The authors would like to thank the UC Davis Mouse Biology Program for breeding the mice. This research was supported by a grant from Merck KGaA, Darmstadt, Germany.

All animal studies and experiments were conducted under a protocol approved by the University of California, Davis Institutional Animal Care and Use Administrative Advisory Committee.
1. MUC1.Tg Mouse Breeding and Propagation
<ol>
	<li>The UC Davis Mouse Biology Program (MBP) breeds wild type C57BL/6 male mice with heterozygous MUC1.Tg C57BL/6 female mice to establish our breeding colony. MUC1.Tg offspring are delivered for studies as needed.</li>
	<li>MBP personnel clip the toes of the offspring in a defined pattern (0-99) for mouse identification, and when applicable clip the tail. The toe or tail tissue is processed for genotyping using standard DNA extraction and Polymerase Chain Reaction (PCR) analysis.</li>
</ol>
2. Study Design
<ol>
	<li>Study Group Assignments
	<ol>
		<li>Weigh each mouse separately on a balance and record the weight in grams for each mouse.</li>
		<li>This part of the procedure involves the methodology and design of the study. For the first part of the study, assign age-matched MUC1.Tg and wild type mice to start bladder cancer induction with OH-BBN. Euthanize mice 8 weeks after the last OH-BBN dose to confirm the presence of bladder tumors by histology.</li>
		<li>For the second part of the study, randomize male MUC1.Tg mice into the appropriate number of treatment and control groups. These mice will be monitored for serum cytokines and T-cell immune responses during induction and tumor development, and then at termination of the study 8 weeks after the last OH-BBN dose.</li>
	</ol>
	</li>
	<li>Bladder Cancer Induction
	<ol>
		<li>OH-BBN is a carcinogen and highly toxic. Please read and follow the guidelines of the material safety data sheet when handling and storing this chemical.</li>
		<li>Calculate the dosing solution concentration needed to deliver the appropriate dose (3 mg), in a volume of 100 &#956;l, to each mouse based on the average weights and number of mice in each study and group.</li>
		<li>Dilute the OH-BBN in 100% ethanol. Bring the final concentration to 30 mg/ml with sterile water. The final ethanol:water concentration should be 20:80, v/v.</li>
		<li>Administer the OH-BBN orally using stainless steel, 20 G&#160;gavage needles daily, 5 days/week for&#160;12 weeks, based on the treatment group assignment starting at the age of 8 weeks.</li>
	</ol>
	</li>
	<li>Vaccination Treatments
	<ol>
		<li>Reconstitute each vial of lyophilized peptide vaccine in 600 &#956;l of 0.9% sterile saline and thoroughly resuspend by drawing the solution through a 0.5&#160;inch 27 G&#160;needle 6x. Using saline, adjust the concentration so that the desired dose is delivered in a volume of 100 &#956;l.</li>
		<li>Starting in Week 20, after the last dose of OH-BBN, administer the vaccine on a weekly basis for an eight-week cycle by subcutaneous injection of 100 &#956;l using a 25 G&#160;needle (week number corresponds to the age of the mice).</li>
	</ol>
	</li>
	<li>Monitoring and Sample Collection
	<ol>
		<li>Weigh all mice and palpate for the presence of new tumors once each week. Euthanize any mice that have lost &#8805;20% of body weight or if there are palpable tumors, blood in the urine, and/or urinary retention.</li>
		<li>Prior to the first OH-BBN dose and then at 4&#160;week intervals thereafter, collect whole blood via submandibular bleeds. Collect the blood in serum clotting tubes (BD Microtainer), and allow 30 min for the blood to clot.</li>
		<li>Centrifuge the blood samples in a microcentrifuge at 3,500 x g for 10 min. Carefully transfer the serum to screw cap cryotubes using a pipette.</li>
		<li>Flash freeze in liquid nitrogen, and store at -80 &#176;C until further analysis by multiplex.</li>
		<li>Eight weeks after the last dose of OH-BBN, euthanize all mice by CO2 asphyxiation.</li>
		<li>Place each mouse on a dissection board and pin down by all four limbs.</li>
		<li>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 gently separate the skin from the underlying tissue with the help of forceps.</li>
