The Role of Schwann Cells in Perineural Invasion

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The Role of Schwann Cells in Perineural Invasion

Heidi Kristin Nielsen

Literature study by the faculty of medicine UNIVERSITY OF OSLO

February 2019

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The Role of Schwann Cells in Perineural Invasion

Heidi Kristin Nielsen

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The Role of Schwann Cells in Perineural Invasion Heidi Kristin Nielsen

http://www.duo.uio.no/

Trykk: Reprosentralen, Universitetet i Oslo

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Abstract

Cancer is the second leading cause of death in the world, by which metastasis constitutes 90%. Perineural invasion (PNI) is the ability of cancer cells to invade in, around and trough nerves. PNI can be used as a prognostic marker in some cancer patients and the nerves are a potential route of metastasis for some tumor types. Schwann cells (SCs) are the most

abundant cell type in the peripheral nervous system. They are involved in maintenance of axons, play a crucial role in axonal regeneration and SCs seem to be a major player in the process of PNI. A structural search using the following search strings in PubMed:

((perineural) AND invasion) AND Schwann cells, ((perineural) AND spread) AND Schwann cells, (((neurotropic) AND carcinomatous) AND spread) AND Schwann cells, and (((neural) AND invasion) AND Schwann cell) AND cancer, yielded 53 hits,18 articles fitted the criteria.

SCs have been shown to change cancer cell morphology and increase motility, migration and invasion. SCs have been reported to both induce epithelial-to-mesenchymal transition and Schwann-like differentiation of cancer cells, and de-differentiation of SCs have been observed in the presence of cancer cells. The communications between cancer cells and SCs were shown to involve CCL2, L1CAM, NCAM1, integrin A6B1, NTRK2, NTRK1, NGFR, CXCL12, CXCR7, CXCR4, MMP-2, MMP-9, GDNF, NGF and activation of MAPK and ERK signaling pathways in cancer cells. Not all mechanisms are applicable to all cancers and more investigation must be performed to find the true place of SCs in the process of PNI.

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1 Introduction

Cancer is the second leading cause of death in the world. It is estimated that 9.6 million deaths were caused by cancer in 2018, by which metastasis constitutes 90%. (1). Two common and well known routes of metastatic spread are through the blood and lymphatic system. One less known route of metastatic spread is through the nerves. Cancer cells are found in the area surrounding the nerves (Fig. 1), a histologic phenomenon called perineural invasion (PNI), and may represent the first step of distant metastasis. For some tumors, PNI may even be the only route of metastatic spread, and can be observed in the absence of lymphatic and vascular invasion in some tumors. Even though PNI has been described in the literature since the mid-1800 (2), it is only during the last decade that the interest of the role of nerves in cancer progression has exploded. A PubMed search showed over 1000

publications on PNI since the beginning of 2015 until the end of 2018, compared to only 59 publications in the five-year period from 1985-1989 (Fig. 2).

1.1 Perineural invasion: what it is and its clinical implications

PNI was defined by Batsakis in 1985 as the ability of cancer cells to invade in, around and through nerves (3). The true definition of PNI is, however, debated (2). Liebig et al. suggest new criteria for the identification of PNI in histological specimens: “finding tumor cells within any of the 3 layers of the nerve sheath, or tumor foci outside of the nerve with involvement of ≥33% of the nerve’s circumference” (2). Several studies have shown that cancer cells in the perineural space have reduced rate of apoptosis and increased proliferation, thus providing a survival advantage (4-7).

PNI is frequently observed in several cancer types such as prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, and adenoid cystic carcinoma. A systematic review and meta-analysis on PNI in pancreatic ductal adenocarcinoma conducted by Schorn et al. revealed that PNI appeared to be an independent prognostic marker for overall survival, disease-free survival and progression-free survival (8). Patients

demonstrating PNI in colorectal cancer have been shown to have decreased survival (9) and poor prognosis (10). The presence of PNI in squamous cell carcinoma has also been found to

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be a prognostic factor for local recurrence and decreased survival (11-13). Duraker et al.

concluded that PNI was not an independent prognostic factor in gastric cancer (14). In a systematic review conducted by Dantas et al. PNI was associated with local tumor recurrence in adenoid cystic carcinoma (15). The prognostic significance of PNI in prostate cancer remains controversial; while some studies have found that PNI is an independent prognostic factor after investigating radical prostatectomy specimens of prostate cancer (16-18) other studies have failed to find the same (19-21). The prognostic significance of PNI in prostate cancer biopsies is also controversial because there is conflicting evidence weather PNI found in prostate cancer biopsies correlate with extraprostatic extension or disease progression after treatment. The issue is nicely summarized in a systematic review by Harnden et al. (22). They conclude that the weight of evidence suggests that PNI is a significant prognostic indicator in prostate cancer biopsies. This is important since PNI status in prostate cancer needle biopsies could influence treatment decisions thus PNI status in prostate cancer biopsies should be included in the pathology report (23).

The increasing focus on PNI in tumors and the observations of association to local recurrence and decreased survival in patients with PNI have led to a change in treatment strategies in several types of cancer. In the treatment of head and neck malignancies, PNI status is a rationale for changing therapy, e.g. surgical strategies and adjuvant treatment (24), and some patients with colorectal cancer are offered adjuvant therapy after resection of PNI-positive tumor. Treatment of other cancers, such as in pancreatic cancer, remains unchanged by PNI status (2). However, there are studies that suggest that PNI should be considered when planning surgical treatment of pancreatic cancer (25).

1.2 The composition of a nerve

All areas of the nerve are critical for normal neural function, and the nerve consists of several anatomical structures and different cell types that make up the neural micromilieu. To

understand what PNI is and the mechanisms behind it, we need to have a clear understanding of the structure of the peripheral nerve, and the cells that it consists of.

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3 1.2.1 Three layers of connective tissue

A peripheral nerve consists of multiple bundles of axons, called fascicles, held together by three distinct connective tissue layers; epineurium, perineurium and endoneurium (Fig. 3A).

The epineurium is the outermost connective tissue layer which surrounds bundles of multiple fascicles. There are two distinct layers within the epineurium: an outer one consisting of areolar connective tissue with vascular components, and an inner layer of collagen fibers arranged in a wavy pattern and straight longitudinally arranged elastic fibers interspaced between the dense wavy layers of collagen (26). Each nerve fascicle is protected by a surrounding multilayered cellular tube; the perineurium (27, 28), which shields the nerve fibers from unwanted cells and molecules, making an effective diffusion barrier around the endoneurial space (28). The perineurium consists of perineural cells and some collagen fibers and fibroblasts between the perineural cells (29). The collagen fibers associated with

perineurium shows a similar wavy appearance as the inner epineurial layer (26). Perineural cells are flat, have spindle-shaped nuclei, and are attached to each other with desmosomes, arranged in concentric layers around the nerve fascicle, covered by basal lamina of variable thickness on either side (27, 29). The number of layers varies in different areas, but appears to be related to the size of the fascicles (27). The fascicles surrounded by the perineurium consist of the endoneurium, axons, Schwann cells, immune cells, fibroblasts and capillaries. The endoneurium is a collagen-rich, vascularized extracellular matrix (28). The collagen fibers are loosely arranged lying parallel to the nerve fibers (30), and are in variable numbers (27).

1.2.2 Schwann cells

Schwann cells (SCs) are the most abundant cell type in peripheral nerves originating from the neural crest (28). There are two distinct types of SCs; myelin and non-myelin (Remak) SCs (Fig. 3B-C). Myelin SCs encircles one axon (Fig. 3B), and non-myelin (Remak) SCs

encircles multiple axons (Fig. 3C). Myelination insulates the axons which significantly boost the conduction velocity. SCs are involved in the maintenance of axons, and are crucial for neuronal survival (31), and play an important role in axonal regeneration after nerve injury, especially in the early phases (32-34). There is a large body of evidence implying that SCs are capable of producing and secreting a variety of cytokines, which can act as immune

modulators, and that SCs can function as antigen presenting cells and thus contribute to the local immune network (35).

