The Analysis Of Macrophages And Angiogenic Factors In Bronchoalveolar Lavage Fluid From Lung Cancer Patients

Lung cancer, with high incidence and mortality, is one of the most common malignanttumors among human beings. Early invasion and distant metastasis, which are dependent ongrowth and/or pro-angiogenic factors such as vascular endothelial growth factor (VEGF)and interleukin8(IL-8), have been identified as important risks for poor prognosis anddeath in lung cancer patients. Increasing evidence has been emerging that theabovementioned factors can be produced by cancer cells and infiltrating inflammatory cellsin parenchyma and/or mesenchyma of tumor tissues. In fact, infiltration of inflammatorycells and production of pro-angiogenic factors play an important role in tumor immunity oflung cancer. However, the distributions of those cells and their contributions to theproduction of pro-angiogenic factors as well as the activation phenotype of macrophages inbronchoalveolar lavage fluid (BALF) of lung cancer patients remain unclear. In this study,we detected the number and types of distinct inflammatory cells and the activationphenotype of macrophages together with the levels of VEGF and IL-8within BALF from54smoking lung cancer patients including36squamous cell carcinoma (SCC),9adenocarcinoma (AC), and9small cell lung cancer (SCLC) in comparison with those from13non-smoking and7smoking patients with nonspecific chronic inflammation and8non-smoking normal controls and analyzed its clinical significance. Main results andconclusion are as following:1. CD163+macrophages are the predominant subset of macrophages that exist in theBALF from lung cancer patients.(1) All the patients [54smoking lung cancer patients (36squamous cell carcinoma,9adenocarcinoma, and9small cell lung cancer),13non-smokingand7smoking patients with nonspecific chronic inflammation] and8non-smoking normalcontrols with infirmed consent were diagnosed at Southwest Hospital, Third MilitaryMedical University. Informed consents were obtained from all the participants and the studywas approved by our institutional review board.(2) In order to profile inflammatory cellswithin BALF, we first applied liquid-based cytology to identify the types, numbers and percentages of infiltrating cells in BALF in addition to cancer cells. A lower percentage ofmacrophages among the inflammatory cells and a higher percentage of neutrophils in BALFwere seen from cancer and nonspecific chronic inflammation patients than that from normalcontrol (p<0.01). Further analysis showed a significantly lower percentage of macrophages(p<0.05) and higher percentage of neutrophils (p<0.05) in the BALF from the non-smokingpatients with nonspecific chronic inflammation as compared with those with AC cases andSCLC cases. Interestingly, a significantly higher percentage of lymphocytes were alsoobserved in the BALF from AC cases. Additionally, more neoplastic cells were found in theBALF from squamous cell carcinoma (SCC) cases than that from AC or SCLC cases.(3)We then attempted to identify the immunophenotypes of macrophages in the BALF byusing CD68and CD163antibodies. We found that all the identified macrophages in HEstaining were positive for CD68, and some of them were also positive for CD163(37.4%±28.1%), indicating that nearly half of themacrophageswere accounted for the totalinfiltrating ones. Importantly, CD163+macrophages were detected in81%of cancerpatients as compared to70%of controls. Moreover, CD163+macrophages were also foundhigher in SCLC cases.2. The analysis of levels of pro-angiogenic factors in BALF from lung cancer patients.(1) We measured the levels of IL-8and VEGF in BALF from lung cancer patients usingELISA. We found a significantly higher level of IL-8protein in the BALF from lung cancerpatients (5.0±5.3ng/ml) as compared with that from normal ones (0.8±0.7ng/ml, p<0.05)and non-smoking patients with nonspecific chronic inflammation (1.5±1.6ng/ml, p<0.05).More precisely, when the level of IL-8was determined as RIL-8, i.e., the ratio of IL-8concentration (pg/ml) to the corresponding total protein level of BALF (mg/ml), RIL-8inBALF from the patients with lung cancer was significantly higher than that from normal andnon-smoking patients with nonspecific chronic inflammation [(9.1±8.9)×106vs.(1.7±1.1)×106and (4.2±4.0)×106, both p<0.05].(2) Similarly, the levels of VEGF proteinand RVEGFin lung patients were significantly higher than those in normal subjects (0.4±0.3ng/ml vs.0.2±0.1ng/ml,[(8.3±4.2)×107vs.(4.9±2.3)×107, respectively, both p<0.05].(3) In addition to the difference in the level of IL-8between BALF from SCC andthat from AC cases, no statistical significant difference was found in the levels of IL-8andVEGF between histological types of lung cancer. 3. The correlation analysis between the concentration of pro-angiogenic factors and theproportions of BALF cells.(1) We found a significantly positive correlation between theconcentration of IL-8and the number of neutrophils in the BALF from non-SCLC patients(p<0.01). Furthermore, a significantly positive correlation between the concentration of IL-8and the number of lymphocyteswas seen in the BALF fromAC patients (p<0.05).(2)Futhermore, a significantly positive correlation was found between VEGF level and thenumber of cancer cells (p<0.05), which was further confirmed by means ofimmunohistochemistry in lung cancer tissue.(3) However, the levels of IL-8and VEGFwere not related to the percentage of M2macrophages.In summary, Our results suggest that the detection of infiltrating inflammatory cellsand pro-angiogenic factors in BALF to reflect the local immune milieu will be helpful fordiagnosis of cancerous inflammation in the lungs.