		<li>Make a vertical incision from the horizontal incision following the middle axis towards the anterior end of the mouse. Separate the skin from the rib cage, and using a 1&#160;ml syringe and a 22 G&#160;needle, puncture the heart and collect blood with a smooth and steady draw.</li>
		<li>Refer to step 2.4.3 and 2.4.4 for serum isolation and storage.</li>
		<li>Using forceps and scissors, cut and peel back the rest of the epidermal layer. Cut through the abdominal wall and peritoneum and aseptically remove bladder tumor for Immunohistochemistry (IHC) and Western blot.</li>
		<li>Collect spleen for cell viability analysis (Muse) and ELISpot. For IHC, place bladder tumor specimen in tissue cassette and fix in chilled formalin overnight at room temperature.</li>
		<li>The following day, replace the formalin with 70% ethanol.</li>
		<li>For Western blot analysis of tumor, homogenize the tumor, add protein extraction buffer plus Halt protease inhibitors and transfer to 1.5&#160;ml microcentrifuge tubes.</li>
		<li>Vortex for 30-60 sec and hold on ice for 5 min. Flash freeze in liquid nitrogen and thaw in ambient water. Repeat the process of vortexing, freezing and thawing twice.</li>
		<li>Centrifuge the samples at 10,000 x g for 10 min at 4 &#176;C and transfer the cellular extracts to new labeled tubes.</li>
		<li>Quantify the concentration by performing a Bicinchoninic Acid Protein Assay (BCA) for protein measurements. Store samples at -80 &#176;C until ready for Western blot analysis.</li>
	</ol>
	</li>
</ol>
3. Molecular Biology/Western
The following procedures were performed to verify the expression of MUC1 in mouse bladder tumor tissue using standard Western Blot protocol (data not shown).
<ol>
	<li>Separate the protein extracts by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and then transfer onto PVDF membrane using semidry apparatus.</li>
	<li>Block the protein transferred membrane with 5% nonfat milk in 0.1% Tween-20 in Phosphate Buffered Saline (PBS-T) pH 7.4 for 1 hr on an orbital shaker at room temperature.</li>
	<li>Incubate the membrane for 1 hr at room temperature on a shaker with anti-MUC1 or anti-&#946;-actin antibody in 0.1% PBS-T.</li>
	<li>Wash the membrane by decanting the solution and adding 0.1% PBS-T. Swirl the membrane on the shaker for 5 min and decant. Repeat this step twice.</li>
	<li>Incubate the membrane for 1 hr at room temperature with a horse radish peroxidase (HRP) conjugated secondary antibody.</li>
	<li>Wash 3x (refer to step 3.4).</li>
	<li>Follow the protocol for the Enhanced Chemiluminesence (ECL) kit to activate and read the fluorography.</li>
</ol>
4. Multiplex Fluorometric Microbead Immunoassay
<ol>
	<li>Setup and Calculations
	<ol>
		<li>Using a 96-well plate map, assign blanks, standards, controls, and unknowns to the wells. Calculate the number and volume of analytes, capture antibodies and Streptavidin-Phycoerythrin (SA-PE) needed for the assay.</li>
		<li>Allow all buffers and diluents to equilibrate to room temperature. Prepare the standards and sample dilutions using a blank 96-well plate.</li>
		<li>Reconstitute the serum matrix and lyophilized standard according to the manufacturer&#39;s protocol and make 1:5 serial dilutions of the standard with the appropriate diluent in the 96-well plate. For the blank wells, use the appropriate diluent.</li>
		<li>Dilute all controls and unknown samples 1:2 in the appropriate diluent in the rest of the 96-well plate.</li>
		<li>For the bead mix, pipette the required volume of the appropriate diluent into a 15&#160;ml tube. Vortex each bead vial for 20 sec and pipette the required volume of each analyte into the 15&#160;ml tube.</li>
		<li>Always protect the beads from light to avoid photobleaching. Do not place the 96-well plate on absorbent material to avoid loss of sample through wicking.</li>
	</ol>
	</li>
	<li>Assay Protocol
	<ol>
		<li>Prewet the 96-well filter bottom plate with 200 &#956;l of Assay Buffer and allow for complete soaking of the filter. Gently drain by using the 96-well plate vacuum apparatus and blot dry the bottom of the plate with a paper towel.</li>
		<li>Using a multichannel pipette, pipette 25 &#956;l of serum matrix to the wells assigned for the blanks and standards and pipette 25 &#956;l of Assay Buffer to the wells assigned for the controls and unknowns.</li>
