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 ...

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 ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

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) ...

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 ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Behavior

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

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

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

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

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 ...

Medicine

Targeted Cancer Therapies

The targeted cancer therapies, also known as &#8220;molecular targeted therapies,&#8221; take advantage of the molecular and genetic differences between the cancer cells and the normal cells. It needs a thorough understanding of the cancer cells to develop drugs that can target specific molecular aspects that drive the growth, progression, and spread of cancer cells without affecting the growth and survival of other normal cells in the body.
There are several types of targeted therapies against specific molecular targets to treat different kinds of cancers.
<h4>Angiogenesis inhibitors </h4>
Angiogenesis plays a huge role in the tumor microenvironment by providing necessary oxygen and nutrients to the growing tumor cells. Therefore, the use of specific inhibitors, such as bevacizumab that can bind circulating vascular endothelial growth factors and block the formation of new blood vessels, can help to restrict the growth of tumor cells.
<h4>Monoclonal antibodies</h4>
Another commonly used strategy against cancer is the use of monoclonal antibodies, such as alemtuzumab, trastuzumab, and cetuximab, that can directly target tumor cells. While some of these antibodies target a specific marker on the cancer cells, others just improve the immune response in the body.
<h4>Proteasome inhibitors</h4>
The ubiquitin-proteasome pathway plays a crucial role in apoptosis, cell survival, cell-cycle progression, DNA repair, and antigen presentation in eukaryotic cells. Inhibitors of this pathway, such as bortezomib, carfilzomib, and ixazomib, are successfully used to treat myeloma and mantle-cell lymphoma (MCL).
<h4>Signal transduction inhibitors</h4>
Most cancer cells have abnormal signal transduction pathways that lead to their uncontrolled cell growth, proliferation, and survival. The development of drugs that can inhibit the aberrant signal transduction elements in the cancer cells, such as the surface receptors or the downstream effectors, such as kinases, is a promising avenue for targeted therapy. For example, epidermal growth factor receptor or EGFR is a transmembrane receptor tyrosine kinase that is abnormally expressed in some cases of cancer. Gefitinib, an FDA-approved drug, is an EGFR inhibitor that is successfully used for the treatment of non-small cell lung cancer.

Targeted Cancer Therapies: Imatinib Mesylate and Trastuzumab

The targeted cancer therapies, also known as &#8220;molecular targeted therapies,&#8221; take advantage of the molecular and genetic differences between ...

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

Summary

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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

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

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

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

Comparative Lesions Analysis Through a Targeted Sequencing Approach

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

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

Targeted Cancer Therapies