This work is supported by grants from the National Key R&#38;D Program of China (2018YFC1704300 and 2020YFE0201600), the National Nature Science Foundation (81973877 and 82174408), the research projects within the budget of the Shanghai University of Traditional Chinese Medicine (2021LK047), and the Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation.

Six to eight week old male BALB/c athymic mice (n = 10) were housedin individually ventilated mice cages (5 mice/cage) in a specific-pathogen-free (SPF) animal room under the conditions of 12 h light/dark cycle, with free access to SPF feed and sterile water. Mice were adaptively fed for a week before the experiments.All animal experiments were approved by the animal welfare committee of Shanghai University of Traditional Chinese Medicine.
1. Cell preparation
<ol>
	<li>On the day of prostate cancer cell injection, wash 80%-90% confluent luciferase labeled PC-3 cells (PC-3-luciferase) cultured in a 10 cm cell culture dish with pre-cold sterile PBS (pH 7.4) twice. Trypsinize for 3 min with 1.5 mL of 0.25% trypsin, and collect the cells into a 15 mL centrifuge tube after quenching the trypsin with 6 mL of F-12 medium containing 10% serum. 
	NOTE: The PC-3-luciferase cell line is derived from the PC-3 cell line after being transfected with the pLV-luciferase vector15.</li>
	<li>Use an automatic cell counter and calculate the cell concentration by transferring 20 &#181;L of the cell suspension to the cell counting plate (see Table of Materials).</li>
	<li>Centrifuge the cells at 800 x g at room temperature (RT) for 5 min.</li>
	<li>Use a pipette to discard the supernatant and resuspend the cell pellet in F-12 medium (see Table of Materials) to a final cell density of 1 x 107 cells/mL.</li>
	<li>Keep the cells on ice at all times until injection.</li>
	<li>Bring the cells to the surgery room and finish the cell injection within 2 h. 
	NOTE: Prepare additional cell suspensions (usually doubled volumes as required) to ensure accurate injection doses (for example, to avoid the dead spaces inside the syringes).</li>
</ol>
2. Surgery for intra-cardiac injection of the human prostate cancer cells
NOTE: The surgical apparatus used for intra-cardiac injection is a 1 mL syringe (Figure 1).&#160;Provide thermal support throughout the procedure until the animal&#8217;s recovery from anesthesia.
<ol>
	<li>Keep the mouse in the SPF animal room. Perform all the procedures with sterilized apparatuses within an aseptic cabinet.</li>
	<li>Anesthetize the mouse using 2% isoflurane with 98% oxygen under a 2 L/min oxygen flow rate. 
	NOTE: Smear a small quantity of ophthalmic ointment on the mouse&#39;s eyes to avoid dryness during the anesthesia.Perform the whole surgery in a well-ventilated area. Make sure that the mouse is deeply anesthetized by pinching the mouse&#39;s toes before cell injection. If responses (e.g., a jerk or twitch) remain, wait for additional time for the anesthesia to take effect.</li>
	<li>Place the mouse in a supone position and immobilize both upper limbs perpendicularly stretched away from the midline (Figure 2A).</li>
	<li>Immobilize the mouse from the abdomen down with surgical adhesive tape. Avoid pressing hard and displacing the internal organs (Figure 2B). 
	NOTE: Maintaining the topographic anatomy (i.e., fixing with adhesive tape) is essential since the intra-cardiac injection is blind (i.e., the accurate injection site on the heart is invisible to the naked eye).</li>
	<li>Disinfect the skin of the thorax with a 70% ethanol swab.</li>
	<li>Identify the mouse xiphoid process in the depression at the lowest end of the mid-sternum by palpating down the middle of the anterior chest wall. Identify the mouse jugular notch in the central depression at the upper border of the manubrium by palpating up from the middle of the anterior chest wall.</li>
	<li>Label the most inferior point of the xiphoid process and jugular notch with a marker pen (Figure 2A). Make a third mark in the middle of these two landmarks and slightly to the right (animal&#39;s left) just over the heart in the third intercostal space. 
	NOTE: The site of injection is 1-2 mm left of the midline, and the injection point is between the third and fourth ribs of the mouse (Figure 2A).</li>
	<li>Load 200 &#181;L of the cell suspension into a 1 mL syringe. 
	NOTE: Keep an air bubble in the syringe, as shown in Figure 2C, to ensure that the blood is pulsating. The cell suspension must be thoroughly mixed before loading.</li>
	<li>Vertically insert a 26 G needle through the injection site (Figure 2D).</li>
