We would like to acknowledge the generous contributions of the patients who provided tumor samples for the development of PDXs. Emidio Pistilli acknowledges the significant prior contributions of Hannah Wilson, MD/PhD. This research was supported by the following organizations: National Institutes of Arthritis, Musculoskeletal and Skin Diseases (NIAMS) under award number R01AR079445 (Pistilli); the WVU Genomics Core Facility (U54GM104942); the WVU Animal Model and Imaging Facility (P20GM121322, U54GM104942, P20GM144230, P30GM103488); National Institutes of General Medical Sciences under award number P20GM121322 (Lockman). Figure 1 was created with BioRender.com.

This protocol adheres to the guidelines established by the West Virginia University (WVU) Institutional Animal Care and Use Committee. Female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, aged 8 weeks or older and weighing approximately 25 g, were used for tumor cell injections. Breast cancer tumor tissue was procured at West Virginia University Cancer Institute (Morgantown, WV) and by the NCI Cooperative Human Tissue Network under approved WVU Institutional Review Board protocol and WVU Cancer Institute Protocol Review and Monitoring Committee. Informed written consent was obtained from the patients. All procedures were conducted under aseptic conditions within a Class II biological safety cabinet, following appropriate personal protective equipment (PPE) protocols. The details of the animals, reagents, and equipment used in this study are provided in the Table of Materials.
1. High-throughput enzymatic dissociation of tumor tissue
<ol>
	<li>Thaw and combine enzymes H, R, and A from the human tumor dissociation kit in a sterile C-tube. Add 200 &#181;L of enzyme H, 100 &#181;L of enzyme R, and 25 &#181;L of enzyme A. Bring the final volume to ~5 mL by adding 4.7 mL of sterile RPMI 1640 medium to the C-tube. 
	NOTE: Dulbecco&#39;s Modified Eagle Medium (DMEM) can be used in place of RPMI 1640.</li>
	<li>Tumor fragments can be cut and frozen as described in existing protocols9. Transfer thawed tumor fragments and accompanying freezing medium onto one half of a sterile 100 mm culture dish. Using sterile forceps, transfer the tumor fragments to a sterile 5 mL microtube. 
	NOTE: A sterile hypodermic needle may be used as an alternative to forceps. A 35 mm dish can be used in place of a 5 mL microtube.</li>
	<li>Wash the tumor fragments with 1-3 mL of sterile phosphate-buffered saline (PBS, 1x). Manipulate the fragments in the solution using forceps to ensure thorough removal of the residual freezing medium.</li>
	<li>Transfer the washed fragments to a 2 mL microtube using forceps. Apply 1 mL of triple antibiotic (TAB) solution to the tumor fragments and incubate for 10 min at room temperature. 
	NOTE: This step may be skipped if desired. 10x TAB stock can be made by dissolving 1% gentamicin, 1% clindamycin, and 0.5% polymyxin B sulfate in sterile 1x PBS. Dilute to 1x TAB solution in sterile 1x PBS.</li>
	<li>Using sterile&#160;forceps, transfer the disinfected fragments to a new 5 mL microtube. Perform a second wash with 1-3 mL of sterile PBS. Transfer the washed fragments to the dry half of the 100 mm culture dish from step 1.2.</li>
	<li>Using two sterile No. 10 scalpel blades, thoroughly mince the tumor tissue. Transfer the minced tumor fragments onto one blade and deposit them into the C-tube containing the prepared enzyme solution. Securely fasten the tube lid until a final click is felt.</li>
	<li>Invert the C-tube several times to ensure an even distribution of tumor fragments within the enzyme solution.</li>
	<li>Place the C-tube in the mechanical dissociator. Invert the tube and ensure all contents are submerged in the medium. The tube will be in an inverted position in the machine.
	<ol>
		<li>Secure the tube in a slot with the indented part facing the operator. If applicable, place the heater around the tube.</li>
	</ol>
	</li>
	<li>Select the 37C_h_TDK_3 program from the touchscreen interface. This program operates for 60 min at 37 &#176;C. For softer tumors, 37C_h_TDK_1 or 2 can be used. 
	NOTE: For the greatest efficiency, it is recommended that the mice be prepared for injection during this dissociation period by applying depilatory cream to the injection sites.</li>
	<li>Upon completion of the dissociation program, inspect the tube contents. No visible tumor fragments should remain. 
	NOTE: If fragments are present, additional dissociation time may be required. In such cases, run the program for an additional 20-30 min.</li>
	<li>Once adequate dissociation is achieved, centrifuge the C-tube at 200 x g for 5-8 min at 4 &#176;C to pellet dissociated cells. Carefully remove the supernatant using vacuum aspiration or a pipette.</li>
	<li>Resuspend the cell pellet in 5-10 mL of fresh RPMI medium. Transfer the cell suspension to a 15 mL conical tube for easier manipulation. Centrifuge the cell suspension at 200 x g for 5-8 min at 4 &#176;C. Carefully aspirate the supernatant. 
	NOTE: This step may be omitted if the cell pellet is small. In such cases, the cells are resuspended directly in the desired volume of media and then combined with basement membrane extract (Matrigel) to minimize cell loss.</li>
