Semaphorin SEMA3F localization in malignant human lung and cell lines: A suggested role in cell adhesion and cell migration

Abstract

Semaphorins/collapsins are a family of secreted and membrane-associated proteins involved in nerve growth cone migration. However, some are expressed widely in adult tissues suggesting additional functions. SEMA3F/H.SemaIV was previously isolated from a 3p21.3 homozygous deletion region in human lung cancer. We studied SEMA3F cellular localization using our previously characterized anti-SEMA3F antibody. In normal lung, SEMA3F was found in all epithelial cells at the cytoplasmic membrane and, to a lesser extent, in the cytoplasm. In lung tumors, the localization was predominantly cytoplasmic, and the levels were comparatively reduced. In non-small-cell lung carcinomas, low levels correlated with higher stage. In all tumors, an exclusive cytoplasmic localization of SEMA3F correlated with high levels of vascular endothelial growth factor and was related to the grade and aggressiveness. This suggests that vascular endothelial growth factor might compete with SEMA3F for binding to their common receptors, neuropilin-1 and -2 and might contribute to SEMA3F delocalization and deregulation in lung tumor. In parallel studies, SEMA3F distribution was examined in cell cultures by confocal microscopy. Marked staining was observed in pseudopods and in the leading edge or ruffling membranes of lamellipods or cellular protrusions in motile cells. SEMA3F was also observed at the interface of adjacent interacting cells suggesting a role in cell motility and cell adhesion.

Figure 1.

SEMA3F and VEGF levels and localization in normal lung and lung tumors. SEMA3F (A–C, E, G–L) and VEGF (D, F, H) were detected with a secondary biotinylated anti-rabbit antibody. In normal lung (A, B), SEMA3F was detected in all epithelial cells (score 3) with strong membranous staining in bronchial epithelial cells (A) and type II pneumocytes (B). Three examples are given for lung tumors (C–H) for: a squamous cell carcinoma with SEMA3F score 0 to 1 (C) and VEGF score 3 (D); for another squamous cell carcinoma with SEMA3F score 2 (E) and VEGF score 3 (F); and for an adenocarcinoma with SEMA3F score 3 (G) and VEGF score 0 to 1 (H). Note in C and E the diffuse cytoplasmic localization of SEMA3F, and in G the membranous localization of SEMA3F. SEMA3F was located at the basally oriented membrane toward tumor stroma in carcinoma suggesting a function in cellular interactions (I). Cells disseminated in the stroma expressed higher levels of SEMA3F (J) than in lobules. SEMA3F staining was intense and membranous in low grade neuroendocrine carcinoid (K) but diffuse and cytoplasmic in high grade neuroendocrine SCLC tumors (L).

Figure 2.

Real-time quantitative RT-PCR analysis for SEMA3F mRNA. A: Electrophoresis using a 2% agarose gel showing PCR products after 40 cycles, with SEMA3F-f and -r primers from cDNA of positive cells (Kasumi-1, K562, HeLa, H661), negative cells (H740 and H1450, which are homozygously deleted for SEMA3F), along with normal lung and lung tumors. The positive control was SEMA3F cDNA cloned into pBS and amplified with the primers. The DNA size is indicated in bp on the left. A specific product was obtained for all positive samples. The corresponding G3PDH products are shown in bottom panel. B: Linearity of the SEMA3F amplification was tested using various dilutions of the cloned SEMA3F cDNA. The PCR cycle (C_t) at which the product level (determined by fluorescence) crossed an arbitrary threshold during the exponential phase of amplification was measured as function of template concentration (in arbitrary units). The C_t for SEMA3F-f and -r primers over nine serial template dilutions demonstrated a linear fit on a semilog plot, indicating the quantitative nature of the process. For each concentration, the amplifications were performed in triplicate. C: Fluorescence versus cycle number for three replicate reactions each using G3PDH and SEMA3F-f and -r primers in one tumor. The difference between the C_t for G3PDH and the C_t for SEMA3F corresponds to the relative expression level of SEMA3F.

Figure 3.

SEMA3F immunostaining in tumor cells detected by confocal microscopy. SEMA3F was detected with a FITC-conjugated goat-anti-rabbit IgG secondary antibody. Detection of SEMA3F in NSCLC NCI-H661 cells showed a localization in the cytoplasm (A, B) and at the interface of adjacent cells (B: arrows). Polarized lung adenocarcinoma Calu-3 cells, growing in islets, confirmed the major distribution of SEMA3F in the apical domain of lung epithelial cells (C). In cervical carcinoma HeLa cells (D, E) and neuroblastoma SK-N-SH cells (F), SEMA3F was detected in the cytoplasm but also in motile regions (D, E, F: arrows). In D, the inset showed the edge of lamellipodia. In E and F, the intensity of SEMA3F staining was quantified by an arbitrary color scale from blue (no SEMA3F) to red (highest level of SEMA3F). In G and H, SEMA3F and F-actin were double stained in HeLa cells. SEMA3F was detected with a FITC-conjugated goat-anti-rabbit IgG secondary antibody (G), and F-actin was labeled with TRITC-conjugated phalloidin (H). SEMA3F colocalized with actin microfilaments in ruffling regions. Scale bar, 10 μm.