Urethane and cyclophosphamide were purchased from Sigma-Aldrich® (St. Louis, MO). Diethylstilbestrol was purchased from Medisca (Plattsburgh, NY). L-BLP25 vaccine and the peptides used in ELISpot (BP25 and BP1-424) were provided by Merck Serono Research, Merck KGaA, Darmstadt, Germany.
A total of 179 MUC1.Tg and 10 wild type C57BL/6 mice were supplied by our breeding colony maintained by the UC Davis Mouse Biology Program and housed at the UC Davis Center for Laboratory Animal Science vivarium. To establish the colony, heterozygous hMUC1 female transgenic founder mice were purchased from Mayo Clinic (Scottsdale, AZ). The mice were kept in cages (4 mice/cage) maintained at constant temperature and humidity with a 12-h light/12-h dark cycle. All mice received free access to water and Purina Laboratory Rodent Diet (LabDiet® 5001, PMI® Nutrition International, St. Louis, MO). All animal studies were conducted under a protocol approved by the University of California Davis Institutional Animal Care and Use Administrative Advisory Committee. UC Davis is an Association for Assessment and Accreditation of Laboratory Animal Care accredited institution. For all mouse studies, the week number refers to the age of the mice in each respective study.
For DNA extraction, toe clippings from two-week old mice were placed in a 96-well plate. Next, 40 μl of 50 mM NaOH was added to each well, and plates were heated to 94°C for 10 min. Extracted DNA was neutralized with 20 μl of 1 M Tris (pH 8) per well. Plates were sealed, vortexed briefly, centrifuged (6000 rpm × 2 min), and stored at −20°C until use. Polymerase chain reaction was used to identify MUC1.Tg mice using an ABI Prism 7900HT Sequence Detection System (AB Applied Biosystems, Carlsbad, CA). MTag forward and reverse primers were 5′-TTGGAGAATGTTTTTGTCTTGAA TG and 3′-CAGCACATCTCGGGTTGGT. The MTag TaqMan probe (ACATGCAATGGTTTGGAA) carrying a 6′ FAM reporter label and a 3′ MGBNFQ quencher group was used. For MUC1, forward and reverse primers were 5′-CACTCTTCCCCCAACCTTAAGTG and 3′-GGGTG GGTGGTGGTCATG. The MUC1 TaqMan probe (ACC AGTCCCTCCCTACG) carrying a 5′ VIC reporter label and a 3′ MGBNFQ was used. The amplification program consisted of one cycle of 2 min at 95°C and 40 cycles of 15 sec each at 60°C and 95°C.
The lungs, left kidney and spleen were harvested from each mouse at the time of sacrifice, which was performed by CO2 asphyxiation. The lungs were filled with 10% neutral buffered formalin prior to excision. All tissue samples were fixed in 10% buffered formalin overnight, followed by 70% ethanol until processed. Tissues were then paraffin embedded and step-sectioned at 4 μm for immunohistochemical analysis. Immunohistochemistry was performed using a MUC1 antibody (CD227, 550486; 1:400; BD Pharmingen) which recognizes the tandem-repeat region. The Animal Research Kit peroxidase (ARK; K3954; Dako) was used to minimize reactivity of secondary mouse antibody with endogenous immunoglobulin present in the tissue. Lung whole mounts were prepared using standard protocols.
Multiplex cytokine assays
The Mouse Cytokine 20-plex Panel (Invitrogen; cat. #LMC0006, Carlsbad, CA) was used to analyze the levels (pg/mL) of Th1/Th2 and inflammatory cytokines in all serum samples except for those from the studies described under “Different Schedules of L-BLP25”, for which a 25-plex Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA) was used. The 20-plex panel consisted of interleukin (IL) -1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, interferon gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10), monokine induced by IFN-γ (MIG), keratinocyte derived cytokine (KC), monocyte chemotactant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1α), granulocyte/macrophage colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-α), and basic fibroblast growth factor (FGF-basic). In addition to the analytes listed for the 20-plex panel, with the exception of VEGF, MIG, and FGF-basic, the 25-plex panel included granulocyte colony-stimulating factor (G-CSF), IL-7, IL-9, IL-12 (p40/p70), IL-15, MIP-1β, MIP-2, and regulated on activation, normal T-cell expressed and secreted (RANTES). The assays were performed according to their respective manufacturer’s instructions. The concentration of each cytokine was calculated relative to respective standard curves. For 20-plex analyses, cytokine concentrations were acquired on a BioPlex System using BioPlex software version 5.0 (BioRad, Hercules, CA, USA). The 25-plex analysis was performed on a Luminex 100/200 system running xPonent software version 3.1 (Luminex Corporation, Austin, TX).
