Ganoderma Spore Lipids Inhibit Perineural Invasion in Pancreatic Cancer by Downregulating NGF-TrkA Pathway

Abstract

Perineural invasion (PNI) is an important factor leading to the recurrence of pancreatic cancer (PanCa). The NGF–TrkA pathway is related to PNI progression. Ganoderma spore lipid (GSL) is a drug with anti-cancer properties. In this study, we find out whether GSL can prevent PNI of PanCa by inhibiting NGF–TrkA pathway. In vitro, wound healing assays, transwell-based assays and three-dimensional tumor-nerve cell co-culture system showed that GSL significantly inhibited the migration and invasion capacity of PanCa cells. Inhibiting the NGF–TrkA pathway is considered an effective approach for treating PanCa. We showed that GSL effectively inhibited NGF-TrkA pathway via immunofluorescence assays and western blotting analysis. The supplement of recombinant NGF reversed the GSL inhibitory effect on the migration and invasion of PANC-1. In vivo, a sciatic nerve invasion animal model was constructed using BALB/c mice. GSL significantly suppressed tumor growth and suppressed the expression of TrkA, NGF, and vimentin and upregulated the level of the epithelial marker E-Cadherin. Moreover, GSL reduced the expression of S100 and PGP9.5. These findings suggested that GSL effectively inhibited the PNI of PanCa cells by downregulating the NGF–TrkA pathway, which may provide a new adjuvant for PanCa treatment.

Introduction

Pancreatic cancer (PanCa) represents one of the most aggressive malignancies in the digestive tract. ¹ Despite advancements in PanCa diagnosis and treatment in recent decades, the 5-year survival rate remains at a mere 13%. ² One contributing factor to the unfavorable prognosis is the absence of early detection techniques.^3,4 PanCa often progresses to metastasis and invasion when it is discovered.^3,4 Only about 25% can have surgery because most cases are too late for surgery. Even after undergoing tumor resection, PanCa patients exhibit a high rate of recurrence. Perineural invasion (PNI) serves as a crucial pathway for PanCa cell migration and invasion. ⁵ PNI is defined as the pathological process of tumor cell infiltration and spread along nerve fibers or within the neural membrane. ⁶ The incidence of PNI in PanCa is exceptionally high, affecting nearly all patients because of the abundance of neural tissue in the pancreas. ⁶ Research indicates that the PNI serves as an independent prognostic factor. A high PNI significantly correlates with early recurrence. ⁷ Furthermore, nerve invasion by cancer cells can lead to severe neuropathic pain, significantly impacting quality of life. ⁸ Consequently, the development of effective interventions targeting PNI is essential for PanCa treatment. Currently, the mechanism of PNI remains incompletely understood. Recent advancements suggest that the process of PNI involves intricate signaling interactions between tumors and nerves in the tumor microenvironment, which are mediated by various signaling molecules, such as neurotrophic factors, chemokines, and neurotransmitters.^9,10

Nerve growth factor (NGF) plays an important role in PNI. ¹¹ As a member of the neurotrophic factor family, NGF exerts its effects by binding to its specific high-affinity receptor tropomyosin-receptor kinase A (TrkA). ¹¹ NGF regulates neuronal growth and development in normal tissues. ¹² However, pancreatic cancer (PanCa) cells significantly express NGF and TrkA.^13,14 These cancer cells activate multiple intracellular signaling cascades through the autocrine NGF-TrkA pathway, facilitating the acquisition of metastatic ability. ¹⁵ Additionally, NGF secreted by cancer cells promotes the growth of peripheral nerves, further promoting the development of PNI. ¹⁵ Therefore, the NGF-TrkA pathway is a promising target for PNI inhibition and PanCa treatment.

