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.

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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 border="1">
	<tbody>
		<tr>
			<td>Reagent&#160;</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 &#38; 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 &#38; 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&#38;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 &#38; 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></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Equipment and Software</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

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Medicine

21.1K 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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...