Mechanisms of neural infiltration-mediated tumor metabolic reprogramming impacting immunotherapy efficacy in non-small cell lung cancer

Abstract

Background: Current evidence underlines the active role of neural infiltration and axonogenesis within the tumor microenvironment (TME), with implications for tumor progression. Infiltrating nerves stimulate tumor growth and dissemination by secreting neurotransmitters, whereas tumor cells influence nerve growth and differentiation through complex interactions, promoting tumor progression. However, the role of neural infiltration in the progression of non-small cell lung cancer (NSCLC) remains unclear.

Methods: This study employs the techniques of immunohistochemistry, immunofluorescence, RNA sequencing, molecular biology experiments, and a murine orthotopic lung cancer model to deeply analyze the specific mechanisms behind the differential efficacy of NSCLC immunotherapy from the perspectives of neuro-tumor signal transduction, tumor metabolism, and tumor immunity.

Results: This study demonstrates that nerve growth factor (NGF) drives neural infiltration in NSCLC, and 5-hydroxytryptamine (5-HT), which is secreted by nerves, is significantly elevated in tumors with extensive neural infiltration. Transcriptome sequencing revealed that 5-HT enhanced glycolysis in NSCLC cells. Pathway analysis indicated that 5-HT activated the PI3K/Akt/mTOR pathway, promoting tumor metabolic reprogramming. This reprogramming exacerbated immunosuppression in the TME. Neutralizing 5-HT-mediated metabolic reprogramming in tumor immunity enhanced the efficacy of PD-1 monoclonal antibody treatment in mice.

Conclusions: The findings of this study provide a novel perspective on the crosstalk between nerves and lung cancer cells and provide insights into further investigations into the role of nerve infiltration in NSCLC progression.

Keywords: 5-hydroxytryptamine; Neural infiltration; Non-small cell lung cancer; Tumor metabolic reprogramming; Tumor microenvironment.

Fig. 1

Neural infiltration promotes the development and progression of NSCLC (A) The TCGA database was used to screen neuro markers to evaluate the prognosis of non-small cell lung cancer (NSCLC). (B) Immunohistochemistry (IHC) staining for PGP9.5 was conducted on 85 pairs of NSCLC tissues, and representative images were presented, ranging from the weakest (-, Group 1) to the strongest (+++, Group 4), based on increasing staining intensity. Representative staining results were shown for the effective (CR + PR) and progressive (PD + SD) groups. (C) The degree of tumor tissue and paraneoplastic nerve infiltration in patients with NSCLC was statistically analyzed. (D) Statistical analysis of the degree of tumor tissue and paraneoplastic nerve infiltration in patients with NSCLC. (E) The impact of protein gene product 9.5 (PGP9.5) expression level on survival in patients with NSCLC was assessed, revealing that high PGP9.5 expression was correlated with a worse prognosis compared with low PGP9.5 expression (p = 0.028)

Fig. 2

NSCLC secretes NGF to promote axon formation, with nerve invasion degree positively correlated with 5-HT secretion. (A) Co-culture experiments were performed over 3–5 days in Transwell Boyden chambers, with NSCLC cells in the upper membrane and the neuronal PC12 cells in the lower membrane. Quantitative statistics of axonal growth in nerve cells showed that PC12 had neurites at least twice the size of the cell body that were considered differentiated. (B) Expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3/4 (NT-3/4) at the mRNA level in NSCLC cell lines. (C) Representative in vivo imaging results of tumor formation in the two mouse groups. (D) Statistical graph of tumor fluorescence intensity in two mouse groups. (E) Detection of neurotransmitter levels in mouse tumors using ELISA. (F) Detection of 5-hydroxytryptamine (5-HT) levels in tumor tissues of two mouse groups using ELISA. (G) Tumor tissues from mice were i-serially sectioned and examined for PGP9.5 and 5-HT expression through IHC

Fig. 3

5-HT exhibits a protective effect on NSCLC cells through the HTR1D receptor (A) Effect of 5-HT on NSCLC and normal lung epithelial cell viability. Cells were treated with dimethyl sulfoxide (DMSO) or 5-HT for 24 h, and cell viability was detected using the cell counting kit-8 kit. (B) Effect of 5-HT on colony formation in NSCLC cells. (C) The effect of 5-HT on the invasion of NSCLC cell lines. (D) The effect of 5-HT on the migratory ability of NSCLC cell lines. (E) The expression of 5-HTR at the mRNA level in NSCLC cell lines stimulated with 5-HT. (F) Effect of 5-HT on the proliferation of HTR1D-knockout cells. (G) Effect of 5-HT on the migration of HTR1D^KO NSCLC cells. The t-test in GraphPad Prism8 software was used to assess the statistical significance of differences between groups in panels. * The difference between the two groups was significant (p < 0.05). Data represent at least three independent experiments (n = 3; error bars, SD)

Fig. 4

Transcriptome analysis reveals significant enrichment of glycolytic pathways in NSCLC cells after 5-HT action (A) Heat map showing the differentially expressed genes (DEGs) modulated by treatment with 5-HT. (B) Differential metabolite volcano plots of the control and 5-HT-treated groups. (C) Pathway enrichment analysis of DEGs shows a significant enrichment of central carbon metabolism in cancer. (D) Gene set enrichment analysis plots based on the gene expression profiles of NSCLC cells. NES represents the normalized enrichment score