To establish the in-house control for this assay, wildtype C57BL/6 mice were challenged with lipopolysaccharide (LPS). Mice were injected intraperitoneally (i.p.) with 200 μg LPS from Escherichia coli serotype O111:B4 (Sigma-Aldrich) dissolved in 1X PBS. At 4–5 h post-injection, mice were euthanized by CO2 asphyxiation. Whole blood was collected by cardiac puncture and placed in a clotting tube for isolation of serum by centrifugation. The serum was flash frozen in liquid nitrogen and stored at −80°C until analysis by multiplex assays on Luminex system.
Lung cancer model development in MUC1.Tg mice
For this study, 38 male MUC1.Tg mice (5 weeks of age) were divided into five groups: Control (vehicle only) (n = 8); Urethane (n = 10); Urethane + DES 7 mg/kg (n = 10); and Urethane + DES 14 mg/kg (n = 10). A second urethane group composed of 10 wild type males was included to compare human MUC1 expression with the transgenic mice. Urethane (0.75 mg/g) or sterile water (control) was administered by i.p. injection weekly for 10 weeks using a 25-gauge needle. All injection volumes were 100 μl. Starting 24 h after urethane dose 4, DES (7 mg/kg or 14 mg/kg) or corn oil (control) was administered weekly for a total of 8 weeks by subcutaneous (s.c.) injection in a volume of 100 μl using a 25-gauge needle. Mice were monitored until termination of the study in Week 38. The lungs, spleens, and left kidneys were collected and analyzed from a total of 46 mice. The presence of hMUC1-expressing lung adenoma was confirmed by blinded IHC evaluations performed by a pathologist.
Cytokine response in MUC1.Tg mice
We studied the polarization of cytokine response in the hMUC1.Tg model during the induction and progression stages of lung tumor development. A total of six male hMUC1.Tg mice were bled 24 h after the 5th, 8th, and 10th doses of urethane (Weeks 10, 12, and 15, respectively) and thereafter at approximately four-week intervals (Weeks 20–40). Urethane administration was performed starting at approximately 4 weeks of age, as described under Lung Cancer Model Development in MUC1.Tg mice. Whole blood was collected from mice via submandibular bleeds, pooled and allowed to clot for 30 min, and then serum was isolated by centrifugation for 10 min at 3500 × g. Serum was flash frozen in liquid nitrogen and stored at −80°C until analysis by Luminex assay.
Effect of single dose of Cyclophosphamide on Th1/Th2 cytokine response
To determine the effect of low-dose CPA on the L-BLP25-induced immune response, and whether low-dose CPA induces a Th2/Th1 cytokine shift in this model, a total of 103 MUC1.Tg mixed sex C57BL/6 mice were divided into five treatment groups: Control (n = 20), CPA 100 mg/kg (n = 21), CPA 300 mg/kg (n = 20), L-BLP25 alone (n = 21) and CPA 100 mg/kg + L-BLP25 (n = 21). Each group contained 13 male and 8 female mice, except for the control and CPA 300 mg/kg groups, which each contained seven females. All mice underwent 10 weeks of urethane dosing at 0.75 mg/g weekly starting at 4 weeks of age. At Week 14, four days after urethane dosing was completed, mice were administered a single dose of CPA at either 100 or 300 mg/kg by i.p. injection according to treatment group assignment. Whole blood was collected from mice in each treatment group via submandibular bleeds 24 h following CPA administration. In our L-BLP25 mouse studies we have been using a CPA dose of 100 mg/kg, which is equivalent to the 300-mg/m2 dose used in humans . Three days after CPA administration, mice were given s.c. injections of 10 μg L-BLP25 at rotating sites according to treatment group using a 25-gauge needle once each week for eight weeks, with the 8th dose being administered in Week 21. The lyophilized L-BLP25 was reconstituted in sterile 0.9% saline to a concentration of 100 μg/ml and delivered in a volume of 100 μl. Twenty-four hours after the 4th and 8th doses of L-BLP25, whole blood was collected from mice in each treatment group via submandibular bleeds. Blood was pooled within a treatment group and serum was isolated by centrifugation. The serum was flash frozen in liquid nitrogen and stored at −80°C until analysis by Luminex assay. In order to assess the antitumor effects of CPA when combined with L-BLP25, this study was continued through Week 41 as described below in the two-cycle dosing study.