		<li>Pipette 25 &#956;l of the blank, standards, controls, and unknowns to the respective assigned wells. Vortex the bead mix for 20 sec and transfer the beads to a reservoir.</li>
		<li>Pipette 25 &#956;l of the bead mix into each well. Cover the plate with aluminum foil or an opaque plate lid to protect from light.</li>
		<li>Shake the plate at 500 rpm for two hours at room temperature on a plate shaker. Drain and wash the plate with 200 &#956;l of 0.1% Tween 20 in Phosphate Buffered Saline (PBS-T) twice. Drain and blot dry.</li>
		<li>Prepare the capture antibody solution. Pipette the required amount of 0.1% PBS-T and capture antibody into a 15&#160;ml tube and vortex for 10 sec. Transfer the capture antibody mix to a reservoir and pipette 25 &#956;l into each well.</li>
		<li>Shake the plate at 500 rpm for one hour at room temperature on a plate shaker. Drain and wash the plate with 200 &#956;l of 0.1% PBS-T twice. Drain and blot dry.</li>
		<li>Prepare the SA-PE solution. Pipette the required volume of 0.1% PBS-T and SA-PE into a 15&#160;ml tube and vortex for 10 sec. Transfer the solution to a reservoir and pipette 25 &#956;l into each well.</li>
		<li>Shake the plate at 500 rpm for 30 min at room temperature on a plate shaker. Drain and wash the plate with 200 &#956;l of 0.1% PBS-T twice. Drain and blot dry.</li>
		<li>Pipette 100 &#956;l of 0.1% PBS-T and shake on a plate shaker at 500 rpm for at least two minutes to resuspend the beads. Read and analyze the plate on the Luminex Lx200 machine.</li>
	</ol>
	</li>
</ol>
5. IFN-&#947;/IL-4 ELISpot Preparation and Analysis
<ol>
	<li>In a biological safety cabinet, process the spleens through 100&#160;&#956;m Nylon tissue sieves into 5 ml sterile Phosphate Buffered Saline (PBS) in sterile Petri dishes. Layer the splenocytes onto 3 ml of lymphocyte separation medium in sterile 15-ml tubes.</li>
	<li>Centrifuge the tubes at 600 x g for 15 min to separate the lymphocytes from the red blood cells. Transfer the layered lymphocytes above the gradient to new sterile 15-ml tubes.</li>
	<li>Adjust the volume to 10 ml with sterile PBS. Centrifuge the suspension at 600 x g for 10 min to pellet the cells.</li>
	<li>Aspirate the supernatant and resuspend the cells in 1 ml of PBS for cell viability and count with the Muse analyzer. Follow the Muse Count &#38; Viability Kit protocol.</li>
	<li>Make serial dilutions of the lymphocytes in 1.5&#160;ml screw cap centrifuge tubes at dilution factors of 1:10, 1:20, and 1:40 (or as necessary) in Count &#38; Viability Reagent (minimum total volume 300 &#956;l). Pipette each dilution up and down several times to mix and analyze on the Muse.</li>
	<li>Prepare a plate map of the samples and conditions for the ELISpot plate. Prepare the ELISpot plate according to the manufacturer&#39;s protocol and pipette 100 &#956;l of medium, peptide (10 &#956;g/ml), or scramble peptide (10 &#956;g/ml) to each well.</li>
	<li>Pipette 100 &#956;l of cell suspension, delivering 1.0 x 106 cells to each well and incubate the plate at 37 &#176;C overnight.</li>
	<li>Follow the manufacturer&#39;s protocol for the ELISpot assay. Analyze the developed ELISpot plate using a dissection microscope.</li>
	<li>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 (SFC) in each well.</li>
</ol>
6. Immunohistochemistry (IHC) and Hematoxylin &#38; Eosin (H&#38;E) Staining
<ol>
	<li>Take bladder tumor tissue preserved as described above (Section 2.4.11), embed in paraffin and step-section at 4 &#956;m for immunohistochemical analysis.</li>
	<li>Perform IHC using a MUC1 antibody that recognizes the tandem repeat region of MUC1. Use the Animal Research Kit peroxidase to minimize the reactivity of the secondary mouse antibody with endogenous immunoglobulin present in the tissue.</li>
	<li>Perform H&#38;E staining using standard protocols.</li>
</ol>
7. Statistical Methods
For the Multiplex Fluorometric Microbead Immunoassay, use a two-tailed Student&#39;s t-test to compare the average observed serum cytokine concentrations between the treatment and control groups. For ELISpot, use a one-way ANOVA to compare the spot forming colonies between the media control, scrambled peptide and peptide groups. Use Dunnett&#39;s Multiple Comparison Test to lessen the likelihood of a false positive result. A p-value of &#8804;0.05 is considered significantly different for all analyses.

