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PERSPECTIVES

substantial nerve sprouting occurs during the


OPINION
process of tumorigenesis16,17, it is plausible
that cancer cells proliferate in response to
Mechanisms of cancer dissemination the same factors that participate in nerve
regeneration.
along nerves Tumour growth induces neural
sprouting towards the tumour, which in
turn initiates cancer cellnerve crosstalk18.
Moran Amit, Shorook Naara and Ziv Gil Subsequently, a perineural inflammatory
response develops19,20. Recently, we
Abstract | The local extension of cancer cells along nerves is a frequent clinical demonstrated macrophage infiltration
finding for various tumours. Traditionally, nerve invasion was assumed to occur around intrapancreatic nerves invaded by
via the path of least resistance; however, recent animal models and human cancer, in pathological specimens excised
studies have revealed that cancer cells have an innate ability to actively from patients with pancreatic ductal
migrate along axons in a mechanism called neural tracking. The tendency of adenocarcinoma (PDAC, see figure1 in
REF.20) that also include infiltration of
cancer cells to track along nerves is supported by various cell types in the Tcells and mast cells around the invaded
perineural niche that secrete multiple growth factors and chemokines. We nerve. These findings may explain the
propose that the perineural niche should be considered part of the tumour visceral hypersensitivity in the pancreas19.
microenvironment, describe the molecular cues that facilitate neural tracking Whether these inflammatory cells contribute
and suggest methods for its inhibition. to pancreatic cancer neural invasion awaits
further investigation.
Neural tracking depends on a continuous
Solid tumours disseminate in three provides an unopposed pathway to the balance between nerve injury and repair,
well-known ways: direct invasion of central nervous system6. The distensibility which is maintained by various cells in
surrounding tissue, lymphatic spread and of the perineural space and the endoneurial the perineural niche. Neuronal damage
haematogenic spread. However, a fourth space enables nerve fibres to remain triggers a complex series of biochemical and
route of cancer spread, that of dissemination intact despite tumour invasion, such that molecular pathways that eventually results
along nerves1, is frequently disregarded and degeneration of the fibres owing to pressure in neuronal cell death2123. It is possible
less well understood. Perineural invasion occurs only in confined spaces in the late that nerves invaded by cancer simulate
(PNI) has emerged as a key pathological stages of disease. This distensibility accounts dying nerves, by secreting growth factors
feature of many malignancies, including for the minimal symptoms observed in and chemokines that contribute to the
those of the head and neck, pancreas, colon patients with PNI until late in their disease7. inflammatory response and to cancer cell
and rectum, prostate, biliary tract and However, ultrastructural studies have shown invasion.
stomach1. For many of these malignancies, that multiple layers of collagen and basement Identification of the early events involved
PNI is associated with substantial morbidity membrane compose the nerve sheath and in neural tracking and of the mechanisms
and is a marker of poor outcome and a make this path highly resistant 8,9. This responsible for PNI would have substantial
harbinger of decreased survival1,2 (TABLE1). indicates that the PNI phenomenon is more implications for risk stratification and
Occasionally, perineural spread makes like invasion than simple diffusion. Indeed, treatment of many tumours. In this Opinion
complete tumour removal with safe margins until recently, the mechanisms driving article, we discuss how cancer cells subvert
difficult at the time of diagnosis, and hence cancer cell dissemination along nerves a process that normally triggers neural
optimal resection in such cases is rare3,4. remained largely unknown. Recent data and regeneration in the perineural niche. Once
Since PNI was first described more than a the development of new invasion models the signals that drive neural tracking by
century ago, there has been a paradigm shift have enabled the identification of specific cancer cells are identified, their activity
in our understanding of its pathogenesis. molecular signals in the perineural niche could be targeted pharmacologically as
The predominant theory was that tumour (that is, the microenvironment in the space an innovative approach to prevent cancer
cells spread passively along planes of least surrounding a nerve) that can induce cancer dissemination. For inoperable primary
resistance in the connective tissues that invasion1013. However, there is much that is tumours or for patients with a high
cover the nerve (the sheath)5. This theory speculated from data about general invasion likelihood of nerve invasion at the time
was formerly accepted because the perineural mechanisms; PNI-specific mechanisms still of surgery, such treatment is also likely
space between the nerve and its sheath, remain to be fully understood. to decrease neuropathic pain and reduce
extending from the smallest branches of Nerves and the perineural niche can morbidity. Earlier publications on PNI have
a nerve to the central nervous system, is initiate cues that induce cancer survival, focused on the clinical implications and
composed of loose tissue that, once reached, invasion and motility 12,14,15. Furthermore, as have described the interaction between

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Table 1 | Clinical implications of cancerous neural tracking


Cancer type Associated nerves Percentage Effect on survival Other clinical Refs
of patients outcomes
with PNI
Pancreatic ductal Coeliac ganglia 70100 The median OS for patients without Abdominal pain 3,
adenocarcinoma Superior mesenteric plexus PNI was 56 months compared with Back pain 124,125
28 months for patients with PNI
Gastric carcinoma Vagus nerve 60 OS of patients with PNI is Increased vascular 126
Coeliac ganglia significantly worse than that of invasion and lymph node
Superior mesenteric plexus patients without PNI metastasis
Cholangiocarcinoma Coeliac ganglia 7585 PNI is a significant predictor of OS Associated with locally 127129
and bile duct carcinoma invasive tumours
Prostate cancer Superior and inferior 75 Five-year progression-free survival Increased positive 130,131
hypogastric plexus rates were 70% for patients with surgical margins and
PNI compared with 90% for patients extracapsular invasion
without PNI Higher T stage
Head and neck cancer Trigeminal ganglion 80 Three-year OS for patients with Trigeminal neuralgia 132135
Facial nerve PNI is 23% compared with 49% for Facial nerve paralysis
patients without PNI in SCC
Local recurrence rate for patients
with PNI is 23% compared with 9%
for patients without PNI
Disease-specific mortality for
patients with PNI is 54% compared
with 25% for patients without PNI
Colorectal cancer Superior and inferior 33 Five-year OS for patients with PNI Higher metastatic rate at 136139
mesenteric plexus is 25% compared with 72% for the time of resection
patients without PNI
Five-year disease-free survival for
patients with PNI is 16% compared
with 65% for patients without PNI
OS, overall survival; PNI, perineural invasion; SCC, squamous cell carcinoma.