Different schedules of L-BLP25
Two studies were conducted in order to determine the effects of L-BLP25 on the development of lung tumors in this mouse model following either one or two cycles of treatment. In the first study, 32 MUC1.Tg male C57BL/6 mice were divided into four treatment groups: Untreated; CPA 100 mg/kg; L-BLP25 alone; and L-BLP25 + CPA 100 mg/kg (n = 8, all groups). All mice received 10 weekly i.p. injections of 0.75 mg/g urethane starting at four weeks of age as described under model development. Cyclophosphamide was then prepared and administered as described above, 24 h following the final urethane dose (Week 13). A single eight-dose cycle of weekly L-BLP25 was begun three days following CPA administration (Week 14). L-BLP25 was prepared as described above and administered weekly for a total of eight 10-μg doses by s.c. injection at rotating sites using a 25-gauge needle (injection volume 100 μl). Mice were then followed for 20 additional weeks. At the conclusion of the study in Week 41, all mice were euthanized by CO2 asphyxiation. Whole blood was then collected by cardiac puncture, and lung whole mounts were prepared to compare average lung tumor numbers between groups. Serum was isolated from whole blood and stored as described above.
In the two-cycle L-BLP25 study, which was a continuation of the low-dose CPA study described above, a total of 65 male and female MUC1.Tg C57BL/6 mice in four treatment groups were utilized. To reiterate, the treatment groups were Untreated (n = 18), CPA 100 mg/kg (n = 17), L-BLP25 alone (n = 14), and L-BLP25 + CPA 100 mg/kg (n = 16). The CPA 300 mg/kg group was excluded. This study was designed as described for the single-cycle study, except that 11 weeks following the end of the first eight-dose cycle of L-BLP25 (Week 32), these mice began a second cycle of treatment that continued weekly until the conclusion of the study in Week 41. The second cycle consisted of a total of nine 10-μg doses of L-BLP25, which was prepared and administered as described above. Three days prior to beginning the second cycle of L-BLP25, another 100-mg/kg dose of CPA was administered. At the end of the study, mice were euthanized by CO2 asphyxiation, and whole blood was collected by cardiac puncture. Serum was isolated and stored as described above for Luminex system analysis. Following blood collection, lung whole mounts were prepared to compare total average tumor numbers between treatment groups. IFN-γ/IL-4 ELISpot analysis was performed as detailed below on four mice from each treatment group to assess immune response.
To assess the immune response to L-BLP25 following one and two cycles of treatment, splenocytes from L-BLP25-treated and untreated mice were examined for Th1/Th2 polarization by analyzing two key cytokines: IFN-γ for Th1 and IL-4 for Th2 polarization. Dual color ELISpot mouse IFN-γ/IL-4 (R&D Systems, Minneapolis, MN) kits were used for this assay. Spleens were removed aseptically, processed through 100-μm nylon tissue sieves (Becton Dickinson, Franklin Lakes, NJ) into sterile PBS, and the cell suspensions were layered over lymphocyte separation medium (Lonza, Walkersville, MD). Lymphocytes were isolated by centrifuging at 600 × g for 15 min, washed in PBS, and then resuspended in improved minimum essential medium (Invitrogen) containing 10% fetal bovine serum (HyClone, Logan, UT), 50 μg/ml streptomycin and 50 U/ml penicillin (Invitrogen) prior to counting and viability assessment using an Auto T4 Cellometer™ (Nexcelom Bioscience, Lawrence, MA). BP25 peptide and scrambled peptide (BP1-424) were prepared at a final concentration of 5 μg/ml in culture medium. Lymphocytes (1.0 × 106/well) were incubated with either no peptide (medium only), BP25, or scrambled peptide in triplicate at 37°C overnight. ELISpot plates were developed according to the manufacturer’s instructions.
Differences in tumor incidence between experimental groups in the model development study were evaluated for statistical significance using Fisher’s exact test (two-tailed). In the one- and two-cycle L-BLP25 studies, a one-way ANOVA was used for comparisons between average number of lung tumors between the various treatment groups. Bonferroni’s adjustment for multiple comparisons was used to lessen the likelihood of a false positive result. GraphPad Prism® 5 software was used for all statistical analyses. A p-value of ≤ 0.05 was considered significant for all analyses.