{'fraction_non_alphanumeric': 0.03628929210324559, 'fraction_numerical': 0.019882443138257093, 'mean_word_length': 5.1451005025125625, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) constitutes a leading cause of cancer death [1,2], primarily owing to the lack of effective early detection methods and poor efficacy of existing therapies. Moreover, even among the 10% to 20% of patients who received a diagnosis of surgically resectable PDAC, most ultimately die of recurrent and metastatic disease [3]. These low survival rates are attributed in part to the fact that PDAC metastases have often progressed to the point where surgical removal cannot provide a cure. In order to improve the cancer mortality, detection and treatment in the early phase are necessary. Toward this end, analyses of PDAC pathological specimens and of genetically engineered PDAC mouse models have suggested that PDAC develops from pancreatic intraepithelial neoplasia (PanIN) [4,5].
Similar to other CRMP family proteins, CRMP4 is highly expressed in the central nervous system. CRMP4 expression is also observed in malignant tumors originated from various organs including the intestine, liver, pancreas, and prostate [14,[38][39][40][41][42][43][44][45]. However, the roles of CRMP4 in tumorigenesis or tumor progression remain unknown. Overexpression of CRMP4 suppresses the invasion ability and inhibits tumor metastasis of prostate cancer cells [14]. Consistent with this, lower expression of CRMP4 mRNA in hepatocellular carcinoma tissues is associated with shorter recurrencefree survival and subsequent adverse prognosis [42]. In contrast, high expression levels of CRMP4 mRNA in gastric cancers are significantly associated with shortened recurrence-free survival [39]. Previously, we reported that CRMP4 expression is associated with poor prognosis through the promotion of liver metastasis of pancreatic cancer [43]. In addition, CRMP4 knockdown using siRNA reduces the cellular invasion of Capan-1 cells, a human pancreas adenocarcinoma cell line. CRMP4 expression was also found to be enhanced in the pancreatic parenchyma and the infiltrated lymphocytes in pancreatic tissue of a pancreatitis mouse model [46]. In turn, a meta-analysis of chronic pancreatitis has shown a relative risk of 13.3 for developing malignancy [47], and chronic pancreatitis is considered to have a strong relationship with carcinogenesis and pancreatic cancer [48]. Together, these findings suggest that CRMP4 is involved in the pathogenesis of pancreatic cancer, although direct causality has not been demonstrated. To further clarify the role of CRMP4 in in pancreatic carcinogenesis, in this study, we examined the role of CRMP4 in the progression of PanIN in a genetically engineered mouse model of pancreatic cancer.

Ethics Statement
All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals (Japanese Association for the Laboratory Animal Science) and the Guide for Yokohama City University. Specific approval for the mouse experiments was obtained from the Institutional Animal Care and Use Committee of Yokohama City University School of Medicine with the protocols F-A-15-042 and F-A-16-019 "Biological function of CRMP4 in metastasis and invasion with pancreatic cancer model mice." All surgical procedures were performed under isoflurane (Pfizer, New York, NY) and pentobarbital sodium (Kyoritsu seiyaku, Tokyo, Japan) anesthesia, and all efforts were made to minimize the number of animals used and their suffering.

Animals
Wild-type (WT) C57BL/6JJmsSLc male mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). All mice were maintained at the animal care facility of Yokohama City University under a 12-hour light/12-hour dark cycle at 23°C ± 1°C, with free access to water and food. All mice were fed commercially available MF feed (Oriental Yeast Kogyo Co., Tokyo, Japan).

