Vascular Endothelial Growth Factor Mediates the Sprouted Axonogenesis of Breast Cancer in Rat
2020; Elsevier BV; Volume: 191; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2020.12.006
ISSN1525-2191
AutoresHongxiu Han, Chunxue Yang, Yuan Zhang, Changhao Han, Guohua Zhang,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoNerve infiltration into the tumor is a common feature of the tumor microenvironment. The mechanisms of axonogenesis in breast cancer remain unclear. We hypothesized that vascular endothelial growth factor (VEGF), as well as nerve growth factor (NGF), is involved in the axonogenesis of breast cancer. A N-methyl-N-nitrosourea (MNU)-induced rat model of breast cancer was used to explore the presence of axonogenesis in breast tumor and the involvement of VEGF, as well as NGF, in the axonogenesis of breast tumor. Nerve infiltration into the tumor was found in MNU-induced rat model of breast cancer including the sensory and sympathetic nerve fibers. Nerve density was increased following the growth of tumor. The sensory neurons innervating the thoracic and abdominal mammary tumors peaked at T5 to T6 and L1 to L2 dorsal root ganglions, respectively. Either VEGF receptor inhibitor or antibody against VEGF receptor 2, as well as NGF receptor inhibitor, apparently decreased both the nerve density and vascular density of breast tumor. The reduced nerve density was correlated with the decreased vascular density induced by these treatments. In cultured dorsal root ganglion neurons, phosphatidylinositol 3 (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK), and p38 inhibitors significantly attenuated VEGF-induced neurite elongation. These findings provide direct evidence that VEGF, as well as NGF, may control the axonogenesis of breast cancer. Nerve infiltration into the tumor is a common feature of the tumor microenvironment. The mechanisms of axonogenesis in breast cancer remain unclear. We hypothesized that vascular endothelial growth factor (VEGF), as well as nerve growth factor (NGF), is involved in the axonogenesis of breast cancer. A N-methyl-N-nitrosourea (MNU)-induced rat model of breast cancer was used to explore the presence of axonogenesis in breast tumor and the involvement of VEGF, as well as NGF, in the axonogenesis of breast tumor. Nerve infiltration into the tumor was found in MNU-induced rat model of breast cancer including the sensory and sympathetic nerve fibers. Nerve density was increased following the growth of tumor. The sensory neurons innervating the thoracic and abdominal mammary tumors peaked at T5 to T6 and L1 to L2 dorsal root ganglions, respectively. Either VEGF receptor inhibitor or antibody against VEGF receptor 2, as well as NGF receptor inhibitor, apparently decreased both the nerve density and vascular density of breast tumor. The reduced nerve density was correlated with the decreased vascular density induced by these treatments. In cultured dorsal root ganglion neurons, phosphatidylinositol 3 (PI3K)/Akt, extracellular signal-regulated protein kinase (ERK), and p38 inhibitors significantly attenuated VEGF-induced neurite elongation. These findings provide direct evidence that VEGF, as well as NGF, may control the axonogenesis of breast cancer. The tumor microenvironment, consisting of fibroblasts, immune and inflammatory cells, blood, lymphatic vessels, and nerves, has various influences on tumor development and patients' survival. There has been an increasing interest in the biological phenomenon of active axonogenesis that occurs in tumor, and provides insight into therapeutic implications of neural regulation of tumor progression. Accumulating evidence suggests that axonogenesis occurs in cancer located in human prostate,1Ayala G.E. Dai H. Powell M. Li R. Ding Y. Wheeler T.M. Shine D. Kadmon D. Thompson T. Miles B.J. Ittmann M.M. Rowley D. Cancer-related axonogenesis and neurogenesis in prostate cancer.Clin Cancer Res. 2008; 14: 7593-7603Crossref PubMed Scopus (165) Google Scholar,2Olar A. He D. Florentin D. Ding Y. Ayala G. Biologic correlates and significance of axonogenesis in prostate cancer.Hum Pathol. 