	<li>Resuspend the cell pellet in a volume of RPMI 1640, equivalent to half the desired final injection volume. RPMI 1640 and Matrigel will be combined in a 1:1 v/v ratio, with a final injection volume of 100 &#181;L per injection site. 
	NOTE: For bilateral injection in 4 mice, the total required volume is 800 &#181;L. Therefore, resuspend the cells in at least 400 &#181;L of RPMI 1640. To account for potential volume loss, adding an additional 20% to the final volume is advisable, maintaining the 1:1 v/v ratio. Alternatively, DMEM or 1x PBS can be used in place of RPMI 1640.</li>
	<li>Count the resuspended cells using a hemocytometer or automated method. Dilute as needed to achieve the desired concentration for injection. A starting point of approximately 1-2 million cells per injection site is suggested.</li>
	<li>In a 2 mL round-bottom microcentrifuge tube, combine the desired amount of diluted cell suspension in RPMI with an equal volume of Matrigel. Gently mix the suspension by repeated pipetting to ensure homogeneity. 
	NOTE: Following the previous step, add 400 &#181;L of diluted cell suspension to 400 &#181;L of Matrigel. Consider the number of injection needles to be used and factor in associated volume losses. Keep Matrigel on ice at all times. Do not thaw it until cells are resuspended, as it will solidify quickly. Recommended to maintain small aliquots (e.g., 120 &#181;L) for quick thawing and combination in 2 mL tube.</li>
	<li>Maintain the cell suspension on ice until ready for injection.</li>
</ol>
2. Orthotopic inguinal mammary fat pad injection of dissociated PDX
<ol>
	<li>Determine the mass of each mouse using a calibrated balance. Apply a thin layer of depilatory cream to the target injection site(s). Using a sterile cotton swab, massage the cream in alternating clockwise and counterclockwise motions to facilitate rapid fur removal.
	<ol>
		<li>Remove excess fur and depilatory cream using a sterile saline-soaked gauze sponge. Ensure complete hair removal at the injection area. 
		NOTE: Do not exceed 60 s of depilatory cream exposure to prevent skin irritation. Mice can be shaved prior to the application of depilatory cream to further minimize exposure time. This step can be performed during tumor dissociation to optimize time efficiency and minimize the duration of cell suspension storage on ice.</li>
	</ol>
	</li>
	<li>Conduct a thorough visual inspection of each mouse to assess fitness for injection. Evaluate for any signs of pain, distress, illness, or other contraindications.</li>
	<li>Using a 1 mL syringe without an attached needle, gently aspirate the cell suspension up and down to ensure homogeneity. Attach a 26 G needle to the 1 mL syringe and withdraw the desired injection volume. Eliminate any air bubbles from the syringe by gentle tapping or flicking. 
	NOTE: It is recommended not to exceed 600 &#181;L per syringe to facilitate single-handed injection.</li>
	<li>Place the prepared syringe horizontally on top of the ice container, with the needle oriented away from the operator. Position the ice container and needle on the same side as the operator&#39;s dominant hand or preferred injection hand. Remove the mouse from its housing and disinfect the injection area using an alcohol cleansing pad.</li>
	<li>Securely restrain the mouse by grasping the dorsal skin firmly at the nape and back. Ensure sufficient immobilization to clearly expose the mammary fat pads. The inguinal mammary fat pad spans from the nipple to the top of the hip. It can be observed as soft, pinkish tissue in the described area underneath the skin. 
	NOTE: An assistant operator may use flat-sided forceps to pinch and elevate the skin containing the fat pad. This technique helps ensure that the operator performing the injection successfully targets the fat pad.</li>
	<li>Place the mouse in a supine position. Insert the needle (bevel up) at a 45-degree angle, pointing toward the hip approximately 0.5-1 cm caudal to the fat pad, adjusting for needle length.
	<ol>
		<li>Advance the needle into the fat pad. In a controlled manner, inject the desired volume of cell suspension. Efficient injection speed will help minimize animal agitation from prolonged restraint. 
		NOTE: To minimize animal distress, the maximal duration of continual manual restraint should not exceed 30 s. If the restraint duration exceeds 30 s, the animal should be released into the cage to recover before a second attempt.</li>
	</ol>
	</li>
	<li>Post-injection, rotate the needle 180 degrees (bevel down). Maintain needle position briefly before slow withdrawal to minimize cell loss from the injection site. If any bleeding occurs, apply standard gauze or hemostatic gauze as necessary.</li>
	<li>Repeat steps 2.6 and 2.7 for the contralateral fat pad if bilateral injections are required. For multiple animals, repeat steps 2.2-2.7 as needed.</li>
</ol>
3. Follow up procedures
<ol>
	<li>Dispose of all sharps in designated sharps containers. Dispose of all other contents in proper waste containers.</li>
	<li>If bleeding occurs during injection, monitor mice for at least 1 h afterward to ensure hemostasis.</li>
	<li>Monitor body mass on a weekly basis. Assess tumor volume once growth becomes palpable.</li>
</ol>