Morphological Analysis of PanIN
Subsequent to euthanasia with an excessive dose of isoflurane and pentobarbital, mice were perfusion-fixed with 4% (wt/vol) paraformaldehyde in PBS. To obtain macroscopic and microscopic findings of both KC-Crmp4 WT mice and KC-Crmp4 KO mice, the pancreas was removed together with the spleen and duodenum. Collected pancreases were fixed in 4% paraformaldehyde in PBS for 12 hours at 4°C.
Tissues were embedded in paraffin. Paraffin-embedded pancreas sections (3 μm thick) were deparaffinized in xylene and rehydrated sequentially in ethanol. Slides were washed in deionized water, stained with hematoxylin (131-09665, Wako, Osaka, Japan) and eosin (051-06515, Wako) in 60% ethanol, then dehydrated sequentially in ethanol, cleared with xylenes, and mounted with Entellan new (Merck KGaA, Darmstadt, Hessen, Germany). All slides were analyzed by a pathologist (I.K.) for findings of PanIN development. The pathologist was blinded to the tissue genotype. Each slide was classified as normal acinar/ductal cells; acinar-toductal metaplasia; PanIN-1A, -1B, -2, or -3; or PDAC based on the classification consensus [55]. Briefly, PanIN-1 constitutes flat (1A) or papillary (1B) lesions of the columnar epithelium with basally oriented, round nuclei. PanIN-2 represents papillary lesions with nuclear hyperchromasia, crowding, and pseudostratification. PanIN-3 indicates papillary, micropapillary, or cribriform lesions with nuclear pleomorphism, frequent loss of nuclear polarity, and mitoses [56,57]. The tissue samples were evaluated at high optical power (objective, 40) for all PanIN lesions in a randomly selected microscopic field. PanINs grade were presented as incidence of the highest grade of PanINs in the tissue regions examined. The grades of PanINs defined were roughly correlated with the density of PanINs in the tissue regions.
For immunohistological evaluation of CRMP4, two investigators, I. K. and K. Y., independently assessed the stained sections. These investigators were blinded to the sources of sections. The intensity of cytoplasmic CRMP4 immunoreactivity was scored as follows: 0, no staining; 1, mild; 2, moderate; and 3, strong. In statistical analyses, scores 0 and 1 were defined as CRMP4 negative, whereas 2 and 3 were defined as CRMP4 positive. For measurement of colocalization between CRMP4, and αSMA or CD3, we quantitated the number of CRMP4-, αSMA-, and CD3-positive cells using serial sections, respectively (n = 3).

Statistical Analyses
Data are presented as the means ± standard deviation and were analyzed with the built-in Student's t test using SPSS computer software package version 26.0 for Windows (SPSS Inc., Chicago, IL). P values of <.05 were considered significant. The relationship between the progression of PanIN grade and the expression of CRMP4 was analyzed using Fisher's exact test in R software for Windows version 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria). In all cases, a group size was chosen that produced statistically unambiguous results.

Low-Grade PanIN Progression in KC-Crmp4 KO Compared to KC-Crmp4 WT Mice
Gross pathological findings included swelled pancreas with hard nodular lesions in KC-Crmp4 WT and KC-Crmp4 KO mice. No obvious difference was observed between WT and KO mice (Figure 2, A and B). Histological examination revealed pathological lesions in the pancreatic tissues of WT and KO mice that were similar to those in human PanIN (Figure 2, C and  D). Notably, PanIN-2 lesions were mainly observed in KC-Crmp4 WT mice ( Figure 2C, Table 1), which showed a papillary structure with pseudostratification and enlarged nuclei. Conversely, PanIN-1 lesions were mainly observed in KC-Crmp4 KO mice ( Figure 2D, Table 1), which consisted of tall columnar cells with basally located nuclei and abundant supranuclear mucin. Table 1 shows the grade of PanIN in both genotypes. High-grade PanIN lesions (PanIN-2 and -3) were observed more frequently in KC-Crmp4 WT mice than in KC-Crmp4 KO mice ( Table 1). The acinar-to-ductal metaplasia was similarly observed in both groups. The observation period was 139.2 ± 41.2 days for KC-Crmp4 WT and 126.0 ± 23.1 days for KC-Crmp4 KO mice (Student's t test, P = .340).

Increased Expression of CRMP4 in PanIN Lesions
We first validated the sensitivity and specificity of the anti-CRMP4 antibody. Western blot analysis using brain lysate revealed that the band detected in Crmp4 +/− samples was absent in Crmp4 −/− ( Figure 3A). In immunohistochemistry using pancreatic tissues, CRMP4 expression was detected in the ductal and acinar cells in Crmp4 +/+ but not in Crmp4 KO  (Figure 3, E-H). In WT mice, weak expression of CRMP4 was detected in some of the nuclei of the ductal and acinar cells ( Figure 3F). CRMP4 expression was also detected in the cytoplasm of cells in PanIN lesions ( Figure  3, G and H) and in the stroma surrounding PanIN lesions (Figure 3, G and H, black arrowheads). Preabsorption of anti-CRMP4 antibody with antigen peptide reduced the immune signal (data not shown). These findings supported the specificity of the anti-CRMP4 antibody used.

