Clinical characteristics of NSCLC patients
In the present study, the curative effects of 6 patients reached PR, and the curative effects of eight patients reached SD, while that of two patients reached PD after palliative radiotherapy. There was a distant metastasis (Stage IV) in twelve patients from sixteen NSCLC patients which included ten adenocarcinoma and six squamous carcinoma patients. Seven lung cancer patients received radiation to the lungs, five to the brain, and four to the bone. Clinical characteristics of NSCLC patients were presented in Table 1. The median age of eight heath controls (4 females and 4 males) was 54.5 (ranging from 49 to 62).
The percentages of peripheral MDSCs and CD68 + CD163 + M2-like macrophages were significantly elevated in NSCLC patients
The percentages of MDSCs and CD68+CD163+M2-like macrophages were analyzed by flow cytometry in healthy control (HC) and NSCLC patients (Fig. 1A, B). We found that the percentages of peripheral MDSCs and CD68+CD163+M2-like macrophages were significantly elevated in NSCLC patients in pre-RT and RT compared to HC group, while the proportions of MDSCs and CD68+CD163+M2-like macrophages were decreased in NSCLC patients in post-RT compared to in RT. Otherwise, there were obviously more peripheral MDSCs and CD68+CD163+M2-like macrophages in NSCLC patients in RT than in pre-RT (Fig. 1C, D).
MDSCs were correlated positively with CD68 + CD163 + M2-like macrophages in NSCLC patients
In HC, we found that MDSCs were correlated negatively with CD68+CD163+M2-like macrophages, but their correlation was not significant (Fig. 2A). Interestingly, the correlation between MDSCs and CD68+CD163+M2-like macrophages was not significant in NSCLC patients in pre-RT (r = 0.072, p = 0.791) (Fig. 2B), but peripheral MDSCs were positively correlated with CD68+CD163+M2-like macrophages in NSCLC patients in RT (r = 0.5548, p = 0.0257) and post-RT (r = 0.51, p = 0.0435) (Fig. 2C, D). These data indicated that ionizing radiation (IR) could cause the expansion of peripheric MDSCs and CD68+CD163+M2-like macrophages in NSCLC patients.
The value of MDSCs and CD68 + CD163 + M2-like macrophages in NSCLC patients with various clinical characteristics
To further investigate the value of MDSCs and CD68+CD163+M2-like macrophages of NSCLC patients in pre-RT, RT, and post-RT, we reviewed and classified the clinical data of these patients. In this study, we found that MDSCs as well as CD68+CD163+M2-like macrophages of NSCLC patients in RT and post-RT were elevated in NSCLC patients without metastasis but were not significantly increased in NSCLC patients with metastasis. Furthermore, there were higher percentages of MDSCs of NSCLC patients with metastasis in pre-RT than NSCLC patients without metastasis in pre-RT (Fig. 3A, B). The selection of radiotherapy area could affect the response to radiotherapy. However, in this study, the percentages of MDSCs and CD68+CD163+M2-like macrophages did not show clear difference in NSCLC patients with lung, brain and bone radiation. Hence, we speculated that the alteration of the percentages of MDSCs and CD68+CD163+M2-like macrophages of NSCLC patients in RT were irrelated with radiotherapy area (Fig. 3C, D). After radiotherapy (post-RT), the percentages of MDSCs in NSCLC patients with brain radiation were significantly less than NSCLC patients with lung and bone radiation, suggesting that MDSCs could be a more sensitive marker in NSCLC patients with brain radiation. We analyzed the percentages of MDSCs and CD68 + CD163 + M2-like macrophages in non-small cell lung squamous carcinoma and lung adenocarcinoma patients, finding that in radiotherapy, the proportions of MDSCs were markedly increased in non-small cell lung adenocarcinoma patients but not in non-small cell lung squamous carcinoma patients; however, the proportions of CD68+CD163+M2-like macrophages were markedly elevated in non-small cell lung squamous carcinoma patients but not in non-small cell lung adenocarcinoma patients (Fig. 3E, F).
The increasing MDSCs and CD68 + CD163 + M2-like macrophages improved the prognosis of NSCLC patients
After radiotherapy, the curative effects of NSCLC patients reached PR, SD and PD. We found that as for the NSCLC patients who reached PR, the frequencies of MDSCs and CD68+CD163+M2-like macrophages in RT were more than in pre-RT and post-RT. Furthermore, the percentages of MDSCs and CD68+CD163+M2-like macrophages of NSCLC patients reached PR in RT were higher than NSCLC patients reached SD or PD in RT. (Fig. 4A, B). By the correlation analysis between MDSCs and CD68+CD163+M2-like macrophages of NSCLC patients in pre-RT, RT, and post-RT at every curative effect group, we found that these two kinds of peripheral cells of PR group in RT had an obvious relation, demonstrating that radiotherapy could simultaneously increase their percentages in peripheral blood (Fig. 4C, D). This part of data illustrated that MDSCs and CD68+CD163+M2-like macrophages also act as a prognostic marker for treatment responses in NSCLC patients.
