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
<ol>
	<li>Transfer the cryovial with tumor fragments from liquid nitrogen to a 37 &#176;C water bath.</li>
	<li>Agitate the cryovial with an occasional gentle flick during thawing.</li>
	<li>After the tissue is thawed, take the cryovial out of water bath and mix by gentle inversion.</li>
	<li>Dry off the outside of the cryovial and spray with 70% ethanol. Transfer it to a biosafety hood.</li>
	<li>Transfer the thawed tumor tissues into a 15 mL conical tube filled with 10 mL of cold Dulbecco&#39;s modified Eagle medium (DMEM). Mix well by inverting the tube. Allow the tissue fragments to settle to the bottom of the tube.</li>
	<li>Aspirate the supernatant and resuspend in 10 mL of cold DMEM. Mix well by inverting the tube. Allow the tissue fragments to settle to the bottom of the tube.</li>
	<li>Aspirate the supernatant and resuspend in 10 mL of cold DMEM. Place the tube on ice. 
	NOTE: The tissue is ready for transplantation.</li>
</ol>
2. Collection and preparation of fresh mammary tumor for transplantation
<ol>
	<li>Euthanize the breast tumor-bearing mouse. 
	NOTE: PDX host may be SCID/Beige, NSG or NRG female mice while in the current study female Balb/c mice aged 3&#8722;5 weeks are used.</li>
	<li>Spray the tumor region of the euthanized mouse with 70% ethanol. 
	NOTE: Try to avoid hair with the tumor sample which might cause contamination of tumor fragments for cryostorage or transplantation.</li>
	<li>With serrated forceps to pinch and lift up the skin surrounding the tumor, use scissors to make a short incision.</li>
	<li>Separate the tumor from the skin with the scissors to dissect the whole tumor from the host mouse. Trim off any remaining mouse fat pad tissue from the outer surface of the tumor. Place the tumor in a 15 mL conical tube filled with 5 mL of cold DMEM. 
	NOTE: Use tumors at a maximum size of 1 cm diameter since larger tumors are likely to contain necrotic cores.</li>
	<li>In a biosafety hood, transfer the dissected tumor to a sterile 10 cm Petri dish containing enough DMEM to prevent drying.</li>
	<li>Cut the tumor into 1 mm3 fragments with scalpel or blade under aseptic conditions. 
	NOTE: The scalpel or blade should be exposed under UV in the biosafety hood for at least 20 min prior to use.</li>
	<li>Transfer the tumor fragments to a 15 mL conical tube filled with cold DMEM. Place the tube on ice. 
	NOTE: The tissue is ready for transplantation. The dissected tumor fragments from step 2.4 can be, 1) snap frozen in liquid nitrogen for protein and RNA/DNA extraction, 2) fixed with 4% paraformaldehyde (PFA) or 10% neutral buffered formalin (NBF) for hematoxylin and eosin (H&#38;E) and immunohistochemistry (IHC) analysis, or 3) cryopreserved in 1.25 mL freezing medium (10% dimethyl sulfoxide [DMSO], 40% DMEM and 50% fetal bovine serum [FBS]) by slow freezing in a -80 &#176;C freezer overnight and then transferring to liquid nitrogen for long-term storage.</li>
</ol>
3. Prepare animal for surgery
<ol>
	<li>For animal pain management, subcutaneously inject buprenorphine sustained release 60 min prior to the surgery at a dose of 1 mg/kg or follow institution&#39;s guide for 72 h of analgesic coverage.</li>
	<li>Set up the surgical suite according to institutional guidelines for aseptic surgeries.</li>
	<li>Anesthetize a 4-week old female in an induction chamber of the isoflurane anesthesia machine at the rate of ~11.25 mL/h. Transfer the mouse to procedure area, onto the sterile (silicone rubber) surgery board, where it will receive anesthesia through a small facemask. Put the mouse on its back and tape the legs down in their natural positions. 
	NOTE: SCID/Beige, NSG or NRG are used for PDX and Balb/c is used for GEMM transplant models.</li>
	<li>Apply ophthalmic ointment to the eyes to prevent dryness.</li>
	<li>Confirm the appropriate level of sedation by toe pinch. 
	NOTE: No response/movement of the animal indicates that the animal is sufficiently anesthetized and ready for surgery.</li>
	<li>Shave the mouse on the lower abdomen, especially the region around the fourth nipple where the surgery will take place.</li>
	<li>Using a circular motion and starting in the center of the surgery site, work toward the outer edges with povidone-iodine surgical scrub, followed by removal of povidone-iodine with a 70% isopropyl ethanol pad. Repeat two additional times.</li>
</ol>
4. Transplantation of tumor fragments into the fourth (inguinal) mammary fat pad
<ol>
	<li>Use a sterile surgical drape to cover the body of the animal except the incision site. 
	NOTE: Confirm the appropriate level of sedation by toe pinch before making the incision.</li>
	<li>Using serrated forceps pinch and lift up the skin at the #4 nipple.</li>
	<li>With the blunt side down, use scissors to make a short (about 1 cm), parasagittal incision, from the #4 nipple towards the head.</li>
	<li>Using a cotton tipped applicator, separate the skin from the peritoneum on the medial side of the incision.</li>
	<li>While holding the lateral side of the incision, gently peel the #4 mammary fat pad from the skin using the same swab.</li>
	<li>Once the fat pad is separated, pin down the skin with a 27 G needle close to the animal&#39;s body.</li>
	<li>If it is experimentally necessary to clear the fat pad of endogenous mouse mammary epithelium perform the following steps.
	<ol>
		<li>Using the serrated forceps, gently extend the fat pad away from the body and locate the inguinal lymph node, which is beneath the intersection points of the major vessels in the gland (close to where the forceps will be holding the gland).</li>
		<li>Carefully cauterize the vessels medial to the lymph node and the fat joining the fourth and fifth fat pads which forms a triangular area. 
		NOTE: Temporarily turn off the oxygen source for this step.</li>
		<li>Using micro dissecting scissors, cut each cauterized vessel one at a time (to ensure there is no bleeding after each cut) until the section of the fat pad that contains the lymph node is removed.</li>
		<li>Discard the &#34;cleared&#34; fat pad tissue.</li>
	</ol>
	</li>
	<li>Hold the fourth mammary fat pad using blunt separation forceps. With the other hand, insert the closed tip of the angled fine forceps into the middle of the fat pad above the remaining vessel and close to the wall of the peritoneum. Slowly open the forceps to make a small pocket. Remove the angled fine forceps from the fat pad.</li>
	<li>Using the angled fine forceps, take a piece of tumor fragment and insert it into the pocket.</li>
	<li>Slowly open the forceps tips to release the tumor fragment into the fat pad pocket.</li>
	<li>Withdraw the angled fine forceps carefully. 
	NOTE: Look at the pocket to confirm that the tumor fragment remains in the pocket of the mammary fat pad when withdrawing the forceps. Tumors may be implanted into both contralateral fat pads when excess tumor tissue is needed for experiments or banking. Animals with double-sided transplants are not recommended for treatment studies because of known interactions between contralateral tumors. Alternatively, a trocar device might be used for the implantation process.</li>
	<li>Starting from the base of the incision, collect the skin on each side using the claw and serrated forceps. Bring the two sides together and lift slightly to prepare the skin for wound clip application. Uncurl the edges of the incision with the claw forceps and pinch the edges together to form a continuous surface at the top.</li>
	<li>Holding the two sides together with serrated forceps, use the wound clip applicator to place a wound clip in the center of the incision. If necessary, apply tissue adhesive to the ends of the incision in order to keep them closed and secured.</li>
	<li>Place the animal into a clean cage that is on a warming surface. Monitor for bleeding, signs of dehiscence, and pain during the postsurgical period. The animal should be up and moving within minutes postsurgery.</li>
	<li>Pay close attention to the incision site and overall health of the animals for at least 3 days following surgery. Follow institutional guidelines for pain management.</li>
	<li>Clean the surgical tools for 10 s in a glass beads sterilizer before continuing with the next transplant. Wait for the tools to cool down prior to use.</li>
	<li>Repeat steps 4.1&#8722;4.15 until all mice are transplanted. 
	NOTE: Estrogen supplementation is required for ER+ tumors, which can be supplied through water or slow release pellets59.</li>
</ol>
5. Monitoring tumor growth in response to drug treatment
NOTE: Palpable tumors of established transplantable PDXs and p53-null tumors takes between 2 weeks and 8 weeks to develop after surgery, depending on intrinsic tumor growth rates.
<ol>
	<li>Measure tumors in two dimensions using calipers twice a week. Calculate the volume using the formula: 
	Tumor volume (mm3) = W2 x L/2 
	where W is width and L is length of the tumor.</li>
	<li>Start drug treatment when the tumor volume reaches 150&#8722;300 mm3. 
	NOTE: Depending on the property of the drugs, oral gavage or intraperitoneal injection may be used to deliver the drugs.</li>
	<li>Collect tumor samples and perform IHC staining60, protein and RNA/DNA isolation and immune phenotyping61 for various purposes. Collect blood and perform immune phenotyping or use it for pharmacokinetic/pharmacodynamic studies.</li>
</ol>

