Introduction

The tumor microenvironment (TME) is a complex acidic environment consisting of tumor and non-tumor cell types [1, 2], which plays a crucial role in the development and progression of tumors [3]. As an important part of the non-tumor element in the TME, the role and mechanism of tumor innervation have been increasingly investigated in various tumors including lung cancer [4, 116,117,118,119,120].

Parasympathetic Innervation in PDAC

General Background

In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate tumor growth in an organ-specific way. The stomach is innervated predominantly by the parasympathetic nervous system, where choline can stimulate the gastric epithelium to overexpress NGF, which leads to further enlargement of the enteric nerve and promotes canceration [39]. Acetylcholine can also promote the self-renewal and immune escape of CD133+ thyroid cancer cells through activation of the CD133/PI3K/Akt pathway [14]. In human prostate cancer cell lines and mouse models of prostate cancer, cholinergic signals are transduced in the tumor stroma through the muscarinic cholinergic receptor 1 (CHRM1) to promote tumor invasion [121]. The ability of muscarinic agonists to stimulate growth and muscarinic receptor antagonists to inhibit tumor growth has also been demonstrated for breast, melanoma, lung, colon, ovarian, and brain cancer [122].

Mechanisms in PDAC

In PDAC, over-expressed parasympathetic and cholinergic receptors have been detected in tumor tissue from patient and mouse models [123, 124]. Patients with PDAC and high parasympathetic density showed higher tumor budding and earlier recurrence rates than patients with low parasympathetic density [123]. The cholinergic signal enhances tumor growth by inhibiting the T cell response in the orthotopic PDAC model. When the parasympathetic nerve is stimulated, acetylcholine is released from the postganglionic fibers. Acetylcholine inhibits the recruitment of CD8+ T cell infiltration to PDAC through histone deacetylase 1-mediated CCL1, and directly inhibits CD8+ T-cell production of IFNγ in a concentration-dependent manner, reducing the Th1/Th2 ratio in the TME. In contrast, in tumor-bearing mice, vagotomy blockade not only reduces PNI but also increases CD8+ T cell infiltration and mouse survival [125] (Fig. 4A). Nicotine also promotes the metastasis of pancreatic cancer via the activation of the nicotinic acetylcholine receptor/JAK2 /STAT3 downstream signaling cascade and the upregulation of MUC4 expression [126] (Fig. 4B). However, Renz and colleagues showed that subdiaphragmatic vagotomy accelerates tumorigenesis and a muscarinic agonist suppresses tumorigenesis via MAPK and PI3K/AKT signaling [127] (Fig. 4C), suggesting that parasympathetic innervation may play distinct roles during the initiative and progressive stages of PDAC.

Fig. 4
figure 4

Parasympathetic innervation and crosstalk with PDAC cells. A PDAC cells release the neurotrophic factor NGF, which combines with TrkA on the parasympathetic nerve and promotes the proliferation of the parasympathetic nerve and the innervation of PDAC, resulting in an increase in the level of acetylcholine (ACh) and promotes the growth of PDAC cells. In addition, parasympathetic nerves can also promote the transformation of Th1 to Th2 immune cells by releasing chemokines and inhibiting the release of IFNγ from CD8 + T cells, resulting in immunosuppression. B The activation of the α7 subunit of nAChRs by nicotine increases the expression of MUC4 through JAK2/STAT3 downstream signaling and in cooperation with the MEK/ERK1/2 pathway. MUC4 upregulation further promotes the metastasis of PDAC via the activation of downstream effectors, such as HER2, c-Src, and FAK. C Activation of ACh receptors by muscarinic agonists inhibits downstream EGFR/MAPK and PI3K/AKT signaling pathways and inhibits the proliferation of PDAC cells. Abbreviations: PDAC, pancreatic ductal adenocarcinoma; NGF, nerve growth factor; TrkA, tropomyosin receptor kinase A; nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine

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Therapeutic Implications

Blocking parasympathetic innervation with bilateral subdiaphragmatic vagotomy improves the survival of PDAC mice [47]. Similarly, abrogation of cholinergic input by vagotomy or chemical denervation inhibits the growth of gastric cancer by blocking the M3 receptor-mediated Wnt pathway [39]. It also enhances the therapeutic effect of systemic chemotherapy and prolongs survival. The inhibitory effect induced by denervation is related to the inhibition of Wnt signaling and stem cell expansion [128]. Carbachol is a selective CHRM3 agonist, which enhances prostate cancer growth via the CaM/CaMKK-mediated phosphorylation of Akt. Blocking CHRM3 by darifenacin treatment inhibits prostate cancer growth and castration resistance in vitro and in vivo [129]. In this line, other studies have also reported that CHRM1 is involved in regulating the migration and invasion of prostate cancer through the Hedgehog signaling pathway. The selective CHRM1 antagonist pirenzepine inhibits the migration and invasion of cancer cells [121]. Furthermore, the application of the CHRM inhibitors Pirenzepine [17] and Darifenacin [129] reduces migration and invasion, thereby suppressing cancer cell proliferation.

