The work reported here was supported by the French National Research Agency (ANR-18-CE19-0029). The authors warmly thank S.M. Bardet for her contribution to the generation of a stable GCaMP6f cell line and D. O&#39;Connor for her help with the in ovo model.

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The authors declare that they have no competing financial interests.

The approach described in this work enables brain tumor models with an integrated PEF delivery system to study the effect of PEFs at different levels of biological organization. The microfabrication protocol consists of standard thin-film processes, which provide a large degree of freedom in electrode design that can be adapted to the specific application. Sometimes, an additional thermal annealing step can be useful at the end of the fabrication, to reduce bending of the electrodes that occurred during manufacturing.
The use of a stable glioblastoma cell line expressing a fluorescent calcium indicator avoids all complications linked with dye delivery and retention, especially in 3D tumors that are very dense16. Indeed, a high expression level is observed over a long period compared to standard chemical fluorescent calcium indicators17. This protocol can be applied to various cell lines, as it is commonly used for imaging neural activity11. Here, human and murine cell lines were used (U87 and Gl261 for implantation in immunodeficient or immunocompetent mice, respectively). Indeed, recent studies showed that the U87 cell line is different from that of the original cells as many mutations were acquired over years of cell culture, affecting experimental reproducibiliy18. The method used for the preparation of 3D tumors is high-throughput, reproducible, and allows the generation of spheroids of a specific size depending on the cell line, the number of cells at seeding, and the time of growth19. However, these spheroids are dense, which presents a disadvantage when imaging at the core of the tumor.
The in ovo model is useful as a first approach to study the effect of PEF on 3D tumors and their vasculature, without interactions with other cell types that are present in the brain. This model is inexpensive, fast, high-throughput, and raises fewer ethical issues than animal models. It is important to maintain the integrity of the embryo throughout the entire experiment, as it could affect its survival and the quality of the imaging. Special care must be taken while opening the quail egg, to avoid damages to the embryonic membrane. The graft and the placement of the flexible electrodes must also be performed carefully, to avoid bleeding that could kill the embryo. Injection of fluorescent dye in the blood vessels allows simultaneous visualization of the tumor cells and vascularization with fluorescence microscopy. The intraocular injection must be performed carefully to avoid dye leaking into the embryonic liquid, which could cause a residual fluorescence in the background that degrades the quality of the imaging. This model can also be used for following drug uptake, as it allows access to the circulatory system. However, experiments are limited by the 12-day survival time of the embryo, thus allowing 7 days of observation, which is significantly shorter than the in vivo model21.
The in vivo brain tumor&#160;model can be monitored for 4 to 5 weeks before animals reach an ethical experimental endpoint determined by a sudden 20% weight loss. It is well tolerated and remains in place if the connecting tail of the electrode is not too long. Otherwise, animals tend to scratch the flipping connector, which might ultimately be torn, hence preventing subsequent connection to the stimulator. This 4-week period is nevertheless valuable to cover the different stages of glioblastoma development. When comparing the tumor cell densities in the same volume of interest at different time intervals, the evolution of the tumor growth kinetics can be observed. In particular, enhanced tumor growth was observed at the time of the immune switch22. A similar study in the presence of a stimulating electrode would inform on the effect of PEF on tumor proliferation rate and tumor sensitivity to immune elimination. In comparison to in ovo model, the in vivo model can be seen as a valuable preclinical model to study the impact of immune cells on tumor progression and their contribution to the therapeutic effect of PEF. This protocol is adapted from a previous article with the addition of a flexible electrode device on the tumor before placing a cranial window14. Both the acute and chronic bioelectric treatments of tumors can be characterized by direct and subsequent observations with two-photon microscopy given that initial stimulation is expected to induce cell death and to trigger lasting dysregulation of the immune response.
The connections of the flexible probe are easily accessible under the two-photon microscope. Electrical stimulation parameters can thus be adjusted in real-time based on the observed effect on the neural tissue and/or the targeted cells, similar to how a medical doctor would perform interventional procedures while observing MRI or CT images of his patient. A final consideration is the importance of careful sealing of the electrode on the brain with superglue and silicone glue to prevent tissue regrowth.
In conclusion, the protocol described here represents an innovative model to study the effect of PEF therapy with flexible organic polymer electrodes for glioblastoma tumor models. The two models exhibit different levels of complexity such that cellular, vascular, or immune effects can be separated for a better understanding of the mechanisms of action. Conformal, superficial electrodes reduce the iatrogenic damage while enabling disruption of the tumor microenvironment, triggering vasoconstriction or dysregulation of intracellular calcium15.

