Abstract
The pathological role and mechanism of psychological stress in cancer progression are little known. Here we show in a mouse model that psychological stress drives pancreatic ductal adenocarcinoma (PDAC) progression by stimulating tumour nerve innervation. We demonstrate that nociception and other stressors activate sympathetic nerves to release noradrenaline, downregulating RNA demethylase alkB homologue 5 (Alkbh5) in tumour cells. Alkbh5 deficiency in these cancer cells causes aberrant N6-methyladenosine (m6A) modification of RNAs, which are packed into extracellular vesicles and delivered to nerves in the tumour microenvironment, enhancing hyperinnervation and PDAC progression. ALKBH5 levels are inversely correlated with tumour innervation and survival time in patients with PDAC. Animal experiments identify a natural flavonoid, fisetin, that prevents neurons from taking in extracellular vesicles containing m6A-modified RNAs, thus suppressing the excessive innervation and progression of PDAC tumours. Our study sheds light on a molecular mechanism by which crosstalk between the neuroendocrine system and cancer cells links psychological stress and cancer progression and raises a potential strategy for PDAC therapy.
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Data availability
RNA-seq and ATAC-seq data of isolated tumour cells from orthotopic tumour tissue have been deposited in the Gene Expression Omnibus under accession codes GSE261937 and GSE261936, respectively. The H3K27ac and Chd4 CUT&Tag-seq data have been deposited in the Gene Expression Omnibus under accession code GSE278755. The data from m6A-seq in KPC cells and KPC EVs, RNA-seq data of DRG neurons and Snd1 PAR-CLIP-seq data have been deposited in the Gene Expression Omnibus under accession code GSE241850. Mass spectrometry data of isolated tumour cells from orthotopic tumour tissue and EVs secreted from KPC cells have been deposited in ProteomeXchange with the primary accession codes PXD061963 and PXD061967, respectively. The human pancreatic cancer data were derived from the TCGA Research Network (http://cancergenome.nih.gov/). The TCGA-PAAD dataset used in this study was accessed and downloaded from the UCSC Xena platform (https://xenabrowser.net/datapages/?cohort=TCGA%20Pancreatic%20Cancer%20(PAAD)). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
Code availability
In the process of data analysis, only publicly available tools were used and the parameters have been described in relevant sections of the Methods and Nature Portfolio Reporting Summary. The analysis codes are available at Zenodo (https://zenodo.org/records/15055598)61.
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Acknowledgements
This study was supported by the National Key Research and Development Program of China (2021YFA1302100 to J. Zheng), National Natural Science Foundation of China (82325037 and 82072617 to J. Zheng, 82272694 to X. Huang.), Cancer Innovative Research Program of Sun Yat-sen University Cancer Center (CIRP-SYSUCC-0002 and PT13010201 to D.L.), Young Talents Program of Sun Yat-sen University Cancer Center (YTP-SYSUCC-0015 to J. Zheng and YTP-SYSUCC-0068 to X. Huang.) and Guangdong Basic and Applied Basic Research Foundation (2023B1515040006 to J. Zheng).
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Contributions
J. Zheng, X. Huang and D.L. conceived of and designed the entire project. Z.C., Y.Z. and C.X. supervised and performed most of the experiments. Z.C., C.X., L. Zeng and Z.X. prepared all of the samples for ATAC-seq of isolated tumour cells, RNA-seq of neurons and tumour cells, CUT&Tag sequencing and m6A-seq in KPC cells or EVs. Y.Z., S.D. and X.W. performed bioinformatics analyses of the data from RNA-seq, ATAC-seq, Snd1 PAR-CLIP-seq and m6A-seq. H.Z., X. He and Shaoqiu Liu performed the animal experiments. J.L. and S. Zhao performed the immunofluorescence and immunohistochemical staining of tumour tissue. M.L. and S. Zhang contributed to PDAC sample preparation and histopathological examination. X.P. and Shuang Liu designed and performed the vector construction and lentivirus production. L. Zhuang and S.W. performed the cell proliferation, migration and invasion assays. R.B. and J. Zhang provided technical support. Z.C., X. Huang, J. Zheng and D.L. prepared the manuscript. All authors reviewed the manuscript.