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1.2.3 Immune cells

Macrophages are distributed throughout the endoneurium, and these are called endoneurial macrophages (36). They represent a subset of tissue-resident macrophages that inhabit normal peripheral nerves, which are distinct from the hematogenous macrophages that are derived from monocytes recruited under various pathological conditions (37), such as during

inflammation (38), and account for up to 9% of the entire endoneurial cell population (36). It is reported that endoneurial macrophages appear triangular in shape; sometimes with tiny, slim processes in cross-section and that they either fill the gaps between myelinated axons and seem to attach to the myelin sheaths, or are close to endothelial cells of endoneurial capillaries (36). Endoneurial macrophages are known to form a part of the local immunity in peripheral nerves, and may act as a first line of defense until sufficient recruitment of macrophages from the systemic circulation occurs (35). They are known to participate in cellular defense and regeneration, however it is reported that they can produce growth factors that promote cancer proliferation and invasion (39).

1.2.4 Fibroblasts

Fibroblasts are located in the endoneurium and epineurium (27, 30, 40). In the endoneurium, fibroblasts are diffusely scattered between nerve fibers, frequently located near blood vessels and under the perineurium where they are usually arranged parallel to perineural cells (40).

They appear spindle-shaped with triangular or rectangular cell bodies, and their processes extend through the endoneurium (30, 40). They are often called endoneurial fibroblast like- cells and may represent approximately 2% to 9% of the endoneurial cells (40). The fibroblasts of the epineurium are numerous, and most of them are oriented parallel to perineural cells (40). After nerve injury the number of fibroblast increases (41).

1.2.5 Blood vessels

There are blood vessels adjacent to the perineurium, but they can, in some cases, also be seen within the perineurium or between layers of perineural cells (27). Small blood vessels can be found in the center of the fascicles, in the endoneurium, and are called endoneurial blood vessels (30).

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1.3 Theories of PNI

1.3.1 Perineural lymphatics

In the early history of PNI, there was a theory that the metastatic spread of cancer cells along nerves was an extension of lymphatic metastasis; through lymphatic vessels located in the perineurium or endoneurium, called perilymphatic spread or endolymphatic spread (3, 42, 43). But later, several studies failed to show the presence of lymphatic vessels in the

perineurium and endoneurium (27, 29, 41, 44) and today the theory of perineural lymphatic and endoneurial lymphatics has largely been discarded.

1.3.2 Low resistance path

A popular hypothesis is that PNI is a result of cancer cells following the path of least resistance (3, 25, 27, 41). In hematoxylin and eosin slides, it has been observed a separation of connective tissue from nerves, with production of a cleft-like space without a cell lining.

Rodin et al. suggest that this indicates an area of relatively little tissue cohesion immediately surrounding nerve, leaving an open path for cancer cells to spread (44). However, this theory does not explain how cancer cells grow through the perineurium and into the endoneurium, which is an issue stressed by Liebig et al. (2). There are multiple layers of collagen and basement membrane that separate the inside of the nerve from the surrounding lesion; this is not a low resistance path. Evidence is emerging indicating that the PNI phenomenon is more a invasion process than simple diffusion (2). After observing lysosome-like bodies in the

cytoplasm of cancer cells in the leading margin of the cancer tissue partially surrounding the peripheral nerves, Takubo et al. hypothesize that cancer cells produce enzymes that induce degeneration of perineural cells, making a path of less resistance for tumor cells to invade (29). A special pattern of circumferential tumor tissue around nerves is often observed in PNI (29, 41). This may be caused by the arrangement of the perineural cells in concentric layers encircling a nerve, meaning that when cancer cells infiltrate and destroy only a part of the perineural cells, the perineurium is destroyed circumferentially (29).

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1.3.3 Nerve communication/axonal migration

PNI may involve reciprocal signaling interactions between tumor cells and nerves, and that the invading tumor cells may have acquired the ability to respond to pro-invasive signals within the peripheral nerve milieu (2). Ayala et al. demonstrated in a PNI model, using mouse dorsal root ganglia co-cultured in Matrigel with prostate cancer cells that prostate cancer colonies facilitates nerve growth, and recruit nerves and migrate along neurites towards the ganglia, resulting in PNI (2).

1.3.4 Schwann cells

There is an emerging body of evidence that points out SCs as a major player in the PNI process. Since they are the most abundant cell type in the peripheral nervous system and play a major role in nerve regeneration, we aimed to investigate what is known about SC and cancer cell interaction, and which molecular mechanisms that may be involved in their communication. This might illuminate if SCs is important for the metastatic process of PNI, and what potentially can be done in the future to stop the spread of cancer cells along nerves.

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2 Methods

I conducted a search in PubMed after the available literature using the following search strings: ((Perineural) AND invasion) AND Schwann cells, ((Perineural) AND spread) AND Schwann cells, (((Neurotropic) AND carcinomatous) AND spread) AND Schwann cells, and (((Neural) AND invasion) AND Schwann cell) AND cancer. The search was ended on December 31. 2018.

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3 Results

The PubMed search yielded 53 hits. 7 articles were duplicates, 3 could not be found, and 1 was not written in English. 10 articles were books, review and commentaries. 14 articles were read in full and did not fit the thesis; there were articles about nerve sheet tumors, benign PNI, and the enteric nervous system with more. The search is illustrated in Fig. 4, and list over all the included articles can be found in Table 1, and Table 2 shows all the articles that were excluded.

3.1 Morphology

3.1.1 Human cancer specimens

When investigating 12 prostate cancer specimens from patients treated with radical prostatectomy Hassan et al. found PNI in 6 lesions. Cancer cells grew close to the nerve fibers, sometimes encircling the nerve. Electron microscopy revealed that cancer cells usually were outside the perineurium, but in some cases Hassan et al. could not see the perineurium, and the cancer cells seemed to invade into the nerve (27).

Carter et al. investigated 250 patients with squamous cell carcinoma that was surgically resected and found PNI in 36% of the samples. 20 necropsy specimens showed a 90%

incidence of PNI. Morphological investigation of PNI showed that cancer cells invade the perineural space and less frequently into the fascicles. The longest distance between the primary tumor and cancer cells within PNI area was 6 cm. In some areas SCs and axons were grossly degenerated and other areas they were lacking myelin (45).

Takubo et al. investigated 129 cases of esophageal squamous cell carcinoma. Using light microscopy, PNI was observed in 23% of the cases and there was a positive association of PNI and the depth of cancer cell infiltration. Invasion of cancer cells in the endoneurium was rarely observed, they were frequently limited to the perineural spaces, often completely encircling the nerves. Electron microscopy revealed that nerves completely encircled by cancer cells showed markedly degenerated SCs and axons, the perineurium was thinner and completely gone in some areas. Further investigation revealed that the perineurium was

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destroyed by cancer cells. They also observed cytoplasmic projections through the basal lamina between the perineurium and cancer cells (29).

When Bockman et al. investigated samples of tissue in light microscopy from 37 patients after surgery for pancreatic cancer they observed a difference in morphology between cancer cells that are in contact with the nerves compared to those which were not. Cancer cells in direct contact with the nerves were well-differentiated adenocarcinoma, with the morphology of mucin-producing cells, with secretion product and cellular debris in the lumen. The cancer cells also had considerably reduced height compared to cancer cells not interacting with nerves. The natural geometry of the nerves, with typically round or ovoid cross section, was distorted when invaded, and electron microscopic observation revealed that the nerves invaded by pancreatic cancer cells were almost completely unmyelinated. The perineurium was destroyed, and replaced by cancer cells, and thus, cancer cells were in direct contact with SCs and axons, and there was severe damage and loss of neural elements (25).