2014; 45: 1358-1364Crossref PubMed Scopus (21) Google Scholar colon,3Albo D. Akay C.L. Marshall C.L. Wilks J.A. Verstovsek G. Liu H. Agarwal N. Berger D.H. Ayala G.E. Neurogenesis in colorectal cancer is a marker of aggressive tumor behavior and poor outcomes.Cancer. 2011; 117: 4834-4845Crossref PubMed Scopus (73) Google Scholar breast,4Zhao Q. Yang Y. Liang X. Du G. Liu L. Lu L. Dong J. Han H. Zhang G. The clinicopathological significance of neurogenesis in breast cancer.BMC Cancer. 2014; 14: 484Crossref PubMed Scopus (22) Google Scholar and pancreas,5He D. Manzoni A. Florentin D. Fisher W. Ding Y. Lee M. Ayala G. Biologic effect of neurogenesis in pancreatic cancer.Hum Pathol. 2016; 52: 182-189Crossref PubMed Scopus (19) Google Scholar,6Zhang L. Guo L. Tao M. Fu W. Xiu D. Parasympathetic neurogenesis is strongly associated with tumor budding and correlates with an adverse prognosis in pancreactic ductal adenocarcinoma.Chin J Cancer Res. 2016; 28: 180-186Crossref PubMed Scopus (19) Google Scholar which indicates axonogenesis is viewed as a risk factor for poor prognosis of cancer. For example, Albo et al3Albo D. Akay C.L. Marshall C.L. Wilks J.A. Verstovsek G. Liu H. Agarwal N. Berger D.H. Ayala G.E. Neurogenesis in colorectal cancer is a marker of aggressive tumor behavior and poor outcomes.Cancer. 2011; 117: 4834-4845Crossref PubMed Scopus (73) Google Scholar demonstrated that axonogenesis of tumor plays a key role in colorectal cancer progression. The authors' previous study demonstrated that nerve density was associated with tumor grade, angiogenesis, and patient survival.4Zhao Q. Yang Y. Liang X. Du G. Liu L. Lu L. Dong J. Han H. Zhang G. The clinicopathological significance of neurogenesis in breast cancer.BMC Cancer. 2014; 14: 484Crossref PubMed Scopus (22) Google Scholar Olar et al2Olar A. He D. Florentin D. Ding Y. Ayala G. Biologic correlates and significance of axonogenesis in prostate cancer.Hum Pathol. 2014; 45: 1358-1364Crossref PubMed Scopus (21) Google Scholar reported that nerve density had a positive correlation with lymph node status. In addition, some studies provide direct evidence that infiltration of tumors by growing nerves contributes to cancer progression. Magnon et al7Magnon C. Hall S.J. Lin J. Xue X. Gerber L. Freedland S.J. Frenette P.S. Autonomic nerve development contributes to prostate cancer progression.Science. 2013; 341: 1236361Crossref PubMed Scopus (560) Google Scholar demonstrated that sympathetic nerves are involved in the early phase of tumor development, whereas parasympathetic nerves are responsible for the cancer dissemination. Zhao et al8Zhao C.-M. Hayakawa Y. Kodama Y. Muthupalani S. Westphalen C.B. Andersen G.T. Flatberg A. Johannessen H. Friedman R.A. Renz B.W. Sandvik A.K. Beisvag V. Tomita H. Hara A. Quante M. Li Z. Gershon M.D. Kaneko K. Fox J.G. Wang T.C. Chen D. Denervation suppresses gastric tumorigenesis.Sci Transl Med. 2014; 6: 250ra115Crossref PubMed Scopus (288) Google Scholar have shown that denervation of the stomach significantly attenuates tumor incidence and progression. Axonogenesis impacts cancer progression; thus, it must be asked what drives axonogenesis in cancer. Although the latest studies indicate that neural progenitors leave the subventricular zone, reach the primary tumor through the blood, and differentiate into new neurons, much attention has been paid to the fact that the neurotropic growth factors such as nerve growth factor (NGF) and granulocyte colony-stimulating factor in the tumor microenvironment promote the axon sprout from pre-existing nerves into the tumors.9Mauffrey P. Tchitchek N. Barroca V. Bemelmans A.P. Firlej V. Allory Y. Roméo P.H. Magnon C. Progenitors from the central nervous system drive neurogenesis in cancer.Nature. 2019; 569: 672-678Crossref PubMed Scopus (87) Google Scholar, 10Dobrenis K. Gauthier L.R. Barroca V. Magnon C. Granulocyte colony-stimulating factor off-target effect on nerve outgrowth promotes prostate cancer development.Int J Cancer. 2015; 136: 982-988Crossref PubMed Scopus (38) Google Scholar, 11Hayakawa Y. Sakitani K. Konishi M. Asfaha S. Niikura R. Tomita H. Renz B.W. Tailor Y. Macchini M. Middelhoff M. Jiang Z. Tanaka T. Dubeykovskaya Z.A. Kim W. Chen X. Urbanska A.M. Nagar K. Westphalen C.B. Quante M. Lin C.-S. Gershon M.D. Hara A. Zhao C.-M. Chen D. Worthley D.L. Koike K. Wang T.C. Nerve growth factor promotes gastric tumorigenesis through aberrant cholinergic signaling.Cancer Cell. 2017; 31: 21-34Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 12Pundavela J. Demont Y. Jobling P. Lincz L.F. Roselli S. Thorne R.F. Bond D. Bradshaw R.A. Walker M.M. Hondermarck H. ProNGF correlates with Gleason score and is a potential driver of nerve infiltration in prostate cancer.Am J Pathol. 2014; 184: 3156-3162Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar One study demonstrated that NGF production is associated with the infiltration of nerve fibers in breast cancer.13Pundavela J. Roselli S. Faulkner S. Attia J. Scott R.J. Thorne R.F. Forbes J.F. Bradshaw R.A. Walker M.M. Jobling P. Hondermarck H. Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer.Mol Oncol. 2015; 9: 1626-1635Crossref PubMed Scopus (52) Google Scholar Intriguingly, vascular endothelial growth factor (VEGF) plays important roles in neurogenesis as well as angiogenesis.14Guaiquil V.H. Pan Z. Karagianni N. Fukuoka S. Alegre G. Rosenblatt M.I. VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions.Proc Natl Acad Sci U S A. 2014; 111: 17272-17277Crossref PubMed Scopus (75) Google Scholar, 15Schlau M. Terheyden-Keighley D. Theis V. Mannherz H.G. Theiss C. VEGF triggers the activation of cofilin and the Arp2/3 complex within the growth cone.Int J Mol Sci. 2018; 19: 384Crossref PubMed Scopus (8) Google Scholar, 16Castillo X. Melo Z. Varela-Echavarría A. Tamariz E. Aroña R.M. Arnold E. Clapp C. Martínez de la Escalera G. Vasoinhibin suppresses the neurotrophic effects of VEGF and NGF in newborn rat primary sensory neurons.Neuroendocrinology. 2018; 106: 221-233Crossref PubMed Scopus (12) Google Scholar The authors' previous study indicated that axonogenesis is correlated with angiogenesis in human breast cancer.4Zhao Q. Yang Y. Liang X. Du G. Liu L. Lu L. Dong J. Han H. Zhang G. The clinicopathological significance of neurogenesis in breast cancer.BMC Cancer. 2014; 14: 484Crossref PubMed Scopus (22) Google Scholar There is documented evidence showing that VEGF activates numerous cellular transduction signals such as phosphatidylinositol 3 (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) in neuron functions.17Rosenstein J.M. Mani N. Khaibullina A. Krum J.M. Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons.J Neurosci. 2003; 23: 11036-11044Crossref PubMed Google Scholar,18Fournier N.M. Lee B. Banasr M. Elsayed M. Duman R.S. Vascular endothelial growth factor regulates adult hippocampal cell proliferation through MEK/ERK- and PI3K/AKT-dependent signaling.Neuropharmacology. 2012; 63: 642-652Crossref PubMed Scopus (114) Google Scholar Here, the contribution of VEGF, as well as NGF, to the axonogenesis of breast cancer was investigated using a rat model established by the carcinogen N-methyl-N-nitrosourea (MNU). Female Sprague-Dawley rats aged 7 weeks (n = 150) and 14 days (n = 27) were purchased from Shanghai Jiao Tong University School of Medicine Animal Center and used for in vivo and in vitro studies, respectively. Animals were housed at 24°C with a 12:12-hour light/dark cycle. Standard rat chow and water were provided ad libitum. All of the experimental protocols were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. The most common experimental model of breast cancer in female rats is the induction of DNA alkylation by the carcinogenic agent MNU.19Imaoka T. Nishimura M. Doi K. Tani S. Ishikawa K. Yamashita S. Ushijima T. Imai T. Shimada Y. Molecular characterization of cancer reveals interactions between ionizing radiation and chemicals on rat mammary carcinogenesis.Int J Cancer. 2014; 134: 1529-1538Crossref PubMed Scopus (11) Google Scholar In the current study, breast cancer was established by i.p. injections of 50 mg/kg MNU (Sigma-Aldrich, St. Louis, MO) dissolved in 0.9% saline in female Sprague Dawley rats twice (every other month). Tumors were monitored by manual palpation at weekly intervals after the second injection. The rats (61/150, 40.6%) with tumor initially induced between 3 and 4 months after the first injection of MNU were utilized in the current study. Breast cancer was histologically determined using hematoxylin and eosin staining. The paraffin-embedded breast tumor tissues were sectioned at 3 to 5 μm. The sections were stained using hematoxylin solution and counterstained in eosin solution. For immunohistochemical detection of pan-neuronal marker protein gene product 9.5 (PGP9.5) and blood vessel marker CD34, 3-μm tumor tissue sections were stained with rabbit anti-PGP9.5 antibody (1:200, ab27053; Abcam, Cambridge, MA) or rabbit anti-CD34 antibody (1:500, ab185732; Abcam). The sections were then incubated with goat anti-rabbit antibody followed by 3,3′-diaminobenzidine chromogen (DAB; Dako, Carpinteria, CA). All sections were counterstained with hematoxylin. Digital images were captured with the Olympus BLISS HD virtual microscopy system (Olympus Corp., Tokyo, Japan) at ×400 magnification. Nerve density was determined by aggregating the pixel density of PGP9.5+ nerve bundles in 10 representative fields (0.6 mm2/field) from three to five sections of each animal using ImageJ software version 1.50i, Java 1.6.0 (NIH, Bethesda, MD; http://imagej.nih.gov/ij) and then converted to the positive nerve area (μm2). Vascular density was assessed by counting the number of CD34+ blood vessels in 20 randomly selected fields from two to three sections of each animal. To investigate the kinds of afferent and efferent nerve fibers sprouting in breast tumors, the expression of specific sensory nerve marker calcitonin gene-related peptide (CGRP), sympathetic nerve marker vesicular monoamine transporter 2 (VMAT2), and parasympathetic nerve marker vesicular acetylcholine transporter (VAChT), in addition to PGP9.5, was detected by immunofluorescence. Eight rats with breast cancer were transcardially perfused with saline and then with 4% paraformaldehyde on month 2 after induction of breast tumors. Fifteen micron sections were incubated with the following antibodies including rabbit anti-PGP9.5 antibody (1:1000; Abcam), rabbit anti-CGRP antibody (1:500, ab47027; Abcam), rabbit anti-VMAT2 antibody (1:500, ab81855; Abcam), or rabbit anti-VAChT antibody (1:200, ab235201; Abcam) and then incubated with secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA). Staining obtained without primary or secondary antibody was used as a negative control. To investigate the spatial distribution of sprouted nerves in the breast tumors, multiplex immunofluorescence was used to identify nerve, blood vessel, and tumor epithelium with rabbit anti-PGP9.5 antibody (1:1000; Abcam), rabbit anti-CD34 antibody (1:500; Abcam), and mouse anti-cytokeratin (1:500, 4545; Cell Signaling Technology, Beverly, MA) on a single 3-μm formalin-fixed, paraffin-embedded tissue section, respectively. To confirm the accuracy of the PGP9.5 antibody in detecting nerve densities in the breast tumor, another mouse anti-neurofilament (NF) antibody (1:500, 7794; Abcam) was used to determine the colocalization with PGP9.5. Primary antibody was visualized by using tyramide signal amplification linked to a specific fluorochrome including fluorescein isothiocyanate, Cy3, or Cy5 for each primary antibody. A stripping procedure with microwave heating was performed between uniflex immunofluorescence staining. The images were captured using fluorescence confocal microscopy (Leica TCS SP8 STED 3×; Leica Microsystems, Wetzlar, Germany) with the appropriate filter set. On months 1, 2, and 3 after induction of breast tumor, the tumor tissues were harvested from six rats for each stage and homogenized with lysis buffer [20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP-40, 1 mmol/L PMSF] containing protease inhibitor cocktail and phosphatase inhibitor (all from Sigma-Aldrich). Thirty micrograms of proteins were separated on 4% to 20% Tris-glycine ready gels (Bio-Rad Laboratories, Hercules, CA), and then transferred to nitrocellulose membranes (Bio-Rad Laboratories). Blots were incubated with rabbit antibody against PGP9.5 (1:500; Abcam) followed by horseradish peroxidase–conjugated secondary antibody (1:1000; Bio-Rad Laboratories). The VEGF and NGF levels in breast tumors from six rats for each stage were measured using rat VEGF enzyme-linked immunosorbent assay kit (ELR-VEGF-001; RayBiotech, Peachtree Corners, GA) and rat beta-NGF enzyme-linked immunosorbent assay kit (ELR-BNGF-001; RayBiotech), respectively, according to the user's