bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.948638; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice Hsieh-Fu Tsai,1 Camilo IJspeert,1 and Amy Q. Shen1, a) Micro/Bio/Nanofluidics Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa JAPAN (Dated: 18 February 2020) Transformed astrocytes in the most aggressive form cause glioblastoma, the most common cancer in central nervous system with high mortality. The physiological electric field by neuronal local field potentials and tissue polarity may guide the infiltration of glioblastoma cells through the electrotaxis process. However, microenvironments with multiplex gradients are difficult to create. In this work, we have developed a hybrid microfluidic platform to study glioblastoma electrotaxis in controlled microenvironments with high through- put quantitative analysis by a machine learning-powered single cell tracking software. By equalizing the hydrostatic pressure difference between inlets and outlets of the microchannel, uniform single cells can be seeded reliably inside the microdevice. The electrotaxis of two glioblastoma models, T98G and U-251MG, re- quire optimal laminin-containing extracellular matrix and exhibits opposite directional and electro-alignment tendencies. Calcium signaling is a key contributor in glioblastoma pathophysiology but its role in glioblas- toma electrotaxis is still an open question. Anodal T98G electrotaxis and cathodal U-251MG electrotaxis require the presence of extracellular calcium cations. U-251MG electrotaxis is dependent on the P/Q-type voltage-gated calcium channel (VGCC) and T98G is dependent on the R-type VGCC. U-251MG and T98G electrotaxis are also mediated by A-type (rapidly inactivating) voltage-gated potassium channels and acid- sensing sodium channels. The involvement of multiple ion channels suggests that the glioblastoma electrotaxis is complex and patient-specific ion channel expression can be critical to develop personalized therapeutics to fight against cancer metastasis. The hybrid microfluidic design and machine learning-powered single cell analysis provide a simple and flexible platform for quantitative investigation of complicated biological systems. I. INTRODUCTION electric fields in the brain may play an important role in mediating the glioma tumorigenesis and invasion15{19. Glioma is one of the most common types of brain Cells sense the electric field by bioelectrical activation of cancer and the aggressive form of it, glioblastoma, con- voltage-sensitive proteins, mechanosensing due to elec- tributes to poor prognosis, high mortality, and high prob- trokinetic phenomena, or activated chemical signaling ability of recurrence1,2, due to the infiltration nature of due to electrokinetically polarized membrane receptors the disease. The highly infiltrative ability of glioblas- (Supplementary FIG. S.1). The voltage gradient cre- toma originates from the invasive/migratory ability of ates a large voltage drop at cellular membrane which glioma stem cells or brain tumor initiating cells3,4. Not can directly activate voltage sensitive proteins such as only glioma cells are important, but also the microen- voltage-gated ion channels that are most commonly ex- vironment in the brain helps shaping the heterogeneity pressed on excitable membranes at neuronal synapses 20 of the glioma5. The glioma cells interact with the ex- and neuro-muscular junctions . Among the voltage- tracellular matrix (ECM), glial cells, and immune cells gated ion channels in the brain, calcium channels are in the brain and mediate the formation of peri-vascular, especially important as calcium influx plays a pivotal 21,22 peri-necrotic, and invasive tumor microenvironments6{9. role in cellular signaling . Calcium signaling is also Understanding the molecular mechanisms of the invasive- important in glioma cell proliferation, resistance to ther- 23{27 ness in glioma cells with respect to the tumor microenvi- apy, and metastasis . Whether or not the calcium ronment is vital for developing new therapeutic options signaling in glioma is mediated by electric field is still an and improving patient outcome10,11. open question. In the brain, glial cells are immersed in an electric field Conventional in vitro electrical stimulation systems created by tissue polarity from brain macrostructures as for studying cell responses in electrical microenvi- well as the local field potentials which are established ronments are bulky and the experimental through- from the action