The overall goal of the following experiment is to effectively induce a cytotoxic T lymphocyte response targeting a tumor associated antigen in bladder cancer. This is achieved by inducing bladder cancer in mice that express the human tumor associated antigen mach one. Next, a peptide vaccine is injected into the mice and a mach one specific cytotoxic T lymphocyte response is generated.
Then the serum tumor and spleen are isolated and the tissues are analyzed for cytokines, antigens, and immune response respectively. Utilizing western blot multiplex, fluoro metric microbead, immunoassay immunohistochemistry, and a live spot analysis allows us to test the efficacy of a peptide vaccine against bladder cancer. Hello, I'm Dr.Gregory Wetz, a research scientist at the University of California Davis Health System in Sacramento, California.
The main advantage of this technique compared to existing methods like xenografts, is that xenografts require immunocompromised hosts, whereas our model utilizes an immune intact transgenic mouse that expresses a human tumor associated antigen that is the target of the peptide vaccine. Demonstrating the techniques with me will be Daniel V and Doctors Chaga and Audrey Gutierrez, all of whom are fellow research scientists, and Dr.Michael de Gregorio's laboratory and the uc Davis Health System. Before administering the first dose of N butyl, N four hydroxy, butyl NITROSAMINE, or O-H-B-B-N to induce bladder cancer, perform a submandibular bleed on each mouse to collect whole blood and blood clotting tubes and allow 30 minutes for the blood to clot.
Beginning at eight weeks using stainless steel 20 gauge gavage needles, administer the O-H-V-B-N orally five days per week for 12 weeks. At week 20, use 600 microliters of 0.9%sterile saline to reconstitute each vial of lyophilized peptide vaccine and thoroughly resuspend by drawing the solution through a 0.5 inch 27 gauge needle six times using saline. Adjust the concentration so that the desired dose is delivered in a volume of 100 microliters after the last dose of O-H-B-B-N and administer the vaccine on a weekly basis for an eight week cycle by using a 25 gauge needle to inject 100 microliters of the vaccine eight weeks after the last dose of O-H-B-B-N.
After euthanizing all the mice by carbon dioxide asphyxiation, place each mouse on a dissection board and pin down all four limbs. Next, using forceps and scissors, make a horizontal incision in the upper abdominal region. Insert the scissors into the incision between the epidermal layer and abdominal wall, and with the help of forceps, gently separate the skin from the underlying tissue.
Make a vertical incision from the horizontal incision following the middle axis towards the anterior end of the mouse. Then separate the skin from the rib cage and using a one milliliter syringe and a 22 gauge needle, puncture the heart and collect blood with a smooth and steady draw using forceps and scissors. Cut and peel back the rest of the epidermal layer inside a biological safety cabinet.
Cut through the abdominal wall and peritoneum and aseptically. Remove the bladder tumor for immunohistochemistry or IHC and Western blot for IHC. Place the bladder tumor specimen in a tissue cassette and fix it in chilled formin overnight.
At room temperature, collect the spleen for cell viability analysis and a lie spot to carry out the multiplex fluoro metric microbe immunoassay. Use 200 microliters of assay buffer to pre-wet. A 96 well filter bottom plate, allowing the filter to completely soak.
Then use a 96 well plate vacuum apparatus to gently drain the filter and use paper towels to blood dry the bottom of the plate using a multichannel pipette pipette 25 microliters of serum matrix into the wells assigned for the blanks and standards and pipette 25 microliters of assay buffer into the wells assigned for the controls and unknowns. Next pipette 25 microliters of the blank standards, controls and unknowns to the respective assigned wells. Vortex the bead mix for 20 seconds and transfer the beads to a reservoir.
Then pipette 25 microliters of the bead mix into each. Well cover the plate to protect it from light and shake the plate on a shaker at 500 RPM for two hours at room temperature, drain the plate and use 200 microliters of PBST to wash it twice before draining and blotting it dry. To prepare the detection antibody solution, combine the required amount of 0.1%PBST and detection antibody in a 15 milliliter tube and vortex for 10 seconds.