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Extended data
Extended Data Fig. 1 Effects of stress on tumor progression of KPC mice.
a, Experimental scheme of stress on PDAC mice. CUS, chronic unpredictable stress; RTX, Resiniferatoxin. b, Experimental procedures of stress on KPC mice. c and d, Pro-cancer effect of restraint stress (c) or CUS (d) on KPC mice indicated by PanIN and tumor area and pancreatic tumor burden (n = 5 KPC mice). e−f, Restraint stress (e) and CUS (f) enhance tumor cell proliferation indicated by Ki67 in tumor tissue (n = 5 KPC mice). g−i Representative immunofluorescence images show promoting effects of nociception stress on tumorous sympathetic nerve (g, TH+), sensory nerve (h, Trpv1+) but not parasympathetic nerve (i, VAchT+) innervation (n = 5 KPC mice). j−m, Immunofluorescence images showing promoting effects of restraint stress on tumor pan-nerve innervation (j, β3-tubulin+) including sympathetic nerve (k, TH+), sensory nerve (l, Trpv1+) but not parasympathetic nerve (m, VAchT+) innervation (n = 5 KPC mice). n−q, Immunofluorescence images show promoting effects of CUS on tumor pan-nerve innervation (n, β3-tubulin+) including sympathetic nerve (o, TH+), sensory nerve (p, Trpv1+) but not parasympathetic nerve (q, VAchT+) innervation (n = 5 KPC mice). r and s, The increment of plasma noradrenaline (left) and corticosterone (right) levels in KPC mice after exposure to restraint stress (r) or CUS (s) for 1 h (n = 5 KPC mice). t, Light-dark box test shows behavioral change of KPC mice after exposure to nociception stress (n = 5 KPC mice). u and v, Open-field test (u) and Light-dark box test (v) showing behavioral change of KPC mice after exposure to restraint stress. Left panel shows tracks of KPC mice in the test and right panel is quantitative statistics (n = 5 KPC mice). w and x, Open-field test (w) and Light-dark box test (x) showing behavioral change of KPC mice after exposure to CUS (n = 5 KPC mice). Scale bars, 2 mm in c and d and 200 μm in e−q. Bar chart data are means ± s.e.m. from 5 mice, with P value by two-tailed Student’s t-test in c−m, o−x and by Wilcoxon rank-sum test in n.
Extended Data Fig. 2 Effects of stress on mouse allograft tumor progression.
a and b, Restraint stress (a) and CUS (b) significantly enhance the growth of KPC cell-derived allograft in C57BL/6J mice. Left panel shows bioluminescence imaging of tumors and right panel is quantitative statistics (n = 5 mice). c and d, Restraint stress (c) and CUS (d) enhance tumor cell proliferation indicated by Ki67 in orthotopic tumor tissue (n = 5 mice). e–h, Nociception stress significantly promotes tumor nerve innervation (e, β3-tubulin+) including sympathetic nerve (f, TH+), sensory nerve (g, Trpv1+) but not parasympathetic nerve (h, VAchT+) innervation in mouse allograft tumors. Left panel shows immunofluorescence images and right panel shows quantitative statistics (n = 5 mice). i–l, Restraint stress significantly promotes tumor nerve innervation (i, β3-tubulin+) including sympathetic nerve (j, TH+), sensory nerve (k, Trpv1+) but not parasympathetic nerve (l, VAchT+) innervation in mouse allograft tumors (n = 5 mice). m–p, CUS significantly promotes tumor nerve innervation (m, β3-tubulin+) including sympathetic nerve (n, TH+), sensory nerve (o, Trpv1+) but not parasympathetic nerve (p, VAchT+) innervation in mouse allograft tumors (n = 5 mice). q–s, Nociception stress (q), restraint stress (r) and CUS (s) cause significant increment of plasma noradrenaline (Left) and corticosterone (Right) in orthotopic PDAC mice (n = 5 mice). t and u, Open-field test (t) and Light-dark box test (u) show behavioral change of orthotopic PDAC mice after exposure to nociception stress (n = 5 mice). v and w, Open-field test (v) and Light-dark box test (w) show behavioral change of orthotopic PDAC mice after exposure to restraint stress (n = 5 mice). x and y, Open-field test (x) and Light-dark box test (y) show behavioral change of orthotopic PDAC mice after exposure to CUS (n = 5 mice). Scale bars, 200 μm (c–p). All bar chart data are means ± s.e.m. from 5 mice, with P value by two-tailed Student’s t-test in a, b, d–y and by Wilcoxon rank-sum test in c.
