Clinicopathological Significance of Nerves in Esophageal Cancer
2020; Elsevier BV; Volume: 190; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2020.05.012
ISSN1525-2191
AutoresNathan Griffin, Christopher W. Rowe, Fangfang Gao, Phillip Jobling, Vanessa Wills, Marjorie M. Walker, Sam Faulkner, Hubert Hondermarck,
Tópico(s)Brain Metastases and Treatment
ResumoNerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell–released NGF. Nerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell–released NGF. Nerves in the microenvironment of solid tumors now are recognized as drivers of cancer progression.1Faulkner S. Jobling P. March B. Jiang C.C. 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These pioneering studies developed the concept of nerve dependence in cancer, where molecular crosstalk between cancer cells and nerves participate in the initiation and progression of the disease.9Boilly B. Faulkner S. Jobling P. Hondermarck H. Nerve dependence: from regeneration to cancer.Cancer Cell. 2017; 31: 342-354Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar For example, in pancreatic cancer, sympathetic nerves release noradrenaline, stimulating cancer cell proliferation through β receptors,7Renz B.W. Takahashi R. Tanaka T. Macchini M. Hayakawa Y. Dantes Z. Maurer H.C. Chen X. Jiang Z. Westphalen C.B. Ilmer M. Valenti G. Mohanta S.K. Habenicht A.J.R. Middelhoff M. Chu T. Nagar K. Tailor Y. Casadei R. Di Marco M. Kleespies A. Friedman R.A. Remotti H. Reichert M. Worthley D.L. Neumann J. Werner J. Iuga A.C. Olive K.P. Wang T.C. Beta2 adrenergic-neurotrophin feedforward loop promotes pancreatic cancer.Cancer Cell. 2018; 33: 75-90.e7Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar and in gastric cancer, cholinergic nerves release acetylcholine, which stimulates cancer growth via muscarinic receptors.4Hayakawa 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 (163) Google Scholar In addition, in prostate cancer, noradrenalin released from sympathetic nerves is a potent stimulator of angiogenesis.10Zahalka A.H. Arnal-Estape A. Maryanovich M. Nakahara F. Cruz C.D. Finley L.W.S. Frenette P.S. Adrenergic nerves activate an angio-metabolic switch in prostate cancer.Science. 2017; 358: 321-326Crossref PubMed Scopus (150) Google Scholar Interestingly, a feed-forward loop exists wherein the stimulation of tumors via locally released neurotransmitters induces the release of nerve growth factor (NGF) from cancer cells, further promoting tumor innervation.4Hayakawa 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 (163) Google Scholar,7Renz B.W. Takahashi R. Tanaka T. Macchini M. Hayakawa Y. Dantes Z. Maurer H.C. Chen X. Jiang Z. Westphalen C.B. Ilmer M. Valenti G. Mohanta S.K. Habenicht A.J.R. Middelhoff M. Chu T. Nagar K. Tailor Y. Casadei R. Di Marco M. Kleespies A. Friedman R.A. Remotti H. Reichert M. Worthley D.L. Neumann J. Werner J. Iuga A.C. Olive K.P. Wang T.C. Beta2 adrenergic-neurotrophin feedforward loop promotes pancreatic cancer.Cancer Cell. 2018; 33: 75-90.e7Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,11Pundavela 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 (45) Google Scholar In esophageal cancer, the presence and potential impact of nerves in the tumor microenvironment is unclear. In fact, knowledge of the specific innervation of the normal human esophageal mucosa is limited. Evidence suggests that the upper esophagus is more sensitive to mechanical and chemical stimuli than the lower esophagus.12Krarup A.L. Olesen S.S. Funch-Jensen P. Gregersen H. Drewes A.M. Proximal and distal esophageal sensitivity is decreased in patients with Barrett's esophagus.World J Gastroenterol. 2011; 17: 514-521Crossref PubMed Scopus (31) Google Scholar,13Thoua N.M. Khoo D. Kalantzis C. Emmanuel A.V. Acid-related oesophageal sensitivity, not dysmotility, differentiates subgroups of patients with non-erosive reflux disease.Aliment Pharmacol Ther. 2008; 27: 396-403Crossref PubMed Scopus (40) Google Scholar In addition, it has been reported that afferent nerves in the mucosa of the proximal esophagus are located more superficially (cell layers closest to the lumen) than the distal esophagus.14Woodland P. Aktar R. Mthunzi E. Lee C. Peiris M. Preston S.L. Blackshaw L.A. Sifrim D. 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Luo R.Z. Yun J.P. Xie D. Cai M.Y. The prognostic effect of perineural invasion in esophageal squamous cell carcinoma.BMC Cancer. 2014; 14: 313Crossref PubMed Scopus (41) Google Scholar However, other data have suggested that PNI is not associated significantly with cancer-specific survival and does not possess independent prognostic value in esophageal cancer.24Tachezy M. Tiebel A.K. Gebauer F. Kutup A. Tharun L. Pantel K. Izbicki J.R. Vashist Y.K. Prognostic impact of perineural, blood and lymph vessel invasion for esophageal cancer.Histol Histopathol. 2014; 29: 1467-1475PubMed Google Scholar,25Khan O.A. Alexiou C. Soomro I. Duffy J.P. Morgan W.E. Beggs F.D. Pathological determinants of survival in node-negative oesophageal cancer.Br J Surg. 2004; 91: 1586-1591Crossref PubMed Scopus (49) Google Scholar In the present study, the clinicopathologic significance of nerves in the tumor microenvironment of esophageal cancer was investigated. Nerves were detected in a significant proportion of esophageal cancers and were associated more specifically with squamous cell carcinoma histologic subtype. An association between high production of NGF by cancer cells and the presence of nerves was observed. Furthermore, in vitro, squamous esophageal cancer cells induced neurite outgrowth via the release of NGF, thus pointing to a possible NGF-driven nerve infiltration in esophageal cancer. High-density tissue microarrays (TMAs) were obtained from Biomax, Inc. (Derwood, MD). The TMAs used (catalog numbers ES804, ES2001a, and HEso-Squ180Sur-03) included a total of 303 esophageal cancer tissues (of adenocarcinoma and squamous subtypes), 40 matched lymph node metastases, and 137 normal adjacent tissues. The following clinicopathologic information was available: patient age and sex, histologic subtype, tumor size, grade, lymph node status, and survival status. Biomax, Inc., quality controls are described as follows. Each single tissue spot on every array slide was examined individually by certified pathologists according to World Health Organization published standardizations of diagnosis, classification, and pathologic grade.26Nagtegaal I.D. Odze R.D. Klimstra D. Paradis V. Rugge M. Schirmacher P. Washington K.M. Carneiro F. Cree I.A. WHO Classification of Tumours Editorial BoardThe 2019 WHO classification of tumours of the digestive system.Histopathology. 2020; 76: 182-188Crossref PubMed Scopus (439) Google Scholar Each specimen collected was consented to by both the hospital and the individual. Discrete legal consent was obtained and the rights to hold research uses for any purpose or further commercialized uses were waived. The study was approved by the Human Research Ethics Committee of the University of Newcastle (approval H-2012-0063). Immunohistochemistry was performed as previously described.27Faulkner S. Jobling P. Rowe C.W. Rodrigues Oliveira S.M. Roselli S. Thorne R.F. Oldmeadow C. Attia J. Jiang C.C. Zhang X.D. Walker M.M. Hondermarck H. Neurotrophin receptors TrkA, p75(NTR), and sortilin are increased and targetable in thyroid cancer.Am J Pathol. 2018; 188: 229-241Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar After deparaffinization and rehydration of the TMA slides following standard procedures, heat-induced epitope retrieval was performed in a low-pH, citrate-based, antigen-unmasking solution (catalog number H-3300; Vector Laboratories, Burlingame, CA) using a decloaking chamber (Biocare, West Midlands, United Kingdom) at 95°C for 20 minutes. Immunohistochemistry then was performed using an ImmPRESSTM horseradish peroxidase IgG (peroxidase) Polymer Detection Kit (Vector Laboratories), as per the manufacturer's recommendations. Briefly, after inactivation of endogenous peroxidases with 0.3% H2O2, and