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>

<ol>
	<li>Siegel, R. L., Miller, K. D., Fuchs, H. E., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cancerstatistics,+2021.">Cancer Cancerstatistics, 2021.</a> CA: A Cancer Journal for Clinicians. 71 (1), 7-33 (2021).</li><li>Sung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2020:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).</li><li>Coleman, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+complications+of+malignancy.">Skeletal complications of malignancy.</a> Cancer. 80, 1588-1594 (1997).</li><li>Macedo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+metastases:+An+overview.">Bone metastases: An overview.</a> Oncology Reviews. 11 (1), 321 (2017).</li><li>Rea, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+in+prostate+cancer+translational+research:+From+xenograft+to+PDX.">Mouse models in prostate cancer translational research: From xenograft to PDX.</a> BioMed Research International. 2016, 9750795 (2016).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+GFP+intravital+imaging+of+the+differences+in+cellular+and+angiogenic+behavior+of+subcutaneous+and+orthotopic+nude-mouse+models+of+human+PC-3+prostate+cancer.">Real-time GFP intravital imaging of the differences in cellular and angiogenic behavior of subcutaneous and orthotopic nude-mouse models of human PC-3 prostate cancer.</a> Journal of Cellular Biochemistry. 117 (11), 2546-2551 (2016).</li><li>Stephenson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+model+for+human+prostate+cancer+using+orthotopic+implantation+in+nude+mice.">Metastatic model for human prostate cancer using orthotopic implantation in nude mice.</a> Journal of the National Cancer Institute. 84 (12), 951-957 (1992).</li><li>Simmons, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bone+metastasis.">Animal models of bone metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J., Purchio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+human+breast+cancer+cell+lines+that+permit+rapid+and+sensitive+in+vivo+detection+of+mammary+tumors+and+multiple+metastases+in+immune+deficient+mice.">Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice.</a> Breast Cancer Research: BCR. 7 (4), 444-454 (2005).</li><li>Corey, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+osseous+prostate+cancer+models:+intra-tibial+injection+of+human+prostate+cancer+cells.">Establishment and characterization of osseous prostate cancer models: intra-tibial injection of human prostate cancer cells.</a> The Prostate. 52 (1), 20-33 (2002).</li><li>Andersen, C., Bagi, C. M., Adams, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-tibial+injection+of+human+prostate+cancer+cell+line+CWR22+elicits+osteoblastic+response+in+immunodeficient+rats.">Intra-tibial injection of human prostate cancer cell line CWR22 elicits osteoblastic response in immunodeficient rats.</a> Journal of Musculoskeletal &amp; Neuronal Interactions. 3 (2), 148-155 (2003).</li><li>Sudhan, D. R., Pampo, C., Rice, L., Siemann, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+L+inactivation+leads+to+multimodal+inhibition+of+prostate+cancer+cell+dissemination+in+a+preclinical+bone+metastasis+model.">Cathepsin L inactivation leads to multimodal inhibition of prostate cancer cell dissemination in a preclinical bone metastasis model.</a> International Journal of Cancer. 138 (11), 2665-2677 (2016).</li><li>Jinnah, A. H., Zacks, B. C., Gwam, C. U., Kerr, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+and+established+models+of+bone+metastasis.">Emerging and established models of bone metastasis.</a> Cancers. 10 (6), 176 (2018).</li><li>Simmons, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+prostate+cancer+cell+line+(Probasco)+produces+osteoblastic+metastases+in+vivo.">Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo.</a> The Prostate. 74 (13), 1251-1265 (2014).</li><li>Lamar, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRC+tyrosine+kinase+activates+the+YAP/TAZ+axis+and+thereby+drives+tumor+growth+and+metastasis.">SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis.</a> The Journal of Biological Chemistry. 294 (7), 2302-2317 (2019).</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrine+inhibits+prostate+cancer+via+activation+of+the+unfolded+protein+response/endoplasmic+reticulum+stress+signaling+and+reversal+of+epithelial+to+mesenchymal+transition.">Matrine inhibits prostate cancer via activation of the unfolded protein response/endoplasmic reticulum stress signaling and reversal of epithelial to mesenchymal transition.</a> Molecular Medicine Reports. 18 (1), 945-957 (2018).</li><li>Arguello, F., Baggs, R. B., Frantz, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+experimental+metastasis+to+bone+and+bone+marrow.">A murine model of experimental metastasis to bone and bone marrow.