manuals. The levels of VEGF and NGF were normalized to the total protein level. Rats at month 2 or 3 after induction of tumor were anesthetized with 2% to 4% isoflurane in oxygen. A skin incision was made, and 10 μL of 0.5% 1,1′-diocadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (D384; Thermo Fisher Scientific), a fluorescent neural tracer, was injected into the tumor. DiI was applied using a 25-ga needle into 10 closely spaced sites. The animals were monitored for 5 to 7 days to recover. Breast tumors, T3 to T10 and T12 to S1 DRGs, were collected from rats with tumor grown in thoracic and abdominal mammary glands, respectively. Every sixth section was mounted on one slide to avoid repeat counting of cells. Ten sections were counted for each ganglion. DiI-labeled DRG neurons and nerve fibers of breast tumors were observed under fluorescence microscope (Leica DM2500) and images were captured using Leica application suite version 4.3 software (Leica Microsystems). To investigate the involvement of VEGF as well as NGF in axonogenesis of breast cancer, 24 rats with breast tumors were randomly assigned to be treated with either VEGF receptor (VEGFR; Flk-1) inhibitor SU5416 (1 mg/kg, 3037; Tocris, Ellisville, MO), NGF receptor (NGFR; TrkA) inhibitor AG879 (1 mg/kg, 2617; Tocris), anti-VEGFR2 antibody (5 μg/kg, ab10972; Abcam), or vehicle (10% dimethyl sulfoxide) after at least one tumor in the animal reached a diameter >1 cm. SU5416/AG879 and anti-VEGFR2 antibody were intraperitoneally given every other day and twice a week for 1 month, respectively. The doses used were based on the authors' pilot experiments. Breast tumors were harvested on day 1 after the last treatment. Subsequently, they were frozen and embedded in paraffin to examine the expression of PGP9.5 by Western blot (WB) and immunohistochemistry, respectively. The thoracolumbar DRGs were quickly isolated from young Sprague Dawley female rats (approximately 14 days old) as previously described.20Han H. Liang X. Wang J. Zhao Q. Yang M. Rong W. Zhang G. Cannabinoid receptor 1 contributes to sprouted innervation in endometrial ectopic growth through mitogen-activated protein kinase activation.Brain Res. 2017; 1663: 132-140Crossref PubMed Scopus (8) Google Scholar In brief, DRG neurons were dissociated by digestive solution. The cells were seeded onto 18-mm coverslips coated with poly-d-lysine in a culture dish at 37°C. DRG neurons were cultured in 2 mL neurobasal media (Gibco; Thermo Fisher Scientific) for 24 hours. The cells were then incubated with VEGF (10, 50, and 100 ng/mL, SRP4365; MilliporeSigma, Burlington, MA), VEGF (100 ng/mL) + SU5416 (10 μmol/L) overnight. To investigate the effects of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) on VEGF-induced neurite outgrowth, the cells were treated overnight with PI3K/Akt inhibitor LY294002 (10 μmol/L, 19142; Sigma-Aldrich), extracellular signal-regulated protein kinase (ERK) indirect inhibitor PD98359 (10 μmol/L, 2243; Tocris), p38 inhibitor SB203580 (10 μmol/L, 1202; Tocris), or c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μmol/L, 1496; Tocris) plus VEGF (100 ng/mL). To assess the neurite outgrowth, the tubulin immunostaining of DRG neurons was performed according to the authors' previous study.20Han H. Liang X. Wang J. Zhao Q. Yang M. Rong W. Zhang G. Cannabinoid receptor 1 contributes to sprouted innervation in endometrial ectopic growth through mitogen-activated protein kinase activation.Brain Res. 2017; 1663: 132-140Crossref PubMed Scopus (8) Google Scholar The DRG neurons were fixed with 4% paraformaldehyde after treatment and then immunostained using mouse antibody against β-tubulin (1:1000, T5076; Sigma-Aldrich). For each treatment, 10 sampling fields were counted, and a total of 100 cells were assessed. Three independent culture experiments were set up for each condition. The longest neurite length measurement of neurons was performed with Leica application suite version 4.3 software (Leica Microsystems). SPSS version 13 software (IBM SPSS, Chicago, IL) was used for statistical evaluation. The data are presented as means ± SD for in vivo experiments and as means ± SEM for in vitro experiments. Analysis of variance with post-hoc Turkey's multiple