Then pipette 25 microliters into each. Well shake the plate at 500 RPM for one hour at room temperature. Then drain and wash the plate with 200 microliters of 0.1%PBST twice drain and blot dry pipette 25 microliters of the freshly prepared strep, AVID and FICO eryn or SAPE to each, well place the plate on the plate shaker and incubate for 30 minutes at room temperature at 500 RPM.
Drain and wash with PBST twice. To suspend the beads, add 100 microliters of 0.1%PBST to each well and shake at 500 RPM for at least two minutes. Use Luminex LX 200 machine to read and analyze the plate in a biological safety cabinet.
Process the mouse spleens through 100 micrometer nylon tissue sieves into five milliliters of sterile PBS in sterile Petri dishes. Layer the PLE aytes onto three milliliters of lymphocytes separation medium in sterile 15 milliliter tubes. Centrifuge the tubes at 600 GS for 15 minutes.
To separate the lymphocytes from the red blood cells, transfer the layered lymphocytes above the gradient to new sterile 15 milliliter tubes in 1.5 milliliter screw cap centrifuge tubes. Use countin viability reagent to make serial dilution of the lymphocytes. Then analyze on the muse.
Prepare a plate map of the samples and conditions for the ally spot plate by preparing the plate according to the manufacturer's protocol and pipette 100 microliters of medium peptide or scramble peptide into each. Well add 100 microliters of cell suspension and incubate the plate at 37 degrees Celsius overnight. Follow the manufacturer's protocol for the ALI spot assay and use the dissection microscope to analyze the developed Aly spot plate.
Quantify the results by counting the number of colored spots corresponding to each analyte in each well. The spots correspond to the number of spot forming cells in each well in our transgenic mouse model induction with the chemical carcinogen. O-H-B-B-N resulted in a high rate of bladder cancer incidents of predominantly transitional cell carcinoma or TCC with some squamous cell carcinoma or SCC, which is similar to bladder cancer in humans as shown in this figure.
Eight weeks following O-H-B-B-N induction bladder cancer incidence rate for both mach one transgenic or TG and wild-type mice was 67%Hematin and eosin staining confirmed the presence of both TCCs and SCCs with TCCs predominating at a two to one ratio. Among these, we observed a range of low and high grade non-invasive to high grade invasive tumors. All Mach one bladder cancer specimens were positive for mach one expression by IHC.
During model development, the serum levels of inflammatory cytokines were monitored serially between weeks eight to 28. We observed that inflammatory cytokine levels increased over time from induction through the end of the study. This cytokine pattern is very similar to what we observed previously in our lung cancer model, which strongly suggests that increasing inflammatory cytokine levels may correlate with tumor development.
To assess the th one serum cytokine response to the peptide vaccine, 15 vaccinated and 14 placebo treated mach one TG mice were euthanized and blood was collected at the end of the study. 24 hours after the last vaccine treatment. Multiplex analysis shows increased th one serum cytokine levels of TNF alpha interferon gamma IL two IL 12, and IL 17 in the vaccine group.
Compared to the placebo group levels of TNF alpha interferon gamma and IL 17 were significantly higher in the vaccine treated mice. These results suggest a th one polarized cytokine response to the peptide vaccine in order to evaluate the TH one, TH two immune response to the peptide vaccine. SP cytes were assessed by interferon gamma IL four ALI spot shown here is an assessment of isolated lymphocytes for viability after seeding ALI spot plates with viable lymphocytes, the results in this experiment were clear and specific interferon gamma response to the peptide, which confirms the TH one immune response to the peptide vaccine After its development.
This technique paved the way for researchers in the field of cancer immunology to explore new immunotherapeutics utilizing a preclinical model of advanced bladder cancer.