</a> Cancer Research. 48 (23), 6876-6881 (1988).</li><li>Brylka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spine+Metastases+in+immunocompromised+mice+after+intracardiac+injection+of+MDA-MB-231-SCP2+breast+cancer+cells.">Spine Metastases in immunocompromised mice after intracardiac injection of MDA-MB-231-SCP2 breast cancer cells.</a> Cancers. 14 (3), 556 (2022).</li><li>Rahman, M. M., Veigas, J. M., Williams, P. J., Fernandes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DHA+is+a+more+potent+inhibitor+of+breast+cancer+metastasis+to+bone+and+related+osteolysis+than+EPA.">DHA is a more potent inhibitor of breast cancer metastasis to bone and related osteolysis than EPA.</a> Breast Cancer Research and Treatment. 141 (3), 341-352 (2013).</li><li>Park, S. I., Kim, S. J., McCauley, L. K., Gallick, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+mouse+models+of+human+prostate+cancer+and+their+utility+in+drug+discovery.">Pre-clinical mouse models of human prostate cancer and their utility in drug discovery.</a> Current Protocols in Pharmacology. , (2010).</li><li>Wright, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+breast+cancer+bone+metastasis.">Murine models of breast cancer bone metastasis.</a> BoneKEy Reports. 5, 804 (2016).</li><li>Fearon, K. C., Glass, D. J., Guttridge, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cachexia:+mediators,+signaling,+and+metabolic+pathways.">Cancer cachexia: mediators, signaling, and metabolic pathways.</a> Cell Metabolism. 16 (2), 153-166 (2012).</li><li>Waning, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excess+TGF-&#946;+mediates+muscle+weakness+associated+with+bone+metastases+in+mice.">Excess TGF-&#946; mediates muscle weakness associated with bone metastases in mice.</a> Nature Medicine. 21 (11), 1262-1271 (2015).</li><li>Talbot, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+body-weight+reduction+as+a+humane+endpoint:+a+critical+appraisal.">Defining body-weight reduction as a humane endpoint: a critical appraisal.</a> Laboratory Animals. 54 (1), 99-110 (2020).</li><li>Paget, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+secondary+growths+in+cancer+of+the+breast.">The distribution of secondary growths in cancer of the breast.</a> Cancer Metastasis Reviews. 8 (2), 98-101 (1989).</li><li>Yin, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+blockade+inhibits+PTHrP+secretion+by+breast+cancer+cells+and+bone+metastases+development.">TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development.</a> The Journal of Clinical Investigation. 103 (2), 197-206 (1999).</li><li>Schneider, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=turnover+mediates+preferential+localization+of+prostate+cancer+in+the+skeleton.">turnover mediates preferential localization of prostate cancer in the skeleton.</a> Endocrinology. 146 (4), 1727-1736 (2005).</li><li>Padalecki, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+18+suppresses+prostate+cancer+metastases.">Chromosome 18 suppresses prostate cancer metastases.</a> Urologic Oncology. 21 (5), 366-373 (2003).</li></ol>

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><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>&amp;nbsp;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>&amp;nbsp;430790, Corning</td><td></td></tr><tr><td>EDTA solution</td><td>Wuhan Xavier Biotechnology Co., Ltd</td><td>G1105</td><td>&amp;nbsp;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&amp;nbsp;</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>&amp;mu;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,&amp;nbsp; and &amp;mu;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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

4.4K 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 Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

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

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

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

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Medicine

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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 ...

Intra-prostatic Injection of Cancer Cells: A Technique to Deliver Cancer Cells for Establishing Orthotopic Prostate Cancer Mouse Model

Source: Anker, J. F. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57301" target="_blank">Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the procedure for administering intra-prostatic injection of prostate cancer cells into a mouse model. Orthotopic murine models can be used to study the disease origin and response to treatment therapies in a non-invasive and clinically relevant environment.

Source: Anker, J. F. et al. Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate ...