Perineural Invasion In Pancreatic Cancer Research Articles

P-0039 - Paracrine Sonic Hedgehog Signalling Pathway Promotes Perineural Invasion Via the Activation of Stromal Cells in Pancreatic Cancer

Background: Pancreatic cancer (PCa) is characterised by stromal desmoplasia and perineural invasion (PNI). Clinical data has shown that PCa recurrence rate in patients with PNI are significantly higher than those without PNI. Sonic hedgehog (SHH) has been associated with the progression of PCa, which impacts on proliferation, invasion and metastasis. Currently, the mechanism of PNI in PCa, especially the interaction of the various factors in the tumour microenvironment remains unclear.

P-0040 - Hypoxia Promotes Pancreatic Cancer Perineural Invasion Through the Activation of Hedgehog Signaling

Background: Pancreatic cancer often experience low oxygen tension environments, which is associated with enhanced tumor invasiveness, angiogenesis and distant metastasis. Perineural invasion (PNI) is one of the most important causes of local recurrence and poor survival in pancreatic cancer. However, the exact mechanism of PNI is still not clear. In this study, we sought to identify the reciprocal signaling interactions between pancreatic cancer cells and peritumoral nerves and the underlying mechanisms.

Neurotransmitter Substance P Mediates Pancreatic Cancer Perineural Invasion via NK-1R in Cancer Cells

Pancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the underlying mechanism is not clear. This study aimed to determine the relationship between Substance P (SP) and pancreatic cancer perineural invasion (PNI) as well as the mechanism of SP mediating pancreatic cancer PNI, which causes pain in patients with pancreatic cancer. Human pancreatic cancer cells and newborn dorsal root ganglions (DRG) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzed using MTT assay and Transwell Matrigel invasion assay, respectively. Alterations in the neurotropism of pancreatic cancer cells were assessed by coculture system, which mimics the interaction of tumor/neuron in vivo. SP is not only widely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3 cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines examined. SP induces cancer cell proliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells, and NK-1R antagonists inhibit these effects. Furthermore, SP promotes neurite outgrowth and the migration of pancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the coculture model. Our results suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, and blocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer.

Pleiotrophin promotes perineural invasion in pancreatic cancer

Perineural invasion (PNI) in pancreatic cancer is an important cause of local recurrence, but little is known about its mechanism. Pleiotrophin (PTN) is an important neurotrophic factor. It is of interest that our recent experimental data showed its involvement in PNI of pancreatic cancer. PTN strongly presents in the cytoplasm of pancreatic cancer cells, and high expression of PTN and its receptor may contribute to the high PNI of pancreatic cancer. Correspondingly, PNI is prone to happen in PTN-positive tumors. We thus hypothesize that, as a neurite growth-promoting factor, PTN may promote PNI in pancreatic cancer. PTN is released at the time of tumor cell necrosis, and binds with its high-affinity receptor, N-syndecan on pancreatic nerves, to promote neural growth in pancreatic cancer. Furthermore, neural destruction leads to a distorted neural homeostasis. Neurons and Schwann cells produce more N-syndecan in an effort to repair the pancreatic nerves. However, the abundance of N-syndecan attracts further PTN-positive cancer cells to the site of injury, creating a vicious cycle. Ultimately, increased PTN and N-syndecan levels, due to the continuous nerve injury, may promote cancer invasion and propagation along the neural structures. Therefore, it is meaningful to discuss the relationship between PTN/N-syndecan signaling and PNI in pancreatic cancer, which may lead to a better understanding of the mechanism of PNI in pancreatic cancer.