<ol>
	<li>Siegel, R., Naishadham, 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=.">.</a> CA Cancer J. Clin. 63, 11-30 (2013).</li><li>Lynch, C. F., Davila, J. A., Ries, L. A. G., Young, J. 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=Chapter+23.+Cancer+of+the+urinary+bladder.">Chapter 23. Cancer of the urinary bladder.</a> SEER Survival Monograph: Cancer Survival Among Adults: U.S. SEER Program, 1988-2001, Patient and Tumor Characteristics. , 07-6215 (2007).</li><li>Jacobs, B. L., Lee, C. T., 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=Bladder+cancer+in+2010:+how+far+have+we+come.">Bladder cancer in 2010: how far have we come.</a> CA Cancer J. Clin. 60 (4), 244-272 (2010).</li><li>Sexton, W. J., Wiegand, L. 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=Bladder+cancer:+a+review+of+non-muscle+invasive+disease.">Bladder cancer: a review of non-muscle invasive disease.</a> Cancer Control. 17 (4), 256-268 (2010).</li><li>Bohle, A., Jocham, 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=Intravesical+bacillus+Calmette-Guerin+versus+mitomycin+C+for+superficial+bladder+cancer:+a+formal+meta-analysis+of+comparative+studies+on+recurrence+and+toxicity.">Intravesical bacillus Calmette-Guerin versus mitomycin C for superficial bladder cancer: a formal meta-analysis of comparative studies on recurrence and toxicity.</a> J. Urol. 169 (1), 90-95 (2003).</li><li>Shokeir, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Squamous+cell+carcinoma+of+the+bladder:+pathology,+diagnosis+and+treatment.">Squamous cell carcinoma of the bladder: pathology, diagnosis and treatment.</a> BJU Int. 93 (2), 216-220 (2004).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+human+tumour+immunology+and+immunotherapy.">Progress in human tumour immunology and immunotherapy.</a> Nature. 411 (6835), 380-384 (2001).</li><li>Joudi, F. N., Smith, B. 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=Final+results+from+a+national+multicenter+phase+II+trial+of+combination+bacillus+Calmette-Guerin+plus+interferon+alpha-2B+for+reducing+recurrence+of+superficial+bladder+cancer.">Final results from a national multicenter phase II trial of combination bacillus Calmette-Guerin plus interferon alpha-2B for reducing recurrence of superficial bladder cancer.</a> Urol. Oncol. 24 (4), 344-348 (2006).</li><li>Lau, S. K., Weiss, L. 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=Differential+expression+of+MUC1,+MUC2,+and+MUC5AC+in+carcinomas+of+various+sites:+an+immunohistochemical+study.">Differential expression of MUC1, MUC2, and MUC5AC in carcinomas of various sites: an immunohistochemical study.</a> Am. J. Clin. Pathol. 122 (1), 61-69 (2004).</li><li>Hollingsworth, M. A., Swanson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+cancer:+protection+and+control+of+the+cell+surface.">Mucins in cancer: protection and control of the cell surface.</a> Nat. Rev. Cancer. 4 (1), 45-60 (2004).</li><li>Scholfield, D. P., Simms, M. 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=MUC1+mucin+in+urological+malignancy.">MUC1 mucin in urological malignancy.</a> BJU Int. 91 (6), 560-566 (2003).</li><li>Devine, P. L., McKenzie, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins:+structure,+function,+and+association+with+malignancy.">Mucins: structure, function, and association with malignancy.</a> Bioessays. 14 (9), 619-625 (1992).</li><li>Jerome, K. R., Barnd, 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=Cytotoxic+T-lymphocytes+derived+from+patients+with+breast+adenocarcinomas+recognize+an+epitope+present+on+the+protein+core+of+a+mucin+molecule+preferentially+expressed+by+malignant+cells.">Cytotoxic T-lymphocytes derived from patients with breast adenocarcinomas recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells.</a> Cancer Res. 51 (11), 2908-2916 (1991).</li><li>Takahashi, T., Makiguchi, 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=Expression+of+MUC1+on+myeloma+cells+and+induction+of+HLA-unrestricted+CTL+against+MUC1+from+a+multiple+myeloma+patient.">Expression of MUC1 on myeloma cells and induction of HLA-unrestricted CTL against MUC1 from a multiple myeloma patient.</a> J. Immunol. 153 (5), 2102-2109 (1994).