Extended Data Fig. 3 Noradrenaline from sympathetic nerve causes Alkbh5 deficiency in PDAC.
a, Bilateral adrenalectomy (ADX) significantly suppressed plasma corticosterone levels (n = 5 mice). b, ADX had no significant effect on Alkbh5 mRNA (left) and protein level (right) in tumor cells isolated from allograft tumors (n = 5 mice). c, Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) significantly restored Alkbh5 mRNA (left) and protein level (right) in tumor cells isolated from allograft tumors (n = 5 mice). d, Activation of sympathetic nerves in allograft tumors of mice significantly decreased Alkbh5 mRNA (left) and protein level (right) in tumor cells isolated from allograft tumors (n = 5 mice). e, Activation of tumor-innervated sympathetic nerves of mice by AAV-TH-NaChBac significantly enhanced phosphorylated Creb (p-Creb) levels in allograft tumors (n = 5 mice). f, Using AAV-TH-NaChBaC in allograft tumors of mice had no obvious effect on activation of TH+ sensory neurons (arrows pointed) in pancreas-specific dorsal root ganglia (T8–T12) indicated by immunofluorescence of c-Fos. g, Noradrenaline (NA, 1.5 mg/kg, i.p.) administration significantly decreased Alkbh5 mRNA (left) and protein level (right) in tumor cells isolated from allograft tumors (n = 5 mice). h, NA administration significantly increased phosphorylated Creb (p-Creb) expression in allograft tumors (n = 5 mice). i, Noradrenaline (1.5 mg/kg, i.p.) administration significantly increased Ki67 expression in mouse allografts (n = 5 mice). j and k, Stressors significantly increase noradrenaline levels in KPC tumor tissue (j) and orthotopic PDAC tumor tissue (k) assessed by ELISA (n = 5 mice). l, Alkbh5 overexpression in KPC cells significantly prolonged NA-reduced animal survival time (n = 8 mice). m, NA (10 μM) or isoproterenol (ISO, 10 μM) treatment significantly decreased ALKBH5/Alkbh5 mRNA levels in PANC-1, MiaPaCa-2 and KPC cells (n = 3 independent experiments). n, Noradrenaline treatment decreased ALKBH5/Alkbh5 protein level in a concentration-dependent manner. Scale bars, 200 μm (e, h, i), 100 μm (f). All bar chart data are means ± s.e.m., with P value by two-tailed Student’s t-test in a, c–e, g–k, m, Wilcoxon rank-sum test in b, or log-rank test in l.
Extended Data Fig. 4 Noradrenaline promotes PDAC progression through ADRB2.