Ganoderma lucidum, a popular traditional Chinese medicine, is widely utilized for health care and disease treatment.^16,17 Many studies have shown that various active components of Ganoderma lucidum, such as polysaccharides and fatty acids, have anti-cancer, anti-inflammatory, and antioxidant properties.^16,17 Using supercritical fluid carbon dioxide technology, Ganoderma spore lipid (GSL), which contains all the active compounds of Ganoderma lucidum, are extracted from germinated Ganoderma spores, resulting in greater pharmaceutical efficacy than that of normal Ganoderma lucidum.^18–20 Ganoderma spore oil can induce apoptosis in hepatoma cells. ¹⁸ However, its potential anti-PNI effect on PanCa remains to be elucidated.

In this study, we investigated the anti-PNI effect of GSL. We revealed that GSL can hinder PNI of PanCa via inhibiting the NGF-TrkA pathway in vitro and in vivo.

Materials and Methods

Reagents and Antibodies

GSL was supplied by Holistol International Ltd (Hong Kong, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Invitrogen (CA, USA). Antibodies against E-Cadherin, vimentin, NGF, TrkA, S100, and PGP9.5 were obtained from Abcam (Massachusetts, USA). Horseradish peroxidase-conjugated secondary antibodies and fluorescence-conjugated secondary antibodies were purchased from Invitrogen. Biotinylated peroxidase-conjugated secondary antibodies were purchased from Aspen (Wuhan, China). Recombinant NGF and the specific TrkA inhibitor GW441756 were purchased from Sigma–Aldrich.

Cell Culture

The PANC-1 cell line (CRL-1469) and PC12 cell line (CRL-1721) were obtained from the American Type Culture Collection (ATCC) (Manassas, USA) and cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. All the cultures were maintained in a standard incubator at 37 °C with a 5% CO₂ atmosphere.

PANC-1 were divided into several groups: control groups (treated with DMEM), GSL groups (treated with 800 µg/ml GSL), negative control groups (NC, treated with DMEM), positive control groups (PC, treated with 50 µM GW441756), low dose GSL groups (LD, treated with 400 µg/ml GSL), and high dose GSL groups (HD, treated with 800 µg/ml GSL). GW441756 (Sigma-Aldrich) is a selective TrkA inhibitor to validate the inhibition of the NGF-TrkA pathway. GW441756 has been previously demonstrated to suppress cancer cell migration and invasion by blocking TrkA phosphorylation.^21,22 Additionally, for the groups treated with NGF, 25 µM recombinant NGF was added.

Wound Healing Assays

Wound healing assays were conducted to examine the migration capacity of PanCa cells (PANC-1). A 20 µl sterile pipette tip was used to produce a wound across the plate. Then, PANC-1 cells were washed with phosphate-buffered saline (PBS). To prevent cell proliferation, 50 µM cytosine arabinoside (Sigma–Aldrich) was added to each culture medium. The PANC-1 cells were allowed to heal for 72 h. Images were captured at 0, 24, 48, and 72 h via an inverted phase microscope (Olympus, Tokyo, Japan). The relative wound healing (migration) rates were calculated using ImageJ software.

Transwell-based Invasion and Migration Assays

Transwell-based assays are capable of detecting cancer cell invasion and migration. For the invasion assay, Matrigel used to mimic the extracellular matrix was coated on the face of the upper chamber, and 10⁴ PANC-1 cells suspended in serum-free medium were seeded on the chamber. Medium containing 10% fetal bovine serum was added to the bottom chamber. After incubation for 48 h, the invading cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Images were taken, and cell counting was performed under a microscope (Olympus). For the migration assay, except for the upper chamber not coated with Matrigel, the other methods and materials used were identical to those used for the invasion assay.

Tumor-nerve Cell co-Culture Assay

A three-dimensional tumor-nerve cell co-culture system was established as previously described. ²³ High-differentiation PC12 cells with a neuronal phenotype and neurite outgrowth were seeded in Matrigel at a density of 10³/µL. Then, the PANC-1 cells were labeled with 25 μM fluorescent CellTracker Red CMTPX (Thermo Fisher) for 1 h and seeded (10⁴/µL) around the Matrigel. After 72 h of co-culture, the Matrigel was washed three times with PBS, and fluorescence images were captured with a microscope (Olympus). Mean fluorescence intensity (MFI) and integrated optical density (IOD) were quantified using ImageJ software.

