Vascular Endothelial Growth Factor Mediates the Sprouted Axonogenesis of Breast Cancer in Rat

Nerve infiltration into the tumor is a common feature of microenvironment. The mechanisms axonogenesis in breast cancer remain unclear. We hypothesized that vascular endothelial growth factor (VEGF), as well nerve (NGF), involved cancer. A N-methyl-N-nitrosourea (MNU)-induced rat model was used to explore presence and involvement VEGF, NGF, tumor. found MNU-induced including sensory sympathetic fibers. density increased following neurons innervating thoracic abdominal mammary tumors peaked at T5 T6 L1 L2 dorsal root ganglions, respectively. Either VEGF receptor inhibitor or antibody against 2, NGF inhibitor, apparently decreased both reduced correlated with induced by these treatments. In cultured ganglion neurons, phosphatidylinositol 3 (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK), p38 inhibitors significantly attenuated VEGF-induced neurite elongation. These findings provide direct evidence may control microenvironment, consisting fibroblasts, immune inflammatory cells, blood, lymphatic vessels, nerves, has various influences on development patients' survival. There been an increasing interest biological phenomenon active occurs tumor, provides insight therapeutic implications neural regulation progression. Accumulating suggests located human prostate,1Ayala G.E. Dai H. Powell M. Li R. Ding Y. Wheeler T.M. Shine D. Kadmon Thompson T. Miles B.J. Ittmann M.M. Rowley Cancer-related neurogenesis prostate cancer.Clin Cancer Res. 2008; 14: 7593-7603Crossref PubMed Scopus (165) Google Scholar,2Olar A. He Florentin Ayala G. Biologic correlates significance cancer.Hum Pathol. 2014; 45: 1358-1364Crossref (21) Scholar colon,3Albo Akay C.L. Marshall Wilks J.A. Verstovsek Liu Agarwal N. Berger D.H. Neurogenesis colorectal marker aggressive behavior poor outcomes.Cancer. 2011; 117: 4834-4845Crossref (73) breast,4Zhao Q. Yang Liang X. Du L. 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Fox J.G. Wang T.C. Chen Denervation suppresses gastric tumorigenesis.Sci Transl Med. 6: 250ra115Crossref (288) have shown denervation stomach attenuates incidence Axonogenesis impacts progression; thus, it must be asked what drives Although latest indicate progenitors leave subventricular zone, reach primary through differentiate new much attention paid fact neurotropic factors such (NGF) granulocyte colony-stimulating microenvironment promote axon sprout from pre-existing tumors.9Mauffrey P. Tchitchek Barroca Bemelmans A.P. Firlej Allory Roméo P.H. Progenitors central nervous system drive cancer.Nature. 2019; 569: 672-678Crossref (87) Scholar, 10Dobrenis Gauthier L.R. Granulocyte off-target outgrowth promotes development.Int 2015; 136: 982-988Crossref (38) 11Hayakawa Sakitani Konishi Asfaha Niikura Tailor Macchini Middelhoff Jiang Tanaka Dubeykovskaya Z.A. Kim Urbanska A.M. Nagar C.-S. Worthley D.L. Koike tumorigenesis aberrant cholinergic signaling.Cancer Cell. 2017; 31: 21-34Abstract Full Text PDF (180) 12Pundavela Demont Jobling Lincz L.F. Roselli Thorne R.F. Bond Bradshaw Walker Hondermarck ProNGF Gleason score potential driver cancer.Am 184: 3156-3162Abstract (59) One production fibers cancer.13Pundavela Faulkner Attia Scott R.J. Forbes J.F. infiltrate invasion cancer.Mol Oncol. 9: 1626-1635Crossref (52) Intriguingly, (VEGF) important roles angiogenesis.14Guaiquil V.H. Pan Karagianni Fukuoka Alegre Rosenblatt M.I. VEGF-B selectively regenerates injured peripheral restores trophic functions.Proc Natl Acad Sci U S 111: 17272-17277Crossref (75) 15Schlau Terheyden-Keighley Theis Mannherz H.G. Theiss triggers activation cofilin Arp2/3 complex within cone.Int Mol Sci. 2018; 19: 384Crossref (8) 16Castillo Melo Varela-Echavarría Tamariz E. Aroña R.M. Arnold Clapp Martínez de la Escalera Vasoinhibin neurotrophic effects newborn neurons.Neuroendocrinology. 