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

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nat Med. 17 (11), 1514-1520 (2011).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+renewable+tissue+resource+of+phenotypically+stable,+biologically+and+ethnically+diverse,+patient-derived+human+breast+cancer+xenograft+models.">A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models.</a> Cancer Res. 73 (15), 4885-4897 (2013).</li><li>Yoshida, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+patient-derived+tumor+xenograft+models+and+tumor+organoids.">Applications of patient-derived tumor xenograft models and tumor organoids.</a> J Hematol Oncol. 13 (1), 1-16 (2020).</li><li>Wilson, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+breast+cancer+xenograft+model+implicates+peroxisome+proliferator-activated+receptor+signaling+as+driver+of+cancer-induced+muscle+fatigue.">Human breast cancer xenograft model implicates peroxisome proliferator-activated receptor signaling as driver of cancer-induced muscle fatigue.</a> Clin Cancer Res. 25 (7), 2336-2347 (2019).</li><li>Wilson, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+reprogramming+by+breast+cancer+regardless+of+treatment+history+or+tumor+molecular+subtype.">Skeletal muscle reprogramming by breast cancer regardless of treatment history or tumor molecular subtype.</a> NPJ Breast Cancer. 6 (1), 18 (2020).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R&#947;null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R&#947;null mice engrafted with mobilized human hemopoietic stem cells.</a> J Immunol. 174 (10), 6477-6489 (2005).</li><li>DeRose, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+models+of+human+breast+cancer:+Protocols+for+in+vitro+and+in+vivo+applications+in+tumor+biology+and+translational+medicine.">Patient-derived models of human breast cancer: Protocols for in vitro and in vivo applications in tumor biology and translational medicine.</a> Curr Protoc Pharmacol. 14, Unit14.23 (2013).</li><li>Whittle, J. R., Lewis, M. T., Lindeman, G. J., Visvader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models+of+breast+cancer+and+their+predictive+power.">Patient-derived xenograft models of breast cancer and their predictive power.</a> Breast Cancer Res. 17 (1), 17 (2015).</li><li>Fleming, J. M., Miller, T. C., Meyer, M. J., Ginsburg, E., Vonderhaar, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+regulation+of+human+breast+xenograft+models.">Local regulation of human breast xenograft models.</a> J Cell Physiol. 224 (3), 795-806 (2010).</li><li>Okano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+implantation+achieves+better+engraftment+and+faster+growth+than+subcutaneous+implantation+in+breast+cancer+patient-derived+xenografts.">Orthotopic implantation achieves better engraftment and faster growth than subcutaneous implantation in breast cancer patient-derived xenografts.</a> J Mammary Gland Biol Neoplasia. 25 (1), 27-36 (2020).</li><li>Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., Ahituv, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+antibiotics+in+cell+culture+with+caution:+Genome-wide+identification+of+antibiotic-induced+changes+in+gene+expression+and+regulation.">Use antibiotics in cell culture with caution: Genome-wide identification of antibiotic-induced changes in gene expression and regulation.</a> Sci Rep. 1 (1), 7533 (2017).</li><li>Amens, J. N., Bah&#231;ecioglu, G., Zorlutuna, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+system+effects+on+breast+cancer.">Immune system effects on breast cancer.</a> Cell Mol Bioeng. 14 (4), 279-292 (2021).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

This article presents an efficient, high-throughput method for orthotopically implanting BC PDXs into the inguinal mammary fat pads of immunodeficient mice. This optimized workflow enables the single researcher to implant PDXs into dozens of mice per day, supporting large-scale and adequately powered studies. The capacity to simultaneously dissociate and implant multiple unique PDXs also enables precision oncology approaches. Beyond time efficiency, the dissociation of the tumors creates a homogenous suspension of cell t...

