Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

The B6.129-Kras^tm4Tyj (01XJ6) and B6. FVB-Tg (Ipf1-cre)1Tuv (01XL5) mice were obtained from the NCI Mouse Models of Human Cancers Consortium (MMHCC) (Frederick, MD, USA). These animals (LSL-Kras^G12D and Pdx1-Cre) were crossed to remove the LSL cassette in order to activate Kras ^G12D (Kras^G12D;Pdx1-Cre/floxed Kras^G12D) allele in the pancreas of the mouse. The F1 progeny was genotyped for Kras as well as Pdx1-Cre by using specific primers for Kras and Pdx1-Cre by Polymerase chain reaction (PCR). Animals that were positive for Kras ^G12D and Pdx1-Cre expressed the mutated Kras ^G12D allele in the pancreas. The floxed Kras ^G12D animals (positive for both Kras and Pdx1-Cre) and their contemporary littermates positive for either LSLKras ^G12D or Pdx1-Cre were euthanized at 7, 10, 25, 30, 40 and 50 weeks of age (eight animals/group/time point). Throughout the experiment, animals were provided with food and water ad libitum and subjected to a 12-h dark/light cycle. Animal studies were performed in accordance with the U.S. Public Health Service “Guidelines for the Care and Use of Laboratory Animals” under an approved protocol by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC).

Animals were tail clipped at 10-14 days of age and DNA was isolated using standard protocol (Maxwell 16 mouse tail DNA purification kit, Promega, Madison, WI, USA). The genotyping of Kras and Pdx1-Cre was performed by PCR using the following primer sequences Kras K006F-5’-CCT TTA CAA GCG CAC GCA GAC TGT AGA-3’, Kras K005R-5’- AGC TAG CCA CCA TGG CTT GAG TAA GTC TGC A-3’ and Pdx1-Cre F-5’-CTG GAC TAC ATC TTG AGT TGC -3’ and Pdx1-Cre R-5’-GGT GTA CGG TCA GTA AAT TTG -3’. The PCR amplification reaction contained 1 μl of genomic DNA (100 ng), 0.3 μl 10 pmol of each primer, 10 μl of 2X PCR master mix (DNA Polymerase, 400 μM each of dATP, dGTP, dCTP, dTTP and 3 mM MgCl₂) and 8.4 μl of autoclaved water. PCR amplification was carried out in a programmable thermal cycler (MJ Research, Minnesota, USA) using the following program: denaturation for 5 min at 95°C, followed by 40 cycles of amplification by denaturation for 1 min at 94°C, annealing at 2 min at 59°C, elongation for 45 sec at 72°C and a final extension of 10 min at 72°C. The PCR products were resolved on 1.5% agarose gel to confirm the genotype of each animal based on the amplification of target regions.

Total RNA was isolated from the pancreas of floxed Kras ^G12D (Kras^G12D;Pdx1-Cre) and unfloxed Kras ^G12D (LSLKras^G12D) by using the mirVana™ miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA). RNA concentration was measured by using a NanoDrop^TM Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA), and the quality was analyzed with a bioanalyzer (Agilent technologies, Waldbronn, Germany). Samples with good integrity were used for cDNA synthesis.

Total RNA was isolated from the pancreas and the cDNA was synthesized by reverse transcription. Reverse transcription of RNA was performed by adding 10 μl of (2000 ng) total RNA, 1 μl of Oligo (dT) and 1 μl of 10 mM dNTP incubated at 65°C for 5 min and immediately chilled on ice. Then, the master mix containing the following components were added (4 μl of (5X) first strand RT buffer, 1 μl of 0.1 M DTT, 1 μl of RNaseOUT (RNase Inhibitor) and incubated at 42°C for 2 min. Finally, 1 μl (50 units) of SuperScript II RT was then added to each tube mix, and incubated at 42°C for 50 min followed by 70°C for 15 min in order to destroy the superscript II RT (Invitrogen, Carlsbad, CA, USA).