cancer cells and nerves after invasive disease the nerves30,34,35, neurogenesis11,16, cancer myeloid-derived suppressor cells (MDSCs),
has been established therefore we do not cell adhesion to nerve sheaths36,37 and, infiltrate the pre-invasive lesions (PanIN)
focus on these aspects here24,25. eventually, nerve invasion38,39 (FIG.2). and persist throughout PDAC44. Tumour-
associated F4/80 (also known as adhesion
Defining the perineural niche Endoneurial macrophages. Perineural G protein-coupled receptor E1 (ADGRE1),
Neural tracking of cancer cells is an inflammation is a major component of a specific murine macrophage marker)+
ongoing process of nerve injury that in the robust desmoplastic response and an CD11b+ endoneurial macrophages secrete
turn promotes neural regeneration. It is invariant and pathognomonic feature of factors that promote angiogenesis and tissue
possible that the injured nerve secretes several neurotropic malignancies such invasion by cancer cells20,45 (FIG.1).
growth factors that further promote as prostate cancer and PDAC19,20,40,41. In a rat model, nerve injury triggered
cancer cell proliferation. Alternatively, Neuritis in PDAC is composed of cytotoxic secretion of chemokine (CC motif) ligand2
upregulation of growth factor secretion by T lymphocytes, macrophages and mast (CCL2) from various cell types including
nerves can occur independently of neural cells19,20. The cancer-related inflammatory Schwann cells and neurons46. CCL2
damage. Damage to the perineurium by response produces hypertrophy and hyper- expression at the perineural niche by various
invading cancer cells induces a cascade of sensitivity of pancreatic afferents42; sensory cell types of the nervous system, including
inflammation and wound repair processes26. fibres may themselves drive inflammation via Schwann cells, macrophages, endoneurial
Cancer cells respond to soluble proteins, neurogenic mechanisms43. Inflammation has fibroblasts and neurons, induces prostate
adhesion molecules and membranous been shown to be a precipitating event that cancer cell neural invasion through CCL2
receptors that are normally involved in can accelerate the transition of precancerous receptor (CCR2)-mediated signalling 35.
neural homeostasis27 to facilitate cancer lesions to cancer 10. For example, disease Ccr2/ mice, which have ablation of
cell survival12, proliferation16, invasion20,2831 progression from normal histology to monocytes and macrophage recruitment,
and motility 32,13 (FIG.1; see Supplementary pancreatic intraepithelial neoplasia (PanIN) showed reduced prostate and PDAC cancer
information S1 (table)). The various to invasive PDAC is accompanied by cell neural invasion compared with wild-type
cellular components of the perineural progressive infiltration of CD45 (also known animals20,35. Recruitment of macrophages is
niche support further cancer dissemination as protein tyrosine phosphatase, receptor induced by cancer-derived colony stimulating
by maintaining the seven hallmarks of type, C (PTPRC))+ leukocytes. Leukocytes, factor 1 (CSF1)20. Treatment of PDAC
neural tracking: cancer cell survival12,14, consisting largely of CD11b (also known cell-conditioned medium with GW2580, a
nerve homeostasis21,33, inflammatory as integrin M (ITGAM))+ macrophages selective CSF1 receptor small-molecule kinase
response19,20, cancer cell chemotaxis towards and GR1 (also known as Ly6G)+CD11b+ inhibitor, significantly reduced the number

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of macrophages that migrated in response a b


to that medium invitro in a dose-dependent
manner (see TABLE2 for preclinical data
supporting possible therapeutic targets
for PNI)20. In our recent work presented
at the American Association for Cancer
Research 2015 Annual Meeting47, we used
LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx1Cre
(KPC) mice, which develop PanIN lesions
and invasive PDAC at well-documented Chemokine:
time points and recapitulate many aspects of CXCL13
human disease, including PNI48. We used the
chemokine (C-X3C motif) receptor 1green c d
fluorescent protein (Cx3cr1GFP) transgene
to track bone marrow-derived cells in KPC
mice. Endoneurial macrophages colocalized
with Cx3cr1GFP, demonstrating that the
endoneurial macrophages originate from the
bone marrow. Transplanting Ccr2/ bone
marrow into KPC mice, or treating the mice
with GW2580 to inhibit trafficking of bone
marrow-derived macrophages, substantially
reduced the number of infiltrating Growth factors:
endoneurial macrophages and neural GDNF NGF Chemokines:
NRTN GFR1 CCL2
invasion severity in KPC mice with fully ARTN NT3 CX3CL1
developedPDAC47.