Immunofluorescence

For immunofluorescence, the cells were washed with PBS, fixed in 4% paraformaldehyde, and permeabilized in PBS supplemented with 0.5% Triton X-100. The cells were incubated with primary antibodies against TrkA overnight at 4 °C. Then, the cells were washed with TBST three times and incubated with fluorescence-conjugated secondary antibodies. Images were obtained via an inverted fluorescence microscope (Olympus).

Animal Model

All experiments involving animals were approved and conducted in accordance with the guidelines of the Institutional Animal Ethics Committee of Shenzhen University Medical School. Twenty 5-week-old male BALB/c mice were obtained from the Animal Facility of Shenzhen University. The mice were housed under standard laboratory conditions (25 ± 2°C, 60% ± 10% relative humidity, and a 12 h light–dark cycle).

A sciatic nerve invasion model was established. The mice were randomly divided into control (n = 10) and GSL groups (n = 10). All the mice were anesthetized, and the left sciatic nerve was exposed. Under the microscope, 2 ml of suspended PANC-1 cells at a concentration of 1 × 10⁷ cells per microliter were injected into the sciatic nerve via a microsyringe. The mice in the GSL group were orally administered 1 g/kg GSL per day, and those in the control group received only the same dose of distilled water per day for 4 weeks. The size of the tumor and the weight of each mouse were measured every week. After 4 weeks of treatment, all the mice were euthanized, and the tumor tissues were separated and harvested. The tumor tissues were subjected to hematoxylin and eosin (H&E) staining, immunohistochemistry, and western blot analysis.

H&E Staining

The excised tumor tissues were fixed with 10% formaldehyde, embedded in paraffin wax, and then cut into 5 μm-thick slices. The sections were then dewaxed and rehydrated in a xylene–alcohol series and stained with hematoxylin and eosin (Sigma–Aldrich). Images were acquired via an optical microscope (Olympus).

Immunohistochemistry

The paraffin-embedded tumor sections were dewaxed and rehydrated in a xylene–alcohol series, microwaved for 8 min in 0.01 mol/L citrate phosphate buffer (pH 6.0) for antigen retrieval, and immersed in 3% H₂O₂ for 15 min to block the activity of endogenous peroxidase. The sections were incubated with primary antibodies against NGF, TrkA, E-Cadherin, and vimentin overnight at 4 °C. Then, the sections were washed with PBS three times and incubated with biotinylated peroxidase-conjugated secondary antibodies for 30 min at 37 °C. After washing with PBS, hematoxylin was used to counterstain the nuclei. The sections were observed under an Olympus optical microscope.

Western Blotting

PANC-1 cells and harvested tumor tissues were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors. After centrifugation at 12,000 rpm for 5 min, the supernatants were collected as total protein samples, and the protein concentration was measured via a bicinchoninic acid assay. Equal quantities of protein were solubilized in sodium dodecyl sulfate (SDS), separated via SDS–polyacrylamide gel electrophoresis, and then transferred onto methanol-activated polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were blocked in Tri-buffered saline Tween containing 5% nonfat milk for 1 h at room temperature. The membranes were then incubated with primary antibodies against NGF, TrkA, E-Cadherin, and vimentin at 4 °C overnight and blotted with appropriate horseradish peroxidase-conjugated secondary antibodies (Sigma–Aldrich) for 30 min at room temperature. Electrochemiluminescence was performed via an Alpha chemiluminescence system (California, USA) according to the manufacturer's instructions.

Statistical Analysis

Our data are presented as the mean ± standard deviation (SD). Statistical differences were assessed via one-way analysis of variance (ANOVA) followed by Tukey's test. A significance level of P < .05 was considered. Each experiment was repeated at least three times.