Association of diabetes and perineural invasion in pancreatic cancer

Diabetes and perineural invasion are frequently observed in pancreatic cancer. In this study, we tested possible relations between diabetes and perineural invasion in patients with resected pancreatic cancer. We conducted a retrospective study in 544 cases of resected pancreatic adenocarcinoma seen at the University of Texas MD Anderson Cancer Center during 1996–2011. Information on tumor characteristics, diabetes history, and survival time was collected by personal interview and medical record review. Patients with diabetes before or at the time of the pancreatic cancer diagnosis were considered diabetes only. Pearson χ2 test was used to compare categorical variables in diabetic and nondiabetic groups. Kaplan–Meier plot, log-rank test, and Cox proportional regression models were applied in survival analysis. The prevalence of diabetes and perineural invasion was 26.5% and 86.9%, respectively, in this study population. Patients with diabetes had a significantly higher prevalence of perineural invasion (92.4%) than those without diabetes (85%) (P = 0.025, χ2 test). Diabetes was not associated with other pathological characteristics of the tumor, such as tumor size, lymphovascular invasion, tumor grade, lymph node metastasis, and resection margin status. Diabetic patients had a significantly lower frequency of abdominal pain (P = 0.01), but a slightly higher frequency of weight loss (P = 0.078) as early symptoms of their cancer. Both diabetes and perineural invasion were related to worse survival and increased risk of death after adjusting for tumor grade and margin and node status (P = 0.036 and 0.019, respectively). The observed associations of diabetes and perineural invasion as well as reduced frequency of pain as early symptom of pancreatic cancer support the hypothesis that diabetes may contribute to pancreatic progression via the mechanism of nerve damage.

GPC-1 may serve as a predictor of perineural invasion and a prognosticator of survival in pancreatic cancer

The purpose of this study was to investigate the expression of glial cell derived neurotrophic factor (GDNF), glypican-1 (GPC-1), and matrix metalloproteinase-9 (MMP-9), and their association with clinicopathologic characteristics as well as prognostic significance in pancreatic cancer. Immunohistochemical assessment of GDNF, GPC-1, and MMP-9 was performed in 62 cases of surgically resected pancreatic cancer. Perineural invasion in pancreatic cancer was observed by marking nerve fiber with S-100, while 16 normal pancreatic tissues were used as normal control. Correlations of GDNF, GPC-1 and MMP-9 expressions with clinicopathologic parameters were analyzed. A survival analysis was performed to find the prognostic significance. The expressions of GDNF, GPC-1 and MMP-9 in pancreatic cancer tissue were significantly higher than of those in normal pancreatic tissues (41/62 vs. 5/16 for GDNF, 35/62 vs. 2/16 for GPC-1, and 37/62 vs. 3/16 for MMP-9; p<0.01, respectively). The overexpression of GDNF, GPC-1, and MMP-9 in pancreatic cancer tissue was significantly related to the perineural invasion (p<0.05). Although the overexpression of these genes was related to poor survival, GPC-1 had an independent prognostic effect on overall survival. GPC-1 is significantly related to the perineural invasion of pancreatic cancer, holding some prognostic significance in patients with pancreatic cancer.

The actin binding protein destrin is associated with growth and perineural invasion of pancreatic cancer

Background/ObjectivesThe small actin-binding protein destrin is one of the key regulators involved in remodeling of the actin cytoskeleton, a process crucial for cytokinesis, cell migration and polarized cell growth as well as for cancer cell migration and invasion. MethodsA novel ex vivo nerve invasion model mirroring perineural cancer cell invasion as a key feature of pancreatic ductal adenocarcinoma has been previously established. Using this model, highly nerve-invasive clones of human pancreatic cancer cell lines have been obtained. Genome-wide transcriptional analyses of these cells revealed up-regulation of destrin in highly versus lowly nerve-invasive pancreatic cancer cells. ResultsIncreased expression of destrin in these nerve-invasive cells was validated using quantitative RT-PCR and immunoblotting; concomitant changes in cell morphology were demonstrated using immunofluorescence analysis. Silencing of destrin by two specific siRNA oligonucleotides in Panc-1 pancreatic cancer cells decreased invasiveness and migration, and reduced proliferation of these cells. ConclusionsDestrin is upregulated in nerve-invasive pancreatic cancer cells and its expression might be related to perineural invasiveness.