Stellate cells. Stellate cells are myofibroblast- Endoneurial Activated endoneurial


Cancer cell Macrophage
macrophage macrophage
like cells that become activated following
exposure to cancer cells, premalignant cells, Preneoplastic
Fibroblast
Stellate Schwann
Neuron
inflammation or hypoxia49. Recent data from epithelial cell cell cell
human PDAC specimens, invitro dorsal root
Figure 1 | Regulation of neural tracking of cancer cells by the perineural niche.
Nature As a malignant
Reviews | Cancer
ganglia (DRG; see BOX1) and invivo sciatic
precursor lesion evolves, the perineural niche starts to assemble to form a cellular and biochemical
nerve tumour implants show that activated microenvironment that can eventually promote cancer cell invasion into the nerve (panel a). At a
stellate cells support PNI in PDAC39. certain point, these neurogenic cues initiate axonogenesis, which is accompanied by recruitment
Activated stellate cells are also a pivotal of stromal cells typical of the perineural niche (panel b). Subsequent malignant transformation
component of the desmoplastic reaction49,50 results in the release of multiple chemotactic cues such as stroma-derived CXC motif chemokine
that correlates with PNI51 and neuropathic ligand 13 (CXCL13), which promotes further recruitment of inflammatory cells to establish the peri
changes52 in PDAC. Activation of stellate neural niche (panel c). Within the niche, an injured nerve serves as a portal for invasion while neural
cells promoted local growth of pancreatic homeostasis including Wallerian degeneration and nerve regeneration is maintained (panel d). At the
tumours in pancreatic stellate cell and cancer perineural niche, the injured nerve maintains regeneration via nerve growth factor (NGF) and
cell cocultures, as well as facilitated spread neurturin (NRTN) stimulation. Axons secrete C-C motif chemokine ligand 2 (CCL2) that further
nourishes the inflammatory response and leads to endoneurial macrophage recruitment.
of pancreatic cancer cells in an orthotopic
Macrophages secrete glial cell derived neurotrophic factor (GDNF), which facilitates neural tracking
model of PDAC using cancer cells alone and cancer cell invasion by activating RET GDNF receptor 1 (GFR1) in the cancer cells. Neuron
or in combination with primary human tumour crosstalk at the niche is further supported by neurally derived C-X3C motif chemokine
pancreatic stellate cells49,53. ligand 1 (CX3CL1), which enhances cancer cell adhesion to the nerve, and neurotrophin 3 (NT3),
Tumour-associated inflammation which modulates Schwann cell and cancer cell interaction. ARTN, artemin.
induces cancer cells to express transforming
growth factor 1 (TGF1), which stimulates
stellate cells26,54,55. Stellate cell stimulation by in a mouse orthotopic model of tongue PDAC cells overexpressing SHH or stellate
TGF1 promotes stellate cells to express high squamous cell carcinoma impaired PNI and cells cultured separately with DRG39. Invivo
levels of galectin 1 (also known as LGALS1), had a dose-dependent inhibitory effect on treatment with cyclopamine, an inhibitor
which further induces stellate cell activation invadopodia formation, ECM degradation of Hedgehog signalling, attenuated PDAC
in an autocrine loop56. Downstream SRC and MMP9 activation38. Sonic hedgehog cell invasion along the sciatic nerve in
signalling in activated stellate cells leads to (SHH) protein secretion from cancer cells subcutaneous tumour xenografts of PDAC
increased transcription of matrix metallo- and subsequent activation of stellate cells cells either alone or with stellate cells39.
proteinases (MMPs)56,57, which are required also participate in MMP expression and
for perineural extracellular matrix (ECM) PNI. Cocultures of DRG in the presence of Schwann cells. Another key component in
degradation and the movement of cancer PDAC cells overexpressing SHH and stellate neural repair and regeneration is the Schwann
cells through tissue barriers58. Targeting cells revealed increased neurite growth cell, a peripheral glial cell and the most
SRC kinase with saracatinib (AZD0530) towards tumour cells compared with either prevalent cell type in peripheral nerves59.

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Cancer cell L1CAM L1CAM L1CAM Neuron


Neurotrophic factors: Adhesion molecules: GFR3 NRTN GFR3 Neurotrophic Adhesion molecules:
GDNF soluble L1CAM factors: soluble L1CAM
NRTN RET RET GDNF Neuropeptide or
ARTN Cell cycle regulation: NRTN
CAV1 neurotransmitters:
NGF GFR1 GDNF GFR1 ARTN GAL
NT3 PIM2 NGF
TGF1 Substance P
Neuropeptide or Cleaved GFR1 aCh
GFR2 ARTN GFR2
neurotransmitters: ECM degradation: Chemotaxis: NA
GAL MMP2 CCL2 Cell cycle regulation:
Substance P MMP9 CXC3CL1 CAV1 and CAV2

TRKA NT3 TRKA


TRKC NGFR
mAChR M3

CX3CR1
GALR2

CX3CR1 GALR2
MUC1
Bystin

SDC2

CCR2
ADR NK1 CXCR5

GFR1 L1CAM
GFR3
CCR2

TRKA
Macrophage
Adhesion
Neurotrophic Neuropeptide or NGFR molecules:
factors: neurotransmitters: soluble L1CAM
GDNF substance P
MAG
Schwann cell

