Name: orthotopic transplantation of breast tumors as preclinical models for breast cancer
Uploaded: 2020-05-18T14:45:00
Description: Watch this Scientific Journal Video about Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer at JoVE.com

This work was supported by the National Institutes of Health (R37CA228304 and R01HL146642 to Xi Chen, CA148761 to Jeffrey M. Rosen), US Department of Defense (W81XWH-19-1-0524 to Xi Chen, W81XWH-19-1-0035 to Xiangdong Lv), American Cancer Society (RSG-18-181-01-TBE to Xi Chen) and Cancer Prevention and Research Institute of Texas (RR150009 CPRIT Scholar in Cancer Research Award to Xi Chen), the Patient-Derived Xenograft and Advanced In Vivo Models Core at Baylor College of Medicine (funding from RP170691 CPRIT Core Facility Award and NCI-CA125123 P30 Cancer Center Support Grant).

All protocols using animals have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The tumor fragments, around 1&#8722;2 mm3 in size, are from viably frozen stock obtained from the Patient-Derived Xenograft and Advanced In Vivo Models Core at Baylor College of Medicine.
1. Preparation of cryopreserved mammary tumor fragments for transplantation
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	<li>Transfer the cryovial with tumor fragments from liquid nitrogen to a 37 &#176;C water bath....

To reduce variations in tumor growth across animals, it is critical to cut the tumor tissue into 1 mm3 fragments for transplantation. Models that grow soft tissue are harder to work with and the tumor fragments need to be cut slightly larger (1&#8722;2 mm3). When placing the tissue into the mammary fat pad pocket take care not to split the tissue into multiple pieces as this will result in multiple small tumors or oddly shaped tumors.
In addition, use fresh tumor for tran...

Most breast cancer deaths can be ascribed to recurrent disease that is resistant to conventional therapies1,2. The inter- and intra-tumor heterogeneity of breast cancers contribute to therapy resistance. Moreover, tumor heterogeneity can impinge on accurate prognosis and challenge disease management3,4. Identification of predictive biomarkers of response will significantly improve clinical outcomes of patients with breast cancer. Even though most breast cancer types are immunologically &#39;cold&#39; tumors that are likely unresponsive to immunotherapy, immune checkpoint inhibitors have shown promise in clinical trials2,5. For example, a phase III trial showed improved disease-free survival (DFS) and preliminary evidence that atezolizumab (monoclonal antibody against PD-L1) combined with nab-paclitaxel may provide an overall survival benefit as compared with nab-paclitaxel alone in tumors with &#8805;1% PD-L1 staining6. Development of therapies that sensitize breast tumors to immunotherapy will revolutionize treatment regimens.
Preclinical models that faithfully recapitulate human breast cancer heterogeneity and drug response are critical to study tumor biology and identify potential biomarkers for targeted therapy. Immortalized cell lines are widely used for breast cancer research since these cell lines are easy to grow and genetically modify to study molecular mechanisms. However, due to the selective pressure from long term cell culture in vitro, genetic drift may occur over time and breast cancer cell lines may carry cell line-specific genomic alterations that are distinct from aberrations in primary breast tumors7,8,9.
Patient-derived xenograft (PDX) tumor chunks are able to recapitulate the heterogeneity of human disease, and are histologically and immunohistochemically similar to the tumor of origin10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. Importantly, PDX models are phenotypically stable across multiple transplantations as evidenced by histology, transcriptome, proteome and genomic analysis10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. PDX models show treatment responses comparable to those observed clinically10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29. PDX models for estrogen receptor positive (ER+), progesterone receptor positive (PR+), epidermal growth factor 2 positive (ERBB2+, HER2+) and triple negative breast cancer (TNBC) PDX models have been established, and provide an excellent platform to test endocrine-, chemo- and targeted therapies. However, one main caveat of PDX models at present is the lack of a functional immune system in the mouse.
The genetically engineered mouse models (GEMM), such as Trp53 homozygous null, cMyc, Wnt1, PyMT, or Her2 overexpression models, allow the study of spontaneous tumor initiation, progression and metastasis in the context of an intact immune system. However, the tumor latency is long, which makes it difficult to conduct preclinical trials with multiple arms30,31. However, GEMM can be transplanted to syngeneic hosts to generate sufficient numbers of tumors that closely recapitulate human tumors32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55. For example, the mammary epithelium from a p53-null BALB/c mouse was transplanted into the cleared fat pads of syngeneic wild-type recipient mice to form primary tumors, which can be further transplanted into syngeneic hosts56,57. The p53-null tumors recapitulated different subtypes of human tumors.
The combination of PDX models and transplantable GEMM provides valuable preclinical tools to investigate breast tumor biology, drug response and anti-tumor immunity. In the current protocol, a method of orthotopic transplantation of PDX and GEMM tumor fragments into the mouse mammary fat pad is described. These models are amenable for serial passages and usually retain a stable phenotype. To mitigate the risk of genetic drift or loss of heterogeneity across passages over time, multiple tissue fragments are cryopreserved at each passage for subsequent transplantation in the event that biological or morphological changes are observed over time29,58.

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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.

Figure 1 shows the equipment (Figure 1A) and key procedures (Figure 1B) of orthotopic transplantation. Figure 2 shows characterization of a transplanted PDX tumor (MC1). Tumor fragments (1 mm3) of MC1 model were transplanted into the #4 fat pad of SCID/Beige mice. One month later, the average tumor size reached around 350 mm<s...