P-0095 Sonic Hedgehog Paracrine Signaling Activates Pancreatic Stellate Cells to Promote The Perineural Invasion of Pancreatic Cancer in Vivo

IntroductionPerineural invasion (PNI) in pancreatic adenocarcinoma (PADC) is a common pathological phenomenon in which cancer cells invade and intimately contact the endoneuria of pancreatic nerves and is thought to contribute to both pain and local disease recurrence. PNI is an important prognostic factor for PADC that increases as the cancer becomes undifferentiated and predominantly existed in the perineurium and perineural space. It has been shown that the tumor-stroma microenvironment is an important regulator of cancer cell behavior. We hypothesized that the adjacent pancreatic stellate cells (PSC) activated by paracrine sonic hedgehog (SHH) which derived from PADC cells play important roles in creating a dynamic environment that supports pancreatic tumor growth and invasion, thus ultimately induced PNI. This study is aimed to evaluate the important function of pancreatic stellate cells in PNI of PADC. MethodsAsPc-1, a human PADC line expressing high Shh protein were stably transfect with SHH shRNA or empty plasmid. Stable transfectants were confirmed by western blot. Six-week-old male athymic nude mice were anesthetized with inhalational methoxyflurane for all procedures and the left sciatic nerve was exposed deep to the femoracoccygeus and biceps femoris muscles. Cells were microscopically injected into the perineurium of the sciatic nerve, distal to the bifurcation of the tibial and common peroneal nerves. 3 µL of AsPc-1 or AsPc-1-SHH- and PSC suspension at a concentration of 1×108/&mgr;L was co-injected slowly. Sciatic nerve–infiltrating tumors were established and animals were observed for up to 6 weeks for assessment of tumor size, nerve function including the gross behavior, the limb function, and sciatic nerve function index. ResultsAfter 3 weeks of injection, the nerve function including the gross behavior, the limb function, and sciatic nerve function index were worse in the AsPc-1 plus PSC group compared with the AsPc-1-SHH- plus PSC or AsPc-1 alone group (P<0.05). Additionally, the tumor diameter at the implantation site was larger in the AsPc-1 plus PSC group compared with the AsPc-1-SHH- plus PSC or AsPc-1 alone group (P<0.05) due to massive tumor growth. Furthermore, the proximal nerve diameter was larger in the AsPc-1 plus PSC group compared with the AsPc-1-SHH- plus PSC or AsPc-1 alone group (P<0.05) due to extensive infiltration by cancer cells. ConclusionThese data provide evidence for SHH, derived from pancreatic tumor epithelia, may be critical in regulating the tumor microenvironment and thereby contributes to PNI of PADC, which may have important implications for potential therapy directed against PNI.

Pancreatic stellate cells promotes the perineural invasion in pancreatic cancer

Perineural invasion is a prominent characteristic of pancreatic cancer. Pancreatic cancer has an extremely high incidence of perineural invasion which has been associated with poor survival. Early studies mostly focus on the interaction between cancer cells and nerves. Recently, the effect of pancreatic stellate cells in progression of pancreatic cancer has been paid more attention. Both in vitro studies and in vivo ones revealed that pancreatic stellate cells can enhance the proliferation, migration and invasion of pancreatic cancer cells. Pancreatic stellate cells can also regulate the expression and effect of molecules involved in perineural invasion. In addition, pancreatic stellate cells seems to associated with the generation of neuronal plasticity in pancreatic cancer. Herein the hypothesis that pancreatic stellate cells play a potential role in promote the perineural invasion in pancreatic cancer through three mechanisms. One is that pancreatic stellate cells enhance the proliferation, migration and invasion directly through releasing a variety of stimuli and providing a suitable microenvironment. Pancreatic stellate cells also regulate the expression and effects of molecules involved in perineural invasion such as nerve growth factor. Another is that pancreatic stellate cells induce neuronal plasticity, which makes nerves more vulnerable to be invaded. We can conclude that pancreatic stellate cells play a central role in regulating the perineural invasion process by producing different effects on cancer cells and nerve. To inhibit the activity of pancreatic stellate cells or block the interaction between pancreatic stellate cells and cancer cells or nerve tissue might reduce the perineural invasion in pancreatic cancer.