</li><li>Choi, C., Witzens, 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=Enrichment+of+functional+CD8+memory+T+cells+specific+for+MUC1+in+bone+marrow+of+patients+with+multiple+myeloma.">Enrichment of functional CD8 memory T cells specific for MUC1 in bone marrow of patients with multiple myeloma.</a> Blood. 105 (5), 2132-2134 (2005).</li><li>Barnd, D. L., Lan, M. 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=Specific,+major+histocompatibility+complex-+unrestricted+recognition+of+tumor-associated+mucins+by+human+cytotoxic+T-cells.">Specific, major histocompatibility complex- unrestricted recognition of tumor-associated mucins by human cytotoxic T-cells.</a> Proc. Natl. Acad. Sci. U.S.A. 86 (18), 7159-7163 (1989).</li><li>Ioannides, C. G., Fisk, B., 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=Cytotoxic+T-cells+from+ovarian+malignant+tumors+can+recognize+polymorphic+epithelial+mucin+core+peptides.">Cytotoxic T-cells from ovarian malignant tumors can recognize polymorphic epithelial mucin core peptides.</a> J. Immunol. 151 (7), 3693-3703 (1993).</li><li>Tang, C. K., Katsara, 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=Strategies+used+for+MUC1+immunotherapy:+human+clinical+studies.">Strategies used for MUC1 immunotherapy: human clinical studies.</a> Expert Rev. Vaccines. 7 (7), 963-975 (2008).</li><li>Mukherjee, P., Ginardi, A. 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=MUC1-specific+cytotoxic+T+lymphocytes+eradicate+tumors+when+adoptively+transferred+in+vivo.">MUC1-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo.</a> Clin. Cancer Res. 7, 848-855 (2001).</li><li>Acres, B., Limacher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC1+as+a+target+antigen+for+cancer+immunotherapy.">MUC1 as a target antigen for cancer immunotherapy.</a> Expert Rev. Vaccines. 4 (4), 493-502 (2005).</li><li>Butts, C., Murray, N., 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=Randomized+phase+IIB+trial+of+BLP25+liposome+vaccine+in+stage+IIIB+and+IV+non-small-cell+lung+cancer.">Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer.</a> J. Clin. Oncol. 23 (27), 6674-6681 (2005).</li><li>Butts, C., Maksymiuk, 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=Updated+survival+analysis+in+patients+with+stage+IIIB+or+IV+non-small-cell+lung+cancer+receiving+BLP25+liposome+vaccine+(L-BLP25),+phase+IIB+randomized,+multicenter,+open-label+trial.">Updated survival analysis in patients with stage IIIB or IV non-small-cell lung cancer receiving BLP25 liposome vaccine (L-BLP25), phase IIB randomized, multicenter, open-label trial.</a> J. Cancer. Res. Clin. Oncol. 137 (9), 1337-1342 (2011).</li><li>Agrawal, B., Krantz, M. 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=Rapid+induction+of+primary+human+CD4++and+CD8++T+cell+responses+against+cancer-associated+MUC1+peptide+epitopes.">Rapid induction of primary human CD4+ and CD8+ T cell responses against cancer-associated MUC1 peptide epitopes.</a> Int. Immunol. 10 (12), 1907-1916 (1998).</li><li>Palmer, M., Parker, 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=Phase+I+study+of+the+BLP25+(MUC1+peptide)+liposomal+vaccine+for+active+specific+immunotherapy+in+stage+IIIB/IV+non-small-cell+lung+cancer.">Phase I study of the BLP25 (MUC1 peptide) liposomal vaccine for active specific immunotherapy in stage IIIB/IV non-small-cell lung cancer.</a> Clin. Lung Cancer. 3 (1), 49-57 (2001).</li><li>Walsh, M. D., Hohn, B. G., 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=Mucin+expression+by+transitional+cell+carcinomas+of+the+bladder.">Mucin expression by transitional cell carcinomas of the bladder.</a> Br. J. Urol. 73 (3), 256-262 (1994).</li><li>Murray, A., Simms, 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=Production+and+characterization+of+rhenium-188-C595+antibody+for+radioimmunotherapy+of+transitional+cell+bladder+cancer.">Production and characterization of rhenium-188-C595 antibody for radioimmunotherapy of transitional cell bladder cancer.</a> J. Nucl. Med. 42 (5), 726-732 (2001).</li><li>Hughes, O., Bishop, 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=Targeting+superficial+bladder+cancer+by+the+intravesical+administration+of+67copper-labelled+anti-MUC1+mucin+monoclonal+antibody+C595.">Targeting superficial bladder cancer by the intravesical administration of 67copper-labelled anti-MUC1 mucin monoclonal antibody C595.</a> J. Clin. Oncol. 18 (2), 363-370 (2000).