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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Pre-operative Mouse Preparation

NOTE: House mice in the university animal facility for at least one week before surgery to allow for adequate adaptation and minimize animal stress. Steps 1.1-1.6 are performed by the surgeon&#8217;s assistant. All subsequent surgical steps are performed by the surgeon using sterile gloves and surgical tools with sterile technique.

<ol>
	<li>Anesthetize the mice with isoflurane (2-5% for induction via chamber, 1-3% for maintenance via nose cone). Verify full induction by the loss of toe pinch reflex.</li>
	<li>Weigh the mice and administer at least 0.05 mg/kg of the analgesic buprenorphine s.c. 
	NOTE: Mouse weight &#8805;20 g is ideal for successful intra-prostatic injections.</li>
	<li value="3">Apply ophthalmic ointment lubrication to both eyes to prevent corneal drying.</li>
	<li>Shave all fur from the abdomen.</li>
	<li>Sterilize the abdomen by three rounds of circular application of surgical scrub using sterile non-adhering pads followed by sterile alcohol wipes. Allow the abdomen to dry. 
	NOTE: For the rest of the surgical procedure, only sterile gloves and surgical instruments must be used to contact the sterilized mouse abdomen.</li>
	<li value="6">Transfer the mouse supine onto a clean surface on a heating pad directly under the objective of a clean surgical microscope.</li>
	<li>Cover the mouse with a sterile drape with a small hole cut over the abdomen.</li>
</ol>


2. Intra-prostatic Injection

NOTE: Perform all surgical steps under sterile conditions. Adjusting the microscope, the mouse placement, or any other non-sterile object must be done with additional assistance.

<ol>
	<li>Perform an approximately 1 cm incision of the outer skin along the midline of the abdomen superior to the penis and the preputial glands (Figure 1D).</li>
	<li>Separate the connective tissue between the outer abdominal skin and the inner abdominal musculature by opening the scissors between the layers.</li>
	<li>Perform a similar incision on the inner abdominal muscles, using forceps to raise the muscles to avoid puncturing the intestines or bladder (Figure 1D).</li>
	<li>Locate one of the bilateral seminal vesicles (Figure 1A, white)/anterior prostate lobes (Figure 1A, black), which are often posterior, lateral, and slightly superior to the bladder (Figure 1A, yellow), but this may vary between mice. The anterior prostate lobes are translucent and attached to the lesser curvature of the white seminal vesicles.</li>
	<li>Gently raise the tip of the seminal vesicle using the Graefe tissue forceps to create tension on the tissue without puncturing the seminal vesicle (Figure 1E).</li>
	<li>Mix the Myc-CaP matrigel solution by gentile pipetting (performed by the surgeon assistant), as cells may have pelleted, and slowly aspirate 30 &#181;L (1x106 cells) into the needle to avoid air bubbles.</li>
	<li>Carefully insert the bevel of the needle parallel to the long axis of the anterior prostate lobe. Slowly inject 30 &#181;L into the lobe and then slowly retract the needle to prevent leakage (Figure 1F). Verify adequate injection by engorgement of the anterior prostate lobe (Figure 1G).</li>
	<li>Carefully hold the seminal vesicle and the prostate lobe outside of the mouse for approximately 30 s to allow the matrigel to partially solidify within the lobe. During this time, collect any cell solution leakage in the abdomen using a sterile polyester-tipped applicator to prevent non-orthotopic tumor development without pressing on the injected prostate lobe.</li>
	<li>Carefully return the seminal vesicle and the injected prostate lobe into the abdomen without putting pressure on the lobe. Replace any externalized tissues.</li>
	<li>Perform continuous sutures to close the inner abdominal muscles with 5-0 vicryl absorbable reverse cutting needle sutures.</li>
	<li>Perform interrupted sutures to close the outer abdominal skin with 4-0 nylon monofilament non-absorbable reverse cutting needle sutures. NOTE: Sterilized 9 mm staples can also be used to close the outer abdominal skin. However, in contrast to sutures, they interfere with any subsequent bioluminescent and fluorescent tumor imaging signal.</li>
	<li value="12">Clean all tools with sterile ethanol wipes (opened by the surgeon&#39;s assistant) and place them in a glass bead sterilizer for 30 s. Allow the tools to dry before use on the next mouse.</li>
	<li>Flush the syringe and needle in sterile saline (opened by the surgeon&#39;s assistant) to prevent clogging by remnant matrigel. 
	NOTE: A surgeon assistant should be present for all operations, performing all non-sterile pre-operative mouse preparation and post-operative mouse care, as well as mixing the Myc-CaP matrigel solution before injections. To minimize time, as each intra-prostatic mouse procedure will require 20-30 min, the surgeon assistant can begin preparing the next mouse as the surgeon is suturing the current mouse.</li>
</ol>
3. Post-operative Mouse Care
<ol>
	<li>Administer 1 mg/kg of the analgesic meloxicam s.c. immediately, 24 h, and 48 h after surgery.</li>
	<li>Allow mice to recover in a cage with no bedding placed halfway on a heating pad. Monitor mice for at least 30 min after surgery until they regain normal mobility and activity, after which place them in a clean cage with food on the floor of the cage.</li>
	<li>Further monitor mice daily for proper wound healing, body weight, grooming, and ambulation after surgery. Separate mice into individual cages upon any evidence of fighting.</li>
	<li>Remove any remaining sutures within 14 days after surgery.</li>
</ol>

