Poster S12 - Mapping Metastatic States in Breast Cancer Cells Using DEPIS-based Out-of-Plane Inkjet-Printed Castellated Chip Arrays

Introduction: The metastatic dissemination of cancer cells, accounting for more than 90% of cancer-related mortalities, underscores the urgent need for innovative approaches in cancer diagnosis, prognosis, and therapy. Circulating tumor cells (CTCs) represent crucial players in this process, simultaneously serving as both propagators of the disease and potential biomarkers for early cancer detection [1]. Despite extensive investigations into CTC behaviour during metastasis, a significant gap persists in understanding the nuanced characteristics of CTCs across their metastatic potential [2]. In fact, accurately characterizing the diverse states of metastatic cells remains a challenge due to CTC's inherent heterogeneity. Dielectrophoretic Impedance Spectroscopy (DEPIS) emerges as a powerful technique combining dielectrophoretic (DEP) and electrochemical impedance spectroscopy (EIS). DEPIS leverages alterations in dielectric and impedance properties influenced by cellular morphology, membrane integrity, and intracellular structures to rapidly isolate and detect dissociated CTC populations from complex samples like blood [4]. Electrode-based devices utilizing castellated arrays, employed in conjunction with DEP for cell sorting, offer a range of significant advantages [5, 6], and are characterized by enhanced surface area and sharp edges, facilitating stronger and more localized dielectrophoretic forces on cells. Such architecture promotes a more uniform electric field distribution, ensuring consistent and predictable DEP forces, which will enhance the reliability and reproducibility of CTC capture and characterization. Consequently, this heightened sensitivity and precision in manipulating cells makes it particularly effective for sorting cells with subtle differences in their dielectric and dielectrophoretic properties, influenced by their metastatic state.

Materials and

Methods: In this study, we design and develop an in-house built out-of-plane inkjet-printed (IJP) castellated interdigitated electrode array tailored for DEPIS applications to map the dielectric properties of MDA-MB-231 breast cancer cell line across different metastatic states. The adapted cell-line model was realized via modulating the expression of connexin 43 (Cx43), a marker associated with poor prognosis and increased metastasis in breast cancer patients [7]. A numerical analysis was carried out using the finite element method (FEM) software ANSYS Maxwell to design the DEPIS castellated interdigitated electrode platform with a static workflow and simulate electrical fields and dielectrophoretic forces (FDEP) for particle trapping and sensing. The DEPIS devices were microfabricated with an inkjet-printer (DMP- 2850, Fujifilm Dimatix, USA) using silver nanoparticle ink, after which the device's performance and surface topography were characterized. For DEP-trapping and DEPIS experiments, the cells of interest were introduced into the wells atop the electrodes and were allowed to settle via gravity for at least 5 minutes before applying the AC (Alternate Current) signal. For DEPIS experiments, a Potentiostat/Galvanostat/ZRA system (Gamry Instruments, USA) was used to measure the DEPIS devices's impedance spectroscopy using the three-electrode method directly after cell trapping by positive DEP.

Results, Conclusions, and Discussions: The DEPIS microelectrode array design comprises 16 interdigitated fingers, with a 120 µm separation between each finger, and a 150 µm gap between individual castellations. ANSYS Maxwell simulations and electrical characterization revealed that the adapted design generates a high-magnitude electrical field at low voltage while minimizing the risk of Joule's heating during DEPIS applications [7,8]. In addition, our simulations show an increase in both electrical field intensity and FDEP as the electrode thickness increases. In fact, a thickness of 3 µm (achievable through inkjet printing) shows FDEP and field intensity increasing by 60% and 20%, respectively, compared to forces and intensities achieved by microfabricated arrays at 200 nm thickness.

After printing and characterizing the DEPIS electrodes electrically and topographically, we dripped the cells atop the electrodes and applied positive-DEP (pDEP). Upon trapping and characterizing the cells post-pDEP, we revealed a significant correlation between the various MDA-MB-231 metastatic subtypes and their respective dielectrophoretic and dielectric properties. Notably, cells with the highest metastatic potential exhibited the highest membrane capacitance (16.88 ± 3.24 mF/m²), followed by the less metastatic cell subtypes with membrane capacitances below 14.3 ± 2.54 mF/m². In addition, highly metastatic cells exhibited lower crossover frequency (25 ± 1 Hz) compared to the non-metastatic subtype (25±1Hz), an important characteristic for cell sorting and differentiating. In addition, our inkjet-printed electrode arrays successfully discerned various concentrations of DEP-trapped MDA-MB-231 cell subtypes (derived from the same cell lineage) via unique impedimetric spectra. Finally, applying EIS measurements post-DEP and fitting the data in an equivalent circuit model show distinct double layer capacitance (CDL) values at 1 kHz between the metastatic subgroups, confirming unique dielectric and dielectrophoretic properties correlated with the metastatic state of the cell. This can be attributed to differences across the metastatic spectrum in membrane composition, cellular morphology, cytoplasmic content, or intracellular signaling pathways [9].

Our study demonstrates the potential of DEPIS as a non-invasive and rapid analytical tool for characterizing the metastatic potential of captured CTCs in a blood sample. These findings provide insights into cancer biology and support the development of personalized therapeutic interventions tailored to distinct metastatic stages.

Acknowledgements (Optional): The authors acknowledge funding from Maroun Semaan research initiative and the University Research Board (URB).

References:
1. Fares, J., et al., Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduction and Targeted Therapy, 2020. 5(1): p. 28.
2. Hope, T.A. and S.E. Iles, Technology review: The use of electrical impedance scanning in the detection of breast cancer. Breast Cancer Research, 2003. 6(2): p. 69.
3. Crowell, L.L., et al., Electrical Impedance Spectroscopy for Monitoring Chemoresistance of Cancer Cells. LID - 10.3390/mi11090832 [doi] LID - 832. (2072-666X (Print)).
4. Turcan, I., et al., Dielectrophoretic and Electrical Impedance Differentiation of Cancerous Cells Based on Biophysical Phenotype. LID - 10.3390/bios11100401 [doi] LID - 401. (2079-6374 (Electronic)).
5. Challier, L., et al., Printed Dielectrophoretic Electrode-Based Continuous Flow Microfluidic Systems for Particles 3D-Trapping. Particle & Particle Systems Characterization, 2021. 38(2): p. 2000235.
6. Kazan, J.M., et al., Cx43 expression correlates with breast cancer metastasis in MDA-MB-231 cells in vitro, in a mouse xenograft model and in human breast cancer tissues. Cancers, 2019. 11(4): p. 460.
7. Pethig, R.R., Dielectrophoresis: Theory, methodology and biological applications. 2017: John Wiley & Sons.
8. Cottet, J., et al., MyDEP: a new computational tool for dielectric modeling of particles and cells. Biophysical journal, 2019. 116(1): p. 12-18.
9. Shim, S., et al., Dielectrophoresis has broad applicability to marker-free isolation of tumor cells from blood by microfluidic systems. Biomicrofluidics, 2013. 7(1).