Abstract 407: Detection of phospho-Stat3-mediated immunosuppressive microenvironment in benign lymph nodes of patients with early stage non-small cell lung cancer
Authors: Zhang Wang , Pal Sumanta K. , Yang Chunmei , Figlin Robert A. , Yu Hua , Reckamp Karen L. 
Published: July 2012

Abstract
Objectives: Surgical resection offers the most promising chance for cure in patients who present with early-stage disease, although the majority will experience recurrence due to microscopic disease at the time of surgery. In this study, we sought to identify markers of metastatic potential within benign regional lymph nodes of patients with completely resected non-small cell lung cancer (NSCLC) with a focus on VEGFR-1, myeloid cells and pStat3.
Methods: Paraffin-embedded benign pulmonary lymph node specimens from 46 lung cancer patients with stage I-III resected disease were stained for CD68 (AbD Serotec, Raleigh, NC), CD163 (AbD Serotec, Raleigh, NC), VEGFR-1 (Santa Cruz, Santa Cruz, CA), phospho-Stat3 (Cell Signaling, Danvers, MA), MMP-9 (Cell Signaling, Danvers, MA) and VEGF-A (Abcam, Cambridge, MA) by immunohistochemistry or immunofluorescence. Anthracosis in the lymph nodes was evaluated using HE-stained sections. The sections were analyzed with Image Pro Plus or evaluated by two independent observers who were not aware of the patient information (double-blinded).
Results: In benign lymph nodes from resected lung cancer patients, CD68 was highly expressed, while VEGFR-1 demonstrated low expression. Furthermore, these cells were associated with anthracotic deposition. Upon additional evaluation, we found that the infiltrating cells with anthracotic pigment display CD68+ CD163+, suggestive of an M2 macrophage phenotype, which are immunosuppressive and cancer promoting. These M2 macrophages expressed MMP-9 and VEGF-A, and in at least 40% of the patient lymph node tissue, pStat3 was elevated in these anthracotic macrophages. The infiltration of CD68+ macrophages was closely correlated with the intensity of anthracosis (P&lt;0.0001, One-way ANOVA). In some lymph nodes, the infiltration of M2 macrophages was dramatically increased. We also observed positive correlations between overall pStat3 level with M2 macrophage infiltration and anthracosis intensity (P&lt;0.05 and P&lt;0.01, respectively, One-way ANOVA). In addition, pStat3 levels in anthracotic cells increased with the intensity of anthracosis in the lymph nodes.
Conclusion: Our results indicate the presence of M2 macrophages and anthracosis in benign lymph nodes of patients with stage I-III NSCLC. The infiltration of M2 macrophages in benign pulmonary lymph nodes might serve as a predictive factor for micrometastasis and patient prognosis. Correlations between M2 macrophage in lymph nodes with smoking status, patient stage and survival will also be evaluated.
Supported by NIH R01 CA115815 (hy) and NIH K12 CA001727 (klr; skp).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 407. doi:10.1158/1538-7445.AM2011-407