Watch this Scientific Journal Video about Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer at JoVE.com

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

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. ...

This protocol describes the orthotopic transplantation of breast tumor fragments into the mammary fat pad and provides valuable approaches for investigating tumor biology, drug responses, and mechanism of drug resistance. This technique is minimally invasive and ensures proper placement of the tumor tissues within the mammary fat pad, accessibility for counting per measurement, and animal mobility maintenance. This technique allows the transplantation of dozens of animals from a single patient derive's inter graft, for the evaluation of multiple candidate cancer therapies simultaneously in a preclinical trial format.This method is superior to subcutaneous tissue implantation for establishing patient derives inter-graph models, and can also be used when orthotopic implants are not possible. The most difficult stuffs are applying the appropriate amount of pressure to peel the fat pad from the body wall and making the tissue pocket. Visualization of the fat pad peeling and the cracked anatomical placement of the tissue fragment, will help you ensure a successful performance of the procedure.To thaw cryopreserved mammary tumor tissue fragments for transplantation. First, thaw a cryo vial from liquid Nitrogen storage containing the tumor fragments of interest in a 37 degrees Celsius water bath, with occasional gentle flicking. When the fragments have thawed, mix the fragments with gentle inversion, and dry the outside of the vial.Spray the vial with 70%ethanol, and place it in a vial-safety hood. Transfer the cryo vial contents to a 15 milliliter conical tube. Then add 10 milliliters of cold DMEM, and invert the tube to mix.Allow the fragments to settle, and replace a supernatant two times with 10 milliliter aliquot, so, fresh cold DMEM. Then place the tube on ice. To collect a mammary tumor tissue sample, spray the tumor region of the tumor bearing mouse with 70%ethanol and use serrated forceps to pinch and lift the skin surrounding the tumor.Use scissors to make a small incision, and separate the tumor from the skin, taking care to avoid hair contamination. Trim off any remaining mouse fat pad tissue from the outer surface of the tumor, and place the tumor in a 15 milliliter conical tube containing five milliliters of cold DMEM. In a bio-safety hood transfer the dissected tumor into a sterile 10 centimeter Petri dish, containing enough DMEM to prevent drying, and use a UV sterilized blade to cut the tumor into one cubed millimeter fragments under aseptic conditions.Then transfer the tumor fragments to a 15 milliliter tube containing cold DMEM on ice. After confirming a lack of response to pedal reflex, apply ointment to the anesthetized recipient mouse's eyes, and shave the abdomen around the fourth memory gland. Then disinfect the exposed skin three times, using a circular motion and starting in the center of the surgery site while moving outward toward the outer edges, with povidone-iodine surgical scrub, followed by scrub removal with a 70%isopropyl alcohol pad.For transplantation of the tumor tissue fragments, into the fourth inguinal mammary fat pad, cover the animal with a sterile fenestrated surgical drape, and use serrated forceps to pinch and lift the skin at the fourth nipple. With the blunt side of the tips facing down, use scissors to make an approximately one centimeter parasagittal incision from the number four nipple toward the head. Use a cotton tip applicator to separate the skin from the peritoneum on the medial side of the incision, and gently peel the number four memory fat pad from the skin.Use a sterile 27 gauge needle to secure the skin close to the animal's body, and use serrated forceps to gently extend the fat pad away from the body. Locate the inguinal lymph node at the intersection of the major vessels of the gland, and carefully cauterize the vessels medial to the lymph node and the fat adjoining the fourth and fifth fat pads. Using micro dissecting scissors, cut each cauterized vessel one at a time, til the section of the fat pad that contains the lymph node is removed, and use blunt forceps to grasp the fat pad.Using the other hand and a curved motion, insert the close tip of a pair of angled fine forceps into the middle of the fat pad above the remaining vessel close to the wall of the peritenoneum. Retract the tips and reinsert the tips into the original hole, opening the forceps to stretch the hole into a small pocket. Remove the forceps from the hole and use them to insert a piece of tumor fragment into the pocket.Slowly open the tips to release the tumor fragment into the fat pad pocket and carefully withdraw the forceps. After confirming that the fragment has been deposited into the fat pad pocket, starting from the base of the incision, collect the on each side of the incision with serrated forceps and lift the skin slightly. Uncurl the edges of the incision with claw forceps and pinch the edges together to form a continuous surface at the top.Holding the two sides with the serrated forceps, use a wound clip applicator to place a clip in the center of the incision, and place the animal in a clean cage on a warming surface with monitoring until full recumbency. Typically, patient derived xenograft or genetically engineered mouse model tumors can be palpated from about two to eight weeks post transplantation. H&E staining can be performed to analyze the tumor pathology while immunohistochemistry can be used to monitor markers for a specific cell lineages of interest, or cell viability and functional status.It is important to make sure that the tumor fragment is placed securely in the pocket, so, that it doesn't fall out and grow within the subcutaneous space. Once the tumors have grown, drug treatment can be administered to assess tumor responses. The tumors can also be removed to determine the metastatic potential of the cancer cells.This basic technique has been used since the 1950s. Our modification provides a more efficient and less invasive manner in which to conduct larger scale developmental biology in cancer studies.

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