When investigating 22 cases of patients with mandibular ameloblastoma, Nakamura et al.

evaluated the relationship between ameloblastomas and the inferior alveolar nerve with

respect to PNI. The inferior alveolar nerve was clearly demarcated from the tumor in 18 cases.

The 4 patients that demonstrated tumor infiltration around the nerve showed a follicular pattern destroying the mandibular canal and invading the connective tissue surrounding the inferior alveolar nerve (46).

Swanson et al. observed in pancreatic ductal adenocarcinoma specimens that cancer cells sometimes cross the perineurium and are in intimate association with the myelin (Fig. 5A), and other times just touch but do not cross the perineurium (Fig. 5B) (47).

Fujii-Nishimura et al. investigated tumor tissue with detectable PNI resected from 168 pancreatic ductal adenocarcinoma patients in light microscopy. They observed what they called “focal differentiation” in 74% of the tumors at PNI sites (Fig. 5C), mostly in tumors where tumor cells were directly contacting the endoneurium. “Focal differentiation” was defined as a “histological feature of carcinoma cells at perineural space showing better- defined glandular formation with epithelial polarity than that in nearby tumor cells in stroma outside the peripheral nerve”. Focal differentiation was strongly associated with tumor grade, so the prognostic effect of focal differentiation was limited. But when comparing low grade

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pancreatic ductal adenocarcinoma tumors, the presence of focal differentiation showed poor overall survival compared to low grade tumors without focal differentiation (48).

3.1.2 In vitro experiments

Using an in vitro Transwell co-culture model system of salivary adenoid cystic carcinoma cells and SCs to mimic tumor-nerve cell interaction, Shan et al. observed that the salivary adenoid cystic carcinoma cell line SACC-83 changed morphology when co-cultured with SCs (Fig. 6B) compared to those cultured alone (Fig. 6A). They claim the cells changed to a mesenchymal morphology described more in detail as a change to “a spindle-shape and a polygon-shape” and they report that the intracellular junction decreased (31).

Using a 3D co-culture in vitro model were SCs and the human pancreatic cancer cells MIA PaCa-2 and PANC-1, Deborde et al. observed that when cancer cells were grown alone they grew as spherical structures (Fig. 6C); in the presence of SC contact there was a dynamic reorganization of the spherical structures into more linear chains of cells (Fig. 6D) (49).

In a non-contact in vitro co-culture system of pancreatic ductal adenocarcinoma cells and SCs, Fujii-Nishimura et al. observed that the cancer cells co-cultured with SCs formed sheet- like layered structures (Fig. 6E), compared to mono-cultured tumor cells that formed piled-up clusters (Fig. 6F) (48).

3.1.3 In vivo experiments

Demir et al. used murine and human pancreatic cancer and colon cancer precursor lesions to identify SCs in the early stages of cancer. They used double immunolabeling using GFAP (glial fibrillary acidic protein, a marker of glial cells) and S100 (marker of neural crest derived cells) and SOX10 (marker of SCs and melanocytic lineages). They observed SCs in areas in both the precursor and overt cancer tissue, but none in the normal neighboring parenchyma. Similar results were obtained in murine colorectal cancer tissue, but SCs were only occasionally detectable in human colon cancer specimens. In pancreatic cancer tissue, stromal nerves were enlarged and lacked an identifiable perineurium (50).

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3.2 De-differentiation of SCs

Investigating tissue from 37 patients undergoing surgery for pancreatic cancer, Bockman et al.

observed that cancer cells that invaded the nerves mostly interacted with unmyelinated nerves. Investigating the same specimens using electron microscopy the authors noticed that, in the sites of PNI, the nerves were almost completely unmyelinated and that the axons are released from the enfolding SCs and damaged, and replaced by cancer cells (25).

SCs are known to dedifferentiate into a non-myelinating and more active subtype of SCs after nerve injury. This is shown by upregulation of GFAP. Deborde et al. investigated tumor tissue from 8 patients with pancreatic adenocarcinoma, and showed that SCs upregulate GFAP in the presence of cancer cells compared to matched control sections. The same observation was made in other human specimens with PNI, such as pancreatic adenocarcinoma, thyroid cancer, salivary duct carcinoma and cutaneous squamous cell carcinoma. They observed that GFAP⁺ SCs surrounded clusters of cancer cells or wrap around individual cells and that myelin⁺SCs did not associate with cancer cells (49).

The observation made by Deborde et al. was further investigated in an in vivo murine model of PNI, where sciatic nerves were injected with the human pancreatic cell line MIA PaCa-2 or PANC-1. Immunofluorescence staining showed higher expression of GFAP⁺ SCs as

compared to nerves injected with PBS. The number of myelin+ SCs was also reduced in the presence of cancer cells. They observed that GFAP⁺ SCs exhibited long projections that were closely associated with large nucleated cancer cells (49).

3.3 Motility-migration-invasion

Demir et al. used a 3D migration assay in which the pancreatic cancer cell lines T3M4 and SU86.86 were simultaneously confronted with SCs on one side and with rat dorsal root ganglion neurons on the other side (Fig. 7A). They observed that SCs, within 24 hours, were the first cell type to migrate, migrating towards the cancer cells. After 48 hours, the pancreatic cancer cells had started to migrate towards the dorsal root ganglion. A 3D SC outgrowth assay using rat sciatic nerve (Fig. 7B) demonstrated that SC grew out of the nerve and migrated towards the pancreatic cancer cells, demonstrating once again that SCs are attracted to cancer cells. Similar results were obtained with the colon cancer cell lines DLD-1 and SW620. (50).

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Shan et al. used a scratch wound healing assay and a Transwell PNI assay to investigate the effect co-culture of the human adenoid cystic carcinoma cell line SACC-83 with SCs. Co- culturing of SACC-83 with SCs significantly increased migration and invasion of SACC-83 cells compared to the solely cultured SACC-83 cells (31).

Deborde et al. investigated the dynamic interaction between cancer cells and SCs using an in vitro model of PNI where cancer cells are co-cultured with dorsal root ganglion (DRG) explants. The DRG explants contain both neurites and associated SCs. Using the human pancreatic cancer cell line MIA PaCa-2 or PANC-1 they show that cancer cells were recruited to neurites by SCs within 24-48 hours and then migrated on and along SCs and neurites toward the center of the DRG in a contact dependent manner. Cancer cells not in contact of SCs or neurites did not move toward the DRG. The importance of SCs were highlighted when Deborde et al. irradiated DRGs with a single dose of 12-Gy radiation dose to deplete the DRGs of SCs. DRGs lacking SCs reduced the recruitment of both MIA PaCa-2 and PANC-1 cells to neurites. This was salvaged when SCs were added to irradiated DRGs (49).

Using a 3D co-culture in vitro model where SCs and the cancer cell lines MIA PaCa-2 and PANC-1 were grown together, Deborde et al. observed that individual cancer cells dissociated from the 3D structures in the presence of SC contact. They noticed that the SCs were

extremely dynamic, making repetitive contact with individual cancer cells. Further

investigation revealed that cancer cells developed protrusions directed toward SCs, at sites of cell-cell contact, and that this determined the direction of cancer cell migration away from the colony. Further, if there were SCs intercalated between cancer cells via long protrusions, interrupting cell-cell contact that facilitated cancer cells dissociation away from the cluster (49).

