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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Research

JoVE Journal

Cancer Research

5.8K Views.  Baylor College of Medicine. 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.

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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

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, ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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 ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...