	<li>Keep the hand holding the syringe stable on the table, or use the other hand to hold it stable. Ensure that the long axis of the syringe is perpendicular to the injection site, and insert the syringe about 2 mm subcutaneously.</li>
	<li>Bright-red blood pulsation is visible in the needle hub and/or cell suspension when the needle tip is correctly inserted into the left ventricle. If the blood pulsation is invisible, the needle tip is not in the heart. Retract and replace the needle (in case the blood clotting inside the needle stops blood pulsation). Insert the needle once again.</li>
	<li>Inject the cells. Inject the cell suspension (100 &#181;L) very slowly over 40-60 s and keep the hand holding the syringe stable all the time. 
	NOTE: Keep a close eye on the cell suspension in the syringe, and do not inject the air bubble inside the syringe into the heart.</li>
	<li>Apply pressure to the injection site with a dry cotton swab for 15 s to ensure hemostasis when the needle is retracted.</li>
	<li>Return the mouse to the clean cage and monitor the animal until it completely recovers from anesthesia.</li>
	<li>Image the mouse with an in vivo bioluminescence system within 24 h post-injection to ensure the cancer cells have entered the systemic circulation. 
	NOTE: Correctly injected cancer cells enter the arterial circulation via the left heart ventricle, which is seen by the bioluminescence signaling visible all over the body (for example, see Figure 3A).</li>
	<li>Perform in vivo bioluminescence imaging.
	<ol>
		<li>Turn on the instrument and the computer. Open the software when the power indicator on the instrument and computer brightens, and then open the Image Acquisition window.</li>
		<li>Identify the in vivo imaging system connected to the computer in the device pane. Place the mouse to be imaged into the imaging chamber in a supine position. Then close the door of the imaging chamber.</li>
		<li>Set the parameters on the Setting pane provided in the Image Acquisition window.
		<ol>
			<li>Set the parameters with Lens &#62; Zoom &#62; 1x, Lens &#62; Focus &#62; 108, and Lens &#62; Iris &#62; F 2.8 to make the images clear.</li>
			<li>Set the shooting channel in Filter&#38;Light: Filter&#38;Light &#62; Filter &#62; Luminescence, Filter&#38;Light &#62; Light &#62; OFF, and Filter&#38;Light &#62; Light Intensity &#62; Middle.</li>
			<li>Set the type of image to be taken: Imaging &#62; Type &#62; Single-Frame, Imaging &#62; Exposure Time &#62; 500 ms, Imaging &#62; HDR Mode &#62; Low GainMode. Click on the Acquire button to acquire the image.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Visualize the metastatic cancer growth with in vivo bioluminescence. 
	NOTE: At each time point of the visualization, intraperitoneally inject 200 &#181;L of the luciferin substrate into a 20 g mouse (150 mg/kg) and wait 15 min before the anesthesia (2% isoflurane mixed with 98% oxygen in an induction chamber). Place the mouse on the bioluminescence imaging system platform. Maintain anesthesia through a nasal mask with 2% isoflurane.</li>
</ol>
3. Pathologic investigation
<ol>
	<li>Four weeks after the cell injection, sacrifice the mouse by CO2 inhalation and then cervical dislocation.</li>
	<li>Immobilize the mouse in a supine position. Make a vertical incision (about 6 cm) from the abdomen to the thorax with surgical scissors to expose and grossly examine all the organs in the abdomen and thorax for cancerous lesions and/or pathological changes. 
	NOTE: For an intra-cardiac cancer cell injection study, cancer lesions in the mediastinum adjacent to the heart suggest misinjected cancer cells (cells not injected into the left cardiac ventricle); therefore, these mice are not included in the final data collection.</li>
	<li>Excise the metastatic tumors with scissors. Measure the long diameters (a) and short diameters (b) of the tumor using calipers to calculate the tumor volume (V) using the formula V = 1/2 &#215; a &#215; b2, and weigh the tumor using an electronic scale.</li>
	<li>Fix the tumor tissues in 10% formalin solution followed by embedding in paraffin. Alternatively, snap-freeze the tumors in liquid nitrogen.</li>
	<li>Excise the bones (hind limbs, vertebrae, and/or ribs) with metastatic lesions. Fix the specimen of each mouse in a 50 mL tube containing 20 mL of 10% formalin solution for 24 h after removing the skin and muscles, followed by decalcification for 14 days in 10% EDTA solution with frequent buffer change (every 3 days).</li>
	<li>Embed the specimen in paraffin and section the samples for the routine histological examination16.</li>
</ol>