Figure 2 | Molecular signalling of neural tracking. Seven main prop- to the perineural niche and enable adhesionNature of cancer cells| Cancer
Reviews to the
erties that are required for neural tracking are maintained by the various nerves. Autocrine signalling of cancer cells activates intracellular path-
cellular components of the perineural niche: cancer cell survival, nerve ways that enhance extracellular matrix degradation, and cancer cell
homeostasis, inflammatory response, cancer cell chemotaxis towards survival, motility and invasiveness. aCh, acetylcholine; ADR,
the nerves, neurogenesis, cancer cell adhesion to nerve sheaths and adrenergic receptor; ARTN, artemin; CAV, caveolin; CCL2, chemokine
eventually nerve invasion. A summary of these properties and the recep- (CC motif) ligand 2; CCR2, CCL2 receptor; CXCR5, CXC chemokine
tors that regulate them is presented. Before contact between a cancer receptor type 5; CX3CR1, C-X3C motif chemokine receptor 1; ECM,
cell and a neuron is established, reciprocal autocrine and paracrine sig- extracellular matrix; GAL, galanin; GALR, G protein coupled receptor;
nalling between cancer cells and neurons mediates homing of cancer GDNF, glial cell derived neurotrophic factor; GFR, GDNF family recep-
cells to the perineural niche. Autocrine signalling (round arrows) from tor; L1CAM, L1 cell adhesion molecule; mAChR M3, muscarinic acetyl-
neurons (beige) activates intracellular pathways that enhance axonal choline receptor M; MAG, myelin associated glycoprotein; MMP, matrix
growth. Chemotactic factors are secreted from neurons and cancer cells metalloproteinase; MUC1, mucin 1; NA, noradrenaline; NGF, nerve
(light blue) to maintain immune cell recruitment and regeneration of the growth factor; NGFR, NGF receptor; NK1, neurokinin 1; NRTN, neurturin;
invaded nerve. Subsequent paracrine cues that are derived from macro NT3, neurotrophin 3; NTR, neurotrophin receptor; SDC2, syndecan 2;
phages (dark blue) and Schwann cells (light purple) attract cancer cells TRK, tropomyosin receptor kinase.

Schwann cells routinely contribute to towards cancer cells, orchestrating the cues types of cancer 63,64. Furthermore, TRKC
neural homeostasis, including Wallerian required for neurogenesis around neoplastic inhibition, using a specific small-molecule
degeneration and nerve regeneration60. In a cells61. Increased interactions between inhibitor AZD7451, suppressed the invasion
similar manner, and in response to nerve cancer cells and Schwann cells and the both invitro and invivo in patient-derived
damage caused by invading cancer cells, accompanying process of axonal sprouting xenografts of adenoid cystic carcinoma,
activated Schwann cells begin to proliferate at the premalignant phase provide the first a highly neuroinvasive tumour 65. Taken
and migrate from nerves towards cancer access of cancer to nerves, leading to neural together, these findings show that it is
cells61. In an ex vivo sciatic nerve model dissemination even at an early disease stage21. possible that TRKC is also involved in PNI
(see BOX1), Schwann cells migrated towards Tropomyosin receptor kinase C (TRKC; by modulation of the Schwann celltumour
pancreatic and colon cancer cells, but also known as NTRK3) and its ligand cell interactions.
not towards benign cells, before cancer neurotrophin 3 (NT3) promote Schwann cell
cell invasion started61. Through direct dispersal by inhibition of peripheral nervous Soluble factors and their receptors
interaction with cancer cells, activated system myelination62. TRKC expression Neurotrophic factors. The nerve micro
Schwann cells delineate axon guidance is strongly correlated with PNI in several environment is abundant in neural growth

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Table 2 | Preclinical data supporting possible therapeutic targets for perineural invasion
Molecular Source Pathway Expression in human tumours Organ Inhibitor Refs
target activation
(receptor)
GDNF GDNF: Tu, N, M or F ERK or AKT Strong staining of GDNF in tumour cells Pancreas PP1 (SRC inhibitor 13,20,
(GFR1RET) GFR1: Tu, N or SC with PNI Bile duct with a high affinity 67,70
RET: Tu or N for RET)
NRTN NRTN: Tu or N ERK or AKT Overexpression of NRTN and GFR2 in Pancreas mAb to NRTN 68
(GFR2RET) GFR2: Tu or N PDAC predominantly in intrapancreatic
RET: Tu or N nerves, islets and ECM
NGF (TRKA, NGF: Tu or N ADRPKA NGF expression is correlated with PNI in Prostate Ro 08-2750 (NGF 61,75,
high affinity) TRKA: Tu, N or SC STAT3 both PDAC and prostate cancer Pancreas inhibitor) 76,81,
ERK In PDAC, a higher ratio of NGF to TRKA is AG490 (STAT3 95
associated with more frequent PNI inhibitor)
NGF (p75NTR, Tu, N or SC Unknown Unknown Pancreas Ro 08-2750 (NGF 61
low affinity) inhibitor)
NT3 (TRKC) NT3: Tu RAS TRKC is highly expressed in adenoid cystic Head and AZD7451 (TRKC 65
TRKC: Tu ERK carcinoma neck inhibitor)
AKT
VEGFR1
CSF1 (CSF1R) CSF1: Tu Unknown Unknown Pancreas GW2580 (CSF1R 32
CSF1R:M small-molecule
kinase inhibitor)
Noradrenaline ADR: Tu ADR PKA Unknown Prostate AG490 (STAT3 95
(ADR) STAT3 Pancreas inhibitor)
L1CAM Tu, N or SC NF-B Expression in pancreatic carcinoma is Pancreas mAb to L1CAM 36,107,
ERK correlated with PNI and pain Colon 140,141
SHH Tu Hedgehog Higher expression in pancreatic carcinoma Pancreas Cyclopamine 39
pathway compared with normal pancreas;
overexpression of SHH in tumours
correlates positively with pancreatic cancer
distant metastasis, TNM stage and PNI
ADR, adrenergic receptor; CSF1, colony stimulating factor 1; CSF1R, CSF1 receptor; CXCL13, chemokine (CXC motif) ligand 13; CXCR5, CXC chemokine
receptor type 5; ECM, extracellular matrix; F, fibroblasts; GDNF, glial cell derived neurotrophic factor; GFR, GDNF family receptor; L1CAM, L1 cell adhesion
molecule; M, macrophages; mAb, monoclonal antibody; N, nerve; NFB, nuclear factor B; NGF, nerve growth factor; NGFR, NGF receptor; NRTN, neurturin;
NT3, neurotrophin 3; PDAC, pancreatic ductal adenocarcinoma; PKA, protein kinase A; PNI, perineural invasion; SC, Schwann cells; SHH, sonic hedgehog; STAT3,
signal transducer and activator of transcription 3; TRK, tropomyosin receptor kinase; Tu, tumour cell; VEGFR1, vascular endothelial growth factor receptor 1.