Results

GSL Inhibited the PNI of PanCa Cells in Vitro

We first investigated the effects of GSL on the migration and invasion of PanCa cells. Wound healing and Transwell-based migration assays revealed that the migration of PANC-1 cells was significantly lower in the GSL treatment group than in the control group (Figures 1 and 2A). Moreover, in the Transwell-based invasion assay, the PC and GSL treatment groups presented lower invasion rates than the control group. Compared with LD GSL, HD GSL had a greater inhibitory effect on cancer cell invasion (Figure 2B). We further used a co-culture model to mimic the invasion of PanCa cells into nerves. PANC-1 were labeled with red fluorescence by fluorescent CellTracker. The reduction in fluorescence intensity indicates fewer invading cancer cells within the nerve clusters. Compared to controls, a significant reduction in fluorescence intensity was observed in the PC and HD groups, whereas the LD group exhibited no significant differences (Figure 2C). Taken together, these results demonstrated that GSL effectively inhibited the PNI of PanCa cells in vitro.

Figure 1.

Wound Healing Assay of Control and GSL Groups at 0 h, 24 h, 48 h, and 72 h. Scale bar, 200 μm. Mean ± SD *P < .05, **P < .01. Data are Representative of Three Independent Experiments (n = 5). GSL Group: Treated with 800 µg/ml GSL.

Figure 2.

A, B, C. Transwell-based Migration Assay (A), Invasion Assay (B), and Tumor-nerve Cell Co-culture Assay (C) in the Negative Control Group (NC), Positive Control Group (PC), Low-dose Group (LD), and High-dose Group (HD). Scale Bar, 50 μm. Mean ± SD *P < .05, **P < .01, Versus NC; #P < .05, ##P < .01, PC and HD Versus LD. Data are Representative of Three Independent Experiments (n = 3). PC: Treated with 50 μM GW441756 (TrkA inhibitor). LD: Treated with 400 µg/ml GSL. HD: Treated with 800 µg/ml GSL.

GSL Suppressed the NGF-TrkA Pathway in PanCa Cells

Given that the NGF-TrkA pathway is one of the most important pathways for driving PNI, ¹¹ we speculated that the NGF-TrkA pathway can be inhibited by GSL. The western blot results revealed that the expression of NGF and TrkA was significantly downregulated in the PC and GSL treatment groups (Figure 3A). Epithelial–mesenchymal transition (EMT) in cancer cells is a pivotal step in PNI progression and is characterized by a decrease in epithelial marker expression and upregulation of mesenchymal marker expression. ²⁴ However, the TrkA inhibitor and GSL significantly increased the expression of E-Cadherin (epithelial marker) and downregulated the expression of vimentin (mesenchymal marker) (Figure 3A). An additional immunofluorescence assay of TrkA revealed that the TrkA inhibitor and GSL reduced the expression of TrkA (Figure 3B). The HD group presented lower TrkA expression than did the LD group. These results indicated that GSL can downregulate the expression of NGF-TrkA and inhibit EMT in PanCa cells.

Figure 3.

A. Western Blot Analysis of NGF, TrkA, E-Cadherin, and Vimentin in the Negative Control (NC), Positive Control (PC), low-Dose (LD), and High-Dose (HD) Groups. B. Immunofluorescence Assay for TrkA in NC, PC, LD, and HD. Scale bar, 50 μm. Mean ± SD *P < .05, **P < .01, Versus NC; #P < .05, ##P < .01, PC and HD Versus LD. Data are Representative of Three Independent Experiments (n = 3). PC: Treated with 50 μM GW441756 (TrkA Inhibitor). LD: Treated with 400 µg/ml GSL. HD: Treated with 800 µg/ml GSL.

GSL Suppress PNI of PanCa Cells by Inhibiting the NGF-TrkA Pathway

To find out whether GSL suppresses PNI mediated by inhibiting the NGF-TrkA pathway, PANC-1 cells were treated with GSL and recombinant NGF. Transwell-based assays demonstrated that NGF could restore the migration and invasion capacity that was inhibited by GSL in PANC-1 cells (Figure 4A and B). In the tumor-nerve cell co-culture system, we observed that the inhibitory effect of GSL on the invasion of PANC-1 cells toward the nerve was negated by recombinant NGF (Figure 4C). These findings indicated that NGF can reverse the effects of GSL. Thus, we concluded that GSL suppressed perineural invasion by inhibiting the NGF-TrkA pathway in pancreatic cancer.

