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 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 prevalent cause of cancer deaths (9%) among males in the United States, next to cancer of the lung and bronchus (28%)1. According to recent data, it is estimated that 220, 800 newly diagnosed prostate cancer cases and 27, 540 deaths will occur in 20151. The five year relative survival rate of early stage prostate cancer is >99% while that of advanced metastatic disease is only 28%1. A major challenge for treatment of advanced metastatic disease is the lack of understanding of molecular mechanisms underlying the propensity of this disease to metastasize to other organs, particularly to the bone, which is a frequent site for prostate cancer. Hence, there is a clear need to study the molecular makeup of these prostate tumors in order to develop effective therapeutic regimens against progression to advanced metastatic disease2,3.
Prostate tumors exhibit high biological heterogeneity without a well-defined pathway to progression. Metastases often occur with no prior indication of tumor invasiveness4. This clinical heterogeneity is attributed to the molecular diversity of prostate cancer. Understanding the molecular makeup of these lethal tumors is the key to design better diagnostic and therapeutic strategies for this disease. Consequently, prostate cancer research is currently focused on understanding and preventing metastasis.
Pre-clinical in vivo mouse models offer a variety of options to understand the molecular mechanisms of prostate cancer progression to advanced metastatic disease. In addition, these models are important for preclinical evaluations of new therapeutic strategies against this disease. The most commonly used animal models include transgenic mouse models, tail-vein injection, intra-cardiac implantation and human orthotopic mouse models. Transgenic studies are time consuming and correlation of prostate cancer development in mice with that of humans have shown variability11. In spontaneous metastatic mouse models, cells are injected directly into the circulation and though, they have rapid turnaround time, they cannot be used to study the primary tumor or the initial steps in the metastatic cascade5. Orthotopic xenograft models have the limitation of developing bone metastatic lesions, the common site of prostate cancer metastasis. Nonetheless, the human orthotopic prostate cancer xenograft mouse model is well characterized and widely used to study the molecular events of primary tumor development, cross-talk between tumor and organ microenvironment, initial phase of the metastatic disease and use of experimental drugs for therapeutic intervention6,7,8-11.
Protocols for all procedures involving animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC). Follow officially approved procedures for the care and use of laboratory animals. Intra-prostatic injection requires open-abdominal surgery and animals should be kept in a pathogen-free environment with a designated surgery room where proper surgical aseptic techniques are used during the entire procedure.
1. Preparation of Cells for Implantation
NOTE: Based on research need, any prostate cancer cell line can be used. Cell lines are cultured according to the supplier's instructions.
2. Preparation of the Surgical Area
3. Implantation of Tumor Cells
4. Monitoring of Animals
5. Non-invasive Bio-imaging of Animals
Following orthotopic implantation of PC3M-Luc-C6 cells into the posterior prostatic lobe, mice were weekly imaged by using a live animal bioluminescence imaging system to monitor the colonization of cells and tumor growth over the course of experiment (Figure 5A–B). Quantification of the bioluminescent signal indicated that PC3M-Luc-C6 cells successfully colonized the prostate lobes. Increased bioluminescence is indicative of increased primary tumor growth over the course of the experiment (Figure 5B). Based on the research goal, mice can be monitored weekly non-invasively by radiography, fluorescence or luminescence imaging to monitor tumor growth and any distant metastatic lesions. Other parameters that can be achieved with this model are: changes in body weight and food consumption over the course of experiment; effect of drug treatment on tumor size and weight; quantification of tumor size and weight at the termination of experiment; extraction of DNA/RNA/protein to determine molecular changes occurring inside the primary tumor after the termination of the experiment.
Figure 1: Abdominal midline incision for intra-prostatic implantation of tumor cells. Abdominal midline incision is approximately 1-2 cm long. Urinary bladder is directly under the incision. Gentle pressing on both sides of the incision helps to protrude the urinary bladder. Please click here to view a larger version of this figure.
Figure 2: Arrangement of seminal vesicles for intra-prostatic implantation of tumor cells. Seminal vesicles are white sac-like organs and are located directly adjacent to the bladder. Seminal vesicles are exteriorized with cotton swabs and arranged left and right with the bladder in the center. Please click here to view a larger version of this figure.
Figure 3: Dorsum of prostate. At the point of insertion, gently tilt back the seminal vesicles towards the penis sheath so that the two dorsal prostate lobes are clearly visible. Use wet cotton swabs to avoid tissue damage. Please click here to view a larger version of this figure.
Figure 4: Intra-prostatic implantation of tumor cells. Tumor cells are injected into the dorsal lobe of prostate. Please click here to view a larger version of this figure.