factors such as glial cell derived neurotrophic animals with pyrazolopyrimidine 1 (PP1), associated with hypertrophy and hyperplasia
factor (GDNF), neurturin (NRTN) and a potent RET inhibitor 73, abolished neural of stromal nerve fibres in PDAC, in both
artemin (ARTN)66. In response to neural tracking towards the spinal cord of cancer human samples and murine models69,80.
damage, these factors are secreted from cells injected into the sciatic nerve, resulting The expression of NGF, TRKB, NRTN and
nerves67,68 or neighbouring stroma20,6870; in reduced paralysis13. Nerve regeneration GFRA2 rise sharply even at a premalignant
they bind to cancer cells through the GDNF is also mediated by nerve growth factor stage of PDAC81, suggesting the existence of
family receptors (GFR13) and activate the (NGF) and its receptors, TRKA (also a neurogenic influence on tumorigenesis.
GFR cognate tyrosine kinase receptor, RET. known as NTRK1) and NGF receptor Common to these neurotrophic
High levels of GDNF and RET expression (NGFR; also known as p75NTR), which are signals is activation of the ERK and AKT
correlate with the presence and severity highly expressed in the perineural niche of pathways20,67. This was first suggested to
of neural invasion in pancreatic13, breast 71 neurotropic tumours61,7476. These factors occur invitro when human PDAC cell lines
and bile duct 70 carcinomas. Furthermore, hold a dual role as prominent regulators were shown to be prone to ERK-dependent
increased expression of NRTN and GFR2 of neurite outgrowth and promoters of invasion upon stimulation with GDNF13,82.
by cancer cells correlates with nerve invasion neural tracking 77. Targeting NGF with Treatment with PP1 effectively inhibited
and pain level in patients with PDAC68. In an the small-molecule inhibitor Ro 08-2750 cancer cell migration towards DRG
alternative pathway, neural damage induces diminished migration of cancer cells along neurons in a dose-dependent manner and
release of soluble GFR1 from neurons67,72, neurites in DRG cocultures61. Newly inhibited ERK phosphorylation in PDAC
facilitating neural tracking regardless of developed nerves at the tumour front cells13. Also, systemic therapy with PP1
cancer cell GFR1 expression67. Cancer are rich in ARTN and NGF relative to suppressed nerve invasion towards the
cells lacking GFR1, cocultured with DRG nerves not expressing the neuroplasticity spinal cord and prevented paralysis in a
(see BOX1), retain their ability to migrate marker, growth-associated protein 43 mouse sciatic nerve invasion model. These
along nerves in DRG from Gfra1+/+ mice, (GAP43)78. ARTN is involved in neural findings suggest a role for GDNF secreted
but this migration is reduced in DRG from repair and axonal projection79, and cancer by nerves in induction of neural tracking by
Gfra1+/ mice67. Accordingly, treatment of cell expression of ARTN was found to be downstream signalling through ERK13,73.

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Chemokines. Nerves express various increased ERK and AKT phosphorylation more resistant to prostate cancer cell neural
chemokines that support the perineural and cancer cell invasion84,85. Recent studies invasion than were wild-type controls35.
inflammatory response34,35. Chemokine have shown that CXCR5 is overexpressed In addition, invivo sciatic nerve injection
(CXC motif) receptor 5 (CXCR5) is in prostate86 and colorectal87 cancers that of CCR2expressing prostate cancer cells
expressed primarily by lymphocytes and show association with neural invasion, and facilitated PNI and neurological functional
controls their migration into and within therefore this pathway could be relevant in deficits compared with cells treated with
lymph nodes83. Paracrine stimulation PNI. AKT and ERK are also activated by short hairpin RNAs targeting CCR2 (REF.35).
of prostate and colorectal cancer cells the binding of CCL2, secreted by neurons, Neurally derived C-X3C motif
expressing CXCR5 by the stroma-derived to CCR2, on cancer cells35. Indeed, DRG chemokine ligand 1 (CX3CL1, also known
CXCR5 ligand, CXCL13, resulted in cultures derived from Ccl2/ mice were as fractalkine) can exist in either a soluble
or a membrane-bound form34. The CX3CL1
receptor, CX3CR1, is highly expressed
Box 1 | Experimental models for neural invasion by PDAC cells30. CX3CL1 induces the
migration of PDAC cells stably expressing
a b CX3CR1 in a dose-dependent manner 30.
Cancer cell Schwann cell
Invivo, subcutaneous xenografts of
DRG
CX3CR1expressing PDAC cells invaded
ECM Sciatic nerves, whereas nerve infiltration was
nerve
never observed in tumours derived
from cells lacking the CX3CR1 (REF.30).
CX3CL1 stimulation of CX3CR1 leads to
colocalization with 1 integrin and focal
adhesion kinase (FAK) redistribution at the
cells leading edge, which stabilizes adhesion
c d to neural cells30.