Figure 4.

A, B, C. Transwell-based Migration Assay (A), Invasion Assay (B), and Tumor-nerve Cell Co-culture Assay (C) in the Control Group, NGF Group, GSL Group, and GSL + NGF Group. Scale Bar, 50 μm. Mean ± SD *P < .05, **P < .01, Versus GSL. Data are Representative of Three Independent Experiments (n = 3). NGF Group: Treated with 25 µM Recombinant NGF. GSL Group: Treated with 800 µg/ml GSL. NGF + GSL Group: Treated with 25 µM Recombinant NGF and 800 µg/ml GSL.

GSL Suppressed PanCa Growth in Vivo

To further explore the effect of GSL, a sciatic nerve invasion animal model was established. Following the injection of PanCa cells into the sciatic nerve, the mice were weighed, and the tumor volume was measured every week. Compared with the control, GSL reduced the growth rate in terms of tumor volume and weight (Figure 5A and B). Furthermore, the harvested gross tumor specimens also revealed that the tumor size in the GSL group was smaller than that in the control group (Figure 5C). Further H&E pathological examination revealed that the tumors treated with GSL presented a lower cancer cell density than the control tumors (Figure 5D). These results demonstrated that GSL significantly suppressed the growth of PanCa.

Figure 5.

A, B. Weekly Tumor Volume (A) and Mouse Body Weight (B) in the Control and GSL Groups Were Measured. C. Gross Images of Harvested PanCa Tissues in the Control and GSL Groups After 4 Weeks of Treatment. D. H&E Staining of PanCa Tissues from the Control and GSL Groups After 4 Weeks of Treatment. Mean ± SD *P < .05, **P < .01. Scale bar, 50 μm. Data are Representative of Three Independent Experiments (n = 3).

GSL inhibited PNI in PanCa in vivo

We next investigated the anti-PNI effect of GSL in PanCa in vivo. To directly assess the anti-PNI effect of GSL, we performed dual immunofluorescence staining for S100 (a perineural marker) and PGP9.5 (a pan-neuronal marker).^25,26 The results showed that in the GSL group, the expression of S100 and PGP9.5 was significantly lower than the control group (Figure 6A). To test the level of tumor growth and PNI, we detect NGF, TrkA, E-Cadherin, and vimentin via immunohistochemical staining and western blotting.^27,28 Compared with the control, GSL significantly decreased the expression of NGF, TrkA, and vimentin, whereas the expression of E-Cadherin in PanCa cells was increased by GSL (Figure 6B and C). In total, these findings suggested that GSL can inhibit PNI in vivo.

Figure 6.

A. Immunofluorescence Assay for S100 and PGP9.5 in PanCa Tissues from Control Group and GSL Group. B, C. Immunohistochemical Analysis (B) and Western Blot Analysis (C) of NGF, TrkA, E-Cadherin, and Vimentin in PanCa Tissues from the Control and GSL Groups. Mean ± SD **P < .01. Scale Bar, 50 μm. Data are Representative of Three Independent Experiments (n = 3).

Discussion

Distant metastasis and invasion are the primary contributors to cancer recurrence and resistance to treatment. ²⁹ The unique anatomical characteristics of the pancreas, along with the lack of early detection methods and limited treatment options, result in a high incidence of metastasis and peripheral invasion in PanCa. ⁴ PNI is a common pathway for metastasis in PanCa and is recognized as a major factor in recurrence. ⁶ A recent cohort study revealed that the PNI is strongly associated with poor overall survival in patients who underwent tumor resection. ³⁰ Therefore, the development of effective strategies to inhibit PNI is crucial for reducing recurrence rates and enhancing the outcomes of PanCa therapy.

