Intratumoral Localization of Aromatase and Interaction between Stromal and Parenchymal Cells in the Non–Small Cell Lung Carcinoma Microenvironment

Abstract

Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non-small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-α, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas. Cancer Res; 70(16); 6659-69. ©2010 AACR.

Prognostic and Therapeutic Implications of Aromatase Expression in Lung Adenocarcinomas with EGFR Mutations

“…Exemestane, an irreversible steroidal inactivator, either alone (41) or in combination with cisplatin (43) showed significant antitumor effects in 2 separate studies. Both letrozole and anastrozole, reversible steroidal inactivators, demonstrated similar antitumor activity in NSCLCs (12,23,44). However, lung cancer cell lines used in almost all of these studies were without EGFR mutations.…”

The advantages of co-culture over mono cell culture in simulating in vivo environment

“…The techniques of co-culture involved varied but this technique has been performed for the purpose of analyzing "cultivation of embryonic stem cells on feeder cells", "examination of immune cell interactions", and "evaluation of mesenchymal-stromal interactions" [5,[35][36][37][38][39]. Direct co-culture can be performed in nearly all cell culture dishes, for instance by layering two cell types one on top of the other.…”

“…In our laboratory, we have employed 6 or 12 well ThinCert TM (Greiner Bio-One, Kremsmünster, Austria) cell culture inserts with translucent membranes (Fig. 4) [5,39]. The membrane material is polyester, which is clear film and can directly examine the cells under the light microscopy.…”

“…Co-cultures are maintained for 24-72 h at 37 • C and 5% CO 2 . After cultivation using this co-culture system, carcinoma and stromal cells were separated and each component was examined by real time RT-PCR, cell count, analysis of enzymatic activity, and cytokine analysis [5,39].…”

“…Human breast carcinoma cell lines, MCF-7 and SK-BR-3 cells were cultured in co-culture chambers in the absence or presence of 32N or 74T cells and were cultivated on the bottom of the plates or dishes. Both 32N and 74T cells were intratumoral fibroblastic stromal cells obtained from breast cancer patients by primary culture [39]. After 24 h cultivation using this co-culture system, both carcinoma and stromal cells were physically separated and each component was examined in the aromatization assay, estrogen production assays or by real time RT-PCR of aromatase.…”