Neurotransmitters. Nerves can also stimulate


cancer cell invasion through the secretion of
Cancer
cell (GFP+) neurotransmitters, such as catecholamines,
acetylcholine and neuropeptides8890.By
stimulating endothelial cells, immune
Fibroblast
cells and fibroblasts, neurotransmitters
broadly affect the tumour microenviron-
Vagal
ment, beyond their direct effect on cancer
nerve cells91. Extensive innervation of tumours by
parasympathetic cholinergic fibres (positive for
vesicular acetylcholine transporter (VAChT);
One main reason that understanding mechanisms of neural tracking is challenging is the lack
Nature Reviews of
| Cancer
also known as SLC183) and increased
reliable and reproducible experimental models for neural invasion. The in vitro dorsal root ganglia invasion and metastasis were observed
(DRG) assay (see the figure, part a), designed for studying the neurotropic ability of cancer cells13,78, in orthotopic and genetically engineered
also enables modulation of paracrine signalling by controlling chemoattractants13,20,35,67 and by (HiMYC model) mouse models of prostate
signalling between cancer cells and the DRG13,20,95. Neurite outgrowth, directionality and Euclidean cancer 89. Pharmacological blockade or
velocity of cancer cells can be measured before cancerneuron contact has been established78. genetic disruption of the cholinergic receptor
However, the importance of the various cellular components in the classical perineural niche, muscarinic 1 (CHRM1) inhibited invasion
including Schwann cells and fibroblasts, cannot be directly addressed in this model. and metastasis and improved animal
The recently presented 3D Schwann cell outgrowth and migration assay (figure, part b)61 assesses
survival89. In bile duct cholangiocarcin
directional outgrowth of Schwann cells by means of an ex vivo sciatic nerve from newborn rats
cultured in an extracellular matrix (ECM) gel suspension. This model enables the early phases of oma, expression of CHRM3 is associated
axonogenesis and mutual influences of Schwann cells and cancer cells to be studied. withPNI88.
A novel neural invasion assay, recently presented by our group at the American Association for Innervation by the sympathetic
Cancer Research 2016 Annual Meeting123 (figure, part c) enables coculture of murine fibroblasts nervous system can also trigger cancer
with Schwann cells on either side of an 8m Millipore cell culture insert membrane with a gelatine development 89,92, and the activation of
separation layer121,122. After 72hours, labelled cancer cells (with green fluorescent protein (GFP), for sympathetic -adrenergic receptors
example) are added on top to migrate and invade for an additional 24hours. This assay enables the expressed by the cancer cells accelerates
control and study of the reciprocal interactions between cancer cells, fibroblasts and Schwann cells. tumour growth and invasion93,94. In prostate
Comparing neurotropic and non-neurotropic cells enables the identification of early genetic cancer, malignant transformation was
and metabolic cellular effects that occur in these cells. An exvivo perineural invasion model
prevented by chemical or surgical removal
(figure, part d) enables the selection of neurotropic subclones using resected rat vagal nerves32.
The chicken embryo chorioallantoic membrane (CAM)DRG model (not shown in the figure), of sympathetic nerves and by genetic
which is used primarily in developmental biology research, includes a rat DRG grafted onto the deletion of stromal 2- and 3adrenergic
chicken embryo, which becomes incorporated into connective tissue. Next, cancer cells are receptors89. Following the inception of
inoculated near the DRG; subsequently, neural outgrowth is assessed. This was recently proposed as PDAC, in vitro continuous stimulation
an in vivo model to address neural outgrowth at tumour inception before cancer cell invasion11. with the sympathetic neurotransmitter