Abstract B152: Modulating the activity of nerve growth factor (NGF) and its receptors tropomyosin-related kinase A (TRKA) and p75 neurotrophin receptor (p75NTR) in perineural invasion (PNI) in pancreatic cancer.

Abstract Pancreatic cancer is the fourth leading cause of cancer-related death in the US with a 5-year survival rate of less than 5%. Even after surgery and chemotherapy, prognosis is dismal due to tumor re-occurrence. Perineural Invasion (PNI) is a process where surrounding pancreatic nerves are invaded by pancreatic cancer cells. This invasive and proliferative ability of pancreatic cancer cells is indicative of their aggressiveness and is shown to be associated with poor prognosis and pain in pancreatic cancer patients. The nerve growth factor (NGF) signaling pathway is implicated in PNI and NGF binds to a high-affinity receptor, tropomyosin kinase A (TRKA), which is differentially expressed in pancreatic tumor tissues and a low-affinity receptor, p75 neurotrophin receptor (p75NTR), whose role in PNI in pancreatic cancer is unclear. In this study, we seek to understand the molecular mechanisms of NGF regulation of PNI in pancreatic cancer. Whether NGF regulates PNI via an autocrine or paracrine manner is not well understood. Signaling via NGF-TRKA pathway induces neurites in the PC-12 neurite extension assay and conditioned media (CM) from several pancreatic cancer cell lines including BxPC3 induced neurites in this assay. Addition of an antibody to neutralize NGF in the CM or an azaoxindole inhibitor (GW441756) of TRKA resulted approximately in a 50% and 40% decrease in neurites respectively in the PC-12 assay. These results could indicate a paracrine action of NGF in promoting neurite extension in the PC-12 assay and perhaps nerve growth as related to PNI. Furthermore, knocking down the expression of NGF, p75NTR, TRKA using siRNA or inhibitor GW441756 of TRKA decreased migration of BxPC3 pancreatic cancer cells in the Boyden chamber assay by 45 − 60%. A similar inhibitory effect was observed in the movement of pancreatic cancer cells towards dorsal root ganglia (DRG) in a DRG-pancreatic cancer cell co-culture assay. Thus, NGF may function in an autocrine manner to increase proliferation and invasiveness of pancreatic cancer cells in PNI. Currently, we are working to further optimize our assays to delineate the interplay between NGF and TrkA (autocrine or paracrine) in PNI and understand the involvement of p75NTR (low-affinity NGF receptor) in NGF signaling and consequently in PNI. Understanding the molecular mechanisms involved in PNI will aid in designing novel therapeutics that prevent PNI and alleviate pain associated with PNI in patients with pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B152.

Perineural invasion in pancreatic cancer: Advanced research in the neuro-cancer interactions

Pancreatic Cancer (PCa) is characterized by prominently local nerve alterations and perineural invasion (PNI), which frequently aff ects the extrapancreatic nerve plexus, causing severe pain and retropancreatic tumor extension. It precludes curative resection, promotes local recurrence, and at the last negatively influences the prognosis of patients. Recent research on PNI in PCa has revealed the critical involvement of numerous nerve- or cancer cell-derived molecules in vitro and in vivo. However, the mechanisms contributing to alteration and invasion of intrapancreatic nerves and the spread of cancer cells along extrapancreatic nerves in pancreatic cancer patients are still poorly understood. This review focuses on perineural invasion in pancreatic cancer and provides an outline of the characteristics and molecular mechanisms of perineural invasion in pancreatic cancer.