Using a quantitative 3D in vitro invasion assay where SCs were cultured in Matrigel and the pancreatic cancer cell line MIA PaCa-2 were added on top Deborde et al. observed that the presence of SC increased cancer cell invasion. No significant invasion was detected when using conditioned medium from SCs or in the absence of cell-cell contact (49).

Sroka et al. used the Cultrex laminin invasion assay to determine the effect of conditioned medium from SCs had on cancer cell invasion. They used two different types of immortalized SCs; one supposedly myelinating type (S16) and one non-myelinating type (S16Y). Using two prostate cancer cell lines; DU145 and PC3 and one pancreatic cancer cell line; CFPAC-1,

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13 they observed that conditioned medium from S16 SCs increased invasion of all cell lines by 1.6-2.0-fold. In contrast, conditioned medium from S16Y SCs decreased invasion of all cell lines by 50-60% (51).

Using a migration assay Fujii-Nishimura et al. investigated the motility of pancreatic ductal adenocarcinoma cells co-cultured with SCs. 3 of 4 pancreatic ductal adenocarcinoma cell lines displayed reduced migration; Capan-2, Capan-1 and PANC-1 cells, while AsPC-1 cells displayed increased migration when co-cultured with SCs (48).

Na’ara et al. investigated the affect SC conditioned medium had on pancreatic

adenocarcinoma cell lines. Using an in vitro migration assay and an in vitro wound healing assay Na’ara et al. showed that pancreatic adenocarcinoma cells treated with conditioned medium from SCs had increased migration and motility. In a multi-layered neural invasion assay where cancer cells were co-cultured with SCs, Na’ara et al. observed increased invasion of the pancreatic ductal adenocarcinoma cells when co-cultured with SCs, compared to when grown alone (32).

3.4 Extracellular microvesicles

Zhang et al. investigated the effect microvesicles derived from salivary adenoid cystic carcinoma had on the pathogenesis of PNI. Microvesicles are small (30-1.000 nm) bilayer lipid membrane-bound vesicles containing a variety of lipids, proteins and nucleic acid. They can originate from multiple cell types, under normal and pathological conditions. It is thought to be a way of intracellular communication between cells (52).

Microvesicles were harvested from conditioned media from salivary adenoid cystic carcinoma cells using ultracentrifugation. Investigations revealed that SCs internalized the microvesicles derived from salivary adenoid cystic carcinoma cells. Using a phospho-spesific antibody microarray for the ERK signaling pathway to investigate SCs exposed to microvesicles compared to unexposed SCs, Zhang et al. demonstrated that 15 sites associated with the ERK signaling pathway were hypophosphorylated and 31 sites were hyperphosphorylated. The regulated proteins were associated with cell growth, angiogenesis, cell differentiation,

inhibition of apoptosis, development of nervous system and signal transduction indicating that the ERK signaling pathway is activated in SCs exposed to microvesicles derived from

salivary adenoid cystic carcinoma cells (52).

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3.5 Inflammatory monocyte recruitment

Bakst et al. used an in vivo model of PNI in which the human pancreatic cancer cell line MIA PaCa-2 or the murine cell line Panc02 was injected into the distal sciatic nerve and observed that inflammatory monocytes accumulate in high frequency in invaded nerves compared to normal nerves, and that the inflammatory monocytes differentiated into mature macrophages already as early as after 18 hours. Immunohistochemical analysis of human pancreatic ductal adenocarcinoma specimens showed accumulation of macrophages around nerves invaded by cancer cells compared to intrapancreatic nerves from benign pancreatic specimens (37).

3.6 MET-EMT

Epithelial-mesenchymal transition (EMT) is thought to be a key biological process in

epithelial tumors invasion and metastasis. It is a likely mechanism by which tumor cells leave the primary site and establish metastasis, often seen as increased ability of migration and invasion (31). Mesenchymal-epithelial transition (MET) is the reverse phenomenon of EMT and it is thought that it is essential to establish metastatic colonies in distant organs (48) Fujii-Nishimura et al. investigated EMT/MET-related markers (E-cadherin, SMAD3 and vimentin) on tumor tissues with detectable PNI using immunohistochemical analysis. 168 pancreatic ductal adenocarcinoma patient specimens were examined and of the 74% which demonstrated focal differentiation they observed a more distinct membrane distribution of E- cadherin, decreased nuclear localization of SMAD3, and decreased expression levels of vimentin, which indicates MET-like change (48)

Using a non-contact in vitro co-culture system of pancreatic ductal adenocarcinoma cells and SCs, and fixating the cancer cells in formalin, Fujii-Nishimura et al. observed, using

immunohistochemical analysis, and validated using real-time quantitative RT-PCR, that pancreatic ductal adenocarcinoma cells had stronger E-cadherin expression, lower nuclear expression levels of SMAD3 and decreased vimentin expression, as seen in the patient specimens (48).

The authors concluded that SCs induce MET-like changes in pancreatic ductal

adenocarcinoma cells, contributing to colonization in pancreatic nerves. In addition, they stated that MET-like changes likely occur just after invasion into the nerve, because the focal

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perineural space (48).

Shan et al. used a Transwell in vitro PNI assay to investigate the effect co-culture of the human adenoid cystic carcinoma cell line SACC-83 with SCs. Using both RT-PCR analysis and western blot analysis, co-culture of SACC-83 cells with SCs revealed a significant decrease in the expression of E-cadherin, and increase in the expression of N-cadherin and vimentin in SACC-83 cells co-cultured with SCs compared to the solely cultured SACC-83 cells, indicating EMT-like changes (31).

3.7 Schwann-like differentiation

There is emerging evidence that Schwann-like differentiation, the process in which cancer cells develop a Schwann-cell like phenotype and express SC markers, might be involved in the process of PNI (31). Several markers has been defines as “SC markers” in previous studies; S100, S100A4, GFAP, nerve growth factor receptor (NGFR, also known as p75^NTR) and beta-1,3-glucuronyltransferase 1 (B3GAT1, also known as Leu-7, CD57 and HNK-1) (31, 53, 54). These are, however, not SC-specific markers.

Investigating histologic specimens from patients treated surgically for salivary adenoid cystic carcinoma, Luo et al. found that 19 of 20 cases showed moderate to strong intensity staining for S100 and 18 of 20 cases showed strong expression of GFAP, indication a Schwann-like differentiation. 11 samples had PNI and all PNI sites were positive for S100 and 10 of 11 cases were positive for GFAP. Statistical analysis revealed that both S100 and GFAP expression positively correlated with PNI. Investigation of the ultrastructural localization of S100 and GFAP in the samples using electron microscopy revealed that the Schwann-like differentiation occurred in a subpopulation of cells; in the myeloepithelial cells of salivary adenoid cystic carcinoma (53).

Luo et al. also investigated the presence of PNI and expression of SC markers in

mucoepidermoid carcinoma. They found no evidence of PNI in mucoepidermoid carcinoma samples, and the cancer cells did not express S100 or GFAP. They hypothesize that the differences in PNI between adenoid cystic carcinoma and mucoepidermoid carcinoma may reflect the different capabilities of Schwann-like differentiation of these tumors (53).

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Chen et al. investigated patient material from patients surgically treated for salivary adenoid cystic carcinoma and acinic cell carcinoma for the presence of PNI and B3GAT1 expression.

PNI appeared in 57.1% of the salivary adenoid cystic carcinoma samples and 10% of the acinic cell carcinoma. 78.6% of the salivary adenoid cystic carcinoma samples had positive staining for B3GAT1, while none of the acinic cell carcinoma samples where B3GAT1 positive. All PNI positive samples from the salivary adenoid cystic carcinoma group stained positive for B3GAT1 and statistical analysis revealed that there was a significant correlation between B3GAT1 expression and PNI. Chen et al. used immunofluorescence staining and electron microscopic examination to reveal which subpopulation of cancer cells that

expressed B3GAT1. It was the myoepithelial cells that expressed B3GAT1, and thus exhibit the Schwann-like differentiation seen in salivary adenoid cystic specimens (54).