Figure 5: In vivo bioluminescence imaging of intra-prostatic implantation model. (A) In vivo bioluminescence images of mice over the experimental time course after luciferase labeled PC3M-Luc-C6 cells were implanted into the dorsal prostatic lobe of nude mice. (B) Quantification of the bioluminescence signal shows that PC3M-Luc-C6 cells successfully colonized the prostate gland with increased orthotopic tumor growth over the course of the experiment. Please click here to view a larger version of this figure.
This manuscript describes a detailed procedure for establishing a human orthotopic prostate cancer xenograft mouse model. This model was established by direct implantation of the human prostate cancer cell line PC3M-Luc-C6 into the dorsal prostatic lobes of immunocompromised mice. Tumors were allowed to develop over the course of the experiment. Tumor growth was monitored weekly by a non-invasive bioluminescence imaging system during the experiment.
The most important factor in establishing xenograft tumor models is to achieve consistency throughout the implantation of tumor cells. To obtain statistically significant results, each experimental group should contain 5-10 mice and tumor size variation should not exceed more than 10% of average tumor size. To achieve this goal, some critical steps within the protocol are important, such as: i) conducting surgery in area that promotes asepsis during surgery; ii) cells should be transplanted as soon as possible after detachment from culture; iii) injection volume should be consistent; iv) careful lifting of internal organs in and out of body cavity during implantation of cells; v) all animals should be injected using the same technique and by one investigator; vi) animals should be randomized into experimental groups after tumor cell implantation.
Some problems that may occur are: i) tumor does not develop at all or tumor nodules develop in the mesentery or body cavity; ii) uneven tumor size is observed among the same experimental group; iii) there may be high surgery-related mortality. These problems can be overcome by taking simple measures such as: i) testing the cell culture for any contamination with mycoplasma etc.; ii) preventing leakage of the tumor cell suspension into the mesentery and abdominal cavity during injection; iii) agitating the cell suspension before each syringe loading; iv) proper anesthesia dosage should be followed and heating pads should be used to maintain body temperature during the procedure.
A wide variety of data can be collected utilizing this model depending on a particular research goal including mouse weight, food consumption, tumor size and weight, genetic and molecular changes in the tumor cells that contribute to tumor growth as well as regional lymph node metastasis10,16. Hoffman and his group developed the technique of surgical orthotopic implantation (SOI) and have extensively used this technique to transplant histologically-intact fragments of major types of human cancers including prostate, bladder and kidney cancers in the rodents17. These orthotopic models have advantage over the transgenic or subcutaneous mouse models as they accurately represent the clinical cancer18,19. These models were also used to transplant the tumors taken directly from the patients to the corresponding organ of the immunodeficient rodents. Orthotopic models are also well suited to examine the effects of drug treatment on tumor growth and lymph node metastasis10. They are also useful for examining the effects of altered gene expression ex vivo, and determining its effect on tumor incidence as well as intra-prostatic growth and metastasis20. However, a limitation of orthotopic prostate cancer model is that no such models have been reported to lead to spontaneous metastasis to the bone which is the most frequent site for prostate cancer metastasis12.
Failure to achieve bone metastases may be due to mice dying of urinary obstruction before any bone metastatic lesions can develop, or because the microenvironment of the mouse fails to recapitulate the human microenvironment, thus failing to develop bone metastases12. Nonetheless, this model does recapitulate the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation and therefore is a valuable tool to study the primary tumor, early process of metastatic transformation and for preclinical evaluations of new therapeutic strategies10,12.
The authors have nothing to disclose.
We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This work was supported by the National Cancer Institute at the National Institutes of Health through grant numbers RO1CA160079, RO1CA138642, UO1CA184966 and VA funded program project number 1P1 BX001604.
PC3 prostate cancer cell line | ATCC | CRL-1435 | |
Minimum Essential Medium (MEM) | GIBCO,Life Technology | 11095-080 | |
PBS | GIBCO,Life Technology | 10010-023 | |
FBS | GIBCO,Life Technology | 10437-028 | |
Zeocin | Invitrogen,Life Technology | R250-01 | |
Trypsin | GIBCO,Life Technology | 25300-54 | |
IVIS | Xenogen-Caliper | ||
Insulin Syringes (300ul, 28.5g) | Becton Dickinson | 309300 | |
Mice | Charles River Laboratories, Inc | ||
Alcohol Swabs | MEDEquip Depot | 326895 BD | |
PVP Iodine Prep Pad | MEDEquip Depot | C12400PDI | |
Surgical CatGut Chromic Suture | Demetech | CC224017F0P | |
Matrigel | Corning | 354248 |