The crossed correlation analysis of CRP and WBCs with MDSCs and CD68 + CD163 + M2-like macrophages in NSCLC patients
Through retrospectively consulting the clinical data of NSCLC patients, we found that C-reaction protein (CRP) of NSCLC patient were increased in RT but decreased in post-RT compared with NSCLC patients in pre-RT (Fig. 5A). White blood cells (WBCs) of NSCLC patients were slightly decreased in post-RT (Fig. 5B). CRP was correlated positively with CD68+CD163+M2-like macrophages of NSCLC patients in RT (r = 0.7571, p = 0.0044), but did not have a linear relation with MDSCs of NSCLC patients in pre-RT, RT, and post-RT (Fig. 5C, D). As for WBCs, our study indicated that they were correlated negatively with MDSCs of NSCLC patients in post-RT (r=-0.5, p = 0.0572), but were irrelevant with CD68+CD163+M2-like macrophages of NSCLC patients in pre-RT, RT, and post-RT (Fig. 5E, F).
The plasma cytokines were examined by multiplex ELISA in HC and NSCLC patients
Measuring 40 inflammation-associated cytokines of NSCLC patients and HC, we found that 8 kinds of cytokines (BLC, Eotaxin, I-309, MCP-1, MIP-1β, RANTES, TNF RI, and TNF RII) were significantly elevated in NSCLC patients compared to HC. Especially, MIP-1β as well as Eotaxin was markedly decreased in NSCLC patients in pre-RT compared with HC group, but was significantly elevated after radiotherapy (Fig. 6A). 17 kinds of plasma cytokines (G-CSF, GM-CSF, ICAM-1, IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, TNF-α, and TNF-β) were obviously decreased in NSCLC patients compared with HC (Fig. 6B). However, 15 kinds of plasma cytokines (Eotaxin-2, IL-1β, IL-1ra, IL-6, IL-6R, IL-11, IL-12p40, IL-16, MCSF, MIG, MIP-1α, MIP-1d, PDGF-BB, TIMP-1, and TIMP-2) hardly changed in NSCLC patients compared with HC (Fig. 6C).
Plasma cytokines were correlated with MDSCs and CD68 + CD163 + M2‑like macrophages of NSCLC patients in RT
Ionizing radiation increased the percentages of MDSCs and M2-like macrophages in NSCLC patients. However, the correlation between MDSCs as well as CD68+CD163+M2‑like and plasma cytokines had barely been illuminated in NSCLC patient in ionizing radiation. In this study, we found that MDSCs were positively correlated with IL-1ra (r = 0.59, p = 0.0161), MIP-1β (r = 0.4939, p = 0.0516), TIMP-2 (r = 0.5575, p = 0.0248), and TNF RI (r = 0.504, p = 0.0465) in NSCLC patients of IR-middle group but were negatively related with IL-11 (r=-0.4055, p = 0.1192) (Fig. 7A). CD68+CD163+M2‑like macrophages were positively related with IL-1ra (r = 0.6527, p = 0.0061), MIP-1β (r = 0.5577, p = 0.0248), and IL-6 (r = 0.5448, p = 0.0291) in NSCLC patients of IR-middle group but were negatively related with TIMP-1 (r=-0.4545, p = 0.0769) and IL-12p40 (r=-0.5128, p = 0.0422) (Fig. 7B).
Plasma cytokines were correlated with MDSCs and CD68 + CD163 + M2‑like macrophages of NSCLC patients in post-RT
In this part, we analyzed the relation of plasma cytokines with MDSCs, finding that MDSCs were negatively related with MIP-1d (r=-0.6431, p = 0.0072) in NSCLC patients in post-RT (Fig. 8A). CD68+CD163+M2‑like macrophages were positively correlated with Eotaxin (CCL11) (r = 0.7316, p = 0.0013) (Fig. 8B) in NSCLC patients in post-RT but were negatively related with TMP-2 (r=-0.4808, p = 0.0594) (Fig. 8C) and TNF RII (r=-0.4525, p = 0.0785) (Fig. 8D).