comparisons test and unpaired t-test were used to analyze variables from different groups. Correlation test quantified the relationship between nerve density and vascular density. P < 0.05 was considered to be statistically significant. Chemically induced breast cancer in female rats by MNU was used in the current study. Almost all of the tumors were initially palpated on months 3 to 4 after the first application of MNU. About 40.6% (61/150) of the total rats developed grossly detectable tumors. Histopathological examination revealed 100% incidence of mammary tumors. One, two, and three tumors per rat were found in 24, 30, and 7 rats, respectively. The majority of rats (88.5%, 54/61) developed one to two breast tumors (Table 1). The tumors were distributed more in the thoracic and abdominal mammary glands (80/105, 76.2%) than in the cervical and inguinal mammary glands (25/105, 23.8%), as shown in Table 1. Figure 1A shows a tumor in the abdominal mammary gland. The average largest diameter of breast tumor was 1.96 ± 0.72 cm (range, 0.5 to 3.3 cm). Figure 1B shows a representative gross view of a tumor with a largest diameter of 2.2 cm. Almost all of the breast tumors were identified as carcinomas (101/105, 96.2%). The majority of the carcinomas were invasive (93/101; 92.1%), as shown in Table 1. Invasive carcinomas were characterized by their characteristic feature of spreading into the surrounding stroma of epithelial cells like finger, duct, or solid sheets (Figure 1D) compared with normal breast (Figure 1C).Table 1The General Characteristics of MNU-Induced Breast CancerTumor characteristicsSamples, n/total (%)Number of tumors/rat 124/61 (39.3) 230/61 (49.2) 37/61 (11.5)Location of tumors Thoracic/abdominal mammary glands80/105 (76.2) Cervical/inguinal mammary glands25/105 (23.8)Histology Adenoma4/105 (3.8) Carcinoma101/105 (96.2)Invasive93/101 (92.1)In situ8/101 (7.9) Open table in a new tab The authors' previous study provides evidence that axonogenesis occurs in human breast cancer.4Zhao Q. Yang Y. Liang X. Du G. Liu L. Lu L. Dong J. Han H. Zhang G. The clinicopathological significance of neurogenesis in breast cancer.BMC Cancer. 2014; 14: 484Crossref PubMed Scopus (22) Google Scholar To explore its mechanism, existence of whether axonogenesis in breast tumor induced by MNU had to be examined. Immunohistochemistry of paraffin-embedded breast tumors revealed the presence of axonogenesis, either individual PGP9.5+ nerve fibers (Figure 1E) or bundles (Figure 1F), mainly in the tumor stroma. Immunofluorescence of the frozen breast tumors showed PGP9.5-immunoreactive nerve bundles, and sensory (CGRP-positive) and sympathetic (VMAT2-positive) nerve fibers present in the tumor, and PGP9.5+ nerve fibers and bundles observed in the normal breast tissue (Figure 2A). Unfortunately, the authors did not find parasympathetic nerves (VAChT-negative) in the tumor (data not shown). Most of the nerves were distributed in the tumor stroma, and some individual fibers accompanied blood vessels (Figure 2B). Another neuronal marker NF was found to be colocalized with PGP9.5+ nerve bundle in the stroma of breast tumor (Figure 2B). The density of neurites in the breast tumor significantly increased with the development of the tumor such that the density of neurites in tumor was greater at month 3 than at month 2, as well as greater at month 2 than at month 1, after the tumor was initially palpated. The effects are observed qualitatively by WB bands and immunohistochemical images, and quantitatively by graphs in Figure 2, C and D. To determine the DRG sensory neurons innervating breast tumor, the DiI-labeled neurons from T13 to S1 and T3 to T10 DRGs were examined. Thus, fluorescent neural tracer DiI, usually used to investigate the origins of sensory innervations of peripheral organs, was injected into the abdominal and thoracic breast tumors, respectively, at month 2 (from rats 1 to 3) and month 3 (from rats 4 to 6) after the induction of tumor. Figure 3, A and B , provides examples of a tumor grown in the abdominal mammary gland at month 3 after the induction of tumor from rat 4 (Figure 3A) and the other tumor grown in the thoracic mammary gland at month 2 after the induction of tumor from rat 1 (Figure 