Immunohistochemical evaluation of S100A4 staining, performed by Shan et al., in salivary adenoid cystic carcinoma specimens revealed that salivary adenoid cystic carcinoma express a higher level of S100A4, as compared to normal salivary gland tissue, and that the staining intensity of S100A4 were enhanced in cancer cells around the peripheral nerves and

correlated with PNI (31). This observation suggests that S100A4 is involved in the cross-talk between nerves and cancer cells. By using an in vitro Transwell co-culture model system of salivary adenoid cystic carcinoma cells and SCs to mimic the tumor-nerve cell interaction in the process of PNI, immunofluorescence staining, RT-PCR analysis and western blot analysis revealed that cancer cells co-cultured with SCs exhibit increased expression of the SC

markers: S100A4 and GFAP, compared to the solely cultured cancer cells. This indicates that SCs promote the Schwann-like differentiation of salivary adenoid cystic carcinoma (31).

Iwamoto et al. investigated tissue specimens from malignant melanoma patients and found that the expression of NGFR in histological specimens was increased in the subtype called desmoplastic melanoma which often exhibit PNI. Desmoplastic melanoma is a subtype of malignant melanoma that is characterized by spindle cells and stromal fibrosis. The increased expression may be an expression of Schwann-like differentiation in this subtype of malignant melanoma.

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3.8 CCR2-CCL2

CCR2 is a chemokine receptor expressed on inflammatory monocytes, and CCL2 is a known ligand for CCR2. The binding of CCL2 on CCR2 on inflammatory monocytes are known to recruit inflammatory monocytes to sites following infection and inflammation (37).

Using a series of elegant in vivo experiment using CCR2 KO mice and CCR2 KO inflammatory monocytes Bakst et al. demonstrate that the expression of CCR2 on inflammatory monocytes is critical for the development of PNI and the recruitment of inflammatory monocytes to PNI sites (37).

To investigate the importance of the ligand for CCR2; CCL2, Bakst et al. used CCL2 KO mice and WT inflammatory monocytes. They show that the local production of CCL2 is important for the development of PNI and inflammatory monocyte recruitment. They also investigated another known ligand for CCRT; CCL7. Loss of CCL7 did not impair the development of PNI and inflammatory monocyte recruitment in vivo (37).

Bakst et al. demonstrate that SCs produce CCL2 in the presence of cancer cells; this was shown using a combination of immunohistochemistry, immunofluorescence staining and RT- PCR on FACS-sorted SCs. Treating mice with anti-CCL2 antibody injected with cancer cells significantly reduced nerve invasion and inflammatory monocyte recruitment (37).

3.9 Cathepsin B

Cathepsin B is a protease with strong associations to cancer progression (37).

Bakst et al. demonstrates in a series of in vivo experiments using a pan-cathepsin inhibitor, cathepsin B KO mice and RT-PCR on FACS sorted cells, that macrophages was the predominant source of cathepsin B production and that inhibition of cathepsin B in vivo markedly reduced nerve invasion of the human pancreatic ductal cell line Panc02. They hypothesize that cathepsin B released from macrophages may disrupt the integrity of the perineurium and thus facilitate PNI (37).

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3.10 EGFR, TGF- α

It is well known that normal pancreatic tissue express epidermal growth factor receptor

(EGFR) and transforming growth factor α (TGF-α). TGF-α is known to interact with EGFR to start a chain of events that enhance cellular proliferation. Both EGFR and TGF-α is known to be upregulated in pancreatic cancer cells and EGFR overexpression amplifies EGF signal transduction (25).

When investigating prostate cancer tissue with immunohistochemistry Bockman et al.

demonstrates that nerves also express TGF-α and EGFR. Cancer cells were also positive for TGF-α, and strongly positive for EGFR as expected. Bockman et al. hypothesize that when cancer cells meet the nerves it provides a growth stimulus to the cancer cells further

enhancing the invasion of nerves. Bockman et al. does not speculate in which part of the nerve that express EGFR and TGF-α (25).

3.11 Integrins

Integrins are transmembrane receptors that facilitate cell-extracellular matrix adhesion.

Ligands for integrins include fibronectin, vitronectin, collagen and laminin. Integrin A6B1 binds to laminin and is expressed by several cancer types such as prostate cancer and

pancreatic cancer. Cancer cells can cleave integrin A6B1 to form a structural variant; A6pB1.

And both expression of integrin A6B1 and A6pB1 are associated with increased tumor cell migration, invasion, and metastasis (51).

Sroka et al. showed, using immunohistochemistry, that prostate and pancreatic cancer cells have a high expression integrin A6, also during PNI. The increased migration that conditioned medium from the SC S16 induce in the prostate cancer cell lines; DU145 and PC3 and the pancreatic cancer cell line CFPAC1, are inhibited when integrin A6p or integrin B1 are blocked using antibodies. When investigating cancer cells incubated with conditioned medium from SC using immunoblot and quantitative analysis, Sroka et al. observed that production of integrin A6p increased when cancer cells were incubated with conditioned medium from the SC S16 and decreased when incubated with conditioned medium from the SC S16Y. Flow cytometry analysis revealed that integrin A6 surface levels were unchanged in tumor cells treated with the conditioned medium (51).

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19 The cleavage of the integrin A6 into A6p is regulated by the serine protease, urokinase-type plasminogen activator (uPA), and its receptor urokinase plasminogen activator receptor (uPAR). Sroka et al. demonstrate that inhibition of uPA resulted in inhibition of the induced production of integrin A6p in cancer cells exposed to conditioned medium from the SC S16.

Sroka et al. hypothesize that components of the uPA:uPAR axis is likely molecular candidates in the conditioned medium from SC altering the production of integrin A6p and invasiveness of cancer cells (51).

3.12 BDNF-NTRK2

Brain-derived neurotrophic factor (BNDF) is one of the most important neurotrophins.

Neurotrophic receptor tyrosine kinase 2 (NTRK2, also called TrkB) is one receptor for BNDF, and together they help to maintain the survival of existing neurons and encourage growth and differentiation of new neurons and synapses. It has been revealed that BNDF and NTRK2 also are involved in the progression of various malignant tumors. Shan et al. has previously found that BNDF and NTRK2 were highly expressed in salivary adenoid cystic carcinoma and associated with PNI (31).

Shan et al. used an in vitro Transwell co-culture model system of salivary adenoid cystic carcinoma cells and SCs to mimic the tumor-nerve cell interaction in the process of PNI.

Investigation of the expression levels of BNDF and NTRK2 by ELISA, RT-PCR and western blot assay revealed that SC produce BNDF and that the production of BNDF increases when SCs are co-cultured with salivary adenoid cystic carcinoma cell lines. Salivary adenoid cystic carcinoma cell lines co-cultured with SCs upregulate the expression of NTRK2 as compared to solely grown cancer cells, the upregulation was inhibited when using a NTRK2 inhibitor.

The levels of NTRK2 in SCs were unchanged (31).

Inhibition of NTRK2 had several effects on the cancer cells, it markedly blocked the change to mesenchymal phenotype, the EMT-like changes and the Schwann-like differentiation observed in these cells and it markedly inhibited the increased motility observed of cancer cells co-cultured with SCs (31).