</li><li>Conn, I. G., Crocker, 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=HMFG-2+as+a+prognostic+indicator+in+superficial+bladder+cancer.">HMFG-2 as a prognostic indicator in superficial bladder cancer.</a> J. Clin. Pathol. 41 (11), 1191-1195 (1988).</li><li>Hughes, O., Denley, 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=MUC1+mucin+expression+in+transitional+cell+carcinoma+of+the+bladder:+a+target+for+diagnosis+and+therapy+with+monoclonal+antibody+C595.">MUC1 mucin expression in transitional cell carcinoma of the bladder: a target for diagnosis and therapy with monoclonal antibody C595.</a> J. Urol. Pathol. 12, 185-197 (2000).</li><li>Rowse, G. J., Tempero, R. 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=Tolerance+and+immunity+to+MUC1+in+a+human+MUC1+transgenic+murine+model.">Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model.</a> Cancer Res. 58 (2), 315-321 (1998).</li><li>Mukherjee, P., Madsen, C. 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=Mucin-1+specific+immunotherapy+in+a+mouse+model+of+spontaneous+breast+cancer.">Mucin-1 specific immunotherapy in a mouse model of spontaneous breast cancer.</a> J. Immunother. 26 (1), 47-62 (2003).</li><li>McCormick, D. L., Ronan, 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=Influence+of+total+dose+and+dose+schedule+on+induction+of+urinary+bladder+cancer+in+the+mouse+by+N-butyl-N-(4-hydroxy-butyl)nitrosamine.">Influence of total dose and dose schedule on induction of urinary bladder cancer in the mouse by N-butyl-N-(4-hydroxy-butyl)nitrosamine.</a> Carcinogenesis. 2 (3), 251-254 (1981).</li><li>Wurz, G. T., Gutierrez, A. 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=Antitumor+effects+of+L-BLP25+antigen-specific+tumor+immunotherapy+in+a+novel+human+MUC1+transgenic+lung+cancer+mouse+model.">Antitumor effects of L-BLP25 antigen-specific tumor immunotherapy in a novel human MUC1 transgenic lung cancer mouse model.</a> J. Transl. Med. 11, 10-1186 (2013).</li><li>Kunze, E., Schauer, 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=Stages+of+transformation+in+the+development+of+N-butyl-N-(4hydroxybutyl)-nitrosamine-induced+transitional+cell+carcinomas+in+the+urinary+bladder+of+rats.">Stages of transformation in the development of N-butyl-N-(4hydroxybutyl)-nitrosamine-induced transitional cell carcinomas in the urinary bladder of rats.</a> Z Krebsforsch Klin. Onkol. Cancer Res. Clin. Oncol. 87 (2), 139-160 (1976).</li><li>Crallan, R. A., Georgopoulos, N. T., 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=Experimental+models+of+human+bladder+carcinogenesis.">Experimental models of human bladder carcinogenesis.</a> Carcinogenesis. 27 (3), 374-381 (2006).</li><li>Samma, S., Uemura, 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=Rapid+induction+of+carcinoma+in+situ+in+dog+urinary+bladder+by+sequential+treatment+with+N-methyl-N'-nitrosourea+and+N-butyl-N-(4-hydroxybutyl)-nitrosamine.">Rapid induction of carcinoma in situ in dog urinary bladder by sequential treatment with N-methyl-N'-nitrosourea and N-butyl-N-(4-hydroxybutyl)-nitrosamine.</a> Gann. 75 (5), 385-387 (1984).</li><li>Bornhof, C., Wolfrath, G., 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=Induction+of+urinary+bladder+urothelial+cancers+in+the+rabbit+by+dibutylnitrosamine+with+an+artificial+bladder+calculus+as+cocarcinogen.">Induction of urinary bladder urothelial cancers in the rabbit by dibutylnitrosamine with an artificial bladder calculus as cocarcinogen.</a> Urologe A. 28 (6), 339-343 (1989).</li><li>Irving, C. C., Tice, A. 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=Inhibition+of+N-n-butyl-N-(4-hydroxybutyl)nitrosamine-induced+urinary+bladder+cancer+in+rats+by+administration+of+disulfiram+in+the+diet.">Inhibition of N-n-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder cancer in rats by administration of disulfiram in the diet.</a> Cancer Res. 39 (8), 3040-3043 (1979).</li><li>Mehta, N. R., Wurz, G. T., 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=L-BLP25+vaccine+plus+letrozole+induces+a+TH1+immune+response+and+has+additive+antitumor+activity+in+MUC1-expresssing+mammary+tumors+in+mice.">L-BLP25 vaccine plus letrozole induces a TH1 immune response and has additive antitumor activity in MUC1-expresssing mammary tumors in mice.</a> Clin. Cancer Res. 18 (10), 2861-2871 (2012).</li></ol>