Fig. 5

5-HT promotes the survival of NSCLC cells by enhancing the Warburg effect (A) The mRNA levels of glycolysis-related enzymes detected in the presence or absence of 5-HT treatment. (B) Ratio of extracellular acidification rate (ECAR) in NSCLC cell lines after 5-HT stimulation. Glu, glucose; O, Oligomycin; 2-DG, 2-deoxyglucose. (C) The ratio of oxygen consumption rate (OCR) of NSCLC cell lines after 10µM 5-HT stimulation. F, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); R/A, Rotenone/Antimycin A. (D) Changes in mRNA levels of glycolysis-related enzymes in HTR1D^KO NSCLC cell lines after 10 µM 5-HT stimulation. (E) ECAR ratio in HTR1D^KO NSCLC cells after 5-HT stimulation. (F) Changes in cellular glucose consumption (left panel) and lactate production (right panel) in NSCLC cells upon 10 µM 5-HT stimulation. (G) Effect of glycolytic inhibitor (2-DG) on the proliferation of NSCLC cells stimulated by 10 µM 5-HT. (H) Effect of 2-DG on the invasiveness of HTR1D^KO NSCLC cells stimulated with 10 µM 5-HT

Fig. 6

5-HT-mediated metabolic reprogramming is via the activation of the PI3K/Akt/mTOR pathway (A) The mRNA expression of hexokinase (HK2) and hypoxia-inducible factor 1-alpha (HIF-1α) was altered under the stimulation of 5-HT. (B) Changes in the expression of HK2 and HIF-1α protein under the stimulation of 5-HT. (C) Effect of LY294002 (10µM) on 5-HT-mediated PI3K/Akt/mTOR signaling pathway activity. (D) Effect of rapamycin (50nM) on 5-HT-mediated PI3K/Akt/mTOR signaling pathway activity. (E) Planting of the miceLLC into the dorsal skin of the C57 mice. When the tumor reached 100 mm³, 5-HT (45ng/mm³, intratumorally, q2d ) and LY294002 (25 mg/kg, ip, q2d) treatments were initiated. The mice were sacrificed on the 21st day of tumor formation, and the tumor was exfoliated and photographed. (F) Measure the tumor volume every 3 days. Evaluation of the difference between the size of mice tumors in different therapeutic groups through the t-test with GraphPad Prism8 software. (G) IHC of tumor tissue in mice. IHC was performed on tumor tissues of mice and classified according to the degree of staining, and reprehensive pictures were captured. Light yellow indicates weak positivity (+), brownish yellow indicates moderate positivity (++), and brownish-black indicates strong positivity (+++). Statistical plots of HK2 and HIF-1α expression in each group are shown on the right panel. * indicates that compared with the control group, p < 0.05, the difference was statistically significant; # indicates that compared with the 5-HT group, p < 0.05, the difference is statistically significant)

Fig. 7

Inhibition of 5-HT-mediated tumor metabolic reprogramming can alleviate immune tolerance in mic (A) Immunofluorescence detection of cells indicates programmed cell death ligand 1 (PD-L1) expression. Fluorescence expression statistics are shown on the right panel. (B) Immunofluorescence detection of cells indicates MHC-I expression. Fluorescence expression statistics are shown on the right panel. (C) The mice LLC was planted into the dorsal skin of the C57 mice. When the tumor reached 100 mm³, 5-HT (500 µg/kg, intratumoral injection, q3d) treatment was started for all groups. After 1 week later, mice were treated in groups using 2-DG (0.03%, w/w) and PD-1mAb (10 mg/kg, ip, twice a week). When the maximum diameter of the tumor exceeded 1.5 cm, the mice were sacrificed through cervical dislocation, and the tumors were exfoliated and photographed. (D) The tumor volume was measured every 3 days. The difference between the mouse tumor sizes in different therapeutic groups was evaluated through the t-test with GraphPad Prism8 software. (E) Detection of IFN-γ and granzyme B secreted by CD8 + T cells in tumor-infiltrating lymphocytes using flow cytometry and statistical analysis (Right panel). (F) IHC revealed PD-L1 expression in mouse tumor tissues and the differences in expression between the Combo and Saline groups. (G) IHC revealed the expression of MHC-I in mouse tumor tissues and the differences in the expression between the Combo and Saline groups

Fig. 8

Mechanisms of the effect of neuroinvasion on immune tolerance in NSCLC NGF released from NSCLC cells induces neuronal cell differentiation and axonogenesis in the tumor microenvironment (TME). Owing to the innervation in the TME, nerve fibers release neurotransmitter 5-HT, which binds to the HTR1D receptor on the surface of NSCLC cells, activates the PI3K/Akt/mTOR pathway, enhances the Warburg effect in NSCLC cells, and induces tumor immunosuppression. Relieving tumor metabolic reprogramming-induced immunosuppression can synergistically enhance the efficacy of PD-1 monotherapy