PNI is a complex pathological process in which cancer cells invade peripheral nerves. ³¹ The increase in cancer cell migration and invasion ability contributes to the occurrence of PNI. ³¹ Inhibiting cancer cell migration and invasion can effectively prevent PNI. Previous research has demonstrated that GSL has the ability to hinder the migration of cholangiocarcinoma cells. ³² In this study, we utilized wound healing assays, Transwell assays, and tumor-nerve co-culture assays to demonstrate that GSL is able to inhibit PanCa cell migration and invasion in vitro. The epithelial–mesenchymal transition is critical for cancer cells to acquire migratory ability. ³³ We observed that GSL significantly reduced the expression of the mesenchymal marker vimentin while increasing the levels of the epithelial marker E-Cadherin both in vitro and in vivo. Moreover, the neural markers S100 and PGP9.5 in tumor tissues were also significantly suppressed, suggesting the anti-PNI effect of GSL. Additionally, GSL effectively reduced PanCa volume and mouse weight, suggesting its potential to reduce cancer cell viability, which is also significant for anti-PNI. Late-stage cancer often leads to cachexia and rapid weight loss, ³⁴ but we did not observe cachexia in the animal experiments. Therefore, the decrease in mouse weight is likely attributable to the ability of GSL to inhibit tumor growth. Collectively, these findings validate the effectiveness of GSL in suppressing the PNI.

PanCa cells drive the occurrence of PNI through complex crosstalk with nerves. ¹⁰ A key mechanism is the NGF-TrkA pathway, which regulates PanCa and nerve cells to promote PNI. ¹⁰ Knocking down TrkA or using TrkA inhibitors significantly inhibits migration and invasion and reverses the EMT phenotype of PanCa cells.^11,35 A recent study revealed that PanCa cell-derived NGF activated the autophagy of Schwann cells via the NGF-ATG7 pathway, which increased their migration and axon guidance toward PanCa and induced the directional growth of PanCa cells toward nerve fibers. ³⁶ Therefore, in addition to targeting the TrkA receptor, reducing NGF is essential for inhibiting PNI. Our study demonstrated that GSL reduced TrkA expression and decreased NGF levels in PanCa cells and PanCa tissue. Interestingly, the inhibition of TrkA can lead to a decrease in NGF expression, suggesting potential positive feedback regulation between TrkA and NGF. ³⁷ Therefore, the downregulation of NGF may be related to TrkA inhibition led by GSL. The NGF-TrkA pathway also facilitates cancer cell migration and neural invasion in various tumor types, including prostate cancer, breast cancer, and head and neck squamous cell carcinomas. ³⁸ Exploring the anti-PNI effects of GSL in different tumors holds clinical significance.

Current therapies for advanced PanCa rely on radiotherapy, chemotherapy, and targeted agents, yet face limitations including severe toxicity, narrow applicability, and drug resistance.^39–41 Immunotherapy shows low efficacy due to the immunosuppressive microenvironment in PanCa. ⁴² Supportive care, including pain management via nerve blocks or opioids and nutritional interventions, is critical to maintaining quality of life but does not address disease progression. ⁴³ These challenges highlight the need for adjunctive therapies that enhance efficacy and reduce toxicity.

In this context, Ganoderma spore lipid (GSL) emerges as a dual-action candidate. Beyond its ability to inhibit perineural invasion via NGF-TrkA suppression, GSL demonstrates chemoprotective effects. Preclinical studies reveal that GSL mitigates chemotherapy-induced toxicity through multiple mechanisms: (1) Scavenging reactive oxygen species and upregulating endogenous antioxidants, thereby reducing oxidative DNA damage^44,45; (2) Suppressing NF-κB signaling and downregulating pro-inflammatory cytokines ⁴⁵ ; (3) Preserving bone marrow mesenchymal stem cells and enhancing hematopoietic recovery.^44,46 Therefore, GSL represents a promising dual-action strategy for advanced PanCa by targeting the NGF-TrkA pathway and mitigating chemotherapy-induced damage.

We observed growth suppression and PNI inhibition by GSL using PANC-1 cells and animal models. However, other experimental models, including patient-derived primary tumor cells, organoids, and other pancreatic cancer cell lines, are required to further validate these effects of GSL.

In summary, our findings indicate that GSL hinders PanCa cell migration and PNI by inhibiting the NGF-TrkA pathway, which offers a potential adjunctive treatment option for PanCa.