| Background and Objection:In the United States, prostate cancer is a major cause of death and the second leading cause of caftcer-related death in males.In2010,191,533new cases of prostate cancer with26,329related deaths were reported in the United States. Although surgery can effectively control prostate cancer in the early stages, distant metastasis is found in many newly diagnosed patients. Despite surgical treatment, androgen deprivation and radiation therapy, disease progression and metastasis still occur in most cases. Therefore, there is an urgent need to understand the mechanisms underlying metastatic progression of prostate cancer and to develop new and effective therapies. Animal models provide the essential link between in vitro experiments and development of novel therapeutic strategies in clinical studies.Animal model is-a important tool for the exploration of the pathogenesis of prostate cancer. In these models, the relationship between the host and the tumor, the processes of tumor invasion and metastasis, and the effectiveness of different treatment measures can be evaluated. Many research studies have presented animal models of prostate cancer, but these models have been unable to simulate the entire process, including the occurrence, development, invasion, and metastasis of human prostate cancer. This may be because cancer-related events and the molecular biology of tumor metastasis have not yet been clarified. In addition, research tools for studying tumor-host interactions are lacking and models reflecting the biological diversity of human cancer at the molecular level are inadequate.The use of immunocompetent mice in prostate cancer research has allowed the implantation of human cancers in an animal model. Recent studies have identified cells of myeloid origin that are potent suppressors of tumor immunity and a significant impediment to tumor immunotherapy. In addition, these studies have demonstrated the importance of immune system regulators, particularly T-cells and myeloid-derived suppressor cells (MDSCs), in antitumor activity. In these studies, changes in serum tumor markers, tumor growth, and metastasis were analyzed within the same model. These results were unique and have not been published elsewhere.This study aimed to generate mouse prostate cancer cell RM1clones that were capable of stable expression of human prostate-specific antigen (protein PSA; KLK3) and luciferase (Luc). This cell line (RM1-Luc-pIRES-KLK3) maybe useful in the development of new preclinical models for prostate cancer in which tumor volume and levels of serum PSA and bioluminescence can be analyzed in real time. Tumor immune cells, such as regulatory T-cells (Tregs) and MDSCs, may also be assayed during the analysis of tumor progression.Materials and Methods:Tumor cell lines:RM1cells were purchased from the typical culture collection of the Chinese Academy of Sciences. The cell lines were cultured in RPMI-1640 with10%fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA) and maintained at37℃in a humidified atmosphere containing5%CO2.Plasmid construction and Transfection:Eukaryotic expression vector pIRES and pGL3-basic vector were purchased from Clontech Laboratories, Inc.(Mountain View, CA, USA). Polymerase chain reaction (PCR) primers were designed according to the GenBank expression of human PSA protein KLK3gene sequences (GI:22208990). Luc gene sequences (GI:13195703) were synthesized by Life Technology Co. Ltd.(Shanghai, China). Using real-time (RT) PCR, PCR amplification, and restriction endonuclease technology, a Luc-pIRES-KLK3plasmid was constructed. This plasmid was transfected into RM1cells for stable expression in monoclonal strains. The firefly KLK3and luciferase genes were transfected into nine prostate cancer RM-1cells, and G418was successfully used to filter out the RM1-Luc-pIRES-KLK3monoclonal cell line.PSA Expression:The cell lines were characterized in vitro to determine expression of the human PSA protein in the cloned cell lines using Western blot analysis. The total protein from cloning cells was extracted and Western blotting was performed as described previously. Proteins were identified using rabbit polyclonal antihuman PSA antibody at1000×dilution as a primary antibody (#5365, Cell Signaling Technology, Inc., Damvers, MA, USA).Bioluminescence detection in vitro:For in vitro bioluminescence imaging, RM1-Luc-pIRES-KLK3cells were diluted in96-well plates (Costar, Corning, NY, USA) that were divided into8groups, each with7wells. Bioluminescent cells were diluted from200,000to6,250cells in the appropriate cell culture media. In the first wells only cells were added as a blank control. D-luciferin (Biosynth, International, Inc., Naperville, IL, USA) at150ug/ml in media was added to all wells except the one with cells only. Cells were incubated in a humidified atmosphere containing5% C02, temperature were controlled at37℃for10-15min, and then imaging under IVIS-100for2min.Imaging of prostate cancer in mice:Male C57BL/6mice (5-7weeks old) were used for this study (permission number SCXK, Guangdong Province;2011-0015). D-luciferin (150mg/kg) was administered by intraperitoneal injection. Mice were anesthetized with a mixture of10%chloral hydrate and an intraperitoneal