Glioblastoma multiforme (GBM) is a highly invasive tumor and therefore difficult to eradicate with standard treatments such as resection, radiotherapy, and chemotherapy. Despite multimodal treatments, prognosis remains very poor and most of the patients experience disease progression within 1 year of diagnosis1,2. Recently, the development of bioelectric treatments has shown great potential to improve existing therapies. These therapies use the delivery of pulsed electric fields (PEF), typically in a single treatment session, to disrupt cellular membrane integrity and the microenvironment of tumors. This cell membrane disruption, also known as electroporation, can be reversible or irreversible depending on the electric field intensity and the number of pulses. Irreversible electroporation (IRE) is applied as a non-thermal tissue ablation technique in which electric pulses cause fatal damage to cellular membranes leading to cell death3. Reversible electroporation is applied in electrochemotherapy (ECT), an established technique that consists of the delivery of PEFs in combination with chemotherapy drugs to enhance drug uptake in cancer cells4. Moreover, recent studies demonstrated calcium electroporation as an alternative to ECT with high efficiency for cancer treatment, which is also inexpensive and induces fewer side effects5. Despite these promising advances, PEFs are generally applied using rigid, metallic electrodes which are known to cause damage to soft tissue6. The brain is particularly sensitive to such invasive devices where the mechanical mismatch induces inflammation and astroglial scarring7.
In this context, a flexible PEF delivery system in combination with 3D models of glioblastoma tumors is presented, from microfabrication to a murine model. Conformal electrodes are made with standard thin-film microfabrication processes, including the use of soft and biocompatible materials such as parylene-C, gold, and PEDOT:PSS8,9. An interdigitated electrode design is used to cover a large surface area while maintaining adequate transparency for imaging between the electrode fingers10. For the tumor model, 3D spheroids of glioblastoma cells expressing a genetically encoded fluorescence reporter are produced using a variation of the liquid-overlay 96-well plate method11. The spheroids are grafted into the chorioallantoic membrane of a quail embryo, resulting in an in ovo model that has been extensively used to study angiogenesis or drug toxicology12,13. Tumors can be grafted and vascularized by the embryo&#39;s vasculature in the absence of an immune system at this stage of embryonic development12. Flexible electrodes are then placed on top of the vascularized tumor to study the effect of PEF delivery on the spheroid and its vasculature. Finally, these effects are investigated on a complete living organism, including tumor microenvironment and immune system, by implanting engineered spheroids into the brain parenchyma of murine models14. Flexible electrodes are placed on top of the insertion site and the craniotomy is sealed with a glass window, allowing repeated two-photon imaging over several weeks.
These methods will be useful for people interested in various domains ranging from microelectronics engineering to oncology applications. The microfabrication protocol can be used and adapted for any application requiring thin-film metal electrodes coated with PEDOT:PSS. Further, the biological models developed for the evaluation of antitumor electrical treatments will be of general interest for the investigation of the differentiation of cellular, vascular, and immune response to implanted materials.

