, supplies, and implanted materials were used to reduce the likelihood of infection.
	<ol>
		<li>Mix the pieces 1:1 with ice-cold culture medium and inject 100 mL intraperitoneally in at least three female SCID mice.</li>
	</ol>
	</li>
	<li>Cut the tumor tissue for subcutaneous engraftment using sterile forceps, scalpel, or surgical scissors into small fragments, roughly 2 x 2 x 2 mm in size, and transfer the fragmented tissues to a pre-chilled Petri dish on ice.
	<ol>
		<li>Add the cold basement membrane matrix into the dish with the fragmented tissue (approximately 200 mL per 10 pieces of tissue), mix well, and let the tissue fragments soak in the cold basement membrane matrix for 10 min.</li>
		<li>Anesthetize 5 female NOD/SCID mice to prepare them for engraftment.</li>
		<li>Inject each mouse intraperitoneally with ketamine (150 mg/kg) and xylazine (10 mg/kg) combination.</li>
		<li>Confirm that mouse is properly anesthetized by pinching the tail tip with atraumatic forceps.</li>
		<li>Remove gently fully anesthetized mouse from the chamber and put on the mouse a nose cone which has input from vaporizer and output from waste gas scavenging system.</li>
		<li>Put vet ointment on mouse eyes to prevent dryness while under anesthesia. Prepare the area where the surgery will be performed. Use aseptic technique to perform the surgery.</li>
		<li>Ensure that the surface on which the surgery is going to take is non-porous, sealed and is disinfected prior to surgery.</li>
		<li>Start surgery with sterile (by autoclave, gas or chemical sterilization) instruments.</li>
		<li>Use gloves to handle instruments and maintain the sterility of the instrument tips throughout the procedure by submerging them in ethanol between surgery steps.</li>
	</ol>
	</li>
	<li>Sterilize the surgical site by applying 3 alternating scrubs of iodine and alcohol. Use sterile surgical scissors and forceps to make a 5-10 mm vertical skin incision on both flanks of a mouse.
	<ol>
		<li>Insert straight forceps gently into the subcutaneous space to create a pocket large enough for a tumor fragment to be placed under the fat pad.</li>
		<li>Use the sterile straight forceps to insert tumor fragments into previously prepared pocket in each of 5 mice. 
		NOTE: Place 3-4 pieces of tumor tissue in one pocket.</li>
		<li>Close the skin incisions using tissue glue.</li>
		<li>Intraperitoneally inject each mouse with 100 mL of Rituximab after implantation to inhibit lymphocyte proliferation.</li>
	</ol>
	</li>
	<li>Place the mouse into a cage under a heat lamp for approximately 20 min until recovered from anesthesia. Monitor the mouse vital signs and ensure its sufficient hydration.</li>
	<li>Return the mouse to the company of other mice after it recovered from anesthesia and started to have food and water. Provide postoperative care and monitoring according to institutional guidelines. Check for signs of pain and distress daily for 3 consecutive days after surgery. 