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noradrenaline enhanced neural invasion Membrane-anchored proteins B (NFB) p65 subunit via enhanced
along DRG neurites through adrenergic Cancer cells interact with the components production of interleukin 1 (IL1)107,108.
receptor-induced signal transducer and of the ECM before invasion to adjacent Within the perineural niche, L1CAM
activator of transcription 3 (STAT3) structures103. During PNI, cancer cells expression by cancer cells and Schwann
activation and upregulation of MMPs in that overexpress membrane-bound cells activates ERK in cancer cells109,110. Our
cancer cells95. Adrenergically induced neural mucin1 (MUC1) have a selective adhesive preliminary data show that treatment of
invasion was inhibited by AG490, a STAT3 advantage once they have invaded the PDACs in KPC mice with a monoclonal
phosphorylation inhibitor, both in DRG endoneurium of nerves in which myelin- antibody against L1CAM significantly
cocultures and invivo using the sciatic associated glycoprotein (MAG) is reduces nerve density and the neural
nerve injection model95. expressed29. This adhesive advantage may invasion severity score in pancreatic
Neuropeptides are small polypeptides contribute to the capacity of pancreatic tumours, compared with control123. These
that function peripherally as paracrine cancer cells to survive and proliferate findings suggest that paracrine regulation
and endocrine factors and act as within nerves29. Increased expression of of axonogenesis and neural tracking is
neuromodulators in the nervous system90. MUC1 promotes cancer cell invasion controlled byL1CAM.
In cancer, they can also act as autocrine and through activation of catenin and Specific cancer cell adhesion to the
paracrine factors that regulate migration induction ofepithelialmesenchymal neuron can also be mediated by the 2
and invasion. Substance P, a neuropeptide transition,which promotes general invasion subunit of voltage-sensitive sodium
synthesized by macrophages and by but might also apply toPNI104. channels (encoded by SCN2B) and laminin
neuronal, endothelial and epithelial cells, The neural L1 cell adhesion molecule binding 111. The 2 subunit is responsible
mediates pain and neurogenic inflammation, (L1CAM) is a transmembrane glycoprotein for a weak association of PDAC cells
and its expression correlates with poor of the immunoglobulin superfamily with nerves. Laminin is one of the most
prognosis in gastric, prostate, breast that is overexpressed by tumour cells abundant ECM molecules in the peripheral
and pancreatic cancers96,97. Recent data and stroma; its expression is associated nervous system and has been implicated as a
demonstrate that substance P may promote with poor prognosis105,106. Furthermore, molecular binding partner in the facilitation
the proliferation, invasion and neurotropism L1CAM expression is associated with both of prostate cancer PNI112. Overexpression of
of pancreatic cancer cells98. In DRGPDAC neuropathic pain and nerve invasion in the 2 subunit by PDAC cells enhances
cocultures, substance P induces DRG patients with PDAC36. Integrin-dependent pancreatic cancer cell migration, invasion
neurite outgrowth and pancreatic cancer L1CAM activation induces phosphorylation and growth on dense laminin matrix but
cell expression of MMP2, neurotropism and and nuclear shuttling of the nuclear factor not on plastic tissue culture dishes111.
invasion; conversely, substance P receptor
(NK1R; also known as tachykinin receptor1
(TACR1)) antagonists L-733,060and Glossary
L-732,138 inhibit these effects98. Desmoplastic reaction Perineurium
The neuropeptide galanin (GAL), is Also known as desmoplasia, the desmoplastic reaction is Peripheral fibres are each wrapped in a
the ligand for three G protein coupled the growth of fibrous tissue secondary to an insult such as protective sheath known as theendoneurium.
a tumour or surgery. These are bundled together into fascicles, each
receptors GALR1, GALR2 and GALR3
surrounded by a protective sheath known as
(REF.99). GALR2 is pro-proliferative99 and Dorsal root ganglia theperineurium.
is overexpressed in neurotropic head and (DRG). Also known as spinal ganglia, DRG are clusters
neck squamous cell carcinoma (HNSCC) of nerve cellbodies(a ganglia) in aposterior root of a Peripheral glial cell
in association with the frequent 11q13 spinal nerve. Schwann cells are the principalperipheral glial cells that
function to supportneuronsin the peripheral nervous
chromosomal translocation100. The Endoneurial space system.
role of GAL in nerve regeneration and The anatomical space between the deepest layer of nerve
axonogenesis in HNSCC was recently covering, called the endoneurium, and the peripheral Sensory fibres
unveiled using the chicken embryo nerve fibres. Nerve fibres that deliver sensory information (for
example, pain), from a peripheral organ to the central
chorioallantoic membrane (CAM)DRG
Euclidean velocity nervous system.
model system11 (see BOX1). In HNSCC, Directional vector of velocity defined not only by
neurally derived GAL initiates nerve magnitude, but also by direction. Sympathetic nervous system
tumour cell crosstalk via paracrine A component of the autonomic nervous system
activation of GALR2, which is expressed Pancreatic afferents responsible for maintaining homeostasis and stimulating
These afferent nerve fibres are sensory neurons extending the body for flight-orfight response. In peripheral nerves,
by cancer cells11,101. GALR2 induces far from the nerve cell body in the coeliac ganglia. They the main postganglionic sympathetic neurotransmitter is
prostaglandin E2 secretion by cancer conduct pain sensation by carrying nerve impulsesfrom noradrenaline, which activates- and -adrenergic
cells, via nuclear factor of activated T cells sensory receptorstowardsthecentral nervous system. receptors.
and cytoplasmic 2 (NFATC2)-mediated
Parasympathetic cholinergic fibres Visceral hypersensitivity
transcription of cyclooxygenase2
These components of the autonomic nervous system are Cancer-associated altered visceral perception caused by
(COX2)11. Downregulation of COX2 responsible for the bodys activities when it is at rest. They hyperexcitability of the neurons in the visceral afferent
expression reduces HNSCC cell are called cholinergic after the main neurotransmitter, nervous system. Characterized by a lowered threshold for
migrationalong DRG11. In a feedback acetylcholine. abdominal pain and discomfort.
loop, HNSCC cancer cells also secrete
Perineural space Wallerian degeneration
GAL, which induces neurite formation11. The anatomical space between the most superficial nerve A process that results after a nerve injury, in which the
Activation of COX2 is also associated covering, also known as the epineurium layer, and the part of theaxonseparated from the cell body of the
withneural invasion in pancreatic cancer 102. middle layer, called the perineurium, in peripheral nerves. neuron degenerates distal to the injury.