<table><tbody><tr><td>Myc-CaP cell line&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CRL-3255</td><td>Verify as mycoplasma-free before use</td></tr><tr><td>Micro-dissecting scissors&amp;nbsp;</td><td>Roboz</td><td>RS-5910</td><td></td></tr><tr><td>Graege forceps</td><td>Roboz&amp;nbsp;</td><td>RS-5135</td><td></td></tr><tr><td>Graefe tissue forceps&amp;nbsp;</td><td>Roboz&amp;nbsp;</td><td>RS-5150</td><td></td></tr><tr><td>Olsen-Hegar needle holder with&lt;br/&gt; suture cutters</td><td>FST&amp;nbsp;</td><td>12002-12</td><td></td></tr><tr><td>50 &amp;micro;L Syringe 705 RN SYR&amp;nbsp;</td><td>Hamilton&amp;nbsp;</td><td>7637-01&amp;nbsp;</td><td>Sterilize by ethylene oxide gas</td></tr><tr><td>28-gauge Needles Small Hub RN H</td><td>Hamilton&amp;nbsp;</td><td>7803-02</td><td>Point style 4, Angle 45&amp;deg;, Length 0.75 in. Sterilized by ethylene oxide gas</td></tr><tr><td>RPMI&amp;nbsp;</td><td>Gibco</td><td>18875-093</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>10437-028</td><td></td></tr><tr><td>Pencillin/Streptomycin&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>15140-122</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>14190-144</td><td></td></tr><tr><td>Matrigel basement membrane&lt;br/&gt; matrix, phenol red-free, LDEV-free</td><td>Corning&amp;nbsp;</td><td>356237</td><td>Thaw on ice in 4&amp;deg;C overnight&lt;br/&gt; before use</td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Henry Schein&amp;nbsp;</td><td>11695-0500-2</td><td>Acquired from Northwestern University Center for Comparative Medicine (CCM)</td></tr><tr><td>Buprenorphine hyrochloride 0.3&lt;br/&gt; mg/mL</td><td></td><td>12496-0757-5&amp;nbsp;</td><td>Controlled substance, acquired&lt;br/&gt; from Northwestern University CCM</td></tr><tr><td>26 5/8-gauge syringe</td><td>BD</td><td>309597</td><td>For meloxicam and buprenorphine injections</td></tr><tr><td>Ophthalmic ointment lubrication&amp;nbsp;</td><td>Akron&amp;nbsp;</td><td>17478-162-35</td><td></td></tr><tr><td>Betadine surgical scrub (povidoneiodine, 7.5%)</td><td>Purdue Products&amp;nbsp;</td><td>67618-151-16</td><td></td></tr><tr><td>Sterile non-adhering pads&amp;nbsp;</td><td>Moore Medical&amp;nbsp;</td><td>10775</td><td></td></tr><tr><td>Sterile alcohol wipes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>22-363-750</td><td></td></tr><tr><td>Surgical microscope Stemi DV4&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Sterile polyester tipped applicators&amp;nbsp;</td><td>Puritan&amp;nbsp;</td><td>25-806 1PD</td><td></td></tr><tr><td>5-0 vicryl absorbable reverse cutting needle sutures</td><td>eSutures&amp;nbsp;</td><td>J493G</td><td></td></tr><tr><td>4-0 nylon monfilament nonabsorbable reverse cutting needle sutures&amp;nbsp;</td><td>eSutures</td><td>699H</td><td></td></tr><tr><td>Glass bead sterilizer&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>18000-45</td><td></td></tr><tr><td>Sterile saline 0.9% sodium chloride&amp;nbsp;</td><td>Hospira&amp;nbsp;</td><td>0409-4888-02</td><td></td></tr></tbody></table>

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.remotexs.ntu.edu.sg/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.

<img alt="Figure 1" src="/files/ftp_upload/20398/20398_Figure1.jpg" /> 
Figure 1: Anterior prostate lobe, draining lymph nodes, and representative technique for intra-prostatic cell injections. Images of the (A) right anterior prostate lobe (black, *), attached right seminal vesicle (white), right testicle and fat pad (green), and bladder (yellow), (B) bilateral prostate-draining para-aortic lymph nodes (orange), (C)...

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. Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate ...

Intra-prostatic orthotopic tumor injection delivers tumor cells matching the prostate tissue histotype directly into the prostate gland of the recipient animal.
To begin, prep an anesthetized male mouse in the supine position. Make a midline abdominal incision and dissect the inner musculature to expose the abdominal cavity. Locate one of the anterior prostate lobes. These prostate lobes are attached to the lesser curvature of the seminal vesicles and lie close to the urinary bladder.
Now, inject a chilled suspension of prostate cancer cells mixed with the desired basement membrane matrix into the long axis of the anterior prostate lobe. Ensure complete delivery of the cell suspension and gently retract the needle. Allow the matrix to solidify and embed the cells within.
Subsequently, place the prostate lobe along with the seminal vesicle back into the abdominal cavity. Suture the surgical incision. Allow the mouse to recover. Check the mouse weekly to observe the growth of orthotopic tumors within the anterior prostate lobe.

intra-prostatic-injection-cancer-cells-technique-to-deliver-cancer

Research

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Animal model studies

2.6K Views. Source: Anker, J. F. et al. Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer. 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. 

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

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

JoVE Journal

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, ...

Medicine

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

Transcript

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

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

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

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer