Structural disarrangement of nerve tissues in CRC patients has been reported by several studies, indicating putative roles of autonomic and enteric nerves in the development of CRC. A retrospective study of 90 CRC patients tested the immunoreactivity of tyrosine hydroxylase and vesicular acetylcholine transporter respectively in the resected tissue to track down sympathetic and parasympathetic nerves. Results denoted the existence of both sympathetic and parasympathetic nerves in proximity to the tumor. Sympathetic nerves are found in the stroma closer to tumor site, while parasympathetic nerves are located away from tumor cells (Zhou et al., 2018). Distribution of β2A receptors appears to be denser in CRC-invaded ganglia and larger nerve bundles, compared to the nervous tissue in non-tumor sections (11). Besides, an increasingly diffuse and dense expression pattern of β2A receptors is observed from the normal colon tissue to G1, G2 and G3 differentiated adenocarcinoma. There is also a significant association between β2 adrenergic receptor expression and tumor size, tumor invasion or lymph node metastasis. Several studies have reported abnormal morphology of enteric nerves. Significant loss of myenteric and submucosal plexuses was noticed in CRC, especially in more progressive types (Godlewski, 2010; Ciurea et al., 2017); in sigmoid and rectal cancer, disruption of normal ENS structures are observed, with an irregular border of the myenteric plexus and deformation of their structures, and, simultaneously, a larger area of extracellular matrix surrounding the myenteric plexuses with more abundant collagen fibers in tumor-infiltrated colon walls (Zauszkiewicz-Pawlak et al., 2017). Myelin-like structures that usually appear in degeneration of nerves are observed near the tumor invasion site. A marked drop in the density of enteric glial cells was also observed corresponding to the increase in tumor grading (Jaiswal et al., 2020).

Sustained efforts have been made in denervation studies to ascertain whether loss of nerve innervation would affect tumor initiation and survival, providing functional insights into the role of nerves in CRC.

Research of parasympathetic denervation mainly considers ablation of muscarinic receptors. Targeting muscarinic acetylcholine receptor 3 (M3R) through selective and non-selective antagonists (p-fluorohexahydro-sila-difenidol hydrochloride and atropine respectively) repressed H508 colon cancer cell proliferation by approximately 40% whilst acetylcholinesterase inhibitors were capable of stimulating tumor growth by 2 to 2.5 fold (Cheng et al., 2008), indicating participation of cholinergic signaling in tumor development. Similar inhibitory effects were also observed in murine colon cancer models as epithelial proliferation, tumor size and quantity were greatly diminished in mice deficient of CHRM3, the coding gene of M3R (Raufman et al., 2008).

Other denervation studies have been focused on enteric nerve denervation. Chagas disease, a trypanosomiasis-related disease that induces megacolon and damage to myenteric neurons, seems to be an existing example of how myenteric neuronal activity negatively associates with colon carcinogenesis. Experiments on Wistar rats suggested that chronic infection with Trypanosoma cruzi led to fewer tumors when treated with dimethylhydrazine(DMH), a specific chemical carcinogen to induce CRC (Oliveira et al., 2001). A review of histopathological findings from 894 chagasic megacolon patients revealed that none of those patients developed colon cancer(Garcia et al., 2003); previous studies on surgical specimens from Chagas patients who underwent megacolon resection confirmed a decline in the density of myenteric neurons and correspondingly reduced risk for colon cancer [(Kannen et al., 2015)]. To prove the tumorigenic effect of myenteric nerves, in-vivo experimental approach has been established to mimic myenteric denervation in chagasic megacolon through benzalkonium chloride (BAC) treatment. Results showed that BAC-treated rats are less susceptible to DMH, since both preneoplastic and neoplastic lesions were reduced compared to those whose myenteric innervation were intact (Garcia et al., 1996; Vespúcio et al., 2008; Kannen et al., 2015).