Sensory Innervation in PDAC

General Background

The role and mechanism of sensory innervation in tumor progression have been increasingly investigated recently. In head and neck cancer, loss of tumor protein 53 leads to adrenergic transdifferentiation of tumor-associated sensory nerves through loss of the microRNA miR-34a, and tumor growth is suppressed by sensory denervation [130]. Melanoma cells interact with nociceptive sensory neurons, leading to increases in their neurite outgrowth and release of CGRP, which may further increase the exhaustion of cytotoxic CD8+ T cells and promote tumor immune escape [131]. In oral mucosa carcinomas, the low-glucose environment drives the production of NGF, which may further promote the release of CGRP from nociceptive nerves. CGRP subsequently induces cytoprotective autophagy in cancer cells that thrive in nutrient-poor environments [132]. CGRP is also an important neurotransmitter in the neural-immune axis, negatively regulating the infection-related immune response [133,134,135]. In CGRP-knockout mice with oral squamous cell carcinoma, the tumor burden is significantly reduced with increased tumor-infiltrating lymphocytes [29].

Mechanisms in PDAC

Neurotrophic factors derived from PDAC cells can induce the proliferation of nerve fibers including sensory nerves. In turn, sensory nerves promote the migration and invasion of cancer cells in vitro and in vivo by releasing neurotrophic factors or chemokines [58, 86, 136]. In the nutrient-poor microenvironment of PDAC, the sprouting sensory nerve could also secret exogenous serine to maintain the survival of cancer cells [103] (Fig. 5A). In PDAC patient samples, high expression of neurotrophic factors has been confirmed to be associated with PNI [86]. Transient receptor potential vanilla 1 (TRPV1) is an ion channel expressed on nociceptive sensory neurons and mediates thermal pain. TRPV1 can be activated by the acidic environment of the TME [137], resulting in increased release of SP and CGRP from nociceptive neurons. In the early stage of primary PDAC formation, MMP1 induces protease-activated receptor-1 (PAR1) expression in DRGs to release SP by activating the AKT pathway, thereby activating PDAC cells expressing neurokinin 1 receptor (NK-1R) and enhancing cell migration, invasion, and PNI through the SP/NK1R/ERK signal. In addition, SP can also induce the expression of MMP2 in tumor cells [138, 139]. Organoid culture experiments have also confirmed that sensory neurons promote the proliferation of pancreatic intraepithelial neoplasms (PanIN)-like organs through SP-NK1-R signaling and STAT3 activation. In the genetically engineered mouse model of PDAC, sensory denervation leads to a loss of STAT3 activation and slows down the progression of PanIN to tumors [140] (Fig. 5B).

Fig. 5
figure 5

Molecular mechanisms by which sensory neurons promote PDAC progression. A PDAC cells release NGF, promote the sprouting of sensory nerves via TrKA, resulting in increased levels of CGRP and SP, and promote the growth of PDAC cells by binding to the SP receptors NK-1Rs on tumor cells. Sensory nerves also secret exogenous serine to maintain the survival of PDAC. B TRPV1 is activated by the acidic environment of TME, resulting in the increasing release of SP and CGRP from nociceptive neurons. MMP1 binding to its receptor PAR1 in DRG neurons mediates PNI of PDAC cells by activating the Akt pathway and induces the release of SP. SP promotes the migration, invasion, and PNI of PDAC cells through NK-1Rs by the activation of downstream ERK signaling. It also fuels the progress of PanIN by activating the STAT3 signaling pathway. Abbreviations: PDAC, pancreatic ductal adenocarcinoma; NGF, nerve growth factor; TrkA, tropomyosin receptor kinase A; NK-1R, neurokinin 1 receptor; CGRP, calcitonin gene-related peptide; SP, substance P; TME, tumor microenvironment; TRPV1, transient receptor potential vanilla 1; PAR1, protease-activated receptor-1; MMP, matrix metalloproteinase; PanIN, pancreatic intraepithelial neoplasms. PNI, perineural invasion

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Therapeutic Implications

Drugs targeting nociceptor nerves, neuropeptides, and their receptor pathways are mainly used for pain treatment. But they now appear to have great potential in treating cancer. In acute myeloid leukemia and Ewing sarcoma, the efficacy of some drugs targeting CGRP and its receptors calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein 1 (RAMP1) has been verified. The CGRP antagonist olcegepant increases differentiation and reduces the burden of leukemia and key stem cell characteristics in mouse models of acute myeloid leukemia, while small molecule inhibitors targeting CGRP receptors reduce the growth of Ewing sarcoma [141,142,143]. Also, TRPV1 is desensitized by capsaicin, and capsaicin or resiniferatoxin has been used as an alternative pharmacological method to block pain by depleting CGRP and SP without stimulation or toxicity. In addition, intravesical injection of resiniferatoxin improves bladder function in patients with an overactive bladder. In the bone cancer model, intrathecal injection of resiniferatoxin effectively relieves pain and improves function without significant long-term side-effects. These suggest the multiple therapeutic effects of targeting sensory nerves [15, 44, 144,145,146,147,148,149,150].

Pain Relief Targeting the Nerves Innervating PDAC

Cancer cells communicate with their surrounding environment [151]. Non-tumor cells in the TME may directly or indirectly interact with cancer cells, affecting the proliferation, migration, invasion, or drug resistance of PDAC. Evidence shows that sympathetic, parasympathetic, and sensory nerves undergo different forms of neuronal remodeling during the development of normal pancreatic tissue into PDAC. This has been confirmed in animal experiments and clinical pathological samples. Interstitial components such as nerve fibers in the TME play a direct or indirect role in promoting neurogenesis and tumor growth through various neurotransmitters, neurotrophic factors, and chemokines. The neural supply of amino-acids (such as serine) to the nutritionally deficient TME is also an important factor in the progression of PDAC [103]. Therefore, targeting nerves may be a promising strategy to treat cancer and immune evasion in the TME [152].

Pain is one of the common clinical symptoms of advanced PDAC. The abdominal pain symptoms can arise from various causes including tissue damage, inflammation, ductal obstruction and infiltration, and/or a direct mass effect on nerves in the celiac plexus [70]. At present, clinical treatments for pancreatic cancer pain mainly depend on opioids and surgery. Commonly-used analgesics are bucinnazine hydrochloride and morphine, but long-term use usually causes drug tolerance and adverse drug reactions. Surgical treatment can be categorized into celiac plexus neurolysis (CPN) and celiac ganglion neurolysis (CGN) [153,154,155,156], which are variations of an interventional technique for the diagnosis and treatment of concealed abdominal pain. Also, botulinum toxin is used as a preventive strategy for precancerous lesions and local treatment of low-risk tumors in prostate cancer, or as an adjunct to tumor treatment to reduce recurrence rates [157]. Neurolytic agents such as ethanol and phenol are used to permanently destroy the celiac plexus. Local anesthetics, most commonly bupivacaine or lidocaine, are used in combination with steroids and ethanol for the sake of reducing pain and the usage of painkillers [158, 159]. However, short-term back pain may occur at the injection site within 72 hours after celiac nerve block [156]. Other common side-effects include postural hypotension and diarrhea, which may be related to blocking or damaging sympathetic signals. Severe postoperative complications include lower limb paralysis and multiple organ failure, pain, and loss of temperature sensation. Other cases have been reported in which celiac trunk thrombosis after celiac artery spasm causes liver and spleen infarction, as well as stomach and proximal small intestine infarction [160]. In a prospective study of patients with unresectable PDAC and abdominal pain, compared with CPN, CGN shortened the median survival time and did not improve pain, quality of life, or frequency of adverse events [161]. Therefore, celiac nerve block should be carefully considered.

To this end, safer and more effective treatments for PDAC-related pain are urgently needed. Deep exploration of cancer-nerve crosstalk may provide potential targets [162, 163], such as neurotransmitters, neurotrophic factors, and chemokines. The effectiveness and safety of these strategies have been verified in preclinical animal models. Drugs currently known to regulate sympathetic or parasympathetic signals, such as the selective or non-selective β-blocker propranolol or metoprolol, or parasympathetic-like drugs, tend to have an antinociceptive effect with promising suppression of PDAC progression [164]. In turn, lidocaine or bupivacaine treatment has proved effective in inhibiting tumor growth and nerve fiber formation as well as cancer pain relief [165, 166]. Similarly, targeted neurotrophic factor therapy has also demonstrated tumor-suppressive effects in triple-negative breast cancer [167]. However, differences in cholinergic responses between cancers such as gastric and pancreatic cancers need to be carefully identified. In addition, capsaicin or resiniferatoxin targeting nociceptor sensory nerves could reduce the production of CGRP and SP, thus inhibiting PDAC growth and attenuating cancer pain. In addition to existing methods, recently developed neural engineering techniques allow the selective manipulation of the specific type of nerve fibers in the TME, in order to control the cancer progression and pain [152, 168].

Conclusions and Perspectives

Here we highlight the crucial role of tumor-innervating nerves as key TME components regulating the initiation and progression of PDAC as well as other cancer types. In addition, sympathetic, parasympathetic, or sensory innervation modulates distinct signaling pathways of tumor survival or immune escape. Selective peripheral nerve blockade or abrogation, and drugs targeting neuropeptides and their receptor pathways may be promising treatments for PDAC and cancer pain. However, it remains unclear how sensory nerves regulate the infiltration and function of immunological components in the TME of PDAC. Moreover, the direct or indirect modulation of cancer cells, stromal cells, and immune cells by tumor innervation interacting as a network in the TME warrants specific identification and detailed illustration. Recently, innervated wild-type or KPC murine pancreatic organoids have been well established, providing an ex vivo model to further study pancreatic neuropathy [169]. Future research is also needed to determine optimal strategies for tumor innervation based on current findings and to explore potential synergistic benefits when combined with chemotherapy or immunotherapy.