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3.13 Nerve growth factor

Neurotrophins are a family of proteins that regulate differentiation, function and survival of neurons. One of these is nerve growth factor (NGF), a member of the NGF-beta family. NGF binds to both NGFR and neurotropic receptor tyrosine kinase 1 (NTRK1, also known as TrkA). There are several ligands that can act on NGFR; NGF, brain-derived neurotropic factor (BDNF), neurotropin-3 and neurotrophin-4/5 (NT-4/5). It is thought that NGF, acting through NGFR, promotes SC migration (55). NGF has two isoforms; beta-NGF and pro-NGF (50).

Investigation of human pancreatic cancer tissue, using RT-PCR, by Demir et al. revealed an increased expression of NGFR and a tendency towards increased expression of NGF and no increase in BDNF expression. Immunoblotting revealed a prominent increase in the detected amount of proNGF in human pancreatic tissue when compared with normal pancreas. In vivo experiments with murine tissue from wild-type normal pancreas to mice with overt pancreatic cancer revealed similar results; there was an increase in the levels of NGF, NGFR and BDNF (50).

Demir et al. observed in co-culture of SCs with the pancreatic cancer cell lines SU86.86 and T3M4, increased levels of NGF expression in both cancer cells and SCs. Further investigation revealed that both beta-NGF and to a lesser extent pro-NGF augmented SC transmigration.

The migration of SCs towards cancer cells was reduced by blocking NTRK1 and NGFR with an inhibitor, or when SCs were lacking NGFR, using siRNA. The migration of pancreatic cancer cell towards dorsal root ganglion was also inhibited when blocking NTRK1 and NGFR with an inhibitor (50).

Iwamoto et al. investigated tissue specimens from malignant melanoma patients and found that the expression of NGFR in histological specimens was increased in the subtype called desmoplastic melanoma which often exhibit PNI. In addition to be a marker from Schwann- like differentiation, Iwamoto et al. suggest another function of this increased expression.

Previous studies have demonstrated that cancer cells expressing NGFR and exposed to NGF have increased migration. Based on previous observations that SCs produce NGF and BDNF, Iwamoto et al. suggest a mechanism of PNI where cancer cells expressing NGFR react to NGF and BNDF released from SCs, perhaps chemotactically, stimulating infiltration of tumor cells along nerves (55).

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3.14 L1CAM

The L1 cell adhesion molecule (L1CAM) is a transmembrane protein, often overexpressed in human cancer, and associated with poor prognosis and metastasis formation. It plays an important role in the development of the nervous system, including neuronal migration and differentiation. Earlier investigations has revealed that L1CAM can activate the MAP kinase (MAPK) signaling pathway and that soluble L1CAM can interact with integrins and stimulate migration (32).

Investigation of histological specimens from patients with invasive pancreatic cancer revealed that L1CAM were mainly expressed by cancer or nerve cells, as expected. Similar results were obtained when investigating a well-established pancreatic ductal adenocarcinoma model in mice. The expression of L1CAM in cancer cells was significantly higher around nerves.

Immunofluorescent staining revealed that L1CAM was expressed at the membrane of both SC and pancreatic ductal adenocarcinoma cells (32).

Cancer cell motility is increased when cancer cells are exposed to soluble L1CAM, since conditioned medium from SCs also increase cancer cell motility, Na’ara et al investigated the level of soluble L1CAM in SC conditioned medium, using immunoprecipitation, which revealed that SCs secrete soluble L1CAM. By blocking ADAM10, major determinants of L1CAM shedding, L1CAM secretion to the media by SCs were blocked. By a series of in vitro experiments using recombinant L1CAM, SC conditioned media, ADAM10 inhibitor and co-culture experiments with cancer cells and SC, knock down of L1CAM, and L1CAM antibody, Na’ara et al. demonstrate that pancreatic ductal adenocarcinoma cells are dependent on soluble L1CAM and L1CAM membrane expression for migration and invasion. In vivo experiments revealed that anti-L1CAM decrease the extent of neural invasion by cancer cells, the tumor size was not altered by anti-L1CAM (32)

Prior studies indicated that homomeric L1CAM activation induces MAPK signaling. Na’ara et al. investigated pancreatic ductal adenocarcinoma cells with recombinant L1CAM or conditioned medium from SCs. Immunoblot analysis revealed that soluble L1CAM induced ERK phosphorylation, indicating an activation of the MAPK signaling pathway (32).

Looking for additional mechanism, Na’ara et al investigated a panel of candidate protein targets using quantitative RT-PCR in L1CAM activated pancreatic ductal adenocarcinoma

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cells. That revealed increased expression of MMP-2, MMP-9, GDNF, and artemin.

Immunohistochemical analysis of pancreatic ductal adenocarcinoma tumors in mice treated with anti-L1CAM revealed reduced expression of MMP-2 and MMP-9, there was no significant difference in the expression of GNDF and artemin. Investigation into the mechanism behind regulation of MMP-2 and MMP-9 by L1CAM, Na’ara revealed that recombinant L1CAM treated cancer cells induced phosphorylation of STAT3, anti-L1CAM treated mice had lower expression of phosphorylated as compared to untreated mice (32).

To summarize, Na’ara et al. claim L1CAM facilitates neural dissemination by two mechanisms: it serves as a chemoattractant along intra pancreatic neural bundles and it facilitates the breakdown of ECM by upregulation of MMP expression along the axon (32).

3.15 NCAM1

Neural cell adhesion molecule 1 (NCAM1) is a member of the immunoglobulin family, a glycoprotein expressed on the surface of neurons, glia and skeletal muscle among others.

During the myelinating differentiation program of SCs NCAM1 among other proteins are lost, when SCs dedifferentiate, NCAM1 is reexpressed. NCAM1 has been reported to play a role in cancer progression and axonal guidance and is expression is correlated with PNI (49).

Deborde et al. generated stable SC lines depleted of NCAM1 using shRNA targeting

LCAM1. The cells ability to proliferate and form tunnels in Matrigel was not altered. Cancer cells showed decreased cancer cell invasion when investigating the NCAM1 depleted SCs using a 3D co-culture in vitro model with SCs and cancer cells. The ability of SC to affect the shape of cancer cell clusters and to promote cancer cell protrusions were significantly

decreased when using the NCAM1 depleted SCs. Depletion of NCAM1 also decreased the ability of SCs to recruit cancer cells DRGs (49).

Investigation of human pancreatic adenocarcinoma specimens and co-culture in vitro experiments revealed that SCs expressed NCAM1 when in direct contact with cancer cells, and that LCAM1 accumulate in areas in which ling filopodia extend outward, contacting cancer cells and other SCs (49).

Using an in vivo model of PNI injecting cancer cells into the sciatic nerves of WT and

NCAM1 KO mice, Deborde et al. observed decreased PNI and a slower progression of sciatic

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23 nerve paralysis in NCAM1 KO mice, consistent with the hypothesis that NCAM1 expression in SC supports cancer cell invasion into the nerve. The depletion of NCAM1 did not prevent the dedifferentiation of SCs into GFAP⁺ cells. Deborde et al. stress that it is the physical contact between SCs and cancer cells that enhance PNI, and not soluble factors released by SCs (49).

3.16 MUC1-MAG

Mucin 1, cell surface associated (MUC1) is a transmembrane mucin that can affect the adhesion, differentiation and growth of cells. MUC1 is often overexpressed in

adenocarcinomas, including pancreas cancer, and it contains a large and extensively O- glycosylated extracellular tandem repeat domain, which is aberrant glycosylated in

malignancies. MUC1 can bind to receptors found on the same cell surface, bind to receptors on opposing cell surfaces or extracellular matrices. Myelin-associated glycoprotein (MAG) is a cell membrane protein in the sialic acid-binding immunoglobulin-type lectin (SIGLEC) family. It is expressed on oligodendrocytes and SCs, believed to be involved in myelination during nerve regeneration and to function as an adhesive molecule to compact myelin and bind it to axons (47).

