Detection of micrometastases in peripheral blood of non-small cell lung cancer with a refined immunomagnetic nanoparticle enrichment assay

Qing Li¹, Hui Qi¹, Han-Xin Zhou¹, Chun-Yan Deng¹, Hai Zhu³, Jin-Feng Li³, Xi-Li Wang³, Fu-Rong Li^1,2
1Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People's Hospital), Jinan University, Shenzhen, People's Republic of China; ²Shenzhen Institute of Gerontology, Shenzhen, People's Republic of China; ³Shenzhen Bioeasy Biotechnologies Co Ltd, Shenzhen, People's Republic of China

Abstract: Fe₃O₄ particles are currently used as the core of immunomagnetic microspheres in the immunomagnetic enrichment assay of circulating tumor cells (CTCs). It is difficult to further improve the sensitivity of CTC detection or to improve tumor cell-type identification and characterization. In the present study, we prepared immunomagnetic nanoparticles with nanopure iron as the core, coated with anti-cytokeratin 7/8 (CK7/8) monoclonal antibody. These immunomagnetic nanoparticles (IMPs) were used in conjunction with immunocytochemistry (ICC) to establish a refined immunomagnetic nanoparticle enrichment assay for CTC detection in non-small cell lung cancer (NSCLC). The assay was compared with nested reverse transcription polymerase chain reaction (RT-PCR) to detect CK19 mRNA and lung specific X protein (LUNX) mRNA. Human lung adenocarcinoma cell line A549 was used for sensitivity and specificity evaluation. Peripheral blood samples were collected from each group for CTC detection. The average diameter of the immunomagnetic nanoparticles was 51 nm, and the amount of adsorbed antibodies was 111.2 µg/mg. We could detect down to one tumor cell in 5 × 10⁷ peripheral blood mononuclear cells. The sensitivity was consistent with that of nested RT-PCR; however, the false positive rate was significantly reduced. The modified assay combined with ICC did not differ from nested RT-PCR in sensitivity, but it had significantly increased specificity. This approach could, therefore, contribute to identification of micrometastases, re-defining clinical staging, and guiding individual postoperative treatments. The technique shows considerable potential clinical value and further clinical trials are warranted.

Keywords: NSCLC, circulating tumor cells, nested RT-PCR, immunomagnetic nanoparticles, immunocytochemistry