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

Summary

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.

Abstract

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-γ)/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.

Introduction

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 disease^3,4. 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 chemotherapy^3,4. 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 used^3,4. 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-γ)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-α 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 cancer^9,10. 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 foreign^11,12. 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 patients^14,15, 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 cells^16-19. 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 benefit^18,20. A MUC1 liposomal vaccine has already been shown to increase survival in lung cancer patients^21,22, generate CTLs capable of killing MUC1-positive tumor cells, and produce a Th1-polarized cytokine response^23,24. With a high level of MUC1 expression^9,11,25, bladder cancer is a logical candidate for testing MUC1-directed immunotherapy^26,27. 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 humans^30,31. 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.

Protocol

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

1. 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.
2. 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.

2. Study Design

1. Study Group Assignments
   1. Weigh each mouse separately on a balance and record the weight in grams for each mouse.
   2. 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.
   3. 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.
2. Bladder Cancer Induction
   1. 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.
   2. Calculate the dosing solution concentration needed to deliver the appropriate dose (3 mg), in a volume of 100 μl, to each mouse based on the average weights and number of mice in each study and group.
   3. 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.
   4. Administer the OH-BBN orally using stainless steel, 20 G gavage needles daily, 5 days/week for 12 weeks, based on the treatment group assignment starting at the age of 8 weeks.
3. Vaccination Treatments
   1. Reconstitute each vial of lyophilized peptide vaccine in 600 μl of 0.9% sterile saline and thoroughly resuspend by drawing the solution through a 0.5 inch 27 G needle 6x. Using saline, adjust the concentration so that the desired dose is delivered in a volume of 100 μl.
   2. 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 μl using a 25 G needle (week number corresponds to the age of the mice).
4. Monitoring and Sample Collection
   1. Weigh all mice and palpate for the presence of new tumors once each week. Euthanize any mice that have lost ≥20% of body weight or if there are palpable tumors, blood in the urine, and/or urinary retention.
   2. Prior to the first OH-BBN dose and then at 4 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.
   3. 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.
   4. Flash freeze in liquid nitrogen, and store at -80 °C until further analysis by multiplex.
   5. Eight weeks after the last dose of OH-BBN, euthanize all mice by CO₂ asphyxiation.
   6. Place each mouse on a dissection board and pin down by all four limbs.
   7. 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.
   8. 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 ml syringe and a 22 G needle, puncture the heart and collect blood with a smooth and steady draw.
   9. Refer to step 2.4.3 and 2.4.4 for serum isolation and storage.
   10. 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.
   11. 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.
   12. The following day, replace the formalin with 70% ethanol.
   13. For Western blot analysis of tumor, homogenize the tumor, add protein extraction buffer plus Halt protease inhibitors and transfer to 1.5 ml microcentrifuge tubes.
   14. 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.
   15. Centrifuge the samples at 10,000 x g for 10 min at 4 °C and transfer the cellular extracts to new labeled tubes.
   16. Quantify the concentration by performing a Bicinchoninic Acid Protein Assay (BCA) for protein measurements. Store samples at -80 °C until ready for Western blot analysis.

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).

1. Separate the protein extracts by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and then transfer onto PVDF membrane using semidry apparatus.
2. 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.
3. Incubate the membrane for 1 hr at room temperature on a shaker with anti-MUC1 or anti-β-actin antibody in 0.1% PBS-T.
4. 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.
5. Incubate the membrane for 1 hr at room temperature with a horse radish peroxidase (HRP) conjugated secondary antibody.
6. Wash 3x (refer to step 3.4).
7. Follow the protocol for the Enhanced Chemiluminesence (ECL) kit to activate and read the fluorography.

4. Multiplex Fluorometric Microbead Immunoassay

1. Setup and Calculations
   1. 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.
   2. Allow all buffers and diluents to equilibrate to room temperature. Prepare the standards and sample dilutions using a blank 96-well plate.
   3. Reconstitute the serum matrix and lyophilized standard according to the manufacturer'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.
   4. Dilute all controls and unknown samples 1:2 in the appropriate diluent in the rest of the 96-well plate.
   5. For the bead mix, pipette the required volume of the appropriate diluent into a 15 ml tube. Vortex each bead vial for 20 sec and pipette the required volume of each analyte into the 15 ml tube.
   6. 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.
2. Assay Protocol
   1. Prewet the 96-well filter bottom plate with 200 μl 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.
   2. Using a multichannel pipette, pipette 25 μl of serum matrix to the wells assigned for the blanks and standards and pipette 25 μl of Assay Buffer to the wells assigned for the controls and unknowns.
   3. Pipette 25 μl 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.
   4. Pipette 25 μl of the bead mix into each well. Cover the plate with aluminum foil or an opaque plate lid to protect from light.
   5. Shake the plate at 500 rpm for two hours at room temperature on a plate shaker. Drain and wash the plate with 200 μl of 0.1% Tween 20 in Phosphate Buffered Saline (PBS-T) twice. Drain and blot dry.
   6. Prepare the capture antibody solution. Pipette the required amount of 0.1% PBS-T and capture antibody into a 15 ml tube and vortex for 10 sec. Transfer the capture antibody mix to a reservoir and pipette 25 μl into each well.
   7. Shake the plate at 500 rpm for one hour at room temperature on a plate shaker. Drain and wash the plate with 200 μl of 0.1% PBS-T twice. Drain and blot dry.
   8. Prepare the SA-PE solution. Pipette the required volume of 0.1% PBS-T and SA-PE into a 15 ml tube and vortex for 10 sec. Transfer the solution to a reservoir and pipette 25 μl into each well.
   9. Shake the plate at 500 rpm for 30 min at room temperature on a plate shaker. Drain and wash the plate with 200 μl of 0.1% PBS-T twice. Drain and blot dry.
   10. Pipette 100 μl 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.

5. IFN-γ/IL-4 ELISpot Preparation and Analysis

1. In a biological safety cabinet, process the spleens through 100 μm 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.
2. 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.
3. Adjust the volume to 10 ml with sterile PBS. Centrifuge the suspension at 600 x g for 10 min to pellet the cells.
4. 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 & Viability Kit protocol.
5. Make serial dilutions of the lymphocytes in 1.5 ml screw cap centrifuge tubes at dilution factors of 1:10, 1:20, and 1:40 (or as necessary) in Count & Viability Reagent (minimum total volume 300 μl). Pipette each dilution up and down several times to mix and analyze on the Muse.
6. Prepare a plate map of the samples and conditions for the ELISpot plate. Prepare the ELISpot plate according to the manufacturer's protocol and pipette 100 μl of medium, peptide (10 μg/ml), or scramble peptide (10 μg/ml) to each well.
7. Pipette 100 μl of cell suspension, delivering 1.0 x 10⁶ cells to each well and incubate the plate at 37 °C overnight.
8. Follow the manufacturer's protocol for the ELISpot assay. Analyze the developed ELISpot plate using a dissection microscope.
9. 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.

6. Immunohistochemistry (IHC) and Hematoxylin & Eosin (H&E) Staining

1. Take bladder tumor tissue preserved as described above (Section 2.4.11), embed in paraffin and step-section at 4 μm for immunohistochemical analysis.
2. 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.
3. Perform H&E staining using standard protocols.

7. Statistical Methods

For the Multiplex Fluorometric Microbead Immunoassay, use a two-tailed Student'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's Multiple Comparison Test to lessen the likelihood of a false positive result. A p-value of ≤0.05 is considered significantly different for all analyses.

Results

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...

Discussion

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...

Materials

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