a, Noradrenaline (NA, 10 μM) treatment only significantly increased ADRB2/Adrb2 mRNA levels but no effect on other β-adrenergic receptors in PANC-1, MiaPaCa-2 and KPC cells (n = 3 independent experiments). b, Human PDAC tumor tissue has higher ADRB2 mRNA levels (n = 179 PDAC samples) compared with normal pancreatic tissue (n = 171 normal samples) based on analysis of TCGA and GTEx datasets. The center line in box represents the median, the lower and upper hinges represent the 25th and 75th percentiles, respectively, and the lines in the upper and lower whiskers indicate 5–95 percentile and whiskers represent 1.5× the interquartile range and the dots beyond the whiskers are outliers. c, The evidence of Adrb2 KO in KPC cells. d, Adrb2 KO in KPC cells significantly inhibited NA-induced allograft tumor growth (n = 5 mice). e, Adrb2 KO in KPC cells significantly prolonged NA-reduced animal survival time (n = 8 mice). f, Adrb2 KO in KPC cells significantly suppressed stress-induced Ki67 expression in allograft tumors. Left panel is representative images of Ki67 staining and right panel is quantitative statistics (n = 5 mice). g, Adrb2 KO in KPC cells significantly restored stress-reduced Alkbh5 expression in allograft tumors (n = 5 mice). Left panel is representative images of Alkbh5 staining and right panel is quantitative statistics. h–j, Adrb2 KO in KPC cells significantly suppressed stress-induced tumor nerve innervation (h, β3-tubulin+) including sympathetic nerve (i, TH+) and sensory nerve (j, Trpv1+) innervation. Left panel is representative images and right panel is quantitative statistics (n = 5 mice). Scale bars, 200 μm (f–j). All bar chart data are means ± s.e.m., with P value by two-tailed Student’s t-test in a, d, f–j, by Wilcoxon rank-sum test in b, or by log-rank test in e.
Extended Data Fig. 5 The NA-ALKBH5 axis mediates m6A-dependent tumor growth.
a, The evidence of forced overexpression of the catalytically inactive mutant Alkbh5 (A5Mut, H205A) or WT Alkbh5 (A5WT). b, A5WT overexpression rather than A5Mut significantly suppressed stress-mediated mouse allograft tumor growth (n = 5 mice). c, A5WT overexpression rather than A5Mut significantly suppressed stress-induced Ki67 expression in allograft tumor tissue (n = 5 mice). Left panel shows immunofluorescence images of Ki67 and right panel shows quantitative statistics. d–g, A5WT overexpression rather than A5Mut significantly suppressed stress-induced tumor pan-nerve innervation (d, β3-tubulin+) including sympathetic nerve (e, TH+) and sensory nerve (f, immunofluorescence images of Trpv1, and g, quantitative statistics) innervation in mouse allograft tumors (n = 5 mice). h, The evidence of forced overexpression of catalytically inactive mutant ALKBH5 (ALKBH5Mut, H204A) or WT ALKBH5 (ALKBH5WT) in human PDAC organoids. i, A5WT overexpression rather than A5Mut significantly inhibited noradrenaline (10 μM) treatment-mediated human PDAC organoid growth (n = 5 independent experiments). j, The evidence of forced overexpression of catalytically inactive mutant ALKBH5Mut or ALKBH5WT in PANC-1 and MiaPaCa-2 cells. k–m, A5WT overexpression rather than A5Mut significantly suppressed NA-mediated PANC-1 and MiaPaCa-2 cell proliferation (k, n = 3 independent experiments), migration (l, n = 5 independent experiments) and invasion (m, n = 5 independent experiments). n–p, Effect of ALKBH5 overexpression and co-treatment with pan-Trk inhibitor (PF-06737007, 1 μM) on NA-mediated PANC-1 (left) and MiaPaCa-2 (right) cell proliferation (n, n = 4 independent experiments), migration (o, n = 5 independent experiments) and invasion (p, n = 5 independent experiments). q and r, Stress or NA treatment increased angiogenesis by immunofluorescence staining of CD31 (vascular endothelial marker) in orthotopic PDAC tumor tissue (n = 5 mice). Scale bars, 200 μm (c–f, q and r) and 100 μm (i). All data are the means ± s.e.m., with P by two-tailed Student’s t-test in b–e, g, i, and k–r.