blocking with 2.5% horse serum, tissues were probed with primary antibodies at the following specified dilutions: anti-ubiquitin C-terminal hydrolase L1 (UCHL1 1/500 dilution (ab15503; Abcam, Cambridge, UK), anti-NGF 1/200 dilution (ab52918; Abcam), anti-neurotrophic receptor tyrosine kinase 1 (NTRK1, also know as TRKA) 1/200 dilution (2510, 12G8; Cell Signaling Technology, Danvers MA), anti-nerve growth factor receptor (NGFR, also known as p75NTR) 1/500 dilution (4201, D8A8; Cell Signaling Technology). The signal was amplified with horseradish peroxidase–conjugated antibodies 711-035-152 anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) 1/400 in saturating buffer for 2 hours at 37°C and shown with the diaminobenzidine peroxidase (horseradish peroxidase) Substrate Kit (SK-4100; Vector Laboratories). Finally, TMA slides were counterstained with hematoxylin (Gill's formulation; Vector Laboratories), dehydrated, and cleared in xylene before mounting in Ultramount #4 mounting media (Thermo Fisher Scientific, Victoria, Australia). Negative controls, using isotype control antibodies (rabbit IgG isotype control 0.8 μg/mL, 3900; Cell Signaling Technology), are shown in Supplemental Figure S1. Double immunostaining was performed using the ImmPRESS Duet Double Staining Polymer Kit (MP-7714; Vector Laboratories) as per the manufacturer's recommendations. Briefly, after deparaffinization, rehydration, inactivation of endogenous peroxidases with H2O2, and blocking with 2.5% horse serum, the mouse ubiquitin C-terminal hydrolase L1 (UCHL1) antibody (ab8189; Abcam) and rabbit NGF antibody (ab52918; Abcam) were combined and applied to the sections. ImmPRESS Duet Reagent (horseradish peroxidase horse anti-mouse IgG/AP horse anti-rabbit IgG; Vector Laboratories) was applied for 20 minutes followed by ImmPACT diaminobenzidine EqV substrate for 2 minutes. Slides then were washed in buffer before ImmPACT Vector Red substrate was applied for 2 minutes. Finally, tissues slides were washed, counterstained with hematoxylin QS nuclear counterstain (H-3404; Vector Laboratories), dehydrated, and cleared in xylene before mounting in Ultramount #4 mounting media (Thermo Fisher Scientific). Quantification of NGF staining intensities was performed as previously described27Faulkner S. Jobling P. Rowe C.W. Rodrigues Oliveira S.M. Roselli S. Thorne R.F. Oldmeadow C. Attia J. Jiang C.C. Zhang X.D. Walker M.M. Hondermarck H. Neurotrophin receptors TrkA, p75(NTR), and sortilin are increased and targetable in thyroid cancer.Am J Pathol. 2018; 188: 229-241Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar using the Aperio AT2 scanner (Leica Biosystems, Victoria, Australia) and the Halo image analysis platform (Indica Labs, Albuquerque, NM) under the supervision of a pathologist (M.M.W.). Pixel intensity values were used to determine the h-scores for each core (index was calculated as the sum of 3 × the percentage of pixels with strong staining + 2 × the percentage of pixels with intermediate staining + 1 × the percentage of pixels with weak staining). NGF intensity was classified as low (h-score, <25), medium (h-score, ≥25 to <50), and high (h-score, ≥50), with the medium category approximately equivalent to the interquartile range of the data. The presence or absence of nerve fibers was recorded for each individual tumor sample under the supervision of a pathologist (M.M.W.). Nerve positivity was defined as the presence of individual nerve fibers within the tumor core. Nerves were identified based on histologic context and positive reaction to anti-UCHL1 antibody. Perineural invasion was defined using the current prevailing definition: the presence of a nerve trunk (large nerve composed of many individual nerve fibers) with cancer cells surrounding a minimum of 33% of the circumference of the nerve or tumor cells within any three layers of the nerve sheath.28Liebig C. Ayala G. Wilks J.A. Berger D.H. Albo D. Perineural invasion in cancer: a review of the literature.Cancer. 