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Another specific target for nervecancer cell of caveolin 1 (CAV1) and CAV2 into the Concluding perspectives
interaction is bystin, an adhesion molecule perineural microenvironment is observed The microenvironment of many solid
associated with early embryogenesis113. along neurons116. CAV1 is an anti-apoptotic tumours is richly innervated. In tumours that
Bystin is overexpressed by cancer cells that protein and its genetic ablation in the DRG have the propensity to invade nerves, neural
use this early embryological mechanism model reduced human prostate cancer cell tracking along peripheral nerves has been
of adhesion to attach to the nerves, and pro-survival activities12. PIM2 is a member associated with shortened patient survival and
prostate cancer cells cocultured with of a family of serine/threonine protein severe morbidity. The study of neural invasion
DRG express bystin exclusively in spatial kinases that function as oncogenes by is of ongoing interest for understanding
proximity to nerves37. This mechanism is inhibiting apoptosis118. Nuclear expression cancer dissemination. The current paucity
not used during other post-embryological of PIM2 is markedly increased in prostate of data is consequent on the lack of a reliable
processes114 and could therefore be targeted cancer 117. PIM2 expression enhances modelling system of the perineural niche,
for cancer treatment. NFB nuclear translocation, leading to both before and immediately after contact
Syndecan2 (SDC2), a pro-tumorigenic activation of the NFB survival pathway, between cancer cells and nerve has been
receptor associated with the migratory increased proliferation and reduced made. However, contemporary invivo and
potential of melanoma cells, is upregulated apoptosis in human prostate carcinoma invitro models, described in detail in BOX1,
in neurotropic subclones of PDAC cell tissues117. Specifically, PIM2 is overexpressed have led to a better understanding of the
lines28. SDC2 is overexpressed around by cancer cells in the vicinity of invaded perineural niche and its impact on tumour
invaded nerves by both cancer cells nerves in human prostate cancer tissue, progression at its earliest stages, even before
and tumour stroma in human PDAC compared with cancer cells located away invasion. Although none of the mechanisms
specimens; knockdown of SDC2 reduces from the nerve117. Concurrent upregulation of PNI are completely understood, it is
KRAS phosphorylation and ERK pathway of NFB and PIM2 has been observed in clear that an aberrant stromal response
activation, and consequently cancer cell PNI-associated cancer cells using the DRG supports tumour invasiveness into nerves.
invasion28. These results lay the foundation model and prostate cancer cell cocultures14. Accumulating data from these novel models
for an indepth analysis of the interference This indicates that PIM2 might be a central suggest that the mechanisms that drive
in deregulated syndecan signalling in regulator of apoptosis inhibition in the cancernerve interaction may overlap
pancreatic cancerPNI. perineural space117. with the mechanisms that underlie neural
High-throughput genetic analysis of degenerationregeneration.
Cell cycle regulation PDAC neurotropic subclones derived from Our understanding of the molecular
Neural damage promotes a microenviron- an exvivo neurotropic cell sorting model interactions that underlie nervetumour cell
ment that activatesfundamental survival (see BOX1) enables identification of genetic interactions carries an opportunity for the
mechanisms in cancer cells andselection alterations in neurotropic clones within development of new treatment modalities that
pressure for a more aggressive behaviour, the same tumour. In this analysis, mitotic will target these processes to inhibit cancer
such as suppression of apoptosis12,15 and kinesin family member 14 (KIF14) was dissemination and treat neuropathic pain
promotion of cell growth14,21. When nerve downregulated and Rho GDP dissociation derived from neural tracking of cancer cells.
injury occurs, a degenerative process inhibitor (ARHGDI) was upregulated Neural invasion inhibition as an adjuvant
begins before regeneration of the nerve is in neurotropic subclones32. These findings therapy would aim to target microscopic
initiated33. However, DNA damage in the concur with an immunohistochemical tumour advancement and reduce local and
neuron triggers the activation of signals analysis of human PDAC, which revealed systemic tumour burden. Each of the hallmark
that suppress neural death, such as cyclin- decreased KIF14 and increased ARHGDI steps of neural tracking (FIG.2) should be
dependent kinase 4 (CDK4) and CDK6, expression in pancreatic cancer cells that addressed in future experiments, with the goal
which ultimately leads to retinoblastoma invaded nerves32. ARHGDI promotes of developing treatment regimens for patients
protein (RB) hyperphosphorylation and invasiveness and inhibits drug-induced with neurotropic cancers.
the release of the transcription factor apoptosis, mediating resistance of cancer
Moran Amit, Shorook Naara and Ziv Gil are at the
E2F22. The phosphatase cell division cycle cells to chemotherapeutic agents such
Laboratory for Applied Cancer Research, Department
25A (CDC25A) increases CDK activity as etoposide and doxorubicin119,120. of Otolaryngology Head and Neck Surgery, Head and
and consequent E2F release115. The ARHGDI was found to be significantly Neck Center, Rambam Healthcare Campus, Clinical
proto-oncogene PIM1 activates CDC25A, upregulated in most of the perineurally Research Institute at Rambam, Rappaport Institute of
Medicine and Research, The Technion-Israel Institute
and is expressed in response to nerve invasive cancer cells of human pancreatic
of Technology, Haalia Street No. 8, Haifa, Israel.
injury and DNA damage (through NFB cancer tissuesinvivo, and its silencing Correspondence to Z.G.
signalling) and was shown to prevent led to reduced PNI capacity exvivo using ziv@baseofskull.org
neuronal death, to keep the perineural niche resected rat vagal nerves32. Conversely, doi:10.1038/nrc2016.38
in a permissive state23. downregulation of KIF14 in pancreatic Published online 6 May 2016
Signals that induce cancer cell resistance cancer cells results in enhanced PNI32.
1. Liebig,C., Ayala,G., Wilks,J.A., Berger,D.H. &
to apoptosis at the perineural niche Both molecules participate in cytoskeletal Albo,D. Perineural invasion in cancer: a review of the
could afford survival advantage to cancer actin organization and cytokinesis, and in literature. Cancer 115, 33793391 (2009).
2. Amit,M. etal. International collaborative validation of
cells outside the primary tumour (FIG.2). recruitment of adhesion molecules to the intraneural invasion as a prognostic marker in adenoid
Several cell cycle regulators were found cell membrane32. The exact mechanism cystic carcinoma of the head and neck. Head Neck 37,
10381045 (2015).
to be associated with reduced cancer by which KIF14 and ARHGDI promote 3. Chatterjee,D. etal. Perineural and intraneural
cell apoptosis around nerves in human PNI is unknown, but their deregulation in invasion in posttherapy pancreaticoduodenectomy
specimens predicts poor prognosis in patients with
samples and DRG cocultures12,116,117. In tumour cells might prove to be essential for pancreatic ductal adenocarcinoma. Am. J.Surg.
response to nerve injury, a robust secretion neural tracking. Pathol. 36, 409417 (2012).

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