Extended Data Fig. 6 The NA-ALKBH5 axis increases RNA m6A to promote tumor growth.
a and b, Immunofluorescence analysis showing ablation of sympathetic nerve (a, TH+) and sensory nerve (b, Trpv1+) innervation in orthotopic PDAC tumor tissue (n = 5 mice). c, Western blot analysis shows that Substance P treatment (100 nM) increased p-Erk level in KPC cells. d, Western blot analysis shows that Alkbh5 KO had no influence on Nk1r (Substance P receptor) and p-Erk level in KPC cells in vitro. e, Western blot analysis shows that Alkbh5 KO increased p-Erk level in isolated tumor cell of orthotopic PDAC tumor tissue and ablation of nerves prevented Alkbh5 KO-induced p-Erk level. f, Image showing necropsy photograph of DRG acquisition under a microscope (white arrowhead, ganglion; black arrowhead, spine). g, Evidence of Alkbh5 KO, and restoring Alkbh5Mut or Alkbh5WT expression in Alkbh5-KO KPC cells. h, NA treatment (10 μM) significantly increased global RNA m6A abundance in PANC-1, MiaPaCa-2 and KPC cells (n = 5 independent experiments) assessed by ELISA. i, Bar plot of hyper-m6A- and hypo-m6A-peak distribution of KPC cell RNAs after Alkbh5 KO or noradrenaline treatment compared with control. FC, fold change (n = 3 biological replicates). j, Overlap of hyper-m6A RNAs from KPC cells after noradrenaline treatment or Alkbh5 KO compared with control (n = 3 biological replicates). k, Pathway enrichment of overlapping hyper-m6A RNAs identified in KPC cells after Alkbh5 KO or NA treatment (n = 3 biological replicates). l, NA treatment significantly decreased Alkbh5 mRNA levels in KPC cells by transcript level analysis of m6A-seq data (n = 3 biological replicates). TPM, transcripts per million. m, Alkbh5 KO increased the stress signature score in KPC cells (n = 3 biological replicates). The stress signature was defined by upregulated genes in tumor cells isolated from tumor tissue of orthotopic PDAC mice after nociception stress exposure both in mRNA and protein level (P < 0.05, Log2FC > 0) in Fig. 1l. GSVA, gene set variation analysis. Bar chart data are the means ± s.e.m. with P by two-tailed Student’s t-test in h, l, m or Wilcoxon rank-sum test in a, b.
Extended Data Fig. 7 EVs promote axonogenesis and PDAC progression.
a, The evidence of Alkbh5 KO, or Alkbh5 and Rab27a double KO in KPC cells. b, Alkbh5 KO had no effect on EV production and Rab27a KO significantly suppressed the EV production in KPC cells (n = 5 independent experiments). c–e, Rab27a KO significantly suppressed Alkbh5 KO-induced tumor nerve innervation (c, β3-tubulin+) including sympathetic nerve (d, TH+) and sensory nerve (e, Trpv1+) innervation in mouse allograft tumors (n = 5 mice). f, Rab27a KO partially suppressed Alkbh5 KO-induced growth of mouse allograft tumors (n = 5 mice). g, Rab27a KO partially prolonged Alkbh5 KO-reduced survival time of mice bearing allograft tumors (n = 8 mice). h, Experimental scheme of EV treatment in Fig. 4d. i and j, Effect of EVs produced by wide-type KPC cells or Alkbh5-KO KPC cells on sympathetic nerve (i, TH+) and sensory nerve (j, Trpv1+) innervation in PDAC tumor tissue of KPC mice (n = 5 KPC mice). Left panel shows immunofluorescence in tumor tissue and right panel shows quantitative statistics. k and l, Effect of EVs produced by wide-type KPC cells or Alkbh5-KO KPC cells on the axonogenesis of DRG neurons (k) and sympathetic neurons (l) from celiac ganglia (n = 5 independent experiments). m, Axonogenesis examination by coculture of Alkbh5-KO or Alkbh5- and Rab27a-double KO KPC cells (left) and DRG (right) in confocal dish at same plane. n, EVs derived from Alkbh5-KO KPC cells treatment had no obvious effects on Alkbh5-KO KPC cell proliferation (left, n = 4 independent experiments), migration (middle, n = 5 independent experiments) and invasion ability (right, n = 5 independent experiments). o, Rab27a KO had no obvious effects on Alkbh5-KO KPC cell proliferation (left, n = 4 independent experiments), migration (middle, n = 5 independent experiments) and invasion ability (right, n = 5 independent experiments). Scale bars, 200 μm (c–e, i, j), 50 μm (k), 20 μm (l). Bar chart data are the means ± s.e.m., with P by two-tailed Student’s t-test in b–f and i–o, or by log-rank test in g.