Although few studies have directly investigated changes in tumors upon sympathetic denervation, many researchers have reported effects of beta receptor antagonists. Studies of multiple CRC cell lines demonstrated that beta blockade inhibits cell viability and proliferation in a dose-dependent manner, probably through EGFR-Akt/ERK1/2 pathway, cell cycle arrest and apoptosis followed by suppression of β2 signaling (Coelho et al., 2015; Chin et al., 2016). Meanwhile, various clinical studies were conducted in Europe to measure the effect of beta receptor blockers on CRC patient outcomes, but no definitive conclusion has been made. Stage-specific benefit of beta blockade at diagnosis (particularly selective beta blockers) was once detected, as overall and CRC-specific mortality markedly decreased in stage IV patients prescribed with beta blockers (Jansen et al., 2014). However, several large population-based studies have reported that long-term beta blocker usage may contribute to increased risk of CRC progression, and little association was found between pre- or post-diagnostic beta blockade and improved clinical outcomes (Jansen et al., 2012; Hicks et al., 2013; Jansen et al., 2017). Perioperative application of beta blockers, on the other hand, seems to be more promising in improving CRC patients’ prognosis. The latest study demonstrated that preoperative beta blocker treatment results in reduced post-operative complications and mortality in rectal cancer (Ahl et al., 2020); it was also previously found that combine perioperative blockade of β receptor and COX2 leads to lower recurrence rate and improvement in tumor biomarkers associated with epithelial-to-mesenchymal transition, immune microenvironment and CRC-related inflammatory pathways (Haldar et al., 2020).

Neurotrophic factors represent a list of proteins binding to tyrosine kinase receptors to activate downstream signaling pathways in the growth and differentiation of nerves, comprising nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). Their high-affinity receptors include tropomyosin-related kinase (Trk), preferring NGF, BDNF and NT-3/4, while GDNF family receptor (GFRα)/RET prefers GDNF. Each ligand-receptor complex, leading to phosphorylation of various downstream pathways including PI3K/Akt and Ras/MAPK, is responsible for recruitment of specific type of nerves during nerve development. NGF/TrkA signaling dominates in the establishment of sympathetic innervation, GDNF/GFRα in parasympathetic innervation and BDNF/TrkB in sensory innervation. Multifaceted interactions between epithelium and neurons in embryonic establishment of glandular innervation are summarized by Zahalka et al. (Zahalka and Frenette, 2020). In physiological conditions, epithelial mesenchyme secrets neurotrophins to bind their specific receptors, forming a ligand-receptor complex to be engulfed by axons and retrogradely transported to the soma, thus stimulating down-stream transcription, axonogenesis, and nerve recruitment to the target organ. Once morphogenesis of their target organ is accomplished, neurotrophic factors cease to accumulate (Klein, 1994).

However, these molecules indicated in embryonic development of nervous system can be re-stimulated in the context of cancer. Neurotrophic factors such as NGF and GDNF can be released either by tumor cells or neurons (Amit et al., 2016). In colorectal cancer, abnormality in TrkA has been recurrently reported. Chromosomal rearrangement of TrkA coding gene NTRK1 produces NTRK1 fusion proteins and upregulates TrkA kinase activity, followed by oncogenic phosphorylation of downstream proteins and hypersensitivity to NGF binding. TPM3-NTRK1 is the most commonly identified fusion gene, as TrkA itself was originally separated from TPM3-TrkA fusion gene of colon carcinoma, with a detection rate of 0.5–1%. Other fusion forms such as TPR-NTRK1 and LMNA-NTRK1 have also been reported. Indeed, TrkA positive patients may represent a minority of patients with satiable response to TrkA kinase inhibitors. In resected colon adenocarcinoma, cytoplasmic immunoreactivity to TrkA was abundantly detected (Ardini et al., 2014). In vitro experiments confirmed the presence of typical TPM3-NTRK1 fusion gene in KM12 human CRC cell line and demonstrated strong effects of TrkA inhibitor on suppressing cancer cell proliferation. TrkA fusion protein was also discovered in a case of liver metastasis of CRC, and application of Entrectinib, a selective Trk inhibitor, managed to achieve clinical partial response (Sartore-Bianchi et al., 2016).

Other neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), have been shown to induce tumor migration via upregulation of VEGF and activation of p38 and PI3K/Akt signaling pathway (Huang et al., 2014; Huang et al., 2015). In-vitro studies in two human colon cancer cell lines, HCT116 and SW480, proved that migratory activity increases with the concentration of BDNF and GDNF respectively, paralleled with increased expression of VEGF, MAPK and PI3K/Akt-associated proteins at transcription and translation level.