<table><tbody><tr><td>(3-Glycidyloxypropyl)trimethoxysilane</td><td>Sigma</td><td>440167</td><td>GOPS</td></tr><tr><td>0.25% Trypsin-EDTA (1X)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>4-Dodecylbenzenesulfonic acid</td><td>Sigma</td><td>44198</td><td>DBSA</td></tr><tr><td>96-well plate</td><td>Falcon</td><td>353075</td><td></td></tr><tr><td>Acetone</td><td>Technic</td><td>530</td><td></td></tr><tr><td>Acrylic resin</td><td>Fischer scientific</td><td>NC1455685</td><td></td></tr><tr><td>agarose</td><td>Sigma</td><td>A9539</td><td></td></tr><tr><td>autoclave</td><td>Tuttnauer</td><td>3150 EL</td><td></td></tr><tr><td>AZ 10XT</td><td>Microchemicals</td><td></td><td>Positive photoresist</td></tr><tr><td>AZ 826 MIF Developer</td><td>Merck</td><td>10056124960</td><td>Metal-ion-free developer for the negative photoresist</td></tr><tr><td>AZ Developer</td><td>Merck</td><td>10054224960</td><td>Metal-ion-free developer for the positive photoresist</td></tr><tr><td>AZ nLof 2070</td><td>Microchemicals</td><td></td><td>Negative photoresist</td></tr><tr><td>Buprenorphine</td><td>Axience</td><td></td><td></td></tr><tr><td>Carprofen</td><td>Rimadyl</td><td></td><td></td></tr><tr><td>Centrifuge Sorvall Legend X1R</td><td>Thermo Scientific</td><td>75004260</td><td></td></tr><tr><td>CMOS camera Prime 95B</td><td>Photometrics</td><td></td><td></td></tr><tr><td>CO2 incubator HERAcell 150i</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>DAC board</td><td>National Instruments</td><td>USB 6259</td><td></td></tr><tr><td>D&amp;eacute;co spray P&amp;eacute;b&amp;eacute;o</td><td>Cultura</td><td>3167860937307</td><td>Black acrylic paint</td></tr><tr><td>Dextran Texas Red 70.000</td><td>Thermofisher</td><td>D1830</td><td></td></tr><tr><td>Die bonding paste &quot;Epinal&quot;</td><td>Hitachi</td><td>EN-4900GC</td><td>Silver paste</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2438</td><td></td></tr><tr><td>Dispensing machine</td><td>Tianhao</td><td>TH-2004C</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium + GlutaMAX&amp;trade;-I</td><td>Gibco</td><td>10567-014</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma</td><td>D6429</td><td></td></tr><tr><td>Egg incubator COUVAD&#039;OR 160</td><td>lafermedemanon.com</td><td></td><td></td></tr><tr><td>Ethylene glycol</td><td>Carl Roth</td><td>6881.1</td><td></td></tr><tr><td>Fertilized eggs of Japanese quail</td><td>Japocaille</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>S181BH</td><td></td></tr><tr><td>Flask</td><td>Greiner</td><td>658170</td><td></td></tr><tr><td>Fluorescence macroscope</td><td>Leica MZFLIII</td><td></td><td></td></tr><tr><td>Gl261</td><td>DSMZ</td><td>ACC 802</td><td></td></tr><tr><td>Gold pellets - Dia 3 mm x 6 mm th</td><td>Neyco</td><td></td><td></td></tr><tr><td>Handheld automated cell counter</td><td>Millipore</td><td>PHCC00000</td><td></td></tr><tr><td>Heating and drying oven</td><td>Memmert</td><td>UF110</td><td></td></tr><tr><td>Hexadimethrine Bromide Sequa-brene</td><td>Sigma</td><td>S2667</td><td></td></tr><tr><td>hot plate Delta 6 HP 350</td><td>S&amp;uuml;ss Microtec</td><td></td><td></td></tr><tr><td>Illumination system pE-4000</td><td>CoolLed</td><td></td><td></td></tr><tr><td>Infrared tunable femtosecond laser (Ma&amp;iuml;-Ta&amp;iuml;)</td><td>Spectra Physics (USA)</td><td></td><td></td></tr><tr><td>Ionomycin calcium salt</td><td>Sigma</td><td>I3909</td><td></td></tr><tr><td>Kapton