MTL is the founder of a limited partner in StemMed, Ltd. and founder and manager in StemMed Holdings, its general partner. MTL is a founder of and an equity holder in Tvardi Therapeutics, Inc. LED is a compensated employee of StemMed, Ltd.

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.

<table><tbody><tr><td>1 mg/mL Buprenorphine-SR</td><td>ZooPharm (via BCM veterinarians)</td><td></td><td>Sterile</td></tr><tr><td>26G syringe</td><td>BD</td><td>148232E</td><td>Sterile</td></tr><tr><td>Betadine Scrub</td><td>Fisher</td><td>19-027132</td><td></td></tr><tr><td>Cotton Swabs</td><td>VWR International Laboratory</td><td>89031-272</td><td>Sterile</td></tr><tr><td>DMEM</td><td>Fisher</td><td>MT 10-013-CM</td><td>Sterile</td></tr><tr><td>Electric shaver</td><td>Oster</td><td>78005-050</td><td></td></tr><tr><td>Glass beads sterilizer (Germinator)</td><td>Roboz Surgical Store</td><td>DS-401</td><td></td></tr><tr><td>Lubricant ophthalmic ointment</td><td>Akorn Animal Health</td><td>17478-062-35</td><td></td></tr><tr><td>Micro Dissecting Forceps; Serrated, Angular (regular forceps)</td><td>Roboz Surgical Store</td><td>RS-5139</td><td>Sterile</td></tr><tr><td>Micro Dissecting Spring Scissors (fat pad cutter)</td><td>Roboz Surgical Store</td><td>RS-5658BT</td><td>Sterile</td></tr><tr><td>Micro Forceps (tissue placing forceps)</td><td>Roboz Surgical Store</td><td>RS-5069</td><td>Sterile</td></tr><tr><td>Petri Dish</td><td>Fisher</td><td>08-757- 100D</td><td>Sterile</td></tr><tr><td>Sterile drape</td><td>Sai Infusion Technology</td><td>PSS-SD1</td><td>Sterile</td></tr><tr><td>Surgery scissors</td><td>Roboz Surgical Store</td><td>RS-5960</td><td>Sterile</td></tr><tr><td>Tissue Forceps (claw forceps)</td><td>Roboz Surgical Store</td><td>RS-5158</td><td>Sterile</td></tr><tr><td>Wound clip applier</td><td>BD Autoclip Wound System</td><td>01-804</td><td>Sterile</td></tr><tr><td>Wound clip remover</td><td>BD Autoclip Wound System</td><td>01-804-15</td><td>Sterile</td></tr><tr><td>Wound clips</td><td>BD Autoclip Wound System</td><td>01-804-5</td><td>Sterile</td></tr></tbody></table>

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. ...

orthotopic-transplantation-breast-tumors-as-preclinical-models-for

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JoVE Journal

Cancer Research

5.9K Views.  Baylor College of Medicine. Patient-derived xenograft (PDX) modellen en transplanteerbare genetisch gemanipuleerde muismodellen getrouw de menselijke ziekte samenvatten en zijn voorkeursmodellen voor fundamenteel en translationeel borstkankeronderzoek. Hier wordt een methode beschreven om borsttumorfragmenten orthotopisch in het borstvetkussen te transplanteren om tumorbiologie te bestuderen en de respons van geneesmiddelen te evalueren.

Video: Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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, ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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 ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Medicine

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

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

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

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

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

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