DPV, GTW, SMG, CJK, AMG, and GKH declare no competing interests. MWD is the Principal Investigator of a grant received from Merck KGaA, and MW is an employee of Merck KGaA.

The successful induction of invasive transitional and squamous cell bladder carcinoma in human MUC1.Tg mice offers a preclinical model for Immunotherapy development. Immunotherapeutic studies require the use of a spontaneous, immune intact model in order to evaluate the inflammatory response to tumor progression over time as well as the immune response to immunotherapy. In a spontaneous tumor development model, the tumor microenvironment remains intact and the tumors develop at a more representative growth rate that allo...

Bladder cancer is the fourth most common form of cancer and the eighth leading cause of cancer deaths in American men. In the United States, an estimated 72,500 new cases and 15,000 deaths from bladder cancer are expected among men and women combined in 20131. The incidence of bladder cancer is approximately three times as high in men compared to women. In the United States, transitional cell carcinomas (TCC) account for over 90% of cases, while squamous cell carcinomas (SCC) have an incidence of less than 2%2. The overall relative 5-year survival rate for papillary TCC is 91.5% compared to only 30.9% for SCC2. Although noninvasive papillary TCCs account for approximately 75% of cases at the time of diagnosis, even with treatment more than 50% of patients will experience a recurrence within 5 years, with up to 30% of these patients progressing to muscle invasive disease3,4. Typical treatment regimens for non-muscle invasive disease include transurethral resection (TUR) followed by intravesical chemotherapy. In patients with high-grade Ta or T1 tumors, a repeat TUR may be performed prior to chemotherapy3,4. For those patients with low-grade Ta recurrences or high-grade Ta or T1 lesions, TUR followed by adjuvant chemotherapy or immunotherapy in the form of Bacillus Calmette-Guerin (BCG) may be used3,4. Intravesical BCG has been shown to be superior to intravesical mitomycin C with respect to time to recurrence5. For T2 muscle invasive disease, radical cystectomy with or without neoadjuvant chemotherapy is the recommended course of treatment3. In patients with SCC, radical cystectomy appears to be the most effective treatment6. Given the very high rates of recurrence despite the best treatments available, there is clearly a need for new, more effective therapies for bladder cancer.
Expanding new immunotherapies for bladder cancer is one possible approach that may hold promise for extending disease-free survival. Historically, BCG has been the only effective immunotherapy for bladder cancer. Its mechanism of action is thought to involve the nonspecific induction of a T-helper 1 (Th1) type immune response via increasing levels of interleukin-2 (IL-2) and interferon gamma (IFN-&#947;)4. Cellular, or Th1 immunity, is critical in cancer immunotherapy as humoral, or Th2, immunity has never been shown to be effective against solid tumors, with the exception of antibodies directed against growth factor receptors7. In an attempt to improve upon the benefits of BCG monotherapy, IFN-&#945; 2B/BCG combination immunotherapy was evaluated in a phase II clinical trial with inconclusive results8. An alternative approach to immunotherapy for bladder cancer may be to target tumor-associated antigens (TAAs), the identification of which has made cancer immunotherapy more specific7.
One such TAA is mucin 1 (MUC1), which is a cell surface glycoprotein overexpressed in many epithelial cell cancers such as bladder, breast, lung, and pancreatic cancer9,10. The expression and modification of MUC1 is also substantially altered during carcinogenesis, such that underglycosylation exposes antigenic sequences of amino acids known as variable number of tandem repeats (VNTR) on the peptide core. While MUC1 is a self-molecule, these immunodominant VNTR regions are not normally exposed due to extensive glycosylation, and thus they are seen by the immune system as foreign11,12. Cytotoxic T-lymphocytes (CTLs) that specifically recognize MUC1 epitopes have been isolated from the tumor-draining lymph nodes of breast cancer patients13, as well as the blood and bone marrow of myeloma patients14,15, making MUC1 a potential target for a cellular immune response. The immunodominant VNTRs of the underglycosylated form of MUC1 are recognized by CTLs, resulting in the destruction of tumor cells16-19. Native cellular and/or humoral immune responses to cancerous MUC1 are, however, not strong enough to eliminate tumors. To augment the already existing weak immune response to MUC1, synthetic immunodominant peptides can be introduced through vaccination to generate a CTL response strong enough to be of clinical benefit18,20. A MUC1 liposomal vaccine has already been shown to increase survival in lung cancer patients21,22, generate CTLs capable of killing MUC1-positive tumor cells, and produce a Th1-polarized cytokine response23,24. With a high level of MUC1 expression9,11,25, bladder cancer is a logical candidate for testing MUC1-directed immunotherapy26,27. Furthermore, MUC1 has potential as a prognostic factor in bladder cancer28, MUC1 expression in TCC is significantly associated with stage and grade, and metastatic TCC has been shown to continue to express MUC129.
In order to evaluate the potential utility of MUC1-directed immunotherapy in bladder cancer, we developed an immune intact human MUC1 (hMUC1)-expressing transgenic (MUC1.Tg) mouse model of bladder cancer congenic on the C57BL/6 background30. Human MUC1 is expressed as a self-protein under the control of its own promoter, resulting in a tissue expression pattern consistent with that observed in humans30,31. The mice were induced with the known bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)32, and then the resulting tumors were evaluated for hMUC1 expression and tumor type and grade. To assess the effect of the carcinogen on Th1/Th2 cytokine levels during tumor development, serum samples were collected periodically for multiplex analysis. Mice were then treated with a MUC1-targeted peptide vaccine, and the serum cytokine and immune responses were evaluated by multiplex fluorometric microbead immunoassay and ELISpot.