Real time primers for all the mouse genes (Muc1, Muc4, Muc5AC, IFN-γ, CXCL1, and CXCL2) were designed using Primer 3 software (Table 1). Real-time PCR was performed on the Light cycler 480 II PCR System, (Roche Applied Science, Indianapolis, IN, USA). Real-time PCR reactions were performed in triplicate, and non-template controls (NTCs) and standard curve were run for each assay under similar conditions. Real time PCR was performed in a 10 μl reaction volume containing 5 μl 2X SBYR green Master mix (Roche applied science, Indianapolis, IN, USA), 3.2 μl of autoclaved nuclease free water, 1 μl of diluted RT product (1:10) and 0.2 μl each of forward and reverse primer (5pmol/μl). The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Gene expression levels were normalized to the level of β-actin expression and were reported relative to the expression level in RNA from corresponding normal controls.

Anti-mouse Muc1 (mouse monoclonal antibody recognizing the cytoplasmic tail of Muc1), and Anti-mouse Muc5AC (mouse monoclonal) antibody were purchased from Abcam® (Cambridge, MA, USA). The anti-Muc4 (4A-rabbit polyclonal) antibody used in this study was designed by us and developed by GenScript (Piscataway, NJ, USA). Rabbits were immunized with a 15 amino-acid peptide specific to the tandem repeat region of mouse Muc4 (CAGYRPPRPAWTFGD). Analysis of tissue sections pre-incubated with the blocking peptide was conducted in order to confirm the specificity of the antibody.

The F1 progeny of (N=8) floxed Kras ^G12D (Kras^G12D;Pdx1-Cre) and unfloxed Kras ^G12D (LSLKras^G12D) animals were sacrificed at 7, 10, 25, 30, 40 and 50 weeks of age. A section of the pancreas from these animals was fixed in 10% formalin (Fisher Scientific, Fair Lawn, NJ, USA). The tissues were then embedded in paraffin and serial tissue sections (4 μm thick) were cut. The sections were deparaffinized using EZ-DeWaxTM (Bio genex, San Roman CA, USA) and dehydrated gradually. Subsequently, the sections were stained with hematoxylin and eosin (H&E) stains and examined under a light microscope as described [27].

Pancreatic tissues isolated from transgenic mice of different ages were embedded in paraffin after being fixed in 10% formalin for at least 48 h. Subsequently, 4 μm sections of paraffin-embedded pancreas were sliced and prepared for histological analysis. After placing the slides in an oven at 56°C overnight, these were deparaffinized after washing several times in xylene (Fisher Scientific, Fair Lawn, NJ, USA). Tissues were then rehydrated with decreasing concentrations of ethanol. After incubating the tissues for 30 min in the presence of 5% H₂O₂ in methanol to block the endogenous peroxidase, tissue sections were blocked in 2.5% horse serum for 2 h. Without washing the tissue sections, the corresponding primary antibodies were added at the optimum concentrations, which were determined after standardization experiments. The corresponding dilutions used in these sections were: 1:200 anti-Muc1, 1:4000 anti-Muc4, 1:400 anti-Muc5AC. Following overnight incubation, sections were washed three times with PBST and the horseradish peroxidase-conjugated secondary antibody was added for 30 min. IHC staining of the respective mucins were developed after colorimetric detection with a 3,3’-diaminobenzidine (DAB) reagent kit (Vector Laboratories, Burlingame, CA, USA) followed by hematoxylin staining. Tissues were then dehydrated with increasing concentrations of ethanol followed by a xylene wash. IHC staining was evaluated by a pathologist after mounting with Permount mounting medium (Fisher Scientific, Fair Lawn, NJ, USA). Expression of each mucin was scored on a scale of 0–3 where 0-negative, 1-weak, 2-moderate and 3- represented strong immunoreactivity to the antibody used. Further the percentage of cells positive for the antibody was scored on a scale of 1–4 where 1: 0–25% cells positive; 2: 26–50% positive; 3: 51–75% positive; and 4: 76–100% positive. The composite score was then obtained by multiplying the staining intensity and the percentage of immunoreactive cells and it ranged from 0 to 12.