106: 221-233Crossref (12) indicated angiogenesis cancer.4Zhao documented showing activates numerous cellular transduction signals (PI3K)/Akt mitogen-activated kinases (MAPKs) neuron functions.17Rosenstein J.M. Mani Khaibullina Krum Neurotrophic organotypic cortical explants neurons.J Neurosci. 2003; 23: 11036-11044Crossref Scholar,18Fournier N.M. B. Banasr Elsayed Duman R.S. Vascular regulates adult hippocampal cell proliferation MEK/ERK- PI3K/AKT-dependent signaling.Neuropharmacology. 2012; 63: 642-652Crossref (114) Here, contribution investigated using established carcinogen (MNU). Female Sprague-Dawley rats aged 7 weeks (n = 150) 14 days 27) were purchased Shanghai Jiao Tong University School Medicine Animal Center vivo vitro studies, Animals housed 24°C 12:12-hour light/dark cycle. Standard chow water provided ad libitum. All experimental protocols approved Care Use Committee Medicine. most female induction DNA alkylation carcinogenic agent MNU.19Imaoka Nishimura Doi Tani Ishikawa Yamashita Ushijima Imai Shimada Molecular characterization reveals interactions between ionizing radiation chemicals carcinogenesis.Int 134: 1529-1538Crossref (11) current study, i.p. injections 50 mg/kg MNU (Sigma-Aldrich, St. Louis, MO) dissolved 0.9% saline Sprague Dawley twice (every other month). Tumors monitored manual palpation weekly intervals after second injection. (61/150, 40.6%) initially 4 months first injection utilized study. Breast histologically determined hematoxylin eosin staining. paraffin-embedded tissues sectioned 5 ?m. sections stained solution counterstained solution. immunohistochemical detection pan-neuronal gene product 9.5 (PGP9.5) blood vessel CD34, 3-?m tissue rabbit anti-PGP9.5 (1:200, ab27053; Abcam, Cambridge, MA) anti-CD34 (1:500, ab185732; Abcam). then incubated goat anti-rabbit followed 3,3?-diaminobenzidine chromogen (DAB; Dako, Carpinteria, CA). hematoxylin. Digital images captured Olympus BLISS HD virtual microscopy (Olympus Corp., Tokyo, Japan) ×400 magnification. aggregating pixel PGP9.5+ bundles 10 representative fields (0.6 mm2/field) three five each animal ImageJ software version 1.50i, Java 1.6.0 (NIH, Bethesda, MD; http://imagej.nih.gov/ij) converted area (?m2). assessed counting number CD34+ vessels 20 randomly selected two animal. To investigate kinds afferent efferent sprouting tumors, expression specific calcitonin gene-related peptide (CGRP), vesicular monoamine transporter 2 (VMAT2), acetylcholine (VAChT), addition PGP9.5, detected immunofluorescence. Eight transcardially perfused 4% paraformaldehyde month tumors. Fifteen micron antibodies (1:1000; Abcam), anti-CGRP ab47027; anti-VMAT2 ab81855; anti-VAChT ab235201; Abcam) secondary conjugated Alexa Fluor 488 (Thermo Scientific, Waltham, MA). Staining obtained without negative control. spatial distribution sprouted multiplex immunofluorescence identify nerve, vessel, epithelium (1:500; mouse anti-cytokeratin 4545; Cell Signaling Technology, Beverly, single formalin-fixed, section, confirm accuracy PGP9.5 detecting densities another anti-neurofilament (NF) 7794; determine colocalization PGP9.5. Primary visualized tyramide signal amplification linked fluorochrome fluorescein isothiocyanate, Cy3, Cy5 antibody. stripping procedure microwave heating performed uniflex fluorescence confocal (Leica TCS SP8 STED 3×; Leica Microsystems, Wetzlar, Germany) appropriate filter set. On 1, harvested six stage homogenized lysis buffer [20 mmol/L Tris-HCl (pH 8.0), 150 NaCl, 1 EDTA, 1% NP-40, PMSF] containing protease cocktail phosphatase (all Sigma-Aldrich). Thirty micrograms proteins separated 20% Tris-glycine ready gels (Bio-Rad Laboratories, Hercules, CA), transferred nitrocellulose membranes Laboratories). Blots horseradish peroxidase–conjugated Bio-Rad levels measured enzyme-linked immunosorbent assay kit (ELR-VEGF-001; RayBiotech, Peachtree Corners, GA) beta-NGF (ELR-BNGF-001; RayBiotech), respectively, according user’s manuals. normalized total level. Rats anesthetized 2% isoflurane oxygen. skin incision made, ?L 0.5% 1,1?-diocadecyl-3,3,3?,3?