So far, it has not been fully revealed in CRC how neurotrophic factors get engaged in perineural invasion(PNI), but accumulating evidence in other types of cancer indicates crucial importance of neurotrophin signaling through the development of nerve invasion. In pancreatic cancer, GDNF signaling is a characterized mechanism of PNI (Gil et al., 2010; He et al., 2014). In-vitro co-culture of dorsal root ganglia and human pancreatic adenocarcinoma cell lines and in-vivo murine sciatic nerve models of PNI showed that nerves release GDNF, and induce polarized neurotrophic migration of cancer cells via downstream pathways of RET. Tumor invasion of nerves is upregulated upon GDNF induction and is greatly diminished when GDNF/GFRa1/RET pathway is suppressed. Similar changes have been reported in prostate cancer, as GFRα-1/RET binding of GDNF potentiates invasion and proliferation of cancer cells (Ban et al., 2017). NGF and GDNF were also found to be more abundantly expressed in patients with PNI and higher Gleason scores (Baspinar et al., 2017). In salivary adenoid cystic carcinoma (SACC), NT-3/TrkC signaling enhances in-vitro cancer cell migration in the presence of Schwann cells: A comparative study of 78 SACC and 25 normal tissue specimens demonstrated higher expression of NT-3 in tumor cells surrounding the nerves and TrkC in tumor-invaded nerves; statistics proved that level of NT-3/TrkC expression is strongly correlated with the occurrence of PNI (Li et al., 2019). BDNF/TrkB-dependent Schwann-like differentiation was also discovered in human SACC cell lines; both TrkB and S100A4, a surface marker of Schwann cell, are found to be significantly associated with PNI (Jia et al., 2015; Shan et al., 2016).

It can be concluded that neurotrophins have multifaceted roles in tumor-nerve crosstalk of PNI, as both tumor cells and neurons are able to release neurotrophic factors. These growth factors might be secreted by neoplastic epithelium in the same way as embryonic neurogenesis to summon newborn nerves to the invasive front, facilitating direct contact between cancer cells and nerves; or they might be secreted by nerves to polarize neurotrophic migration of cancer cells towards resident nerves, as indicated in pancreatic cancer, prostate cancer, SACC, etc. Further research is needed in CRC to unveil the role of neurotrophic factors in bridging tumor and nerve.

As the main effector molecules released by nerve fibers, neurotransmitters may serve as a crucial messenger between tumor and nerve. Upregulated adrenergic signaling has been reported in colorectal cancer. As indicated in several pathology reports of CRC tissue, distribution of beta receptors appears to be denser in the tumor site (Godlewski, 2010; Ciurea et al., 2017), and such phenomenon is usually accompanied by worse prognosis. Some studies further demonstrate a direct relationship between adrenergic transmitters and tumor proliferation in CRC. An in-vitro study of Wong et al. shows COX-2 dependent stimulation of adrenaline on human colon adenocarcinoma HT-29 cell proliferation (Wong et al., 2011), as adrenaline activates COX-2, VEGF, PGE2 and MMP-9 downstream, which can be rescued by COX-2 inhibitors and beta receptor antagonists. Besides, adrenaline-induced COX-2 upregulation has been found to suppress immunity in coordination with inflammatory signals by enhancing expression of IDO and IL-10 in macrophages, thus attenuating the proliferation and IFN-γ production of CD8⁺ T cells and facilitating immune escape in colon cancer (Muthuswamy et al., 2017). Han et al. have revealed the role of epinephrine in colorectal cancer as well. By targeting the CREB1-miR-373 axis and subsequent downregulation of tumor suppressor gene TIMP2 and APC, epinephrine plays a stimulatory role in the proliferation and dissemination of human colon cancer cells in vitro and in vivo (Han et al., 2020).

Cholinergic signaling via acetylcholine (Ach) and its receptors constitutes another regulating pathway of tumor-nerve crosstalk. Mounting evidence has shown that muscarinic receptor participates in the development of CRC. Muscarinic acetylcholine receptor 1 (M1R) and 3 (M3R) are the predominant types distributed in the gut, through which enteric and autonomic nervous system innervates intestinal smooth muscle tone, with M3R notably over-expressed in CRC lesions. Expression of CHRM3, coding gene of M3R robustly increased by 128 fold in colon adenocarcinoma compared with that in adjacent normal epithelium; immunostaining also shows over-expression of M3R in cancerous tissue in contrast to the unaffected epithelium (Cheng et al., 2017). CHRM3-deficient mice develop fewer and smaller colon neoplasm induced by Azoxymethane, whereas CHRM1 knockout or dual knockout didn’t have much visible impact on tumor size or quantity (Cheng et al., 2014).

Due to ubiquitous distribution and multi-target effect of adrenergic and cholinergic transmitters, it would be troublesome to distinguish whether neurotransmitters support cancer development through direct local interaction with tumor or via systemic neuroendocrine signaling activated by other CRC-related events. Allen et al. have identified a feed-forward loop in ovarian cancer cells, that in response to sustained adrenergic signaling, tumor cells secrete BDNF, which then promotes intratumoral innervation via host neurotrophic receptor tyrosine kinase 2 (TrkB) receptors (Allen et al., 2018), denoting that sympathetic pathway might be an upstream inducer of tumor innervation. Parasympathetic signaling through acetylcholine promotes matrix metalloproteinase 1 (MMP1) expression, facilitating the invasion of HT29 and H508 human colon cancer cells, which can be rescued by atropine and anti-MMP1 antibody (Raufman et al., 2011). Potential mechanistic pathway downstream of M3R activation was reviewed in detail by Ali et al. (Ali et al., 2021), which involves stimulation of protein kinase C-α (PKCα), MMP1 and MMP7 transcription, EGFR signaling and PI3K/Akt pathway and ultimately contributes to tumor growth, dissemination and invasion. Thus, neurotransmitters and their receptors might work as a stimulator to trigger invasive acts of CRC cells, yet more studies are required to identify the role of autonomic signaling in tumor-nerve interaction.

During the maturation of the nervous system, a wide range of cell-matrix and cell-cell interactions are required to facilitate neuronal migration, axon/dendrite outgrowth and synaptogenesis, mainly mediated by the immunoglobulin superfamily of neural cell adhesion molecules (CAMs). L1CAM is a crucial family of adhesion molecules comprising L1 and its close homolog (CHL1), NrCAM and neurofascin. Their extracellular domain consisted of immunoglobulin (Ig)-like and fibronectin type III-homologous (FnIII) domains, specializes in modulating intercellular interaction, particularly at synaptic cleft to assist in synapse forming and functioning. L1 also stimulates signal transduction related to neuron migration and neurite outgrowth, either by serving as signaling molecule itself or as co-receptor(Schmid and Maness, 2008; Sytnyk et al., 2017). By interacting with β1 integrin, L1 helps to upregulate cell-matrix adhesion and initiates downstream cascade, resulting in ERK1/2 MAPK signaling and transcriptional activation of a series of proteins that upregulates cell motility, including integrin and Rac1.

A handful of researchers have proposed that L1CAM represents a potential marker for cancer invasiveness and progression of CRC. It was recurrently reported that L1 expression was correlated with increased tendency of metastasis, along with advanced cancer stage, high-risk pathological features (including poor differentiation grade, solid cancer nests and tumor budding) and severely shortened survival (Boo et al., 2007; Kaifi et al., 2007; Tampakis et al., 2020; Tieng et al., 2020). Transcription of L1CAM mRNA was significantly active in the invasive front compared to the center of tumor especially in patients with nodal involvement; besides, expression levels of L1CAM and ERK1/2 in CRC tissue is associated with lymph node metastasis, indicating supportive role of L1CAM-associated in CRC dissemination (Kajiwara et al., 2011; Fang et al., 2020). Regretfully, perineural invasion status was absent in most of these studies, making it difficult to identify whether L1 also helps tumor cells to infiltrate the nerves. Recently, it has been reported that colorectal neoplastic cells preferentially adhere to enteric neurons, supporting further dissemination of tumor cells. Through tumor-nerve adhesion mediated by L1CAM and N-Cadherin, CRC tumor cells can migrate along enteric neurons in vitro, representing a possible route for CRC perineural invasion due to the wide distribution of enteric nerves throughout the colon and rectum (Duchalais et al., 2018).

In pancreatic cancer, there is a strong association between L1CAM expression in cancer tissue and perineural invasion (Ben et al., 2010). By constructing in-vitro model of PNI, it was revealed that L1CAM secreted by Schwann cells attracts cancer cells into perineural niche via MAPK pathway and enhances expression of metalloproteinase (MMP) in cancer cells to facilitate penetration of extracellular matrix (Na’ara et al., 2019). Such process proves to be L1-dependent as neural invasion is greatly impaired after treatment of L1CAM antibody. Considering dual identity of L1CAM in nerve development and neoplastic invasion, more work remains to be done to pinpoint the position of L1CAM in neural invasion of CRC.