potentials fired by the neurons12. A weak put is limited28,29. To overcome these limitations, endogenous electric current has been shown to serve as a robust high-throughput in vitro platform that cre- a guidance cue for neuroblast migration from the sub- ates stable electrical stimulation of cells and inter- ventricular zone in mouse13, a region speculated as the faces with automated microscopy is a prerequisite for origin of glioma tumorigenesis14. Thus, the physiological rapid screening of targets and identifying molecular mechanisms. To this end, we have developed a hy- brid poly(methylmethacrylate)/poly(dimethylsiloxane) (PMMA/PDMS) microfluidic platform to reliably study a)[email protected] glioblastoma single cell migration under high-throughput bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.948638; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 dcEF stimulations that multiple antagonists can be tested simultaneously to identify molecular mecha- nisms. Quantitative single cell migration analysis is carried out by extracting cell migration metrics such as the directedness, orientation, or speed us- ing a robust machine learning-powered cell segmen- tation/tracking/analysis software with stain-free phase contrast microscopy30. Using the hybrid microfluidic platform, the role of voltage-gated calcium channels in calcium signaling pathways of glioblastoma electrotaxis are investigated. II. RESULTS A. Uniform single cell seeding by submerged manipulation in hybrid multiple electric field chip (HMEFC) To analyze single cell migration in microchannels, cells must be seeded sparsely and allowed to adhere and cul- FIG. 1. The results of U-251MG cell seeding inside mi- ture reliably31,32. However, it is known that uneven dis- crochannels by various methods. (a, d, g, j) In tip loading tribution of cells due to fluid flow, convection in suspen- method, cells are introduced by using gravitational flow with sion and vessel movement after seeding can cause aggre- micropipette tips. The cells can flocculate inside the tips and gation and differentiation of cells33. Different cell loading in microchannels as illustrated in (d). The microscopy image of seeded cells is shown in (g) and magnified in (j); (b, e, h, methods affecting cell distribution in single cell migration k) In tip injection method, cells are injected into the channels experiments are investigated, such as tip loading method, and tips are removed. The small hydrostatic pressure differ- tip injection method, and a pressure-balanced submerged ences between the inlet/outlet (shown as ∆h) will contribute cell seeding, as shown in FIG. 1. to hydrodynamic flow and disturb the cell distribution, caus- In tip loading method (FIG. 1.a), the cells flocculate ing non-uniform cell distribution and aggregates as shown in (e). The microscopy image of seeded cells is shown in (h) in the small pipet tip and cannot be dispersed uniformly and magnified in (k); (c, f, i, l) In our pressure-balanced sub- in the microchannel (FIG. 1.d, g, j). In tip injection merged cell seeding method, the hydrostatic pressure differ- method (FIG. 1.b), the cells are originally injected in the ence is eliminated. The injected cells remain uniform through- channels with uniform cell distribution. However, with- out the channel as shown in (f). The microscopy image of out balancing the microchannel inlet/outlet pressure, the seeded cells is shown in (i) and magnified in (l). The uniform minute hydrostatic pressure difference between inlets and and sparse cell seeding method is suitable for many different outlets generates a small pressure-driven flow that dis- applications from single cell tracking, ensembled cell studies places cells which lead to cell aggregates (FIG. 1.e, h, k). to cell assembly. The scale bars in (g, h, i) represent 500 µm. Furthermore, in tip injection method, due to the small The scale bars in (j, k, l) represent 200 µm. dimension of the punched holes at inlet/outlet interfaces, bubbles are easily trapped and may be introduced into microchannels, disrupting fluid advection and chemical B. Glioblastoma electrotaxis requires optimal transport. laminin-containing ECM By submerging inlets and outlets underwater using a reversibly bonded top reservoir and balancing the pres- An effective ECM coating on the substrate is essen- sure between inlets and outlets (FIG. 1.c), air bubbles can tial for cell adhesion and formation of focal adhesions be avoided and pressure-driven flow is prevented from