tape SCOTCH 92 33x19</td><td>3M</td><td></td><td>Polyimide protection tape</td></tr><tr><td>Lab made pulse generator</td><td></td><td></td><td></td></tr><tr><td>Labcoter 2 Parylene Deposition system PDS 2010</td><td>SCS</td><td></td><td></td></tr><tr><td>Lenti-X 293 T cell line</td><td>Takara Bio</td><td>63218</td><td>HEK 293T-derived cell line optimized for lentivirus production</td></tr><tr><td>Lenti-X GoStix Plus</td><td>Takara Bio</td><td>631280</td><td>Quantitative lentiviral titer test</td></tr><tr><td>Mask aligner MJB4</td><td>S&amp;uuml;ss Microtec</td><td></td><td></td></tr><tr><td>Micro-90 Concentrated cleaning solution</td><td>International Products</td><td>M9050-12</td><td></td></tr><tr><td>Microscope slides 76 x 52 x 1 mm</td><td>Marienfeld</td><td>1100420</td><td></td></tr><tr><td>Needles 30G</td><td>BD Microlance 3</td><td>304000</td><td></td></tr><tr><td>PalmSens4 potentiostat</td><td>PalmSens</td><td></td><td></td></tr><tr><td>parylene-c : dichloro-p-cyclophane</td><td>SCS</td><td>300073</td><td></td></tr><tr><td>PCB Processing Tanks</td><td>Mega Electronics</td><td>PA104</td><td></td></tr><tr><td>PEDOT:PSS Clevios PH 1000</td><td>Heraeus</td><td></td><td></td></tr><tr><td>penicillin / streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Petri dish</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>pGP-CMV-GCaMP6f</td><td>Addgene</td><td>40755</td><td>plasmid</td></tr><tr><td>Phosphate Buffer Saline solution</td><td>Thermofisher</td><td>D8537</td><td></td></tr><tr><td>Plasma treatment system PE-100</td><td>Plasma Etch</td><td></td><td></td></tr><tr><td>PlasmaLab 80 Reactive Ion Etcher</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Plastic mask</td><td>Selba</td><td></td><td></td></tr><tr><td>Plastic weigh boat 64 x 51 x 19 mm</td><td>VWR</td><td>10770-454</td><td></td></tr><tr><td>Poly-dimethylsiloxane: SYLGARD 184 Silicone Elastomer Kit</td><td>Dow chemicals</td><td>1673921</td><td></td></tr><tr><td>Polyimide copper film 60 &amp;micro;m (Kapton)</td><td>Goodfellow</td><td>IM301522</td><td></td></tr><tr><td>Propan-2-ol</td><td>Technic</td><td>574</td><td></td></tr><tr><td>Protolaser S</td><td>LPKF</td><td></td><td></td></tr><tr><td>puromycin</td><td>Gibco</td><td>A11103</td><td></td></tr><tr><td>Round cover glass 5 mm diameter</td><td>Fischer scientific</td><td>50-949-439</td><td></td></tr><tr><td>Scepter Sensors - 60 &amp;micro;m</td><td>Millipore</td><td>PHCC60050</td><td></td></tr><tr><td>Silicone adhesive Kwik-Sil</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>spin coater</td><td>S&amp;uuml;ss Microtec</td><td></td><td></td></tr><tr><td>Spin Coater</td><td>Laurell</td><td>WS-650</td><td></td></tr><tr><td>Super glue</td><td>Office depot</td><td></td><td></td></tr><tr><td>tetracycline-free f&amp;oelig;tal bovine Serum</td><td>Takara Bio</td><td>631105</td><td></td></tr><tr><td>Thermal evaporator Auto 500</td><td>Boc Edwards</td><td></td><td></td></tr><tr><td>Two-photon microscope</td><td>Zeiss LSM 7MP</td><td></td><td></td></tr><tr><td>U87-MG</td><td>ATCC</td><td>HTB-14</td><td>Human glioblastoma cells</td></tr><tr><td>Ultrasonic cleaner</td><td>VWR</td><td></td><td></td></tr><tr><td>Vortex VTX-3000L</td><td>LMS</td><td>VTX100323410</td><td></td></tr><tr><td>Xfect single shots reagent</td><td>Takara Bio</td><td>631447</td><td>Transfection reagent</td></tr></tbody></table>

