Human lung carcinomas synthesize immunoregulatory glucocorticoids

Abstract

The need for new options in lung cancer treatment inevitably leads back to basic research. The tumor itself and the tumor environment especially the interaction with the immune system need to be better understood to develop targeted therapies. In the context of lung cancer glucocorticoids (GC) are mainly known as a combination drug to attenuate side-effects of chemotherapies. However, endogenous extra-adrenal GC have been shown to substantially regulate local immune responses within various tissues, including the lung. In this study we investigated whether primary lung tumors have maintained the capacity to synthesize GC and may thereby regulate anti-tumor immune responses. We show that several non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) cell lines express key steroidogenic enzymes and synthesize bioactive GC under steady state conditions. We also show that tumor-derived GC can inhibit splenic T cell activation, thus demonstrating their immunoregulatory potential. Moreover, steroidogenic enzymes were detected by quantitative RT-PCR and immunohistochemistry in tissue sections of different human lung tumors, further strengthening the idea that human lung carcinomas regulate their microenvironment by releasing immunoregulatory GC, which potentially contributes to immune evasion and treatment resistance.

Fig. 1. Human lung cancer cell lines produce immunoregulatory glucocorticoids.

A A simplified scheme of the glucocorticoid (GC) synthesis pathway in humans. B Expression of CYP11A1, CYP11B1 and HSD11B1 was assessed in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines by RT-qPCR. Expression levels were normalized to β-ACTIN. Bars indicate mean values + SD (n = 3). C GC concentration in cell culture supernatant of control or metyrapone-treated cells (MET) was assessed in the GC bioassay. Bars show mean values + SD (n = 3) D Primary murine splenocytes were pretreated with buffer control or RU486 (100 nM) for 1 h, prior to treatment with 0, 3 or 30 ng/ml cortisol, or lung cancer cell line supernatant. After 2 h cells were activated with concanavalin A (1 µg/ml) and CD69 expression in CD4 + T cells was analyzed by flow cytometry after 20 h. Mean values + SD of n = 3 (cortisol titration) or n = 3–5 (cell culture supernatants) experiments are shown. Representative CD69 expression profiles for CD4 + T cells treated with H460 and H510 cell culture SN: dashed light gray plot = activated CD4 + T cells, black plot = treated with cell culture SN, gray plot = treated with cell culture SN plus RU486. Statistical analysis was performed by using Two-way ANOVA multiple comparisons (C), or two-tailed unpaired t-test (D). ** = p < 0.005; *** = p < 0.0005; **** = p < 0.0001; ns not significant, ND not detectable.

Fig. 2. Different types of human lung cancers express steroidogenic enzymes.

A Expression of CYP11A1, CYP11B1 and HSD11B1 was assessed in tissue sections of non-small cell lung carcinoma (NSCLC, SCC = squamous cell carcinoma, AC = adenocarcinoma) and small cell lung carcinomas (SCLC, t.NET = typical neuroendocrine tumors) by RT-qPCR. Violin plots of n = 5–8 tumor samples are shown. B Immunohistochemistry analysis of 11-β hydroxysteroid dehydrogenase 1 (11-βHSD1) and 11-β hydroxylase (11-βOH) in tissue sections of squamous cell carcinoma (NSCLC) and typical neuroendocrine tumor (SCLC). H&E stainings and Rb IgG isotype controls are shown for comparison. Scale bar = 200 µm. Inlays show magnifications.