Swanson et al demonstrates that MAG is physically associated with MUC1 in pancreatic tumor cells and that interaction between these molecules facilitates tumor cell/SC adhesion.

Increased levels of MAG and overexpression of MUC1 enhanced the adhesion of tumor cells to SC. An antibody targeting MAG (anti-MAG antibody clone 513) specifically blocked the adhesion but not completely suggesting that other receptor/ligand interactions contribute to overall cellular adhesion. They postulate that the adhesive advantage would contribute to the capacity of pancreatic cancer cells to survive and proliferate within the nerves. (47)

3.17 CXCL12-CXCR4/CXCR7

C-X-C motif chemokine (CXCL12) is widely expressed in several well-vascularized

mammalian tissues and cancers and is known to regulate homing, proliferation, and survival of bone marrow-derived hematopoietic stem cells and stromal cells. It is known to bind to its cognate receptor CXCR4 and an alternative receptor CXCR7. CXCR7 can heterodimerize

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with CXCR4. CXCR4 is often overexpressed in several cancer types and influence cancer proliferation and metastasis (56)

Demir et al. observed that both CXCR4 and CXCR7 was highly expressed in immune cells, cancer cells and SCs using double-immunolabeling experiments on pancreatic ductal

adenocarcinoma tissue. Human SCs collected from spinal nerves also contained high levels of CXCR4 and CXCR7. CXCL12 expression was upregulated in pancreatic cancer tissue and cell lines. When the human pancreatic cancer cell lines SU86.86 or T3M4 was co-cultured with SCs or dorsal root ganglia the expression of CXCR4 raised in the SCs and in the dorsal root ganglion (56).

Demir et al. investigated if hypoxia would affect the levels of CXCR4 and CXCR7 in SCs.

They observed that hypoxia increased the expression of CXCR4 and CXCR7 in SCs it did not affect CXCL12 expression. When the pancreatic ductal adenocarcinoma cell lines SU86.86 and T3M4 was exposed to hypoxia it resulted in at least fivefold up-regulation of CXCL12 expression and conditioned medium collected from hypoxia treated pancreatic cancer cells enhanced the migration of SCs (56).

Increasing levels of CXCL12 increased the amount of migrating SCs towards cancer cells.

Co-culture experiments with the pancreas cancer cell lines T3M4, MIA PaCa-2, PANC-1, SU86.86 with SC pretreated with CXCR4 or CXCR7 inhibitor resulted in decreased migration of the SCs treated with CXCR7 inhibitor. Inhibition of CXCR4 or CXCR7 of cancer cells did not affect the cancer cells ability to migrate towards dorsal root ganglion neurons. Using a murine model of pancreatic ductal adenocarcinoma where CXCL12 was abrogated, Demir et al. show that there was a prominent reduction in the number of SCs (56).

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4 Discussion

4.1 Morphology

After investigation of cancer tissue from various cancer types it has become clear that not all cancers are attracted to the neural microenvironment. The cancers that do tend to grow along the perineurium, away from the primary tumor, sometimes destroying it and entering the endoneurium. Thus, cancer cells come into physical contact with SCs and other components of the nerve. There the neuronal elements get destroyed, with a reduction of myelin, number of axons and SCs. Some cancers display a different morphology at PNI sites as compared to the rest of the tumor, demonstrated as a mesenchymal phenotype in pancreatic ductal adenocarcinoma (48) and more well-differentiated cells with reduced height (25).

In vitro experiments with cancer cells co-cultured with SCs showed that the salivary adenoid cystic carcinoma cell line SACC-83 changed to a mesenchymal morphology; “a spindle-shape and a polygon-shape” (31). The pancreatic ductal adenocarcinoma cell lines MIA PaCa-2 and PANC-1 changed from spherical structures into more linear chains of cells (49) and the pancreatic ductal adenocarcinoma cell lines Capan-2, Capan-1, PANC-1 and AsPC-1 formed sheet-like layered structures (48). The differences in these observations may be explained by the different cell lines, and experimental models; the first observation was made in a

Transwell co-culture system where the cancer cells were observed from above, the second was a 3D co-culture model and the last a non-contact system where the cancer cells were observed from the side. The authors also used different SCs in their in vitro experiments, which can also explain the differences seen. Shan et al. used SCs isolated from the sciatic nerves of neonatal SD rats (31), Deborde et al. used the human nonneoplastic SC line HEI- 286 (49) and Fujii-Nishimura et al. used the murine SC line SW10 (48).

In vivo experiments with murine and human pancreatic cancer and colon cancer revealed that SCs are present in pre-cancerous and cancerous tissue, outside the nerves, interacting with the cancer cells (50).

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4.2 De-differentiation of Schwann Cells

Immunohistochemistry and electron microscopy of pancreatic cancer patients revealed that at PNI sites the nerves were almost completely unmyelinated and that cancer cells interacted with unmyelinated nerves (25, 49). The SCs present in these areas were of a non-myelinating type and they surrounded cluster of cancer cells. This was validated an in vivo murine model of PNI.

4.3 Motility-migration-invasion

Sroka et al. and Na’ara et al. investigated how conditioned medium from SCs affect different cancer cell lines using different in vitro such as the Cultrex laminin invasion assay, migration assay, scratch wound healing assay, and a multi-layered neural invasion assay. The human MIA PaCa-2 and the murine KPC-989 pancreatic adenocarcinoma cell lines demonstrated both increased migration and invasion (32). The prostate cancer cell lines DU145 and PC3 and the pancreatic cancer cell line CFPAC-1 demonstrated increased invasion. The

observation in DU145, PC3 and CFPAC-1 was only observed when using conditioned

medium from a myelinating SC type, the non-myelinating type decreased invasion of DU145, PC3 and CFPAC-1 (51).

Using various in vitro model systems such as scratch wound healing assay, Transwell PNI assay, co-culture with DRG, 3D co-culture model, 3D invasion assay and migration assay the authors demonstrated that, in general, cancer cells co-cultured with SCs, have increased motility, migration and invasion. The cancer cell lines demonstrating these properties were the human adenoid cystic carcinoma cell line SACC-83 (31), the human pancreatic cell lines MIA PaCa-2 and PANC-1 (49) and the pancreatic ductal adenocarcinoma cell line AsPC-1 (48).

Fujii-Nishimura et al. found that the human pancreatic ductal adenocarcinoma cell lines Capan-2, Capan-1 and PANC-1 had reduced migration when co-cultured with SCs (48).

Deborde et al. did also use PANC-1 in their investigation and found increased migration and invasion when PANC-1 where grown in co-culture with SCs. First of all Fujii-Nishimura et al. used the murine SC line SW10 (48), and Deborde et al used the human nonneoplastic SC line HEI-286 (49). Second Deborde et al. did only observe increased invasion and migration

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27 when PANC-1 where in direct physical contact with SCs (49), while Fujii-Nishimura et al.

used an assay where the cancer cells and SCs were not in direct contact with each other (48), and this is most likely the reason for the different observations.

Demir et al. found that it is the SCs that migrate towards the cancer cells and not the other way around, and that the cancer cells migrate towards dorsal root ganglion neurons (50).