	NOTE: Criteria for pain or distress include necrosis or ulceration, weight loss/body condition scoring, behavioral signs such as activity level, motor function and posture.</li>
	<li>Routinely check the mice for tumor formation bi-weekly until the tumors reach the size of 0.5 cm in diameter as measured by a caliper.</li>
	<li>Assess the health score of each mouse as derived from appearance, behavior, and body condition39. Use scores of &#8804;6 as criteria for moribund mice to be sacrificed by carbon dioxide inhalation.</li>
</ol>
5. Testing responses to genomically identified targets in PDX models
<ol>
	<li>Start the selected targeted treatments when the tumors are palpable and reach 0.5 cm3 as measured by an ultrasound scan.
	<ol>
		<li>Prior to performing an ultrasound scan of the abdomen remove the mouse abdominal fur and apply sterile jelly lubricant.</li>
		<li>Use an ultrasound machine with a transducer to obtain images with the tumor positioned in cross-section. Make 3 measurements per session for each animal and average the value for a more accurate assessment of tumor size.</li>
		<li>Analyze the images using available software40.</li>
	</ol>
	</li>
	<li>Administer chemotherapy consisting of a mixture of carboplatin at 51 mg/kg and paclitaxel at 15 mg/kg intraperitoneally (IP) once a week for a total treatment duration of 4-6 weeks. Make sure that total volume for injection does not exceed 0.2 mL.</li>
	<li>Make a MK-220641 stock solution in 30% cyclodextrin (e.g., Captisol) and deliver via oral gavage at 120 mg/kg daily for 4 consecutive weeks.</li>
	<li>Prepare the mouse for oral gavage by hold it by pinching the skin of the back and pinning it back, so that the head and limbs of the mouse are immobilized.
	<ol>
		<li>Insert the gavage probe down the back of the throat of the mouse until the probe reaches the esophagus. Make sure that the probe is not inserted too far, as the lungs of the mouse may perforate, causing death.</li>
	</ol>
	</li>
	<li>Make a MK-866942 stock solution in ethanol at 25 mg/mL. Dilute it in a vehicle containing 5.2% Tween 80, 5.2% PEG400 in sterile water for IP injections at 10 mg/kg for 5 days every other week, with a total duration of the treatment of 4 weeks. 
	NOTE: Volume injected into mice should be 50-120 mL, depending on animal&#8217;s weight.</li>
	<li>Use 7-8 mice per treatment group to have enough statistical power to detect differences43,44.</li>
	<li>Assess body weight and general condition of the mice in therapy daily. Withhold drugs if an animal&#8217;s weight drops 20% or more from their initial weight.</li>
	<li>Assess the tumor size weekly by using ultrasound scanning. Make sure that the individual performing the monitoring of the tumor growth is blinded to the treatment to ensure the unbiased scoring of responses. 
	NOTE: Smaller laboratories may employ 2 different people to administer treatment and ultrasound monitoring.</li>
</ol>

The authors declare that they have no conflict of interest.

We describe the approach and protocols we used to conduct a &#8220;clinical trial&#8221; in PDX models that takes advantage of molecular characteristics of the tumor as obtained by genomic profiling to determine the best choice of drugs for testing. Multiple sequencing platforms are currently used for genomic characterization of primary tumors including whole genome sequencing, RNAseq and customized gene panels. For high grade serous ovarian carcinoma, MPseq to identify structural alterations, DNA rearrangements and copy...

Patient-derived xenografts (PDXs), which are generated from the implantation of patient tumor pieces into immunodeficient mice, have emerged as a powerful preclinical model to aid personalized anti-cancer care. PDX models have been successfully developed for a variety of human malignancies. These include breast and ovarian cancers, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, and non-small cell lung cancer1,2,3,4,5. Tumor tissue can be implanted orthotopically or heterotopically. The former, considered more accurate but technically difficult, involves transplantation directly into the organ of tumor origin. These types of models are believed to precisely mimic histology of the original tumor due to the &#8220;natural&#8217; microenvironment for the tumor6,7. For example, orthotopic transplantation into the bursa of the mouse ovary resulted in tumor dissemination into the peritoneal cavity and the production of ascites, typical of ovarian cancer8. Similarly, injection of breast tumors into the thoracic instead of the abdominal mammary gland affected the PDX success rate and behavior9. However, orthotopic models require sophisticated imaging systems to monitor tumor growth. Heterotopic implantation of solid tumor is typically performed by implanting tissue into the subcutaneous flank of a mouse which allows for easier monitoring of tumor growth and is less expensive and time consuming7. However, tumors grown subcutaneously rarely metastasize unlike as observed in the case of orthotopic implantation10.
The success rate of engraftment has been shown to vary and greatly depend on the tumor type. More aggressive tumors and tissue specimens containing a higher percent of tumor cells were reported to have better success rates12,13. Consistent with this, tumors derived from metastatic sites were shown to engraft at frequencies of 50&#8211;80%, while those from primary sites engraft at frequencies as low as 14%12. In contrast, tissue containing necrotic cells and fewer viable tumor cells engraft poorly. Tumor growth can also be promoted by the addition of basement membrane matrix proteins into the tissue mix at the time of the injection into mice14 without compromising properties of the original tumor. The size and number of tissue pieces intended for implantation were also found to affect the success rate of engraftment. Greater tumor take-rates were reported for implantation in the sub-renal capsule compared to subcutaneous implantation due to the ability of the sub-renal capsule to maintain the original tumor stroma and provide the host stromal cells as well15.
Most studies use NOD/SCID immunodeficient mice, which lack natural killer cells16 and have been shown to increase the tumor engraftment, growth and metastasis compared to other strains14. However, additional monitoring is required as they may develop thymic lymphomas as early as 3-4 month of age13. In ovarian tumor transplants grown in SCID mice, the outgrowth of B cells was successfully inhibited by rituximab, preventing the development of lymphomas but without impacting the engraftment of ovarian tumors17.
More recently, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, carrying a null mutation in the gene encoding the interleukin 2 receptor gamma chain18, became a frequently used strain for the generation of PDX models. Tumors from established PDX models passaged to future generations of mice are reported to retain histological and molecular properties for 3 to 6 generations19,20. Numerous studies have shown that the treatment outcomes in PDX models mimic those of their corresponding patients2,3,4,21,22,23. The response rate to chemotherapy in PDX models for non-small lung cancer and colorectal carcinomas was similar to that in clinical trials for the same drugs24,25. Studies conducted in PDX models, developed for patients enrolled in clinical trials, demonstrated responses to tested drugs similar to those observed clinically in corresponding patients2,3,4.
High&#8211;throughput genomic analyses of a patient tumor in conjunction with PDX models provide a powerful tool to study correlations between specific genomic alterations and a therapeutic response. These have been described in a few publications26,27. For example, therapeutic responses to the EGFR inhibitor cetuximab in a set of colorectal PDX models carrying EGFR amplification, paralleled clinical responses to cetuximab in patients28.
There are a few challenges associated with the development and application of PDX models. Among those is tumor heterogeneity29,30 that may compromise the accuracy of treatment response interpretation as a single cell clone with higher proliferative capacity within a PDX can outgrow the other ones31, thus resulting in a loss of heterogeneity. Additionally, when single tumor biopsies are used to develop PDX, some of the cell populations may be missed and will not be represented in the final graft. Multiple samples from the same tumor are recommended for implantation to resolve this issue. Although PDX tumors tend to contain all the cell types of the original donor tumor, these cells are gradually substituted by those of murine origin3. The interplay between murine stroma and human tumor cells in PDX models is not well understood. Nevertheless, stromal cells were shown to recapitulate tumor microenvironment33.
Despite these limitations, PDX models remain among the most valuable tools for translational research as well as personalized medicine for selecting patient therapies. Major applications of PDXs include biomarker discovery and drug testing. PDX models are also successfully used to study drug resistance mechanisms and identify strategies to overcome drug resistance34,35. The approach described in the present manuscript allows the researcher to identify potential therapeutic targets in human tumors and to assess the efficacy of corresponding drugs in vivo, in mice harboring engrafted tumors which were initially genomically characterized. The protocol uses ovarian tumors engrafted intraperitoneally but is applicable to any type of tumor sufficiently aggressive to grow in mice2,3,12.

<table><tbody><tr><td>3M Vetbond</td><td>3M, Co.</td><td>1469SB</td><td></td></tr><tr><td>anti-AKT antibody</td><td>Cell Signaling Technologies, Inc.</td><td>9272</td><td></td></tr><tr><td>Anti-GAPDH antibody(G-9)</td><td>Santa Cruz Biotech. Inc.</td><td>sc-365062</td><td></td></tr><tr><td>Anti-MAPK antibody</td><td>Cell Signaling Technologies, Inc.</td><td>9926</td><td></td></tr><tr><td>Anti-phospho-AKT antibody</td><td>Cell Signaling Technologies, Inc.</td><td>9271</td><td></td></tr><tr><td>Anti-mTOR antibody</td><td>Cell Signaling Technologies, Inc.</td><td>2972</td><td></td></tr><tr><td>Anti-Phospho-mTOR antibody</td><td>Cell Signaling Technologies, Inc.</td><td>2971</td><td></td></tr><tr><td>Anti-Phospho-S6 antibody</td><td>Cell Signaling Technologies, Inc.</td><td>4858</td><td></td></tr><tr><td>Anti-Rictor antibody</td><td>Cell Signaling Technologies, Inc.</td><td>2114</td><td></td></tr><tr><td>Anti-S6 antibody</td><td>Cell Signaling Technologies, Inc.</td><td>2217</td><td></td></tr><tr><td>Captisol</td><td>ChemScene, Inc.</td><td>cs-0731</td><td></td></tr><tr><td>Carboplatin</td><td>NOVAPLUS, Inc.</td><td>61703-360-18</td><td></td></tr><tr><td>DMEM</td><td>Mediatech, Inc.</td><td>10-013-CV</td><td></td></tr><tr><td>Easy-A Hi-Fi PCR Cloning Enzyme</td><td>Agilent, Inc.</td><td>600404-51</td><td></td></tr><tr><td>Lubricant</td><td>Cardinal Healthcare</td><td>82-280</td><td></td></tr><tr><td>Matrigel</td><td>Corning, Inc.</td><td>356234</td><td></td></tr><tr><td>McCoy&#039;s media</td><td>Mediatech, Inc.</td><td>10-050-CV</td><td></td></tr><tr><td>MK-2206</td><td>ApexBio, Inc.</td><td>A3010</td><td></td></tr><tr><td>MK-8669</td><td>ARIAD Pharmaceuticals, Inc.</td><td>AP23573</td><td></td></tr><tr><td>Nair Sensitive Skin</td><td>Church &amp;amp; Dwight Co.</td><td>Nair Hair Remover Shower Power Sensitive</td><td></td></tr><tr><td>NOD/SCID mice</td><td>Charles River, Inc.</td><td>NOD.CB17-Prkdcscid/NCrCrl</td><td></td></tr><tr><td>Paclitaxel</td><td>NOVAPLUS, Inc.</td><td>55390-304-05</td><td></td></tr><tr><td>PEG400</td><td>Millipore Sigma, Inc.</td><td>88440-250ML-F</td><td></td></tr><tr><td>Perjeta</td><td>Genetech, Co.</td><td>Pertuzumab</td><td></td></tr><tr><td>Rituximab</td><td>Genetech, Co.</td><td>Rituxan</td><td></td></tr><tr><td>RPMI1640</td><td>Mediatech, Inc.</td><td>10-040-CV</td><td></td></tr><tr><td>SCID mice</td><td>Harlan Laboratories, Inc.</td><td>C.B.-17/IcrHsd-PrkdcscidLystbg</td><td></td></tr><tr><td>SLAx 13-6MHz linear transducer</td><td>FUJIFILM SonoSite, Inc</td><td>HFL38xp</td><td></td></tr><tr><td>SonoSite S-series Ultrasound machine</td><td>FUJIFILM SonoSite, Inc</td><td>SonoSite SII</td><td></td></tr><tr><td>Tween 80</td><td>Millipore Sigma, Inc.</td><td>P4780-100ML</td><td></td></tr></tbody></table>

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.

Tissue from resected ovarian tumors at the time of debulking surgeries were collected in accordance with IRB guidance and used for 1) genomic characterization and 2) engraftment in immunocompromised mice (Figure 1). Mate-pair sequencing protocol36,37 was used to identify structural alterations in DNA including losses, gains and amplifications. A representative genome plot illustrating a landscape of genomic changes in one tumor (desi...

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

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 ...

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7.2K Views.  Mayo Clinic. 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’ tumors into mice and testing responses to corresponding drugs.

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

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 ...

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 ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

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 ...

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 ...

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

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Chapters in this video

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

A Melanoma Patient-Derived Xenograft Model

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

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

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