flexible organic electronic devices for pulsed electric field therapy of glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This protocol allows the application to two glioblastoma models in which a flexible PEF delivery system is integrated. Following microfabrication and packaging steps, flexible electrodes are characterized in saline solution by electrochemical impedance spectroscopy (EIS) to assess and validate their performance. The PEDOT:PSS-coated electrodes show the typical capacitive and resistive dominated regions separated by a cut-off frequency, whereas the uncoated electrodes display only capacitive behavior (Figure 2).
A variation of the liquid-overlay 96-well plate method is used to grow 3D tumors made of transfected glioblastoma cells stably expressing a fluorescent intracellular calcium reporter. The growth of the spheroids can be observed with a bright-field microscope (Figure 3; ED 0). At least 2 or 3 days are needed to obtain spherical and dense spheroids, depending on the cell line and the number of cells seeded.
In the in ovo model, spheroids are grafted in the chorioallantoic membrane of a quail embryo (Figure 3; ED 6). The success of the graft can be assessed by fluorescence microscopy a few days later, as living cells have intracellular calcium, and are thus fluorescent (Figure 3; ED 9). The vascularization of the tumor can be observed under a fluorescence microscope by injecting a fluorescent dye into the blood vessels (Figure 3; ED9). However, it might not always be possible to visualize the blood vessels inside the tumor as the spheroid is very dense. The flexible interdigitated electrodes are placed on top of the vascularized tumor (Figure 3; ED 9) and connected to a pulse generator. The probe must be gently placed to avoid bleeding of the embryo; otherwise, the fluorescent dye can spread, which obstructs any observation by imaging. Correct delivery of the pulse to the biological environment can be verified by measuring the current going through the circuit. Imaging of these in ovo models allows real-time monitoring of the effect of PEFs on the intracellular calcium in a 3D glioblastoma tumor, as well as the vasoconstriction induced on the tumor&#39;s vasculature, avoiding any influence of other cell types including the immune system15.
The study of the PEF effect on glioblastoma can also be performed in a more complete and predictive model. Indeed, the in vivo model described above14 consists of grafting a 3D glioblastoma tumor in the brain parenchyma of a mouse (Figure 4). The injection site of the tumor is plugged by a cross-linked dextran gel hemi-bead, to recapitulate the physiological biophysical constraints during the growth of the tumor. Although described in reference14, it is worth re-emphasizing that it is critically important that the dextran hemi-bead be precisely superglued to the dura mater; otherwise, the tumor can escape through the open dura and completely cover the brain, making the imaging impossible. For any chronic imaging, tissue ingrowth that takes place as the cranial window heals poses a serious barrier, as the new tissue is non-transparent and makes images foggy or non-usable. Therefore, after inserting and gluing the hemi-bead, the sidewalls of the opened cranial window need to be sealed with a thin layer of superglue meticulously placed all around the cavity wall, without letting the superglue slip or flow onto the dura. When the flexible probe is placed on top of the tumor injection site, no bubbles can stay under the probe, for two reasons. Firstly, imaging cannot proceed when bubbles are present. Secondly, bubbles serve as insulators, thus changing the electrical stimulation properties. After taking the precautions described above, the craniotomy is sealed with a glass window cemented to the skull to allow chronic imaging over weeks. As the tumor consists of GCaMP or DsRed expressing cells, the injection can be confirmed with a fluorescence microscope. The electrochemical impedance of the electrodes must be measured to validate the performance after implantation. Compared to the impedance in saline solution, an