IL-12 can target human lung adenocarcinoma cells and normal bronchial epithelial cells surrounding tumor lesions

Abstract

Background: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC.

Methodology/principal findings: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines.

Conclusions: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.

Figure 1. IL-12Rβ2 expression and function in human lung adenocarcinoma.

1A. Histological features and IL-12Rβ2 expression in human bronchioloalveolar lung carcinomas. Non-mucinous bronchioloalveolar carcinoma typically shows columnar neoplastic cells growing along the alveolar septa (a). In 41.4% of adenocarcinomas, neoplastic cells lack IL12Rβ2 expression (b), though a few may sometimes retain it (inset in b). By contrast, alveoli unaffected by malignant process express IL-12Rβ2 (c), as observed in the remaining tumors (d). (×400). 1B. Angiogenic activity of supernatants from one representative lung ADC sample cultured in the presence or absence of hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (left panel). When supernatants from hrIL-12 treated lung ADC sample was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (right panel). These experiments were repeated three times. Original magnification: ×50. 1C. Pooled results from human angiogenesis PCR array performed in three lung ADC samples cultured in the presence or absence of hrIL-12 are shown. Histogram shows fold expression changes of genes in primary samples treated with hrIL-12 vs medium.

Figure 2. IL-12Rβ2 expression and function in human NSCLC cell lines.

2A. IL-12RB2 expression in NSCLC cell lines, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); PC = positive control (total tonsil B cells); different NSCLC cell lines (Colo699, Calu6, Calu1, A549, SK-MES-1, GLC82 and Calu6/β2 cells) are shown. 2B. Left panel. IL-12Rβ2 protein expression in Calu6/β2 cells, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched antibody staining. Right panel. IL-6 intracellular staining in Calu6/β2 cells cultured with medium or hrIL-12 for 48 h, as assessed by flow cytometry. Open profile: IL-6 staining in cells cultured with medium; dark profile: isotype matched antibody staining, dashed line: IL-6 staining in cells cultured with IL-12. 2C. Angiogenic activity of supernatants from Calu6/β2 cells cultured with medium alone or hrIL12. CAM treated with sponges loaded with supernatant from the untreated cells were surrounded by allantoic vessels developing radially towards the implant in a ‘spoked-wheel’ pattern (upper left panel). When supernatants from hrIL-12treated Calu6/β2 cells was tested, a significant reduction (P = 0.001) of the angiogenic response was appreciable (upper right panel). Lower panels show the angiogenic activity of Calu6/β2 cells in the presence of an anti-IL-6 mAb (left panel) or of an anti-VEGF-C mAb (right panel). These experiments were repeated three times. Original magnification: ×50.

Figure 3. Anti-tumor activity of IL-12 on NSCLC in vivo.

3A. Volume of tumors grown after Calu6/β2 cell inoculation orthotopically (left panel) or subcutaneously (right panel) in PBS and hrIL-12 treated animals. Animals injected orthotopically were sacrificed after 23 days, those injected subcutaneously after 14 days. Volume of tumors grown after inoculation orthotopically (left panel) or subcutaneously (right panel) of Calu6 cell transfected with the empty vector hrIL-12 treated animals was also shown (empty vector+IL12). The differences in size between tumors removed from PBS and hrIL-12 treated mice were evaluated by Mann-Whitney U test. Boxes indicate values between the 25^th and 75^th percentiles, whisker lines represent highest and lowest values for each group. Horizontal lines represent median values. 3B. Tumors (developed after s.c injection) injected subcutaneously with Calu6/β2 cells in PBS-treated SCID/NOD mice are mostly formed of nests of undifferentiated, pleomorphic and proliferating cells (mitotic (figures) features indicated by arrows) rapidly infiltrating the underlying muscle layers (arrowheads) (a), and supplied by a distinct microvessel network, as assessed by laminin staining (b). In hrIL-12 treated mice, tumor histology is altered by the appearance of large areas of ischemic-hemorrhagic necrosis (N) (c) associated with defective microvascular architecture (d) (×400). Orthotopical injection of Calu6/β2 cells gave rise, in PBS-treated mice, to tumors with istopathological features (e) similar to those of subcutaneously developed tumors (a) and supplied by a well developed microvascular network (f). As observed in subcutaneous tumors, in orthotopic tumors as well hrIL-12 treatment induced wide necrosis (g) and severe microvascular alterations (h) (×400). 3C. Human Angiogenesis PCR Array on tumors explanted from hrIL-12 vs PBS treated animals 23 days after orthotopic inoculation of Calu6/β2 cells. Histogram shows fold expression changes of genes in tumors from hrIL-12 vs PBC treated mice. 3D. Tumors from PBS-treated mice express VEGF-C (a) and IL-6 (c). Expression of VEGF-C and IL-6 is strongly reduced (b and d, respectively) in tumors from hrIL-12 treated mice. (×400).

Figure 4. IL-12Rβ2 expression and function in normal bronchial epithelial cells.

4A. IL-12RB2 expression in human primary bronchial epithelial cells, as assessed by RT-PCR. From left to right: MW = molecular weight; NC = negative control (Raji cell line); three different NBEC cultures (NBEC #1, #2 and #3) are shown. 4B. IL-12Rβ2 surface expression in human NBEC, as assessed by flow cytometry. Open profile: IL-12Rβ2 staining; dark profile: isotype matched mAb staining. 4C. Short circuit current recordings in normal bronchial epithelial cells. The figure depicts two representative experiments from control (top) and IL-12 treated (bottom) epithelia showing responses to amiloride (10 µM, apical), forskolin (20 µM, apical and basolateral), UTP (100 µM, apical), and CFTR_inh-172 (10 µM, apical). 4D. Cytokine release by human NBEC, as assessed by Bio-Plex Assay. Pooled results from supernatants of three different bronchial epithelial cell suspensions are shown. IL-12 treatment reduced significantly the release of IL-6 (P = 0.0049), IL-8 (P = 0.0071), FGF-b (P = 0.0231), GM-CSF (P = 0.0028), IP-10 (P<0.0001), MCP-1 (P = 0.0002) and RANTES (P = 0.0108).