-tetramethylindocarbocyanine perchlorate (DiI) (D384; Thermo Scientific), fluorescent tracer, injected DiI applied 25-ga needle closely spaced sites. animals recover. T3 T10 T12 S1 DRGs, collected grown glands, Every sixth section mounted one slide avoid repeat cells. Ten counted ganglion. DiI-labeled DRG observed under microscope DM2500) application suite 4.3 Microsystems). cancer, 24 assigned treated either (VEGFR; Flk-1) SU5416 (1 mg/kg, 3037; Tocris, Ellisville, MO), (NGFR; TrkA) AG879 2617; Tocris), anti-VEGFR2 (5 ?g/kg, ab10972; vehicle (10% dimethyl sulfoxide) least reached diameter >1 cm. SU5416/AG879 intraperitoneally given every day week month, doses based pilot experiments. last treatment. Subsequently, they frozen embedded paraffin examine Western blot (WB) immunohistochemistry, thoracolumbar DRGs quickly isolated young (approximately old) previously described.20Han Rong Cannabinoid innervation endometrial ectopic activation.Brain 1663: 132-140Crossref brief, dissociated digestive cells seeded onto 18-mm coverslips coated poly-d-lysine culture dish 37°C. mL neurobasal media (Gibco; Scientific) hours. (10, 50, 100 ng/mL, SRP4365; MilliporeSigma, Burlington, MA), (100 ng/mL) + (10 ?mol/L) overnight. 3-kinase outgrowth, overnight PI3K/Akt LY294002 ?mol/L, 19142; Sigma-Aldrich), (ERK) indirect PD98359 2243; SB203580 1202; c-Jun N-terminal (JNK) SP600125 1496; Tocris) plus ng/mL). assess tubulin immunostaining study.20Han fixed treatment immunostained ?-tubulin (1:1000, T5076; treatment, sampling counted, assessed. Three independent experiments set up condition. longest length measurement SPSS 13 (IBM SPSS, Chicago, IL) statistical evaluation. data presented means ± SD SEM Analysis variance post-hoc Turkey's multiple comparisons test unpaired t-test analyze variables different groups. Correlation quantified relationship density. P < 0.05 considered statistically significant. Chemically Almost all palpated MNU. About 40.6% (61/150) developed grossly detectable Histopathological examination revealed 100% One, two, per 24, 30, rats, majority (88.5%, 54/61) (Table 1). distributed more glands (80/105, 76.2%) than cervical inguinal (25/105, 23.8%), Table 1. Figure 1A shows gland. average largest 1.96 0.72 cm (range, 0.5 3.3 cm). 1B gross view 2.2 identified carcinomas (101/105, 96.2%). invasive (93/101; 92.1%), Invasive characterized their characteristic spreading surrounding stroma epithelial like finger, duct, solid sheets (Figure 1D) compared normal 1C).Table 1The General Characteristics MNU-Induced CancerTumor characteristicsSamples, n/total (%)Number tumors/rat 124/61 (39.3) 230/61 (49.2) 37/61 (11.5)Location Thoracic/abdominal glands80/105 (76.2) Cervical/inguinal glands25/105 (23.8)Histology Adenoma4/105 (3.8) Carcinoma101/105 (96.2)Invasive93/101 (92.1)In situ8/101 (7.9) Open table tab its mechanism, existence whether examined. Immunohistochemistry axonogenesis, individual 1E) 1F), mainly stroma. Immunofluorescence showed PGP9.5-immunoreactive bundles, (CGRP-positive) (VMAT2-positive) present 2A). Unfortunately, authors did not find (VAChT-negative) (data shown). Most stroma, accompanied 2B). Another neuronal NF colocalized bundle neurites greater palpated. qualitatively WB bands images, quantitatively graphs C T13 Thus, tracer DiI, usually origins innervations organs, (from 3) 6) 3, B , examples gland 3A) 3B). Injection labeled fibers, intense red 3C) 3D). After evident lumbar (peaked L2) T6) E F show peak 6 3G. Dil-labeled micrographs F, graph 3H. change examined assay. differed time established. differences illustrated 4, . Evidence fiber cancer,13Pundavela results suggest VEGFR antibody, NGFR treatments 4B). Intraperitoneal VEGFR/NGFR month) starting 4C. SU5416, AG879, weaker those vehicle. drugs F), 4G). hours inhibitor. overnight, dose-dependent manner. As expected, reversed 5, further MAPKs LY294002, ERK PD98059 ?mol/L), JNK Both substantially inhibited VEGF-elevated axon. Similarly VEGF. However, no VEGF-increased C). Alone, none changed specimens other.4Zhao