Fold change in the mRNA expression of various genes were calculated by ΔΔCt method. Mouse β-actin was used for normalization. A change of 2 fold or more (on the log scale 0.3 or more) was considered statistically significant. A Student’s t-test was used to calculate the significance in the staining pattern for each mucin at different stages of PC progression. All p- values <0.05 were considered statistically significant.

The floxed Kras ^G12D animals (i.e. positive for both Kras ^G12D and Pdx1-Cre) and their contemporary littermates harboring either LSLKras ^G12D or Pdx1-Cre were euthanized at 7, 10, 25, 30, 40 and 50 weeks of age (N = 8 for each time point) and individual pancreas was resected and weighed. The average weight of the pancreas in the Kras^G12D;Pdx1-Cre animals was significantly higher (p < 0.0001) than those of age-matched LSLKras^G12D control animals. Importantly, the average pancreas weight increased from 25 weeks (475 mg) to 50 weeks (863 mg) of age in Kras^G12D;Pdx1-Cre while no significant change was observed in control animals (Figure 1A). These differences in the pancreas weight suggested the occurrence of pathological changes in Kras^G12D;Pdx1-Cre mice.

Upon microscopic examination of the H&E stained tissue sections, no lesions were observed in the pancreas of LSLKras^G12D mice (Figure 1B1-1B4), while Kras^G12D;Pdx1-Cre mice pancreas showed the presence of PanIN lesions as early as 10 weeks of age, which progressively developed into PDAC by 50 weeks of age (Figure 1B5-1B8). Specifically, at 10 weeks of age, mostly PanIN-I lesions were observed (Figure 1B5), which progressed to PanIN-II and III lesions at 25 weeks of age, replacing the majority of pancreatic parenchyma (Figure 1B6). At 40 weeks of age, the majority of parenchyma was replaced by advanced PanIN III lesions and extensive desmoplasia (Figure 1B7), and at 50 weeks of age, the pancreas parenchyma was replaced with PDAC (Figure 1B8). Metastatic lesions involving liver, lung and small intestines were observed at 50 weeks of age in 60-70% of the Kras^G12D;Pdx1-Cre mice (Figure 1C-1E).

Previous reports have shown that MUC1 is overexpressed during the progression of human PC and it plays an important role in cancer invasion and metastasis [6]. In this study, real time-PCR analysis showed an increase in the expression of Muc1 from 10 weeks (fold change 2.5) to 50 weeks (fold change 6.9) of age in the pancreas of Kras^G12D;Pdx1-Cre mice in comparison to the LSLKras^G12D control mice (Figure 2A). The pancreas of unfloxed Kras ^G12D mice expressed basal level of Muc1 (Figure 2A). IHC analysis showed an elevated protein expression of Muc1 in the pancreas of Kras^G12D;Pdx1-Cre mice starting from 10 weeks of age (Figure 2B-2G). The intensity of Muc1 expression increased in pancreatic tissues isolated from 10 weeks to 50 weeks of age with an increase in composite score from 3.6 to 11 (p < 0.0001) (Figure 2H). Muc1 protein was predominately localized at the membrane of pancreatic ductal cells. The IHC results are in agreement with real time-PCR data, as a basal level expression of Muc1 was observed in the pancreas of unfloxed LSLKras^G12D mice, which did not increase even in 50 weeks old mice (Figure 2I). Further, Muc1 expression was also observed in the metastatic lesions involving liver, small intestines and lungs at 50 weeks of age in Kras^G12D;Pdx1-Cre animals (Figure 2J-2L).