CRMP4-Positive Cells in Stromal Areas Coincide with αSMA-Positive Cells
To examine the immunohistochemical features of the CRMP4-positive cells in the stromal areas, pancreatic tissues were stained with an antibody against αSMA, the most common marker of fibroblasts/myofibroblasts, and against CD3, a general marker of T cells. Serial sections of PanIN-2 lesions were stained using hematoxylin and eosin along with antibodies against CRMP4, CD3, and αSMA. CRMP4-positive spindle-shaped cells were observed in the stromal areas ( Figure 4). The staining pattern of CRMP4 was similar to that of αSMA but not of CD3 (Figure 4, B and D, black arrowheads). Quantitative colocalization analysis was performed. The percentage of colocalization between CRMP4 and αSMA or CRMP4 and CD3 was 39.0% ± 1.95% or 13.7% ± 0.98%, respectively (n = 3, P = .003, Figure 5, A and B). Immunofluorescence staining of PanIN-2 lesion ( Figure 6, C and D, yellow arrowhead) illustrated strong expression of CRMP4. Moreover, some stromal cells surrounding the PanIN lesions coexpressed CRMP4-and αSMA-positive cells (Figure 6, F, G and H; white arrowhead).

Immunohistochemical Analysis of CRMP4 in PanIN Lesions and Stromal Areas
To examine the relationship between expression of CRMP4 and the PanIN grade, we evaluated CRMP4 expression in the epithelial cells and stromal cells in PanIN lesions in WT mice. The expression levels of CRMP4 in the epithelial cells of PanIN lesions showed no correlation with PanIN development (Table 2, P = .588). In stromal cells, however, highgrade PanINs were observed more frequently in the CRMP4-positive group than in the CRMP4-negative group ( Table 2, P = .023). These results indicated that the expression levels of CRMP4 in the stromal cells of PanIN lesions correlated with the progression of high-grade PanIN in the mouse model of pancreatic cancer. PanIN-1 was found in almost every tissue examined, while PanIN-2 or -3 was rarely seen.