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All the authors declare no competing financial interests.

Intra-cardiac injection of human prostate cancer cells to generate bone metastasis is an ideal mouse model for exploring the functions and mechanisms of prostate cancer bone metastasis and evaluating the therapeutic efficacy. Studies have shown that bone damage most likely occurs in the proximal tibia and the distal femur17, which may be due to their high vascularization and metabolic activity.
Since bone metastasis is a frequently observed metastatic lesion in breast c...

Prostate cancer is the most frequent cancer in men in 112 countries and ranks second for mortality in higher human development index countries1,2. Most deaths in prostate cancer patients are caused by metastasis, and about 65%-75% of the cases will develop bone metastasis3,4. Therefore, prevention and treatment of prostate cancer bone metastases are urgently needed to improve the clinical outcome of prostate cancer patients. The animal model of bone metastasis is an indispensable tool for exploring the multistage process and molecular mechanisms involved in each stage of prostate cancer bone metastasis, thus identifying therapeutic targets and developing novel therapeutics5.
The most common methods to generate experimental animal models of prostate cancer bone metastasis include the orthotopic, intra-diaphysis (such as intra-tibial), and intra-cardiac injection of prostate cancer cells. The bone metastasis model with orthotopic injection is generated by directly injecting prostate cancer cells into the prostate of a mouse6,7. This experimental animal model has very similar clinical characteristics to prostate cancer bone metastasis. However, the metastasis mainly happens in the axillary lymph node and the lung rather than in the bone8,9.The intra-tibial injection model for prostate cancer directly injects prostate cancer cells into the tibia with a hightumor formation rate in the bone (tibia)10,11; however, the bone cortex and bone marrow cavity are easily damaged. Additionally, the tibial injection method cannot stimulate the pathological process of prostate cancer bone metastasis in which the cancer cells colonize the bone through circulation. To investigate the circulation, vascular extravasation, and distant metastasis with a higher bone metastasis rate of cancer cells, an intra-cardiac injection technique has been developed by directly injecting prostate cancer cells into the left ventricle of the mouse8,12,13. This makes it a valuable animal model for bone metastasis research8. The intra-cardiac injection method shows a bone metastasis rate of about 75%9,14, much higher than the orthotopic injection method. Therefore, the intra-cardiac injection is an ideal method to generate an animal model with prostate cancer bone metastasis.
This work aims to describe the process of establishing a mouse model of prostate cancer bone metastases, allowing readers to visualize the model establishment. The current work provides detailed processes, precautions, and illustrative pictures to generate a bone metastasis xenograft model via intra-cardiac injection of human prostate cancer cells in athymic mice. This method provides an effective tool for further exploring the molecular mechanisms and the in vivo therapeutic effects of prostate cancer bone metastasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringes and needles</td>
			<td>Shandong Weigao Group Medical Polymer Co., Ltd</td>
			<td>20200411</td>
			<td>The cells were injected into the ventricles of mice</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>R500IP</td>
			<td>Equipment for anesthetizing mice</td>
		</tr>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>&#160;For counting cells</td>
		</tr>
		<tr>
			<td>BALB/c athymic mice</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd.</td>
			<td>Male</td>
			<td>6-8 week old, male mice</td>
		</tr>
		<tr>
			<td>Bioluminescence imaging system</td>
			<td>Shanghai Baitai Technology Co., Ltd</td>
			<td>Vieworks</td>
			<td>For tracking the tumor growth and pulmonary metastasis if the injected cells are labeled by luciferase</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL, 50 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>&#160;430790, Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Wuhan Xavier Biotechnology Co., Ltd</td>
			<td>G1105</td>
			<td>&#160;For decalcification of bone tissure</td>
		</tr>
		<tr>
			<td>F-12 medium</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>21700075, GIBCO</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>BL539A</td>
			<td>For fixing the specimen of each mouse</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>VETEASY</td>
			<td>For anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>11668027, Thermo fisher</td>
			<td>Plasmid transfection reagent</td>
		</tr>
		<tr>
			<td>PC-3 cell line</td>
			<td>Cell Bank of Chinese Academy of Sciences</td>
			<td>TCHu 158</td>
			<td>Prostate cancer cell line</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Beyotime Biotechnology</td>
			<td>ST447</td>
			<td>Wash the human osteosarcoma cells</td>
		</tr>
		<tr>
			<td>Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>For detaching the cells</td>
		</tr>
		<tr>
			<td>Vector (pLV-luciferase)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>VL3613</td>
			<td>Plasmid for transfection</td>
		</tr>
		<tr>
			<td>X-ray imaging system</td>
			<td>Brook (Beijing) Technology Co., Ltd</td>
			<td>FX PRO</td>
			<td>For obtaining x-ray images to detect tumor growth</td>
		</tr>
		<tr>
			<td>&#956;CT80</td>
			<td>Shenzhen Fraun Technology Service Co., Ltd</td>
			<td>Scanco Medical AG,Switzerland</td>
			<td>For detection of bone destruction. The mico-CT is equipped with 3DCalc, cone reconstruction,&#160; and &#956;CT Ray V3.4A model visualization software.</td>
		</tr>
	</tbody>
</table>

intra-cardiac injection of human prostate cancer cells to create a bone metastasis xenograft mouse model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Bioluminescence imaging offers tremendous advantages in monitoring the metastatic lesion development for an intra-cardiac injection model. Soon after the cancer cell injection (within 24 h), bioluminescence imaging was used to visualize the cancer cells entering the general circulation (Figure 3A). Obvious bioluminescence signaling all over the body will be seen when the cancer cells are injected into the arterial circulation properly. Data from mice showing bioluminescence signals only at t...

Watch this Scientific Journal Video about Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model at JoVE.com

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

intra-cardiac-injection-human-prostate-cancer-cells-to-create-bone

Research

JoVE Journal

Cancer Research

4.3K Views.  Shanghai University of Traditional Chinese Medicine. Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

Video: Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

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 ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

A Melanoma Patient-Derived Xenograft Model

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

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

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

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

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 ...