Study of Lung Cancer Cell Migratory Behavior and Cell Heterogeneity under Physiological Electric Field in Microfluidic Device

利用微流控芯片研究生理電刺激下肺癌細胞遷移行為與細胞異質性

Student thesis: Doctoral Thesis

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Author(s)

  • Yaping LI

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Award date8 Sep 2016

Abstract

Tumor metastasis is the primary cause of death of cancer patients. Metastasis process involves the movement of cells from one site to another, an early and critical step, however, mounting evidences revealed that cells are diverse even within individual tumor, which is called as cell heterogeneity. Despite significant recent interest, the migratory heterogeneity within lung tumor is yet to be defined. Numerous studies have demonstrated that cancer cells could undergo reorientation and migration in a specific direction by application of physiological electric fields, which suggested its potential implications in metastasis. Microfluidic devices have the potential to configure small channels with micrometer dimensions precisely by flexible fabrication, and thus provide controllable microenvironments and enable real-time observation of cell behaviors.
In chapter 2, we systematically evaluated the migration ability of several non-small cell lung cancer cell lines (H460, HCC827, H1299 and H1975) after applying physiological direct current electric field (dcEF) in a PMMA microfluidic device. These cell lines exhibited greatly different response in applied physiological dcEFs (2-6 V/cm). The electrotaxis of H460 cells was reflected in cathodal redirection. Besides cathodal direction, H1975 cell motility was obviously increased under applied dcEFs. Both HCC827 and H1299 cells showed increased motility and re-orientation perpendicular to EF during dcEFs. However, the anodal redirection of HCC827 was not so pronounced as that of H1299 cells. In addition, differentially expressed genes revealed that the involvement of MAPK/ERK, PI3K and Rho family in cancer cell electrotaxis and realignments. All these results suggested that cell responses under dcEFs were associated with the origin of cancer.
To further explore the contribution of cellular heterogeneity to electrotactic migration, in chapter 3, we isolated different functional populations (side population (SP) and major population (MP)) from lung cancer cells based on Hoechst 33342 efflux method. We demonstrated that SP cells displayed decreased sensitivity to clinical drugs, higher sphere formation ability in vitro and tumorigenity in vivo than major population (MP) cells, suggesting enrichment of cancer stem-like cells. Although the migration of SP cells was higher than that of MP cells in wound healing assay, SP cells could not be distinguished with MP cells on migration behavior after applying physiological dcEF stimulation. Both SP and MP cells displayed obviously migratory behavior towards the cathode, which was strength dependent. The increase of intracellular calcium ion ([Ca2+]i) was observed in these two subgroups under dcEF stimulation. Ca channel activity was further modulated pharmacologically using a series of specific calcium channel blockers. The results suggested that in addition to calcium influx through the activation of stretch-activated calcium channels (SACCs), EF-induced Ca- release would preferentially affect the SP cells with different signaling pathways rather than MP cells on tumor migration.
In chapter 4, we developed a polydimethylsiloxane (PDMS)-based microfluidic device for long-term electrotaxis study on a microscope. Three different electric field strengths could be generated in a single channel microfluidic chip. To further study the intrinsic differences in cells responsible for electrotactic heterogeneity, we developed another migration platform integrated cell immobilization structure, stable electric field generator and cell retrieve module in one microfluidic chip to sort different populations based on electrotactic phenotype. After migration, the high electrotactic and low electrotactic cells were harvested separately for further molecular analysis of their differences. The results suggested that the electrotaxis effects of cancer cells could play important roles in the understanding of cellular heterogeneity and its contribution in tumor metastasis. Our microfluidic devices will be useful for cell heterogeneity study in complex microenvironments.