<table><tbody><tr><td>&lt;strong&gt;Reagent&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)</td><td>TCI America</td><td>B0938</td><td></td></tr><tr><td>20 G Gavage Needles</td><td>Popper Sons, Inc.</td><td>7921</td><td>Stainless steel</td></tr><tr><td>Peptide Vaccine</td><td>N/A</td><td>N/A</td><td>investigational agent</td></tr><tr><td>BD Microtainers</td><td>BD</td><td>365957</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Simport</td><td>M490-12</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr><tr><td>Lysis Buffer</td><td>Pierce</td><td>87787</td><td></td></tr><tr><td>Halt Protease &amp;amp; Phosphatase inhibitor cocktail</td><td>Thermo Scientific</td><td>78444</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>Mouse Cytokine 20plex Kit</td><td>Invitrogen</td><td>LMC006</td><td></td></tr><tr><td>Magnetic Microsphere Beads</td><td>Luminex</td><td>MC100xx-01</td><td>xx is the bead region</td></tr><tr><td>Anti-mouse TNF- Capture Antibody</td><td>BD Pharmingen</td><td>551225</td><td></td></tr><tr><td>Anti-mouse TNF- Detection Antibody</td><td>BD Pharmingen</td><td>554415</td><td></td></tr><tr><td>Anti-mouse IFN- Capture Antibody</td><td>Abcam</td><td>ab10742</td><td></td></tr><tr><td>Anti-mouse IFN- Detection Antibody</td><td>Abcam</td><td>ab83136</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Sigma</td><td>P3813-10PAK</td><td></td></tr><tr><td>Tween-20</td><td>Fisher</td><td>BP337-500</td><td></td></tr><tr><td>Assay Buffer</td><td>Millipore</td><td>L-MAB</td><td></td></tr><tr><td>Cytokine Standard</td><td>Millipore</td><td>MXM8070</td><td></td></tr><tr><td>Multi-screen HTS 96well filter plates</td><td>Millipore</td><td>MSBVN1210</td><td></td></tr><tr><td>SA-PE</td><td>Invitrogen</td><td>SA10044</td><td></td></tr><tr><td>100 m Nylon Tissue Sieves</td><td>BD</td><td>352360</td><td></td></tr><tr><td>Splenocyte Separation Media</td><td>Lonza</td><td>17-829E</td><td></td></tr><tr><td>TNF- /IL-4 ELISpot plates</td><td>R&amp;amp;D Systems</td><td>ELD5217</td><td></td></tr><tr><td>Rabbit Anti-MUC1 monoclonal antibody</td><td>Epitomics</td><td>2900-1</td><td></td></tr><tr><td>Goat Anti-actin monoclonal antibody</td><td>Sigma</td><td>A1978</td><td></td></tr><tr><td>Anti-rabbit HRP antibody</td><td>Promega</td><td>W401B</td><td></td></tr><tr><td>Goat anti-mouse HRP antibody</td><td>Santa Cruz Biotechnology, Inc.</td><td>SC-2005</td><td></td></tr><tr><td>PVDF membrane</td><td>BioRad</td><td>162-0174</td><td></td></tr><tr><td>Mini Protean TGX Precast Gels</td><td>BioRad</td><td>456-1083</td><td></td></tr><tr><td>Muse Count &amp;amp; Viability Kit</td><td>Millipore</td><td>MCH100104</td><td></td></tr><tr><td>MUC1 Antibody</td><td>BD Pharmingen</td><td>550486</td><td>IHC antibody</td></tr><tr><td>Animal Research Peroxidase Kit</td><td>Dako</td><td>K3954</td><td>IHC staining</td></tr><tr><td>&lt;strong&gt;Equipment and Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Millipore plate vaccum apparatus</td><td>Millipore</td><td>MSVMHTS00</td><td></td></tr><tr><td>Luminex Lx200</td><td>Millipore / Luminex</td><td>40-013</td><td>Manufactured by Luminex, distributed by Millipore</td></tr><tr><td>Luminex Xponent Software</td><td>Millipore / Luminex</td><td>N/A</td><td>Version 3.1; included with Luminex Lx200</td></tr><tr><td>Milliple Analyst Software</td><td>Milliplex / VigeneTech</td><td>40-086</td><td>Version 5.1</td></tr><tr><td>Muse Cell Analyzer</td><td>Millipore</td><td>0500-3115</td><td></td></tr><tr><td>Muse Software</td><td>Millipore</td><td>N/A</td><td>Version 1.1.0.0; included with Analyzer</td></tr><tr><td>Dissecting Microscope</td><td>Unitron</td><td>Z730</td><td></td></tr><tr><td>Graphpad Prism Software</td><td>Graphpad Software Inc.</td><td>N/A</td><td>Version 5.1</td></tr><tr><td>Mini Protean Tetra Cell Gel apparatus</td><td>BioRad</td><td>165-8001</td><td></td></tr><tr><td>Trans Blot SD Cell and PowerPac</td><td>BioRad</td><td>170-3849</td><td></td></tr></tbody></table>

induction of invasive transitional cell bladder carcinoma in immune intact human muc1 transgenic mice:  a model for immunotherapy development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

The preclinical assessment of the effects of novel immunotherapies and combinations in bladder cancer requires the development of an appropriate animal model. In our transgenic mouse model, induction with the chemical carcinogen OH-BBN resulted in a high rate of bladder cancer incidence of predominantly TCC with some SCC, which is similar to bladder cancer in humans. To determine tumor histology, MUC1 expression status and the immune response to the peptide vaccine treatment, 21 MUC1.Tg and 18 wild type mice were euthani...

Watch this Scientific Journal Video about Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development at JoVE.com

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

induction-invasive-transitional-cell-bladder-carcinoma-immune-intact

Research

JoVE Journal

Medicine

21.2K Views.  University of California, Davis. An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

Video: Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Cancer Research

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Patient-derived Tumor Xenografting: A Technique to Generate Experimental Mouse Model for Evaluating Urothelial Cell Carcinoma

Source: Moret, R. et al. <a href="https://www.jove.com/video/59223" target="_blank">Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019)
This video describes the procedure to generate an experimental xenograft murine model, which can be used for studying urothelial cell carcinoma progression.

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development

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