Extended Data Fig. 8 Fisetin suppresses EV uptake by neurons and PDAC progression.
a, Flow cytometry analysis showing differential neuronal uptake of Dil-labelled EVs derived from KPC cells after pretreatment with inhibitors compared with vehicle. Neurons were pretreated with the indicated inhibitors (10 µM) or vehicle, followed by incubation with Dil-labelled EVs (1 µg/105 cells) derived from KPC cells (n = 3 independent experiments). b, An example that shows the gating strategy for the analysis of Dil+ EV uptake. c and d, Fisetin pretreatment significantly suppressed neuronal uptake of GFP-labelled EVs (c, green) or Dil-labelled EVs (d, Orange) from KPC cells by quantification of the EV uptake area (n = 6 independent experiments). Neurons were pretreated with Fisetin (10 µM) or vehicle, followed by incubation with GFP-labelled EVs (c) or Dil-labelled EVs (d) (1 µg/105 cells) from KPC cells. Representative images of fluorescence and white light were acquired at the indicated times (n = 6 independent experiments). e–g, Fisetin treatment (10 µM) had no significant effect on the migration (e, n = 5 independent experiments), invasion (f, n = 5 independent experiments) and proliferation (g, n = 4 independent experiments) ability of KPC cells. h–j, Fisetin pretreatment (10 µM) did not alter EV uptake of fibroblasts (h, n = 5 independent experiments), macrophages (i, n = 5 independent experiments) and CD3+ lymphocytes (j, n = 5 independent experiments) isolated from orthotopic PDAC tumor tissue. Fibroblasts, macrophages and CD3+ lymphocytes were pretreated with Fisetin (10 µM) or vehicle, followed by incubation with Dil-labelled EVs (1 µg/105 cells) derived from KPC cells. EV uptake was assessed by flow cytometry (n = 5 independent experiments). k, Fisetin treatment (20 mg/kg, i.p.) significantly decreased Ki67 expression on stressed mouse allograft tumors (n = 5). l and m, Fisetin treatment (20 mg/kg, i.p.) significantly decreased stress-induced sympathetic nerve (l, TH+) and sensory nerve (m, Trpv1+) innervation in mouse allograft tumors (n = 5 mice). Scale bars, 200 μm (c, d, k–m). All bar data are the means ± s.e.m., with P by two-tailed Student’s t-test in a, c–m.
Extended Data Fig. 9 SND1 interacts with TSG101 to transport m6A-RNAs into extracellular vesicles.