Association of diabetes with perineural invasion and overall survival in surgically resected patients with pancreatic cancer.

4117 Background: Hyperglycemia has been hypothesized to promote perineural invasion through enhanced proliferation of cancer cell and subsequent production of cytokines such as nerve growth factors. Hyperglycemia may also cause demyelinization and axonal degeneration of nerves, which can form defections to make cancer cells enter nerves with deep invasion. The purpose of this study was to test the associations between perineural invasion, diabetes, and survival in patients with pancreatic adenocarcinoma. Methods: We retrospectively reviewed the medical records of 344 patients who underwent resection of pancreatic cancer between 2004 and 2008 at M. D. Anderson Cancer Center. Data recorded included history of diabetes prior to cancer diagnosis, presence of perineural invasion, and AJCC TNM stage. Overall survival time was calculated from the date of pancreatic cancer diagnosis to the date of death. The association between diabetes and perineural invasion was tested using the chi square test. Median survival (MS) was compared using the Kaplan-Meier plot, log-rank test, and Cox proportional hazard model. Results: Ninety-two (26.8%) of 344 patients studied had diabetes. The frequency of perineural invasion was significantly higher in patients with diabetes (94.6%) than in non-diabetics (86.5%) (p = 0.037). The median survival time (MST) was 30.2 versus 20.1 months for non-diabetic and diabetic patients, respectively (p = 0.024), and 27.2 versus 40.3 months for those with or without perineural invasion, respectively (p = 0.002). Both factors remained as significant independent predictor for survival after adjusting for AJCC TNM stage. The hazard ratio (95% confidence interval) was 1.36 (1.03-1.80) for diabetes (p = 0.032) and 1.67 (1.01-2.76) for perineural invasion (p = 0.047). Conclusions: Our finding supports the hypothesis that diabetes promotes perineural invasion in pancreatic cancer, and both diabetes and perineural invasion predict poor survival. Understanding the mechanisms underlying the association between diabetes and perineural invasion could lead to novel therapeutic targets. No significant financial relationships to disclose.

Consensus transcriptome signature of perineural invasion in pancreatic carcinoma

Perineural invasion, the growth of tumor cells along nerves, is a key feature of pancreatic cancer. The cardinal symptom of pancreatic cancer, abdominal pain often radiating to the back, as well as the high frequency of local tumor recurrence following resection are both attributed to the unique ability of pancreatic tumor cells to invade the neuronal system. The molecular mechanisms underlying the neuroaffinity of pancreatic tumors are not completely understood. In this study, we developed a novel method to monitor ex vivo perineural invasion into surgically resected rat vagal nerves by different human pancreatic tumor cell lines. Genome-wide transcriptional analyses were employed to identify the consensus set of genes differentially regulated in all highly nerve-invasive (nerve invasion passage 3) versus less invasive (nerve invasion passage 0) pancreatic tumor cells. The critical involvement of kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta) in perineural invasion was confirmed on RNA and protein levels in human pancreatic tumor specimens. We found significant up-regulation of KIF14 and ARHGDIbeta mRNA levels in patients with pancreatic cancer, and both proteins were differentially expressed in tumor cells invading the perineural niche of pancreatic cancer patients as detected by immunohistochemistry. Moreover, functional knockdown of KIF14 and ARHGDIbeta using small interfering RNA resulted in altered basal and/or perineural invasion of pancreatic tumor cells. Our work provides novel insights into the molecular determinants of perineural invasion in pancreatic cancer. The established nerve invasion model and the consensus signature of perineural invasion could be instrumental in the identification of novel therapeutic targets of pancreatic cancer as exemplified by KIF14 and ARHGDIbeta.

