Tumor spread along nerves, a phenomenon known as perineurial invasion, is common in various cancers including pancreatic ductal adenocarcinoma (PDAC).
Neural invasion is associated with poor outcome, yet its mechanism remains unclear.
Using the transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we investigated the mechanism of neural invasion in PDAC. To detect tissue-specific factors that influence neural invasion by cancer cells, we characterized the perineurial microenvironment using a series of bone marrow transplantation (BMT) experiments in transgenic mice expressing single mutations in the Cx3cr1, GDNF and CCR2 genes. Immunolabeling of tumors in KPC mice of different ages and analysis of human cancer specimens revealed that RET expression is upregulated during PDAC tumorigenesis. BMT experiments revealed that BM-derived macrophages expressing the RET ligand GDNF are highly abundant around nerves invaded by cancer. Inhibition of perineurial macrophage recruitment, using the CSF-1R antagonist GW2580 or BMT from CCR2-deficient donors, reduced perineurial invasion. Deletion of GDNF expression by perineurial macrophages, or inhibition of RET with shRNA or a small-molecule inhibitor, reduced perineurial invasion in KPC mice with PDAC. Taken together, our findings show that RET is upregulated during pancreas tumorigenesis and its activation induces cancer perineurial invasion. Trafficking of BM-derived macrophages to the perineurial microenvironment and secretion of GDNF are essential for pancreatic cancer neural spread.
Introduction
Perineurial invasion (PNI) by cancer is associated with poor prognosis in patients with carcinomas of the gastrointestinal tract, head and neck, pancreas and prostate.1 Most patients with pancreatic ductal adenocarcinoma (PDAC) undergo palliative treatment rather than curative surgery, due to distant metastases or neural spread along extra pancreatic nerves. Most importantly, these patients tend to suffer from debilitating neuropathic pain and poor quality of life.2
The prevalence of PNI varies considerably among cancer types, and reaches 80–100% in pancreatic cancer.3, 4 Recent studies have suggested that the tumor microenvironment has a role in cancer dissemination along nerves.5, 6 A prominent inflammatory infiltration is present around preinvasive pancreatic lesions and persists through invasive cancer.7 Of relevance, there is evidence that the neural microenvironment of invaded nerves has a unique inflammatory profile.8, 9
Recently, we and others described the presence of immunocytes in the perineurial niche.8, 9, 10 Clinical and experimental studies have demonstrated a strong association between macrophage density and cancer cell metastasis in PDAC. Furthermore, targeting tumor-associated macrophages by inhibiting either the colony-stimulating factor-1 receptor (CSF1R) or chemokine (C-C motif) receptor 2 (CCR2) improves chemotherapeutic efficacy and inhibits metastasis.11, 12, 13, 14 In the normal nerve, macrophages that can be derived from either resident immune cells or from circulating monocytes participate in regeneration of peripheral nerves.9, 15 However, in the nerve-cancer microenvironment, the origin and polarization of these macrophages, as well as their role in PNI, is not well defined.
It has been suggested that glial-derived neurotrophic factor (GDNF) can also promote PNI.16, 17, 18 GDNF expression in human samples was also associated with PNI and reduced survival.19 Immunofluorescent imaging revealed expression of the GDNF receptor, GDNF family receptor α-1 (GFRα1) and its co-receptor RET by pancreatic cancer cells.9 Although previous clinical and experimental data implicated the involvement of RET in PNI, no direct evidence links RET activity with PNI in transgenic animal models or in patients with PDAC. To detect tissue-specific factors influencing neural invasion by cancer cells, we characterized the perineurial environment in human samples and in a series of transgenic mice models expressing mutations in the Kras, p53, GDNF and CCR2 genes.
The current study provides direct evidence that upregulation and activation of RET by perineurial macrophages induce perineurial spread of PDAC.
Results
Macrophages are a prominent component of the perineurial microenvironment
The patterns of inflammatory response secondary to pancreatic tumorigenesis are distinct from those of chronic pancreatitis.8, 11 Furthermore, an immune cell profile is stage-dependent in many types of cancer.20, 21, 22 To investigate the involvement of immunocytes in the perineurial microenvironment during tumorigenesis, we evaluated pancreata excised from 2-, 3- and 6-month-old KPC mice with normal pancreas, PanIN and PDAC, respectively. Immunofluorescent analysis revealed significantly greater infiltration of lymphocytes around nerves in PDAC and PanINs than around nerves in normal pancreas (P<0.01, n=10 per group, Figures 1a and b). Macrophage infiltration was more prominent around nerves invaded by cancer than around nerves in PanIN lesions or normal pancreas (P<0.001, Figures 1a and b). Hence, recruitment of immunocytes to the neural niche occurs during pancreatic carcinogenesis.
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