a, EV RNAs from Alkbh5-KO KPC cells significantly enhanced axonogenesis of sympathetic neurons compared with Alkbh5-WT (n = 5 independent experiments). b, RNA-seq analysis showing expression-upregulated axonogenesis-related genes in neurons after transfection with EV RNAs from KPC cells with or without Alkbh5 KO (3 biological repeats for each setting). c, qPCR assays verify the expression upregulation of the genes in b (n = 5 independent experiments). d, Knockdown Bdnf in DRG neurons partially suppressed the axonogenesis induced by EV RNAs from Alkbh5-KO KPC cells (n = 5 independent experiments). e, Western blot analysis shows that Alkbh5 KO had no significant effect on Snd1 level in KPC cells compared with Alkbh5 WT. f, Immunoprecipitation and immunoblot analysis show that Alix could not interact with Snd1, Hnrnpa1 and Hnrnpa2b1. g and h, Reciprocal immunoprecipitation assays showing the interaction between SND1 and TSG101 in PANC-1 cells. i, Immunofluorescence assays showing that endogenous Snd1 and Tsg101 colocalized in cytoplasm (white arrows) of KPC cells. j, Proteinase protection assays showing that Snd1 and Tsg101 were degraded by proteinase K only after destruction of the membrane structure using Triton X-100 treatment. k and l, Tsg101 Knockdown (KD) decreased Snd1 levels in EVs (k) but not in cells (l). m, Immunofluorescence assays showing that TSG101 and SND1 were co-localized (white arrows) in multivesicular bodies (MVBs) in RAB5AQ79L-transfected ALKBH5-KO MiaPaCa-2 cells. Scale bars, 20 μm (a), 50 μm (d), 5 μm in left picture and 2 μm in right zoom-in picture (i), 2 μm in left picture and 1 μm in right zoom-in picture (m). All bar chart data are the means ± s.e.m., with P by two-tailed Student’s t-test in a–d.
Extended Data Fig. 10 m6A-modified EV RNAs promote axonogenesis.
a, m6A modification significantly promoted the transfer of Snd1-bound and hyper-m6A-modified RNA transcripts (that is, RNA transcript 2 (produced by Fbxo38) and RNA transcript 3 (produced by Ngrn)) into EVs and then neurons, whereas this transport was not occurred in an RNA transcript without m6A modification (produced by Olfr472) (n = 3 independent experiments). b, The evidence of Alkbh5 KO, or Alkbh5 and Snd1 double KO in KPC cells. c and d, Snd1 KO in KPC cells significantly suppressed tumor innervation of sympathetic (c, TH+) and sensory nerves (d, Trpv1+). Left panel is representative images and right panel is quantification in mouse allograft tumors (n = 5 mice). Scale bar, 200 μm. e, m6A modified EV RNAs transfecting significantly promoted axonogenesis of sympathetic neurons compared with non-m6A modified EV RNAs (n = 5 independent experiments). f, Effect of tumorous ALKBH5 level on stress signature score in a patient cohort (n = 89 each group). PDAC patients with low ALKBH5 levels had significantly higher stress signature score than PDAC patients with high ALKBH5 levels based on TCGA datasets. The center line in box represents the median, the lower and upper hinges represent the 25th and 75th percentiles, respectively, and the lines in the upper and lower whiskers indicate 5–95 percentile. Whiskers represent 1.5× the interquartile range and the dots beyond the whiskers are outliers. g, Effect of tumorous stress signature score on survival time (n = 89 each group). PDAC patients with high stress signature score had significantly shorter survival time than patients with low stress signature score based on TCGA datasets. All bar chart data are means ± s.e.m. with P value by two-tailed Student’s t-test in a, c–e, Wilcoxon rank-sum test in f, or log-rank test in g.
Supplementary information
Supplementary Information
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Statistical source data for Supplementary Figs. 1–4.
Supplementary Table 1
252 genes identified by both RNA-seq and protein mass spectrometry as differentially expressed between the stress treatment and control group.
Supplementary Table 2
Clinical information of patients with PDAC whose specimens were used for ALKBH5, β3-tubulin and tyrosine hydroxylase immunohistochemical analysis in this study.
Supplementary Table 3
Primers used in this study.
Supplementary Table 4
Guide, short hairpin and small interfering RNA sequences used in this study.
Supplementary Table 5
Small-molecule compounds used in this study to inhibit EV uptake of neurons.
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Chen, Z., Zhou, Y., Xue, C. et al. Psychological stress-induced ALKBH5 deficiency promotes tumour innervation and pancreatic cancer via extracellular vesicle transfer of RNA. Nat Cell Biol 27, 1035–1047 (2025). https://doi.org/10.1038/s41556-025-01667-0
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DOI: https://doi.org/10.1038/s41556-025-01667-0
