EGFR-specific single-chain variable fragment antibody-conjugated Fe3O4/Au nanoparticles as an active MRI contrast agent for NSCLC.

Overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor prognosis in non-small cell lung cancer (NSCLC), thus making it a promising biomarker for NSCLC diagnosis. Here, we conjugated a single-chain antibody (scFv) targeting EGFR with Fe3O4/Au nanoparticles to form an EGFR-specific molecular MRI bioprobe (scFv@Fe3O4/Au) to better detect EGFR-positive NSCLC tumors in vivo. In vitro, we demonstrated that the EGFR-specific scFv could specifically deliver Fe3O4/Au to EGFR-positive NSCLC cells. In vivo experiments showed that the accumulation of scFv@Fe3O4/Au in tumor tissue was detectable by magnetic resonance imaging (MRI) at the indicated time points after systemic injection. The T2W signal-to-noise ratio (SNR) of EGFR-positive SPC-A1 tumors was significantly decreased after scFv@Fe3O4/Au injection, which was not observed in the tumors of mice injected with BSA@Fe3O4/Au. Furthermore, transmission electron microscopy (TEM) analysis showed the specific localization of scFv@Fe3O4/Au in the SPC-A1 tumor cell cytoplasm. Collectively, the results of our study demonstrated that scFv@Fe3O4/Au might be a useful probe for the noninvasive diagnosis of EGFP-positive NSCLC.

Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL­6/STAT3 signaling pathway.

Increasing evidence indicates that the inflammatory tumor microenvironment can lead to cancer cell metastasis. Shikonin, which is extracted from the Chinese herb Zicao (the dried root of Lithospermum erythrorhizon), possesses various pharmacological effects, but its effect on tumor metastasis in the inflammatory microenvironment remains unknown. In the present study, we aimed to investigate the potential effect of shikonin on tumor metastasis in an inflammatory microenvironment as well as the underlying molecular mechanisms. It was found that, in the inflammatory microenvironment simulated by THP­1 cell conditioned medium (THP­1­CM) in vitro, shikonin significantly inhibited the epithelial­mesenchymal transition (EMT), migration and invasion of human lung adenocarcinoma cell lines A549 and H1299. In addition, we found that interleukin­6 (IL­6), which is expressed in THP­1­CM, promoted the EMT of lung adenocarcinoma cells, and shikonin markedly inhibited IL­6­induced EMT and cell motility. Moreover, shikonin inhibited IL­6­induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), prevented phosphorylated STAT3 (p­STAT3) translocation into the nucleus, and suppressed p­STAT3 transactivation activity. Additionally, it was found that shikonin inhibited lung metastasis, EMT and expression of p­STAT3 of A549 cells in vivo. Furthermore, IL­6 levels in human lung adenocarcinoma tissues were significantly associated with tumor­node­metastasis stage and lymph node metastasis, and its expression was correlated with tumor­associated macrophage (TAM) infiltration. Together, these results suggest that shikonin suppresses the migration and invasion of human lung adenocarcinoma cells in an inflammatory microenvironment involving the IL­6/STAT3 signaling pathway.

HER2-siRNA delivered by EGFR-specific single chain antibody inhibits NSCLC cell proliferation and tumor growth.

Overexpression of human epidermal growth factor receptor type2 (HER2) is closely associated with aggressive progression and poor prognosis in non-small cell lung cancer (NSCLC). Here, we generated an EGFR-scFv-arginine nonamer peptide fusion protein (scFv-9R) as a cargo to deliver HER2 specific siRNA into HER2-positive NSCLC cells both in vitro and in vivo. HER2-siRNAs delivered by scFv-9R effeciently silenced HER2 expression in EGFR-positive NSCLC cells, and consequently resulted in G1 arrest and cell growth inhibition. Importantly, intravenous injection of scFv-9R/HER2-siRNA complex markedly suppressed growth of EGFR-positive NSCLC xenograft in nude mice, resulting from downregulated HER2 expression, reduced cell proliferation and enhanced cell apoptosis. Collectively, our study provides a novel therapeutic strategy for the treatment of EGFR-positive, HER2-overexpressed NSCLC.

siRNA delivered by EGFR-specific scFv sensitizes EGFR-TKI-resistant human lung cancer cells.

The overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor outcome in non-small cell lung cancer (NSCLC), and EGFR is an ideal biomarker for the targeted therapy of NSCLC. Although patients with EGFR-activating mutations respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs), they eventually acquire resistance, which typically results from a secondary EGFR mutation or the activation of other signaling pathways. Novel approaches to overcome or prevent EGFR-TKI resistance are clinically important. In this study, we developed an EGFR-scFv-arginine nonamer peptide fusion protein, s-9R, as an siRNA carrier. Here, we show that s-9R effectively and specifically delivers EGFR-siRNAs, KRAS-siRNA and MET-siRNA into NSCLC cells and silences the expression of target genes. The sensitivity of NSCLC cells to gefitinib was restored after treatment with the s-9R/siRNA complex, and the apoptosis rates of the treated cells were significantly higher than those of the control groups. Furthermore, the co-administration of s-9R/siRNA and gefitinib successfully suppressed the progression of H1975 xenograft tumors and extended the life span of tumor-bearing nude mice. Collectively, the results of this study provide not only a new scFv derivative for delivering siRNA into EGFR-overexpressing, TKI-resistant NSCLC cells but also a novel method for overcoming TKI resistance.