increase of the impedance is expected in vivo at frequencies above 100 Hz due to the presence of a biological environment (Figure 5). Vascularized neural parenchyma and tumor infiltration can be observed and characterized through the transparent substrate over weeks by two-photon microscopy (Figure 6). The use of transgenic animals expressing fluorescent proteins in cells of interest (immune cells and neurons) can, for example, allow demonstration of the minimal inflammatory process induced by electrode implantation alone (Figure 6A) or show the presence of microglia and monocytes 26 days after implantation of a PEF stimulated electrode implanted on top of a growing GBM tumor (Figure 6B1). In the latter case, both peripheral-monocyte-derived cells and brain-resident microglial cells were found around and inside the tumor (Figure 6B2). On the day of PEF delivery, contact pads of the flexible electrodes can be connected to the pulse generator, directly under the two-photon microscope. Overall, this model can be used to investigate the effect of bioelectric treatments over time using various types of cells involved in brain tumor development, up to a depth of around 500 &#181;m.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63527/63527fig01.jpg" /> 
Figure 1: Microfabrication of flexible electrodes. (A) Gold electrode&#160;patterning and Parylene C substrate. (B) Outline opening. (C) PEDOT:PSS coating. (D) Connections and packaging.&#160;<a href="https://www.jove.com/files/ftp_upload/63527/63527fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63527/63527fig02.jpg" /> 
Figure 2: Electrochemical impedance spectroscopy of flexible gold electrodes and PEDOT:PSS coated cold electrodes in a saline solution. <a href="https://www.jove.com/files/ftp_upload/63527/63527fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63527/63527fig03.jpg" /> 
Figure 3: The in ovo model of glioblastoma. ED 0: Spheroid observed with a bright-field microscope. ED 3: Shell less culture of a quail embryo 3 days after opening. ED 6: Tumor implanted in the CAM observed with a bright-field microscope. ED 9: Flexible device placed on the vascularized tumor (tumor in green and blood vessels in red). <a href="https://www.jove.com/files/ftp_upload/63527/63527fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63527/63527fig04.jpg" /> 
Figure 4: The in vivo application. (A) Scheme for in vivo experiments. (B) Probe placement before the application of cover glass and acrylic resin. (C) Completed probe implantation. <a href="https://www.jove.com/files/ftp_upload/63527/63527fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63527/63527fig05.jpg" /> 
Figure 5: Electrochemical impedance spectroscopy of flexible gold electrodes in a saline solution compared to an implanted probe. <a href="https://www.jove.com/files/ftp_upload/63527/63527fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63527/63527fig06.jpg" /> 
Figure 6: Intravital multispectral two-photon imaging through electrodes. (A) Tiled image of the healthy brain surface in a control multifluorescent AMU-Neuroinflam mouse 3 days after electrode implantation. Cyan shows dendritic arborization of layer 5 pyramidal neurons, green shows recruited granulocytes and monocytes, and yellow shows activated microglia and dendritic cells. Pink shows infrared diffusion due to heat accumulation. (B1) Similar image as in A but 26 days after tumor spheroid implantation 200 &#181;m deep inside the cortex immediately followed by electrode implantation. Note the accumulation of green and yellow immune cells. (B2) Similar image as in B1 but 100 &#181;m below the surface of the electrodes. Note the presence of blue neuronal dendritic arborization in the periphery of the red tumor mass itself infiltrated by yellow microglia and dendritic cells. Deep blue shows a second harmonic signal from the peritumoral collagen. (B3) Zoomed view of B2 showing the presence of interneuron somas (indicated by arrows) in the vicinity of the tumor. <a href="https://www.jove.com/files/ftp_upload/63527/63527fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma at JoVE.com