2009; 115: 3379-3391Crossref PubMed Scopus (556) Google Scholar The presence of nerve fibers and PNI in primary tumors (considered as binary variables of absent or present) were each compared with clinicopathologic parameters. Separate logistic regression models were constructed for the presence of nerves, and the presence of perineural invasion (as the dichotomized dependent variable), including model variables of age, sex, tumor size, and tumor grade. Histologic subtype was not included owing to collinearity. Survival data were used to construct Kaplan-Meier survival curves, stratified by the presence or absence of nerves (or PNI). Survival data then were used to determine separate Cox proportional hazard ratios, both unadjusted; and adjusting for potential confounders of age, sex, tumor grade, tumor size, and lymph node status. NGF h-scores were considered both as a continuous, normally distributed variable; and in categories of intensity described above (Digital Quantification of Immunohistochemistry Staining Intensities) and assessed using unpaired t-tests and the χ2 test, respectively. Distribution was assessed using histograms and q-q plots. Analyses were performed using Stata v 14.2 (StataCorp, College Station, TX). Squamous esophageal cancer cell lines KYSE30 and KYSE70 were purchased from the European Collection of Authenticated Cell Cultures. The KYSE30 cell line was maintained in RPMI-1640 and Ham's F-12 Nutrient Mix (1:1) (11765054; Thermo Fisher Scientific), supplemented with 2% (v/v) fetal calf serum (JRH Bio-sciences, Lenexa, KS) and 2 mmol/L l-glutamine in a humidified incubator at 37°C with 5% (v/v) CO2. The KYSE70 cell line was maintained in RPMI-1640 with 10% (v/v) fetal calf serum (JRH Biosciences). Routine Mycoplasma testing was performed using the MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza, Basel, Switzerland). Cells were not maintained in culture for longer than 3 months to ensure the passage number remained fit for purpose. Subconfluent esophageal cancer cell monolayers were lysed with 1% NP40 lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP40, pH 8.0) that contained complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany). Insoluble proteins were removed by centrifugation at 15 × 103 × g for 10 minutes (at 4°C), and the total protein concentration was determined using the microBCA kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions. A total of 24 μg protein lysate was separated by SDS-PAGE with 12% resolving gel and then transferred to 0.4-μm pore nitrocellulose membranes (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA) using a wet transblotter (BioRad, Gladesville, NSW, Australia). Blots were blocked with blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour at room temperature and then probed with anti-NGF antibody (sc-548; Santa Cruz Biotechnology, Dallas, TX) at a dilution of 1:200, diluted in the blocking buffer. β-actin detection (1/5000 dilution, A2066; Sigma-Aldrich, St. Louis, MO) was used as the equal loading control. After washing with phosphate-buffered saline that contained 0.1% Tween 20, membranes were probed with goat anti-rabbit 800 continuous wave–labeled secondary antisera, and then washes were repeated after labeling. Western blot was imaged using the LI-COR Odyssey infrared imaging system (LI-COR). The neurotrophic ability of esophageal cancer cells was tested in co-culture experiments with the neuronal-like PC12 cells and neurite outgrowth was measured. PC12 cells are used extensively for studying neurite outgrowth.29Suter D.M. Miller K.E. The emerging role of forces in axonal elongation.Prog Neurobiol. 