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 th...

<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>

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 ...

This method can help to produce an ideal model that closely mimics the natural progress of bone metastasis of prostate cancer cells in vivo. This technique has the advantage of a bone metastasis and representing the natural bone metastasis process of prostate cancer cells in vivo. This technique benefits the exploration of the molecular mechanisms and investigation of in vivo therapeutic effects for prostate cancer bone metastasis.On the day of the injection, take 80 to 90%confluent luciferase-labeled PC3 cells cultured in a 10 centimeter cell culture dish and start preparing the cells for injection by washing them two times with cold sterile PBS. Trypsinize the washed cells with 1.5 milliliters of 0.25%trypsin for three minutes, following which quench the trypsin by adding six milliliters of F12 medium containing 10%serum and collect the cells into a 15 milliliter centrifuge tube. Transfer 20 microliters of the collected cell suspension into a cell counting plate and calculate the cell concentration using an automatic cell counter.Next, centrifuge the cells at room temperature for five minutes at 800 G.Discard the supernatant using a pipette and resuspend the cell pellet in F12 medium to achieve the desired final cell density. Now carry the cells to the surgery room and perform the cell injection within two hours. Keep the cells on ice until injection.To begin the surgery, place the anesthetized mouse in a supine position and immobilize both upper limbs by perpendicularly stretching them away from the midline. Using surgical adhesive tape, immobilize the mouse from the abdomen down without pressing hard and displacing the internal organs. Disinfect the skin of the thorax using a 70%ethanol swab.Next, palpate down the middle of the anterior chest wall to locate the mouse xiphoid process in the depression at the lowest end of the mid sternum. Label the most inferior point of the xiphoid process. Then by palpating up from the middle of the anterior chest wall, locate the mouse jugular notch in the central depression at the upper border of the manubrium and mark the most inferior point of the jugular notch.Now locate and mark the midpoint between the previous two marks, slightly towards the right and just over the heart in the third intercostal space. This is the injection point. Thoroughly mix the cell suspension before loading 200 microliters of the suspension into a one milliliter syringe mounted with a 26 gauge needle.Leave some air in the syringe between the plunger and the suspension to allow the entry of pulsating blood during the injection. To perform the injection, vertically insert the needle through the injection site. If the needle tip is correctly inserted into the left ventricle, bright red pulsating blood will be visible in the needle hub.Retry injecting if blood pulsation is not visible or blood is clotting. Once the needle is inserted properly, inject 100 microliters of the cell suspension very slowly over 40 to 60 seconds with a stable hand. After completion of the injection, retract the needle while applying pressure on the injection site with a dry cotton swab for 15 seconds to ensure hemostasis.Return the mouse to the clean cage and monitor the animal until it completely recovers from anesthesia. Bioluminescence imaging performed 24 hours after injecting luciferase-labeled PC3 cells into the mouse showed signals from the entire body of the animal indicating the presence of cancer cells in the systemic circulation. Metastatic lesions appeared in the hind limbs of the mouse two weeks after the injection.The metastatic lesions became larger as time progressed and started appearing in other sites. The prostate cancer cell-induced metastatic lesions in the bones were further detected using x-ray imaging which showed bone destruction in the proximal tibia of the animal. The same was also confirmed by micro CT scanning.The metastatic lesions were further confirmed in paraffin embedded tissues by hematin and eosin staining. Accurate injection site is very important to ensure the success rate of this model. This can be achieved by practice.

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3.6K 次观看.  Shanghai University of Traditional Chinese Medicine. 该方法可以帮助产生一个理想的模型，该模型密切模拟体内前列腺癌细胞骨转移的自然进展。该技术具有骨转移的优点，并且代表了前列腺癌细胞在体内的自然骨转移过程。该技术有利于探索前列腺癌骨转移的分子机制和体内治疗效果的研究。在注射当天，取在10厘米细胞培养皿中培养的80至90%融合荧光素酶标记的PC3细胞，并通过用冷无菌PBS洗涤两次开始准备注射细胞。...