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&#39;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 &#38; 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...

Watch this Scientific Journal Video about Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts at JoVE.com

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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7.1K 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 ...

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 ...

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

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

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

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

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

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

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

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

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

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

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 ...

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

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

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

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

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

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

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