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Perineural invasion (PNI) is an ominous form of cancer progression along nerves associated with poor clinical outcome. Glial derived neurotrophic factor (GDNF) interacts with cancer cell RET receptors to enable PNI, but downstream events remain undefined. We demonstrate that GDNF leads to early activation of the GTPase Cdc42 in pancreatic cancer cells, but only delayed activation of RhoA and does not affect Rac1. Depletion of Cdc42 impairs pancreatic cancer cell chemotaxis toward GDNF and nerves. An siRNA library of guanine nucleotide exchange factors was screened to identify activators of Cdc42. ARHGEF7 (β-Pix) was required for Cdc42 activation and chemotaxis toward nerves, and also colocalizes with RET under GDNF stimulation. Cdc42 enables PNI in an in vitro dorsal root ganglia coculture model, and controls the directionality of migration but does not affect cell speed or cell viability. In contrast, Rac1 was necessary for cell speed but not directionality, while the RhoA was not necessary for either cell speed or directionality. Cdc42 was required for PNI in an in vivo murine sciatic nerve model. Depletion of Cdc42 significantly diminished the length of PNI, volume of PNI, and motor nerve paralysis resulting from PNI. Activated Cdc42 is expressed in human salivary ductal cancer cells invading nerves. These findings establish the GDNF–RET–β-Pix–Cdc42 pathway as a directional regulator of pancreatic cancer cell migration toward nerves, highlight the importance of directional migration in PNI, and offer novel targets for therapy. Implications: Cdc42 regulates cancer cell directional migration toward and along nerves in PNI.

Perineural invasion (PNI) is an ominous event strongly linked to poor clinical outcome. Cells residing within peripheral nerves collaborate with cancer cells to enable PNI, but the contributing conditions within the tumor microenvironment are not well understood. Here, we show that CCR2-expressing inflammatory monocytes (IM) are preferentially recruited to sites of PNI, where they differentiate into macrophages and potentiate nerve invasion through a cathepsin B–mediated process. A series of adoptive transfer experiments with genetically engineered donors and recipients demonstrated that IM recruitment to nerves was driven by CCL2 released from Schwann cells at the site of PNI, but not CCL7, an alternate ligand for CCR2. Interruption of either CCL2–CCR2 signaling or cathepsin B function significantly impaired PNI in vivo. Correlative studies in human specimens demonstrated that cathepsin B–producing macrophages were enriched in invaded nerves, which was associated with increased local tumor recurrence. These findings deepen our understanding of PNI pathogenesis and illuminate how PNI is driven in part by corruption of a nerve repair program. Further, they support the exploration of inhibiting IM recruitment and function as a targeted therapy for PNI. Cancer Res; 77(22); 6400–14. ©2017 AACR.

Recent studies have demonstrated a critical role for nerves in enabling tumor progression. The association of nerves with cancer cells is well established for a variety of malignant tumors, including pancreatic, prostate and the head and neck cancers. This association is often correlated with poor prognosis. A strong partnership between cancer cells and nerve cells leads to both cancer progression and expansion of the nerve network. This relationship is supported by molecular pathways related to nerve growth and repair. Peripheral nerves form complex tumor microenvironments, which are made of several cell types including Schwann cells. Recent studies have revealed that Schwann cells enable cancer progression by adopting a de-differentiated phenotype, similar to the Schwann cell response to nerve trauma. A detailed understanding of the molecular and cellular mechanisms involved in the regulation of cancer progression by the nerves is essential to design strategies to inhibit tumor progression.

Nerves enable cancer progression, as cancers have been shown to extend along nerves through the process of perineural invasion, which carries a poor prognosis. Furthermore, the innervation of some cancers promotes growth and metastases. It remains unclear, however, how nerves mechanistically contribute to cancer progression. Here, we demonstrated that Schwann cells promote cancer invasion through direct cancer cell contact. Histological evaluation of murine and human cancer specimens with perineural invasion uncovered a subpopulation of Schwann cells that associates with cancer cells. Coculture of cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner. Upon contact, Schwann cells induced the formation of cancer cell protrusions in their direction and intercalated between the cancer cells, leading to cancer cell dispersion. The formation of these processes was dependent on Schwann cell expression of neural cell adhesion molecule 1 (NCAM1) and ultimately promoted perineural invasion. Moreover, NCAM1-deficient mice showed decreased neural invasion and less paralysis. Such Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead exploited by cancer cells to promote perineural invasion and cancer progression.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

The ability of cancer cells to invade along nerves is associated with aggressive disease and diminished patient survival rates. Perineural invasion (PNI) may be mediated by nerve secretion of glial cell line-derived neurotrophic factor (GDNF) attracting cancer cell migration through activation of cell surface Ret proto-oncogene (RET) receptors. GDNF family receptor (GFR)α1 acts as coreceptor with RET, with both required for response to GDNF. We demonstrate that GFRα1 released by nerves enhances PNI, even in the absence of cancer cell GFRα1 expression. Cancer cell migration toward GDNF, RET phosphorylation, and MAPK pathway activity are increased with exposure to soluble GFRα1 in a dose-dependent fashion. Dorsal root ganglia (DRG) release soluble GFRα1, which potentiates RET activation and cancer cell migration. In vitro DRG coculture assays of PNI show diminished PNI with DRG from GFRα1(+/-) mice compared with GFRα1(+/+) mice. An in vivo murine model of PNI demonstrates that cancer cells lacking GFRα1 maintain an ability to invade nerves and impair nerve function, whereas those lacking RET lose this ability. A tissue microarray of human pancreatic ductal adenocarcinomas demonstrates wide variance of cancer cell GFRα1 expression, suggesting an alternate source of GFRα1 in PNI. These findings collectively demonstrate that GFRα1 released by nerves enhances PNI through GDNF-RET signaling and that GFRα1 expression by cancer cells enhances but is not required for PNI. These results advance a mechanistic understanding of PNI and implicate the nerve itself as a key facilitator of this adverse cancer cell behavior.