3B). Injection of DiI into the breast tumor labeled nerve fibers, with intense red fluorescence from rat 4 (Figure 3C) and rat 1 (Figure 3D). After application of DiI into the abdominal and thoracic breast tumors, neurons labeled with DiI were evident in sectioned lumbar (peaked at L1 to L2) and thoracic (peaked at T5 to T6) DRGs, respectively. Figure 3, E and F show DiI-labeled neurons at peak L1 and T5 DRGs from rat 4 and rat 1, respectively. The number of DiI-labeled neurons at peak DRGs from rats 1 to 6 is shown in Figure 3G. The number of Dil-labeled neurons was much greater at month 3 than at month 2 after induction of tumor, shown qualitatively by micrographs in Figure 3, E and F, and quantitatively by the graph in Figure 3H. To determine the role of VEGF, as well as NGF in the axonogenesis of breast tumors, the change in VEGF and NGF levels within the breast tumor were first examined at months 1, 2, and 3 after induction of tumor using enzyme-linked immunosorbent assay. The protein levels of either VEGF or NGF differed significantly by the time the tumor had established. These differences are illustrated in Figure 4, A and B . There was more of either VEGF or NGF at month 3 than at month 2, and more at month 2 than at month 1 after induction of tumor. Evidence shows that nerve fiber infiltration into the tumor microenvironment is associated with nerve growth factor production in breast cancer,13Pundavela J. Roselli S. Faulkner S. Attia J. Scott R.J. Thorne R.F. Forbes J.F. Bradshaw R.A. Walker M.M. Jobling P. Hondermarck H. Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer.Mol Oncol. 2015; 9: 1626-1635Crossref PubMed Scopus (52) Google Scholar and the results of the current study suggest that axonogenesis is correlated with angiogenesis in human breast cancer.4Zhao Q. Yang Y. Liang X. Du G. Liu L. Lu L. Dong J. Han H. Zhang G. The clinicopathological significance of neurogenesis in breast cancer.BMC Cancer. 2014; 14: 484Crossref PubMed Scopus (22) Google Scholar To investigate whether VEGF and NGF are involved in the axonogenesis of breast tumor, PGP9.5 expression of breast tumor was examined following VEGFR inhibitor, anti-VEGFR2 antibody, and NGFR inhibitor treatments (Figure 4B). Intraperitoneal injection of either VEGFR/NGFR inhibitor SU5416/AG879 (1 mg/kg, every other day for a month) or anti-VEGFR2 antibody (5 μg/kg, twice a week for a month) starting at month 4 after first injection of MNU is illustrated in Figure 4C. All of the densities of PGP9.5 in breast tumors from rats treated with SU5416, AG879, and anti-VEGFR2 antibody were much weaker than those in rats treated with the vehicle. The effects induced by these treatments are indicated qualitatively by WB bands and immunohistochemical images, and quantitatively by graphs in Figure 4, C and D. In addition, all these drugs reduced the vascular density in the breast tumor (Figure 4, E and F), and the decreased nerve density was correlated with reduced vascular density (Figure 4G). To examine the effects of VEGF on neurite outgrowth of DRG neurons, primary DRG neurons were cultured for 24 hours and then incubated with VEGF plus VEGFR inhibitor. In DRG neurons were incubated with VEGF (10, 50, and 100 ng/mL) overnight, the length of the neurite was increased compared with control in a dose-dependent manner. As expected, application of SU5416, the VEGFR inhibitor, reversed the effect of VEGF, as shown in Figure 5, A and B . To further examine the involvement of the PI3K/Akt and MAPKs in VEGF-induced neurite outgrowth, PI3K/Akt inhibitor LY294002, ERK indirect inhibitor PD98059 (10 μmol/L), p38 inhibitor SB203580 (10 μmol/L), or JNK inhibitor SP600125 (10 μmol/L) were given followed by VEGF (100 ng/mL). Both LY294002 and PD98059 substantially inhibited VEGF-elevated length of axon. Similarly SB203580 significantly attenuated neurite outgrowth induced by VEGF. However, SP600125 had no effect on VEGF-increased length of axon (Figure 5, A and C). Alone, none of the inhibitors changed neurite outgrowth (data not shown). The existence of nerves in the microenvironment of breast cancer from human specimens is demonstrated by the authors and other.4Zhao Q. Yang Y. Liang X. Du
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