injection of300mg/kg. Anesthetized animals received2.0×105RMl-Luc-pIRES-KLK3cells via a subcutaneous injection below the dorsal flank. The animals were imaged at a peak time of20min after injection of luciferin via an IVIS-100instrument (Xenogen, Alameda, CA, USA) using appropriate exposure times and sensitivity settings to avoid saturation. Image processing was performed using the Living Image software program (Xenogen) by performing a region-of-interest count of the total number of photons per second for each tumor, with appropriate background subtraction.Flow cytometry:For flow cytometry,100μl of blood from each animal was collected in a plastic tube with ethylenediaminetetraacetic acid. The blood was then incubated with phycoerythrin-labeled antimouse CD11b, CD4antibody (eBioscience, San Diego, CA, USA) and fluorescein isothiocyanate-labeled antimouse Gr-1, Foxp3antibody (eBioscience) for1h at4℃. Labeled samples were washed twice with cold phosphate-buffered saline before used. Then the sample was re-suspended in250μl of cold phosphate-buffered saline and analyzed using a fluorescence-activated cell sorter Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) by gating on lymphocytes.Serum PSA analysis:For the PSA assay,100μl of blood was collected from each mouse in a plastic tube. Using the human PSA enzyme-linked immunosorbent assay kit (EL1005, Anogen, Mississauga, ON, Canada),30ul of serum was analyzed. The optical density was read at450nm using a microtiter plate reader and the data were collected and assayed using SOFTmax Pro version5.3software (Molecular Devices, Sunnyvale, CA, USA).Tumor growth:To study the subcutaneous growth of RM1-Luc-pIRES-KLK3cells in vivo using bioluminescence imaging and serum PSA analysis,1x105cells were used to construct a subcutaneous model in C57BL/6mice. Tumor growth in mice was monitored weekly using bioluminescence imaging and serum PSA analysis. Quantitative bioluminescence imaging analysis (photons/sec) was performed using the IVIS imaging system on days3,7,14, and21after inoculation. The size of the tumor was measured using vernier calipers for21days after cell implantation. Tumor volume was calculated using the following formula:1/2x (shortest diameter)2x (longest diameter).RESULTS:Proliferation was compared in the RM1-Luc-pIRES-KLK3and normal RM1subsets. The proliferation potential was the same in both subsets after3days of cultivation. The total number of cells was counted at3,7days. Using the IVIS imaging system, Luc expression was detected in the RM1-Luc-pIRES-KLK3cell line. A positive correlation was found between cell count and the level of Luc expression.We used Western blot analysis to evaluate the expression of the human prostate cancer biomarker PSA. PSA was demonstrated in the RM1-Luc-pIRES-KLK3cells. An analysis of tumor growth revealed that RMl-Luc-pIRES-KLK3cells grew readily in mice3days after injection. Human PSA levels in the serum of the cancer-bearing mice were determined. Three days after implantation, the mean PSA level was approximately1.5ng/ml. Serum PSA levels in the cancer-bearing mice increased to10.2ng/ml by day21after tumor implantation.The bioluminescence data were compared with data obtained using the traditional methods of monitoring cancer growth on the basis of tumor volume and serum PSA. levels. A positive correlation was observed between endpoint bioluminescence data and endpoint tumor volume data (R2=0.937). A correlation was also observed with endpoint serum PSA levels (R2=0.988). Similar correlations were found between endpoint tumor volume and endpoint serum PSA levels (R2=0.908).Peripheral blood immunological analysis showed that on days7and21, the population of CD4+Foxp3+Tregs was elevated in patients with progressive cancer. Treg elevation has the potential to thwart protective antitumor immunity. Recent human cancer trials have suggested that accumulation of CDllb+Gr-1+MDSCs correlates with increased tumor burden. To explore the potential mechanisms underlying antitumor immunity, the percentage of the CD4+Foxp3+Tregs and CD11b+Gr-1+MDSCs was assessed in the peripheral blood of each mouse. The percentage of CD4+Foxp3+Tregs and CDllb+Gr-1+MDSC cells in the total lymphocytes was quantified by fluorescence-activated cell sorter analysis. The percentage of CD4+Foxp3+Tregs (7.35%vs.9.62%; P=0.0001) increased with tumor growth by day7and day21after tumor implantation. CD11b+Gr-1+MDSC cells increased from1.93%to3.02%on day7and day21after tumor implantation; this increase was also statistically significant (P=0.002). Furthermore, CD4+、 Foxp3+Tregs and CD11b+、Gr-1+MDSC cells were significantly upregulated in tumor-bearing mice compared with those in normal mice(data not shown).CONCLUSION:With improvements in bioluminescence imaging, previously elusive properties of tumor biomarkers have become more detectable and measurable. Monitoring of tumor volume and levels of serum PSA and bioluminescence and analysis of immune status are now possible in immunocompetent mice. The biomarker/imaging-based approach described here may be an improvement over traditional methodologies, enabling practitioners to provide early and sensitive assessment of tumor immune response in the future. |
PDF Full Text Request