Expression of nerve growth factor and tyrosine kinase receptor A and correlation with perineural invasion in pancreatic cancer

Perineural invasion (PNI) is one of the most common routes of invasion in pancreatic cancer and the exact mechanism is still not clear. The aim of the present study was to investigate the effect of nerve growth factor (NGF) and tyrosine kinase receptor A (TrkA) on PNI and to clarify the possible mechanism of PNI in pancreatic cancer. Expressions of NGF/TrkA were examined in 51 human primary pancreatic cancer using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). The molecular findings were correlated with PNI, clinicopathological parameters and expression of Ki-67. Immunohistochemical analysis indicated that the presence and kind of PNI are prognostic parameters (P = 0.002,, P = 0.004). Tumors with high NGF expression exhibited more frequent presence of PNI (P = 0.033). NGF expression was significantly correlated with metastasis of lymph nodes and involvement of surgical margins (P = 0.006, 0.015). TrkA expression was significantly correlated with degree of PNI (P = 0.017). Negative correlations were found between expression of NGF/TrkA and Ki-67. As shown by RT-PCR, mRNA levels of NGF/TrkA with PNI were significantly higher than that without PNI. In pancreatic cancer, overexpression of NGF may contribute to PNI by prompting the hyperplasia of nerves, restraining the apoptosis of tumor cells and specifically combining NGF and TrkA.

Patterns of expression and function of the p75 NGFR protein in pancreatic cancer cells and tumours

Backgrounds/aims Pancreatic carcinoma is one of the most aggressive human malignancies. The aggressive and highly metastatic behaviour of pancreatic carcinoma may partly be attributable to the autocrine and/or paracrine interactions involving altered expression of neurotrophin growth factors and their corresponding receptors. The aim of the present study is to investigate the expression pattern and function of the p75 NGFR protein in pancreatic cancer cell lines and tumours to explain the phenomenon of perineural invasion in pancreatic cancer. Methods The expression of p75 NGFR in 137 pancreatic adenocarcinoma samples and the corresponding adjacent pancreatic samples was examined immunohistochemically using the EnVision Plus System. Then we examined the in vitro chemotaxis behaviour of cancer cells transfected with p75 NGFR plasmid to nerve growth factor (NGF). Results Immunostaining for p75 NGFR was weak or absent in both normal pancreata and pancreatic carcinoma tissues; however, the immunostaining was relatively weaker in the pancreatic carcinoma tissues than in the normal pancreata. It is interesting to note that p75 NGFR expression in the cancer tissues was positively correlated with the degree of perineural invasion (χ 2 = 32.94, P < 0.01). The chemotaxis ability of the p75 NGFR-transfected pancreatic cancer cells to NGF was significantly stronger than that of the non-transfected or vacant vector transfected cells ( P < 0.01). Conclusions Our findings indicate that p75 NGFR expression may be involved in the perineural invasion of pancreatic cancer cells, and the mechanism might be through mediating the chemoattraction of cancer cells for neural tissues.

Hyperglycemia promotes the perineural invasion in pancreatic cancer

The role of hyperglycemia in perineural invasion in pancreatic cancer is not clear. Pancreatic cancer is characterized by extremely high frequency of perineural invasion (can be as high as 90%, even 100%), which has been associated with poorer survival. In previous epidemiologic, the prevalence of diabetes mellitus in pancreatic cancer is 34-40% and more than half of them are new-onset, which means the course of disease of diabetes mellitus is less than 24 months before cancer diagnosis. The association between diabetes mellitus and pancreatic cancer has long been recognized as that long-standing diabetes mellitus is thought to be an etiologic factor for pancreatic cancer and new-onset diabetes mellitus may be a manifestation of the cancer. Long-standing diabetes mellitus can cause peripheral neuropathy. The main morphological features of established neuropathy include a combination of demyelinization and axonal degeneration of myelinated fibers, degeneration with regeneration of unmyelinated fibers and endoneurial microangiopathy, with nerve fiber loss in its final stage. Diabetes mellitus can also induce the high expression of cytokines such as nerve growth factor to repair the damaged nerves. We present the hypothesis that hyperglycemia promotes the perineural invasion in pancreatic cancer through two mechanisms. One is that hyperglycemia enhances the proliferation of cancer cells, which subsequently increase the expression of cytokines such as nerve growth factor. The overexpression of nerve growth factor can enhance the interaction between nerve and cancer cell and neurotropism. The other is that hyperglycemia causes demyelinization and axonal degeneration of nerves, which can form defections to make cancer cells enter nerves with deeply invasion. The above two mechanisms can promote the perineural invasion in pancreatic cancer. Controlling hyperglycemia might reduce the perineural invasion in pancreatic cancer.