2011; 94: 91-101Crossref PubMed Scopus (147) Google Scholar For co-culture experiments, PC12 cells (50,000 cells in 1 mL) were seeded on bottom wells of 12-well Transwell plates (Corning, Corning, NY) coated with rat-tail collagen I (Invitrogen, Carlsbad, CA). After 24 hours, they were serum-starved in Dulbecco’s modified Eagle’s medium containing 1% horse serum. Esophageal cancer cells were grown in Transwell inserts (diameter, 12.0 mm; pores, 0.4 μm; Corning). Differentiation of PC12 cells was allowed for 3 days, with or without anti-NGF mouse monoclonal blocking antibody (alm-006; Alomone, Jerusalem, Israel) and neurite elongation was measured. PC12 cells showing neurites of at least twice the size of the cell body were considered as differentiated. Images were captured using a Zeiss Axiovert 200 inverted microscope fitted with an AxioCam HRm digital camera (Zeiss AG, Oberkochen, Germany). The one-way analysis of variance statistical test (GraphPad Prism 5.01; GraphPad Software, San Diego, CA) was used. The protocol described here was repeated utilizing the immortalized dorsal root ganglion neuronal cell line 50B11 (kindly provided by Dr. Ahmet Höke, John Hopkins University, Baltimore, MD) to corroborate findings in PC12 cells. To investigate the presence of nerves within esophageal tumors, immunohistochemistry against the pan-neuronal marker UCHL1 (alias PGP9.5) was performed. Typical morphologic features corresponding to both nerve trunks (composed of many axons) and isolated individual axons were observed (Figure 1). Nerves were detected in nearly all normal tissues (96% of cases) (Table 1), and all were localized in the submucosal and superficial muscularis layers of the esophageal lining (Figure 1, A and B). No nerves were detected in the stratified squamous epithelial layer of the esophagus. In esophageal cancer, nerve trunks were localized throughout the cancer cell mass of both adenocarcinoma and squamous cell carcinoma histologic subtypes. Both individual axons and larger nerve trunks frequently were identified adjacent to malignant epithelial cells (Figure 1, C–F). Nerves often were localized in close proximity to or surrounding blood vessels (Figure 1E). In addition, individual axons also were detected in matched lymph node metastases (Figure 1F). Overall, nerve infiltration within esophageal tumors was observed in 38% of cancer cases (Table 1). Nerves also were identified in 18% of lymph node metastases.Table 1Demographic and Disease-Specific Parameters for Included CasesParameterBenign esophagusEsophageal carcinoman137260Mean age, y (SD)62 (11)60 (10)Sex, n (%) Female30 (22)62 (24) Male107 (78)198 (76)Histologic subtype, n (%) AdenocarcinomaNA22 (8%) SquamousNA238 (92%)Tumor size, n (%) T1-T2NA52/203∗Cases in which data points were unavailable are indicated by the denominator. (26) T3-T4NA151/203∗Cases in which data points were unavailable are indicated by the denominator. (74)Grade, n (%) 1NA70/248∗Cases in which data points were unavailable are indicated by the denominator. (28) 2NA89/248∗Cases in which data points were unavailable are indicated by the denominator. (36) 3NA89/248∗Cases in which data points were unavailable are indicated by the denominator. (36)Lymph node metastases, n (%) NegativeNA111/210∗Cases in which data points were unavailable are indicated by the denominator. (53) PositiveNA99/210∗Cases in which data points were unavailable are indicated by the denominator. (47)Median survival, monthsNA21 (95% CI, 16–38) (n = 92)Nerves present on TMA core, % (n)96 (131/137)38 (99/260)Perineural invasion present, % (n)NA12 (31/260)Immunostaining for the pan-neuronal marker ubiquitin C-terminal hydrolase L1 (UCHL1) was performed on a cohort of esophageal carcinomas and normal adjacent tissues. Each clinical case was classified as negative/positive for the presence of nerves and for the presence of perineural invasion.NA, not applicable; TMA, tissue microarray.∗ Cases in which data points were unavailable are indicated by the denominator. Open table in a new tab Immunostaining for the pan-neuronal marker ubiquitin C-terminal hydrolase L1 (UCHL1) was performed on a cohort of esophageal carcinomas and normal adjacent tissues. Each clinical case was classified as negative/positive for the presence of nerves and for the presence of perineural invasion. NA, not applicable; TMA, tissue microarray. Nerves were identified in 9
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