Perineural invasion (PNI) by cancer cells is associated with significant patient morbidity and poor prognosis. Despite recognition of the clinical significance of PNI, the mechanisms underlying it remain poorly understood and no targeted therapies exist to date. Modern theories of PNI pathogenesis have placed significant focus on the active role of the nerve microenvironment with PNI resulting from well-orchestrated interactions between cancer and host. Inflammatory monocytes (IMs) play a critical role in mediating peripheral nerve repair and facilitating cancer metastases by differentiating into tumor-associated macrophages. Here we explore their role in PNI. CCR2 expressed by IMs is a receptor for ligands CCL2 and CCL7, and mediates IM recruitment in infections and other cancer models. A murine model of nerve invasion in which pancreatic cancer (Panc02) is injected into the distal sciatic nerve, was utilized in mice genetically deficient in CCL2, CCL7, or CCR2. Validated functional and histopatholgic endpoints were used to quantify PNI. Fluorescence-activated cell sorting (FACS) and immunohistochemistry (IHC) were performed on specimens to assess IM and macrophage recruitment. Adoptive transfer experiments with labeled IMs were performed to elucidate the signaling pathways involved in homing to the nerve. Genetically modified green fluorescent protein (GFP)-tagged reporter mice were utilized to identify cellular sources of the relevant chemokines. IHC was performed on human tumors with PNI to assess for macrophage recruitment. The recruitment of IMs is dependent on CCL2 synthesized within the nerve. IMs recruited to the nerve differentiate into macrophages within 24-72 hours upon arrival. Mice deficient in CCL2 or CCR2 have impaired IM recruitment and reduced macrophage differentiation in the nerve at the site of PNI, and significantly impaired invasion. Furthermore, PNI was restored in CCR2 KO mice after multiple adoptive transfers of wild type IMs with intact CCR2. By contrast, CCL7 KO mice have only a modest, non-significant decrease in PNI. In the presence of cancer, CCL2 but not CCL7, is expressed locally in the nerve, likely by Schwann cells responding to the cancer invasion. IMs recruited to the nerve serve as an alternate source of CCL2. Human tumors with PNI demonstrate CD68+ macrophages at the site of nerve invasion. These results implicate the immune response as a key mediator of PNI and thereby further our mechanistic understanding of this ominous cancer behavior. Our data identify potential novel therapeutic targets for treating PNI. Citation Format: Richard Bakst, Huizhong Xiong, Chun-Hao Chen, Sylvie Deborde, Yi Zhou, William McNamara, Sei Young Lee, Eric Pamer, Richard J. Wong. CCL2 recruits inflammatory monocytes to facilitate perineural invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3167. doi:10.1158/1538-7445.AM2015-3167

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Perineural invasion (PNI) is the ability of cancer cells to invade in, around, and along nerves. PNI is widely recognized as an adverse pathologic feature of many malignancies. Glial-derived neurotrophic factor (GDNF) is secreted by nerve and glial cells, and is important in neural development. We previously showed that GDNF activation of the RET/GFR-alpha tyrosine kinase in cancer cells induces chemotaxis towards nerves and PNI. Cdc42 is a member of the Rho family of G proteins and a key regulator of cell polarity. We demonstrate here that GDNF-mediated activation of RET/GFR-alpha leads to activation of Cdc42 as a mediator of cancer cell chemotaxis and PNI. Exposure of MiaPaCa2 cells to GDNF or dorsal root ganglia (DRG) activates Cdc42. SiRNA inhibition of Cdc42 impairs MiaPaCa2 chemotaxis towards GDNF or DRG in Boyden chamber assays. SiRNA inhibition of RET decreases Cdc42 activity under GDNF stimulation and MiaPaCa2 chemotaxis. To study dynamic interactions between nerves and cancer cells in vitro, we used a DRG and cancer cell co-culture model of PNI in matrigel. Analysis of individual cell trajectories reveals that cell velocity remains unchanged, while directional migration is disrupted with shRNA inhibition of Cdc42. The disruption of directional response impairs the ability of shRNA Cdc42 MiaPaCa2 cells to migrate along neurites, as visualized by time lapse microscopy and measured by area of PNI. MRI imaging of live mice with sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells reveals significantly diminished length and volume of PNI as compared with injected shRNA control cancer cells when matched for equivalent overall tumor volume. Murine sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells exhibited preserved hindlimb and hind paw neurologic function, and diminished perineural invasion by histopathologic examination as compared with shRNA control MiaPaCa2. To identify potential activators of Cdc42 in this model, we performed a comprehensive siRNA screen of guanine nucleotide exchange factors (GEFs), identifying specific GEFs which are necessary for MiaPaCa2 migration towards DRG in Boyden chambers. We identified and validated six GEFs ([FLJ10521][1], DOCK9, ARHGEF7, RASGRF1, ARHGEF5, PLEKHG1) that impair chemotaxis without affecting proliferation. These findings provide novel insights into the molecular mechanisms underlying cancer cell perineural invasion. The RET-GEF-Cdc42 axis may be an attractive target for novel therapies that may potentially interrupt perineural invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 460. doi:1538-7445.AM2012-460 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ10521&atom=%2Fcanres%2F72%2F8_Supplement%2F460.atom