Breast cancer (BC) is diagnosed in over 2 million women worldwide each year1. In order to advance the basic biological understanding of BC and successfully translate novel therapeutics to approved treatments, preclinical animal models that retain the defining features of patient tumors are necessary. Patient-derived xenografts (PDXs) have been demonstrated to recapitulate tumor heterogeneity with high fidelity when implanted orthotopically, including histopathology, receptor expression, and genetic aberrations2,3,4. Importantly, they also retain stromal cells that contribute to the microenvironment of the tumor, such as vasculature, immune cells, and cancer-associated fibroblasts. Albeit, they are gradually replaced by murine host stromal cells with passaging5. PDXs also enable the study of systemic complications resulting from tumor growth, such as fatigue6,7, which is not currently possible using in vitro models.
Successful BC PDX engraftment does, however, require an immunodeficient mouse strain, with the most commonly used strain being NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). NSG mice are devoid of adaptive immunity and exhibit highly defective innate immunity8. BC PDX engraftment has been achieved in other immunodeficient strains, such as SCID/Beige (CB17.Cg-PrkdcscidLystbg-J/Crl), with similar engraftment rates4.
Traditional methods of implanting BC PDXs involve surgically implanting a tumor fragment into the mammary fat pad9, and this approach leads to successful engraftment at favorable rates. However, the need for sterile surgical procedures and anesthetic use makes it time-consuming, thus limiting the number of mice that can be implanted in a certain time window. Given PDXs&#39; potential predictive benefits of drug responses in tumors and use in precision medicine10, having a simple and reproducible workflow for implanting PDXs homogeneously across mice is vital. This article demonstrates the dissociation of human breast tumors into a single cell suspension and subsequent orthotopic injection into mammary fat pads of immunodeficient mice.

<table><tbody><tr><td>1 mL tuberculin syringe</td><td>BD</td><td>305945</td><td>sterile</td></tr><tr><td>1x phosphate buffered saline, pH 7.4</td><td>Gibco</td><td>10010023</td><td>sterile</td></tr><tr><td>2.0 mL round-bottom microcentrifuge tube</td><td>Eppendorf</td><td>0030123620</td><td>sterile</td></tr><tr><td>26 G needle, &amp;frac12; in.</td><td>BD</td><td>305111</td><td>sterile</td></tr><tr><td>5.0 mL microtube</td><td>Eppendorf</td><td>0030119401</td><td>sterile</td></tr><tr><td>8+ week old NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice</td><td>The Jackson Laboratory</td><td>#005557</td><td></td></tr><tr><td>Alcohol prep pads</td><td>Fisher Scientific</td><td>22-363-750</td><td>sterile</td></tr><tr><td>Basement membrane extract (Matrigel)</td><td>Cultrex</td><td>3632-005-02</td><td></td></tr><tr><td>Cotton-tipped applicators, 6 in.</td><td>Fisher Scientific</td><td>22-029-553</td><td>sterile</td></tr><tr><td>Curved Forceps with Medium Non-serrated Tips, 152 mm</td><td>Electron Microscopy Sciences</td><td>50-365-845</td><td>sterile</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACS Octo dissociator with heaters</td><td>Miltenyi Biotec</td><td>130-096-427</td><td></td></tr><tr><td>No. 10 scalpel blades</td><td>Fisher Scientific</td><td>12-000-162</td><td>sterile</td></tr><tr><td>Non-woven gauze sponges, 4x4 inch</td><td>Fisher Scientific</td><td>22-028-558</td><td>sterile</td></tr><tr><td>RPMI 1640 1x + L-Glutamine</td><td>Gibco</td><td>11875093</td><td>sterile</td></tr><tr><td>Tumor dissociation kit, human</td><td>Miltenyi Biotec</td><td>130-095-929</td><td></td></tr></tbody></table>

high-throughput dissociation and orthotopic implantation of breast cancer patient-derived xenografts

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

This study outlines an efficient and non-surgical approach for the orthotopic implantation of patient-derived breast cancer xenografts in mice. Breast tumor tissue was dissociated using human-specific enzymes and mechanical agitation, resuspended in a 1:1 v/v ratio of Matrigel: RPMI 1640, and injected orthotopically into the inguinal mammary fat pads of NSG mice (Figure 1 and Figure 2). To ensure the fidelity of ...

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

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

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Cancer Research

501 Views.  West Virginia University School of Medicine. Het onderzoek in mijn laboratorium is gericht op het begrijpen van de onderliggende mechanismen die spiervermoeidheid bevorderen bij patiënten met borstkanker. In het bijzonder willen we de communicatie begrijpen tussen een tumor gelokaliseerd in het borstweefsel met perifere skeletspieren, en vervolgens de moleculaire reacties in die spier die tot vermoeidheid leiden. De ontwikkeling van 3D-celcultuurmodellen maakt high-throughput studies mogelijk waarbij f...

Video: High-throughput dissociatie en orthotope implantatie van borstkanker Patiënt-afgeleide xenotransplantaten

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 ...

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

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

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

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

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

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

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

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

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

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

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) ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

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 ...

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

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