Previous studies from our lab have shown that MUC4 is aberrantly overexpressed in human PC [6] and has a role in the progression and metastasis of PC cells [5, 20, 28]. We determined the expression pattern of Muc4 glycoprotein during the initiation and progression of PC in the Kras^G12D;Pdx1-Cre mouse model (10-50 weeks) by real time-PCR and IHC. A significant increase in Muc4 transcripts was observed in the pancreas of Kras^G12D;Pdx1-Cre mice from 10 (fold change 2.9) to 50 weeks (fold change 54) of age (Figure 3A). Similar to normal human pancreas, no expression of Muc4 was observed in the pancreas of LSLKras^G12D mice. Similarly, IHC analysis showed a progressive increase in Muc4 protein levels in the pancreas of Kras^G12D;Pdx1-Cre mice from 7 to 50 weeks of age (Figure 3B-3G). These results were in agreement with real time-PCR results as there was a significant (p < 0.0001) increase in the composite score for Muc4 expression in the pancreas of Kras^G12D;Pdx1-Cre mice from 1.6 at 10 weeks to 7.0 by 50 weeks of age (Figure 3H). Muc4 expression was observed in both membrane and cytoplasm of pancreatic ductal cells associated with PanIN lesions, while no expression was detected in the adjoining acinar and stromal cells. The pancreas of LSLKras^G12D mice was completely negative for Muc4 even at 50 weeks of age (Figure 3I). High expression of Muc4 was also observed in the metastatic lesions involving small intestines as well as liver and lungs of 50 weeks old Kras^G12D;Pdx1-Cre mice (Figure 3J-3L).

It has been previously established that the expression of MUC5AC, a gel-forming secretory mucin increases in tandem with the increase in grade of PanIN lesions and PDAC. However no expression of MUC5AC has been detected in the normal human pancreas [19, 29]. In the present study, real time-PCR analysis showed an increase in the expression of Muc5AC in the pancreas of Kras^G12D;Pdx1-Cre mice from 10 weeks (fold change 1.2) to 50 weeks (fold change 3.0) of age when compared to LSLKras^G12D mice (Figure 4A). Real time-PCR analysis in the pancreas of LSLKras^G12D mice showed no change in the expression of Muc5AC across the different age groups (Figure 4A). Similarly, IHC analysis showed a gradual increase in the protein expression of Muc5AC in the pancreas of Kras^G12D;Pdx1-Cre mice (Figure 4B-4G). The composite scores for Muc5AC expression in pancreatic tissues increased from 0.8 (i.e. no expression) at 10 weeks of age to 9.5 (p < 0.0001) in 50 weeks old Kras^G12D;Pdx1-Cre mice. No expression of Muc5AC was detected in the pancreas of age-matched unfloxed LSLKras^G12D mice (Figure 4I). The IHC analysis of metastatic lesions involving liver, small intestines and lungs at 50 weeks of age showed strong Muc5AC expression (Figure 4J-4L).

Oncogenic Kras has been implicated in the activation of the NF-κB pathway which induces inflammatory responses in PC [30] and the production of cytokines from tumor cells which result in the generation of a pro-inflammatory tumor microenvironment in the bronchiolar epithelium [31]. As mucin genes are known to be regulated under inflammatory conditions [32‐34], we wanted to investigate whether immune infiltration occurred early during PC development. There was no inflammation in the pancreas at 7 weeks of age (postnatal), but at 10 weeks of age, mild inflammation reaction was observed in 5% of the pancreatic tissues (Figure 5A). Subsequently, chronic inflammation was observed in 65% of the pancreatic tissues in 25-30 weeks old Kras^G12D;Pdx1-Cre mice which increases to 75% by 40-50 weeks of age with a strong desmoplastic reaction (Figure 5A). This inflammation scoring was further corroborated with the infiltration of macrophages (F4/80) in the cancer tissue (Figure 5G and 5H) with a composite score of 4.5 (p < 0.05) (Figure 5D) compared to 10 weeks of age (Figure 5E and 5F), where mostly PanIN I were observed.

Expression of inflammatory cytokines/chemokines such as IFN-γ, CXCL1 and CXCL2 were measured by performing real time-PCR using total RNA isolated from mouse pancreas collected at 50 weeks of age. We observed a significantly higher expression of CXCL1 (p < 0.00013), CXCL2 (p < 0.085) and IFN-γ (p < 0.0062) in Kras^G12D;Pdx1-Cre animals compared to LSLKras^G12D control animals (Figure 5B). Correspondingly, an increased infiltration of lymphocytes in pancreatic tissues of Kras^G12D;Pdx1-Cre mice correlated with the increased inflammation and increased inflammatory cytokines detected in the pancreas of Kras^G12D;Pdx1-Cre mice (Figure 5C).