The number and microlocalization of tumor-associated immune cells are associated with patient's survival time in non-small cell lung cancer

Fuqiang Dai 0 3 Lunxu Liu 0 Guowei Che 0 Nanbin Yu 0 2 Qiang Pu 0 Shangfu Zhang 0 Junliang Ma 0 Lin Ma 0 Zongbing You 1 0 Department of Thoracic and Cardiovascular Surgery, West China Hospital, Sichuan University , Chengdu 610041 , China 1 Departments of Structural & Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center, LCRC, Tulane Center for Aging, Tulane Center for Gene Therapy, Tulane University School of Medicine , New Orleans, LA 70112 , USA 2 The Third People's Hospital of Zigong City , Sichuan Province , China 3 Daping Hospital, the Third Military Medical University , Chongqing City , China Background: Tumor microenvironment is composed of tumor cells, fibroblasts, endothelial cells, and infiltrating immune cells. Tumor-associated immune cells may inhibit or promote tumor growth and progression. This study was conducted to determine whether the number and microlocalization of macrophages, mature dendritic cells and cytotoxic T cells in non-small cell lung cancer are associated with patient's survival time. Methods: Ninety-nine patients with non-small cell lung cancer (NSCLC) were included in this retrospective study. Paraffin-embedded NSCLC specimens and their clinicopathological data including up to 8-year follow-up information were used. Immunohistochemical staining for CD68 (marker for macrophages), CD83 (marker for mature dendritic cells), and CD8 (marker for cytotoxic T cells) was performed and evaluated in a blinded fashion. The numbers of immune cells in tumor islets and stroma, tumor islets, or tumor stroma were counted under a microscope. Correlation of the cell numbers and patient's survival time was analyzed using the Statistical Package for the Social Sciences (version 13.0). Results: The numbers of macrophages, mature dendritic cells and cytotoxic T cells were significantly more in the tumor stroma than in the tumor islets. The number of macrophages in the tumor islets was positively associated with patient's survival time, whereas the number of macrophages in the tumor stroma was negatively associated with patient's survival time in both univariate and multivariate analyses. The number of mature dendritic cells in the tumor islets and stroma, tumor islets only, or tumor stroma only was positively associated with patient's survival time in a univariate analysis but not in a multivariate analysis. The number of cytotoxic T cells in the tumor islets and stroma was positively associated with patient's survival time in a univariate analysis but not in a multivariate analysis. The number of cytotoxic T cells in the tumor islets only or stroma only was not associated with patient's survival time. Conclusions: The number of macrophages in the tumor islets or stroma is an independent predictor of survival time in NSCLC patients. Counting macrophages in the tumor islets or stroma is more useful in predicting patient's survival time than counting mature dendritic cells or cytotoxic T cells. - Background Tumor microenvironment is composed of tumor cells, resident cells such as fibroblasts and endothelial cells, and infiltrating cells such as macrophages, dendritic cells, and lymphocytes, as well as products of all these cells such as extracellular matrix, growth factors, cytokines, chemokines, enzymes, and various metabolites [1]. The crosstalk between tumor cells and other tumor-associated cells may lead to either blocking tumor formation or enhancing tumor formation and/or progression. The doubleedged-sword nature of many tumor-associated immune cells such as macrophages, dendritic cells, and cytotoxic T cells has been recognized [2-4]. On the one hand, these immune cells may recognize tumor-associated antigen and activate cytotoxic T cells, in order to initiate antitumor immune responses. On the other hand, the same immune cells may establish immune tolerance and even promote tumor growth and metastasis through enhancing angiogenesis and invasion of extracellular matrix. Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death worldwide. The fiveyear survival rate is approximately 67% for the patients with stage IA NSCLC after putatively curative surgery [5]. In order to identify new prognostic factors that can guide clinical practice, we have previously found that the number of tumor-associated macrophages (TAMs) in the tumor islets is positively associated with survival time in the patients with NSCLC [6]. Because TAMs are not the only tumor-associated immune cells, in this study we further investigated the prognostic value of mature dendritic cells and cytotoxic T cells in the patients with NSCLC. Methods Study population This study was approved by the Institutional Review Board of West China Hospital, Sichuan University. The procedures to obtain human lung cancer tissues and follow-up information are in accordance with the Ethical Principles for Medical Research Involving Human Subjects as formulated in the World Medical Association Declaration of Helsinki (revised in 2008). All specimens were obtained from the archives of formalin-fixed, paraffin-embedded tissue blocks in the Department of Thoracic and Cardiovascular Surgery, West China Hospital, Sichuan University. The lung cancer tissues were collected from surgeries performed from August, 1999 to August, 2001. The patients were followed up until December, 2007, through outpatient visits and/or correspondences to family members. Ninety-nine patients were included in this retrospective study. Histological evaluation was based on the World Health Organization criteria [7]. Tumor stage was evaluated according to the International Union against Cancer TNM classification system [7]. The clinicopathological characteristics were summarized in Table 1. Immunohistochemistry Four-m thick tissue sections were de-waxed in xylene and rehydrated through graded alcohols. Antigen retrieval was carried out using microwave at middle-tohigh temperature for 8 min, low-to-high temperature for 5 min, and then cooled down at room temperature for 20 min. Mouse anti-human CD68 monoclonal antibodies (clone KP1, recognizing macrophages), rabbit antihuman CD8 monoclonal antibodies (clone SP16, recognizing cytotoxic T cells), and streptavidin-peroxidase conjugated secondary antibodies (SP-9002) were obtained from Zhongshan Goldenbridge Biotechnology Co., LTD., Beijing, China. Mouse anti-human CD83 monoclonal antibodies (clone HB15a, recognizing mature dendritic cells) were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. Diaminobenzidine (DAB) substrate kit was obtained from Dako North America, Inc., Carpinteria, CA, USA. Immunohistochemical staining of individual markers was performed according to the kit manufacturer's instructions. Sections were then counterstained with hematoxylin and mounted in an aqueous mounting medium. Tissue sections previously stained positively were used as positive control, while tissue sections with primary antibodies replaced by phos (...truncated)