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

flexible-organic-electronic-devices-for-pulsed-electric-field-therapy

Research

JoVE Journal

Cancer Research

2.7K Views.  Mines Saint-Etienne. Dit protocol maakt de levering van pulselektrische velden door flexibele elektronica mogelijk om hun therapeutische effecten op glioblastoom te bestuderen. Dit doen we door de micro-omgeving van de tumor in vivo te visualiseren met beeldvorming. De bio-elektronica integreert in dit protocol met behulp van verschillende modellen van toenemende complexiteit om het effect van pulserende velden op kanker te bestuderen.De microfabricagetechnieken die in dit werk worden gepresenteerd, kunnen...

Video: Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (&#400; &#62; 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Medicine

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Bioengineering

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity during sonication. Unfortunately, current clinical treatments for glioblastoma (GBM) are lacking, leading to low long-term survival rates among patients. SDT is a promising method for treating GBM in an effective, noninvasive, and tumor-specific manner. Sonosensitizers preferentially enter tumor cells compared to the surrounding brain parenchyma. The application of FUS in the presence of a sonosensitizing agent generates reactive oxidative species resulting in apoptosis. Although this therapy has been shown previously to be effective in preclinical studies, there is a lack of established standardized parameters. Standardized methods are necessary to optimize this therapeutic strategy for preclinical and clinical use. In this paper, we detail the protocol to perform SDT in a preclinical GBM rodent model using magnetic resonance-guided FUS (MRgFUS). MRgFUS is an important feature of this protocol, as it allows for specific targeting of a brain tumor without the need for invasive surgeries (e.g., craniotomy). The benchtop device used here can focus on a specific location in three dimensions by clicking on a target on an MRI image, making target selection a straightforward process. This protocol will provide researchers with a standardized preclinical method for MRgFUS SDT, with the added flexibility to change and optimize parameters for translational research.

Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound.

Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Neuroscience

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...

Flexible Multi-Region Neural Recording Device for Epilepsy Research

An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the propagation of epileptic signals through associated neural circuits. Therefore, in vivo recording of local field potential (LFP) from the critical brain regions is&#160;essential for deciphering the circuits involved in seizure propagation. However, current methods for electrode fabrication and implantation lack flexibility. Here, we present a handy device designed for electrophysiological recordings (LFPs and electroencephalography [EEG]) across multiple regions. Additionally, we seamlessly integrated optogenetic manipulation and calcium signaling recording with LFP recording. Robust after-discharges were observed in several separate regions during epileptic seizures, accompanied by increasing&#160;calcium signaling. The approach used in this study offers a convenient and flexible strategy for synchronous neural recordings across diverse regions of the brain. It holds the potential for advancing research on neurological disorders by providing insights into the neural profiles of multiple regions involved in these disorders.

Author Spotlight: Unraveling Neural Communication and Circuit Interactions in Health and Disease

We have developed a simplified and cost-effective approach for electrode fabrication and conducted recordings of signals across multiple regions in freely moving mice. Utilizing optogenetics, alongside multi-region electrophysiology and calcium signal recording, enabled the revelation of neuronal activities across regions in the seizure kindling model.

Epilepsy is a neurological disorder characterized by synchronized abnormal discharges involving multiple brain regions. Focal lesions facilitate the ...

Tumor Treating Fields Application in Human Brain Tumor Models: A Non-invasive Technique to Deliver Alternating Electric Fields to Human Brain for Treating Brain Tumor

Source: Omar, A. I. <a href="https://www.jove.com/v/51638/tumor-treating-field-therapy-combination-with-bevacizumab-for">Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma.</a> J. Vis. Exp. (2014)
This video demonstrates the application of Tumor Treating Fields or TTFields in human brain tumor models. This non-invasive technique delivers low-intensity alternating electric fields directly to the brain, resulting in inhibition of cell division and subsequent apoptosis.

Source: Omar, A. I. Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma. J. Vis. Exp. (2014)
This ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

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