4.4 MET-EMT

Fujii-Nishimura et al. found MET-like changes in the pancreatic ductal adenocarcinoma specimens demonstrating focal phenotype differentiation in PNI sites. MET-like changes was observed also observed in vitro, when co-culturing four human pancreatic ductal

adenocarcinoma cell lines Capan-2, Capan-1, PANC-1 and AsPC-1 with SCs; observed as upregulation of E-cadherin and down-regulation of vimentin and SMAD3 (48). Shan et al.

found, in an in vitro assay, EMT-like changes in the human adenoid cystic carcinoma cell line SACC-83 co-cultured with SCs (31). The differences seen can be explained by the different cancer cell lines. Fujii-Nishimura et al. used an immortalized murine SC line SW10 (48), and Shan et al. used primary cultures of SCs isolated from the sciatic nerves of neonatal SD rats (31). The in vitro assays did not differ much and both were a non-contact system. Indicating that the differences seen between the two have something to do with the different cancer types and SCs used.

4.5 Schwann-like differentiation

Several cancer tissues demonstrate Schwann-like differentiation. This was observed in salivary adenoid cystic carcinoma (31, 53, 54), and desmoplastic melanoma (55).

In vitro experiments using the human adenoid cystic carcinoma cell line SACC-83 co- cultured with SCs demonstrated Schwann-like differentiation, indicating that SCs are responsible of the Schwann-like differentiation seen in salivary adenoid cystic carcinoma (31), and it may be a player in the Schwann-like differentiation seen in desmoplastic melanoma ass well, but further research must be conducted on the matter.

The markers used to identify Schwann-like differentiation in human specimens and cell lines are not SC specific. Many of them are expressed in a wide range of cells. S100 is highly

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expressed in the heart, but also found in fat, salivary gland and thyroid. S100A4 is widely expressed in bone marrow and lung. NGFR in expressed in several tissues like the adrenal gland, gall bladder, spleen and testis. B3GAT1 is highly expressed in the brain and somewhat expressed in the adrenal gland, prostate and thyroid. GFAP is the only marker that has a restricted expression towards the brain.

4.6 Mechanisms

Several molecular pathways have been investigated to find out how SCs facilitate PNI by recruitment of inflammatory monocytes, alters cancer cells morphology, increase motility, migration and invasion, induce EMT-like changes and Schwann-like differentiation.

The development of PNI in in vitro models of PNI was dependent on local production of CCL2 by SCs, cathepsin B released from macrophages (37), the release of L1CAM from SCs (32) and the expression of NCAM1 on SCs (49). Bakst et al. found that the recruitment of inflammatory monocytes to sites of PNI is dependent on the expression of CCR2 on inflammatory monocytes, and the local production of CCL2 by SCs (37).

The increased migration of the human prostate cancer cell lines DU145, PC3 and the human pancreatic cell line CFPAC1 co-cultured with SCs was reduced when integrin A6p or integrin B1 were blocked using antibodies (51), the increased motility of the salivary adenoid cystic carcinoma cell line SACC-83 co-cultured with SCs was reduced when inhibiting NTRK2 (31), The human MIA PaCa-2 and murine KPC-989 pancreatic adenocarcinoma cell lines increased invasion when co-cultured with SCs were reduced when blocking L1CAM using antibody (32), and the increased invasion of the human pancreatic cell lines MIA PaCa-2 and PANC-1 when co-cultured with SCs were reduced when co-cultured with SCs lacking

LCAM1 (49). The migration of SCs towards the prostate cancer cell lines SU86.86 and T3M4 was reduced when NTRK1 and NGFR were blocked in SCs. The same happened to the migration of pancreatic cancer cells towards dorsal root ganglion neurons when NTRK1 and NGFRwere blocked in the cancer cells (50). The migration of SCs towards pancreatic cancer cells was dependent on the cancer cells expression of CXCL12 and the SCs expression of CXCR7 (56).

The human prostate cancer cell lines DU145, PC3 and the human pancreatic cell line CFPAC1 increased production of integrin A6 when co-cultured with SCs (51), the salivary

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29 adenoid cystic carcinoma cell lines SACC-83 and SACC-LM upregulate NTRK2 when co- cultured with SCs (31), the human MIA PaCa-2 and murine KPC-989 pancreatic

adenocarcinoma cell lines exposed to conditioned medium from SCs and soluble L1CAM had an activation of MAPK signaling and increased expression of MMP-2, MMP-9 and GDNF (32). Co-culture of SCs and the pancreatic cancer cell lines SU86.86 and T3M4 ended up with an upregulation of NGF expression in both cancer cells and SCs (50), SCs increased

expression of CXCR4 when co-cultured with the prostate cancer cell lines (56).

SCs exposed to microvesicles derived from the human salivary adenoid cystic carcinoma cell line ACC-2 had increased activation of the ERK signaling pathway (52), when SCs were co- cultured with the salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM they increased production of BNDF (31)

The NTRK2 inhibitor K252a inhibited the Schwann-like differentiation, the mesenchymal phenotype and the EMT-like changes seen in the salivary adenoid cystic carcinoma cell line SACC-83 (31), this was not blocked completely, thus indicating that other signaling pathways may also be involved in the communication between SACC-83 and SCs.

It is becoming clear that the effect of the different molecular mechanisms involved in the interaction between cancer cells and SCs are numerous and that there are differences between different cancers where one molecular mechanism is more important than another. Even though SCs is a major component in the process of PNI, but it is unlikely that they are working alone. The nerve micromilieu consist of several components where SCs are just a piece of the puzzle in understanding the molecular process of PNI. There is probably a complex signaling interactions between the peripheral nerve milieu, tumor stromal elements, and tumor cells. The molecular mechanisms involved between SCs and cancer cells needs to be further investigated to understand the differences between different cancer types in their interaction with SCs, and the importance of different types of SCs in their interaction with cancer cells.

4.7 Future

Several treatment options have been suggested to inhibit the interaction between SCs and cancer cells and thus inhibit the development of PNI and hopefully the spread of cancer. Shan et al. suggested targeting the BNDF/ NTRK2 axis as a potential treatment strategy for

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inhibiting PNI in salivary adenoid cystic carcinoma (31). Na’ara et al. suggested targeting L1CAM in pancreatic ductal adenocarcinoma as a potential treatment strategy against PNI (32). Anti-CCL2 antibody could potentially reduce PNI and the recruitment of inflammatory monocytes and Pan-cathepsin inhibitor could reduce PNI (37)

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5 References

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8. Schorn S, Demir IE, Haller B, Scheufele F, Reyes CM, Tieftrunk E, et al. The influence of neural invasion on survival and tumor recurrence in pancreatic ductal adenocarcinoma - A systematic review and meta-analysis. Surgical oncology.

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The Role of Schwann Cells in Perineural Invasion

The Role of Schwann Cells in Perineural Invasion

Heidi Kristin Nielsen

Literature study by the faculty of medicine UNIVERSITY OF OSLO

The Role of Schwann Cells in Perineural Invasion

Heidi Kristin Nielsen

Abstract

Table of Contents

1 Introduction

1.1 Perineural invasion: what it is and its clinical implications

1.2 The composition of a nerve

1.3 Theories of PNI

2 Methods

3 Results

3.1 Morphology

3.2 De-differentiation of SCs

3.3 Motility-migration-invasion

3.4 Extracellular microvesicles

3.5 Inflammatory monocyte recruitment

3.6 MET-EMT

3.7 Schwann-like differentiation

3.8 CCR2-CCL2

3.9 Cathepsin B

3.10 EGFR, TGF- α

3.11 Integrins

3.12 BDNF-NTRK2

3.13 Nerve growth factor

3.14 L1CAM

3.15 NCAM1

3.16 MUC1-MAG

3.17 CXCL12-CXCR4/CXCR7

4 Discussion

4.1 Morphology

4.2 De-differentiation of Schwann Cells

4.3 Motility-migration-invasion

4.4 MET-EMT

4.5 Schwann-like differentiation

4.6 Mechanisms

4.7 Future

5 References