Discussion
In this study, we demonstrated that PanIN progression was suppressed in KC-Crmp4 KO mice compared with KC-Crmp4 WT mice. CRMP4 expression was increased in both epithelial cells and the stromal areas of PanIN lesions, especially in αSMA-positive cells. These findings suggested that CRMP4 may participate in the development of PanIN. In addition, CRMP4 expression was observed in in the epithelium of PanIN lesions as well as in normal acinar and ductal cells. This expression profile of CRMP4 in the pancreatic tissue of mice coincides with that in human surgical specimens of PDAC [43]. Whether CRMP4 is also involved in PanIN in human pancreatic cancers thus represents an important issue to be addressed in future studies.
Our study revealed that CRMP4 is expressed in both epithelial cells and in stromal areas, especially in αSMA-positive cells (Figures 4 and 6). Previous studies have shown that the formation of PanIN accompanies the accumulation of a desmoplastic stroma and abundant immune infiltrates [48,58]. It has been suggested that the desmoplastic reaction by inflammation participates in the development of PanIN [59,60]. αSMA-positive cells in stromal areas of PanIN lesions are considered to constitute an active type of pancreatic stellate cells [61]. It is generally accepted that the quiescent type of pancreatic stellate cells, which contain vitamin A, become changed to active type by tissue injury or inflammation [62]. The active type of pancreatic stellate cells accelerates the production of extracellular matrix such  as collagen, fibronectin, and laminin, thereby leading to pancreas fibrosis [59,63,64]. Therefore, the interaction between pancreatic stellate cells and pancreatic cancer cells or epithelial cells of PanIN may be involved in the proliferation of PDAC or PanIN through the desmoplastic reaction. Repeated acute pancreatic injury and inflammation serve as contributing factors to the development of pancreatic cancer. In particular, intracellular activation of both pancreatic enzymes and the transcription factor NF-κB comprises an important mechanism that induces acute pancreatitis [65]. Recurrent pancreatic injury owing to genetic susceptibility, along with environmental factors such as smoking, alcohol intake, and conditions such as obesity, leads to increases in oxidative stress, impaired autophagy, and constitutive activation of inflammatory pathways. These processes can stimulate pancreatic stellate cells, thereby increasing fibrosis and encouraging chronic disease development [66]. Fibrosis has a pivotal role in inflammation and carcinogenesis. PDAC is unique among solid tumors because of the extremely dense desmoplastic reaction that surrounds the cancer cell glands of this tumor. The desmoplasia, containing myofibroblastic pancreatic stellate cells, extracellular matrix proteins, and immune cells, modulates the growth of the cancer by providing a scaffold for the cancer cells to grow, along with growth factors, angiogenesis factors, and immune modulators [67]. Considering that activated pancreatic stellate cells underlie the desmoplasia, CRMP4 expression in stellate cells surrounding PanIN lesions may have a critical role in the development of PanIN.
Our previous study demonstrated that CRMP4 was augmented in CD3positive cells in a pancreatitis mouse model [46]. In comparison, our present study showed that CRMP4-positive cells were coincident with αSMApositive cells, whereas the expression of CRMP4 was barely detectable in CD3-positive cells. The discrepancy might be attributed to differences in the genetic background of the mouse models, the period of observation, and other experimental conditions. For example, WT and CRMP4 KO mice were examined in acute pancreatitis models [46], whereas WT and CRMP4 KO with K-ras mutant background mice were examined in our current study. It has been shown that K-ras-expressing pancreatic acinar cells initiate microinflammation and that an interaction exists between PanIN and αSMA-positive cells, which may contribute to the formation and progression of PanIN lesions [68]. Furthermore, in our previous study,   CRMP4/CD3 double-positive cells were observed mainly in the phase of acute pancreatitis [46], whereas in the present study, CRMP4-positive cells were observed in the recovery phase from acute pancreatitis. It is possible that the number of CD3-positive cells may decrease in recovery phase.
CRMP4 may be involved in PanIN or pancreatic cancer pathogenesis through several mechanisms. First, CRMP4 may promote the inflammation pathway in the development of PanIN. It has been shown that K-ras mutation and cell injury mediated by inflammation play important roles in the development of PanIN lesions [53]. Previous studies have demonstrated that CRMP4 is involved in inflammation pathways [46,51]. We reported that both acute pancreatitis and chronic pancreatitis augment CRMP4 expression and its phosphorylation in infiltrated CD3 T-cells [46]. Therefore, CRMP4 may play a pivotal role in pancreatic inflammation. Notably, deletion of CRMP4 in a spinal cord injury mouse model promotes the recovery of locomotion via neuroprotection and limited scar formation because CRMP4 deletion suppresses the activation of microglia or macrophages and reactive astrocytes following injury [51]. Therefore, we speculated that CRMP4 may participate in the development of PanIN through the acceleration of inflammation.
Second, CRMP4 may help to develop PanIN lesions through reconstruction of the cytoskeleton, as this may induce morphologic changes comparable to those observed during PanIN development [69]. In a pancreatitis mouse model, it was found that RAS-related C3 botulium substrate 1 (Rac1), which is an effector molecule of EGFR and K-ras, was necessary for caerulein-induced acinar morphologic changes and filamentous actin redistribution [70,71]. CRMP4 is also known to regulate the actin and microtubule growth cone cytoskeleton in hippocampal neurons [27,28,72]. Moreover, the alternatively spliced short (CRMP4a) and long (CRMP4b) isoforms are known to be involved in many biological processes [73]. In particular, CRMP4a suppresses RhoA activity, leading to reduced cytoskeletal reorganization and cell motility in prostate cancer [38]. However, further studies are needed to clarify the molecular mechanism of CRMP4 in PanIN development.
In conclusion, the incidence of high-grade PanIN was low in the CRMP4-KO mouse model of pancreatic cancer. The expression of CRMP4 in the stroma cells correlated with the progression of PanIN lesions. These findings suggest that CRMP4 inhibition may serve as a therapeutic strategy to prevent PanIN development and PDAC. Relationship between PanIN grade and CRMP4 expression in epithelial cells (Fisher's exact test; two-sided, P = .588) and stromal cells (Fisher's exact test; two-sided, P = .023).