MUC1 Is a Counter-Receptor for Myelin-Associated Glycoprotein (Siglec-4a) and Their Interaction Contributes to Adhesion in Pancreatic Cancer Perineural Invasion

Perineural invasion in pancreatic adenocarcinoma, a common pathologic phenomenon whereby cancer cells invade and intimately contact the endoneurium of pancreatic nerves, is thought to contribute to both pain and local disease recurrence. MUC1, a type I transmembrane mucin that can affect the adhesive properties of cells, contains a large extracellular tandem repeat domain, which is heavily glycosylated in normal epithelia, but is overexpressed and differentially glycosylated in pancreatic cancer. This altered glycosylation includes the shortened core I O-glycans for monosialyl and disialyl T antigens. Myelin-associated glycoprotein (MAG), a membrane-bound protein expressed on oligodendrocytes and Schwann cells, binds myelin to neurons. MAG's preferred ligands are derivatives of the monosialyl and disialyl T antigen. We investigated whether MUC1 is a counter-receptor for MAG and if their interaction contributed to pancreatic perineural invasion. Results showed that MAG binds pancreatic cells expressing MUC1, that this binding is sialidase-sensitive, and that MAG physically associates with MUC1. Heterotypic adhesion assays between pancreatic cancer cells and Schwann cells revealed that increased expression of MUC1 or MAG enhanced adhesion. Conversely, specific inhibition of MAG or sialyl-T MUC1 partially blocked adhesion. Immunohistochemical analysis of pancreatic perineural invasion showed the expression of both MUC1 and MAG. These results support the hypothesis that the adhesive interactions between MUC1 and MAG are of biological significance in pancreatic cancer perineural invasion.

Technical limitations of lymph node mapping in pancreatic cancer

Aim The high incidence of lymphatic and peri-neural invasion in pancreatic cancer results in poor loco-regional control. Radical pancreatico-duodenectomy may achieve better loco-regional control, but is accompanied by increasing morbidity. Our hypothesis was that if intra-operative mapping of pathological lymph nodes (LN) is technically feasible in pancreatic cancer, it would allow for selective radical resection. Methods In an ethically approved and statistically powered feasibility study of 72 (stopped after 20% enrollment) patients with suspected pancreatic cancer undergoing resection, we injected methylene blue dye peri- and intra-tumorally and studied its progress to identify putative ‘sentinel lymph node(s)’. The Kausch–Whipple procedure (or total pancreatectomy, if required) was carried out in addition to radical LN dissection, which was evaluated histopathologically according to the Japanese criteria. Results Over 18 months, 14/16 patients prospectively recruited underwent lymph node mapping and a mean of 20 (range 11–37) LNs per patient were harvested. Methylene blue dye injection identified blue LN(s) in 4/14 patients, none of which were positive for malignant deposits, whilst 10/14 patients had LN metastases. The commonest stations for LN metastasis were 17A or B (9/10), 8A (2/10) and 6 (3/10). The median survival for the 13 patients with cancer was 22.3 months (IQR: 10.4–30 months). Conclusion Sentinel lymph node mapping is not technically feasible in pancreatic cancer.

Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach

Pancreatic cancer (PanCa) is characterized by perineural invasion (PNI), early lymph node and liver metastasis, and poor prognosis. PNI is one of the important causes of local recurrence. Little is known about the mechanism of PNI in PanCa. We presented a novel model system that may shed light on the mystery of PNI in PanCa. In this study, mouse dorsal root ganglia (DRGs) and human PanCa cell line (MIA PaCa-2) were cocultured in Matrigel matrix (BD Biosciences, San Jose, CA) to build this PNI model. MIA PaCa-2 cell line alone (control 1) or DRG alone (control 2) was cultured with Matrigel matrix as controls. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were observed under inverted microscopy and then were photographed and quantitated with the Optimas imaging system (Optimas Corp., Bothell, MA). At day 14, both the experimental and control 2 samples were harvested and subjected to total RNA isolation and fixed in paraffin-embedded blocks. Slides cut from paraffin blocks were studied with Ki-67 immunostaining and TUNEL assay. Gene profiling was performed using complementary DNA microarray. Overexpressed target genes were verified by quantitative reverse transcriptase polymerase chain reaction. The results showed that reciprocity was observed between neurites and MIA PaCa colonies with 24 hours of coculture. Neurite outgrowth was stimulated in the presence of pancreatic carcinoma cells, which showed 2-fold more area than did control 2. After 72 hours, MIA PaCa colonies cocultured with DRG exhibited 58% more colony area than did control 1. The Ki-67 index of the DRG/MIA PaCa cells (mean, 5.02%) was significantly higher than that in control 1 (mean, 1.18%) (P < .05); in contrast, the apoptotic index in the DRG/MIA PaCa cells was significantly lower (mean, 0.45%) than that in the control 1 (mean, 1.85%) (P < .001). Prosurvival genes MALT1 and TRAF were increased 2-fold in DRG/MIA PaCa compared with controls. We demonstrated that neural-epithelial interaction is a mutually beneficial process for the growth of nerves and PanCa cells. It is possible that oncogenes and growth factors might act synergistically in promoting proliferation and/or inhibiting apoptosis, a survival strategy crucial to the development of PNI in PanCa.

Establishment of Perineural Invasion Models and Analysis of Gene Expression Revealed an Invariant Chain (CD74) as a Possible Molecule Involved in Perineural Invasion in Pancreatic Cancer

Perineural invasion causes frequent local recurrence even after resection and a poor prognosis for pancreatic cancer. We established perineural invasion models and analyzed the molecular mechanism of perineural invasion in pancreatic cancer. Seven pancreatic cancer cell lines with or without human peripheral nerves were s.c. implanted in nonobese diabetes/severe combined immunodeficient mice. We compared expression profiles among high and low perineural invasion cell lines by using an oligonucleotide microarray. We examined up-regulation of the invariant chain (CD74) in high perineural invasion cell lines in mRNA and protein levels and surgical cases immunohistochemically. Four of seven pancreatic cancer cell lines (CaPan1, CaPan2, CFPAC, and MPanc96) showed perineural invasion to s.c. transplanted human peripheral nerves. Moreover, CaPan1 and CaPan2 (high perineural invasion group) also resulted in a high frequency of perineural invasion to mouse s.c. peripheral nerves, whereas three pancreatic cancer cell lines HPAFII, AsPC1, and Panc1 (low perineural invasion group) did not show perineural invasion to either human or mouse nerves. We identified 37 up-regulated genes and 12 down-regulated genes in the high perineural invasion group compared with the low perineural invasion group. Among them, CD74 was up-regulated in the high perineural invasion group in mRNA and protein levels. Furthermore, immunohistochemical expression of CD74 in clinical cases revealed its significant overexpression in pancreatic cancer with perineural invasion (P < 0.008). This is the first report of perineural invasion models using human pancreatic cancer cell lines. In combination with gene expression profiling, it was indicated that CD74 could be a candidate molecule involved in perineural invasion. These models provide new approaches for study of perineural invasion in pancreatic cancer.