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.

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#233;co spray P&#233;b&#233;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 &#34;Epinal&#34;</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&#8217;s Modified Eagle&#8217;s Medium + GlutaMAX&#8482;-I</td>
			<td>Gibco</td>
			<td>10567-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator COUVAD&#39;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&#252;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&#239;-Ta&#239;)</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&#252;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 &#181;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 &#181;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&#252;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&#339;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

2.6K Views.  Mines Saint-Etienne. 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.

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

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% ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Application of Hemostatic Devices in Laparoscopic Hepatectomy

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. Despite its non-invasive aspect, bleeding and bile leakage occur in liver parenchyma tissue resection during the operation or in the post-operation period, indicating the requirement for high-grade hemostatic devices, such as ultrasonic surgical aspiration, bipolar electrocoagulation, etc. The lack of availability of these high-grade hemostatic devices prevents laparoscopic hepatectomy from becoming a generalized procedure in basic medical organizations. In view of the situation mentioned above, a suite of simple and easy hemostatic devices is developed in this protocol, which includes a harmonic scalpel, monopole electrocoagulation, and a single lumen catheter, to innovatively perform liver parenchyma tissue resection. First of all, the porta hepatis or hepatic pedicle is occluded intermittently by a single lumen catheter, followed by clamping for 15 min and releasing for 5 min. Subsequently, using the harmonic scalpel, clamping and crushing of the liver are done to cut off the hepatic parenchyma tissue and to reveal the intrahepatic arteries, veins, and bile ducts. Lastly, the bleeding spots are coagulated by using monopole electrocoagulation at each spot. Intrahepatic pipeline structures are then visible by using these methods, which could stop bleeding easily, reduce the incidence rate of bile leakage, and improve the safety and feasibility of laparoscopic hepatectomy. Therefore, the simple and easy hemostatic devices shown here are suitable for conducting procedures in primary medical institutions.

High-grade hemostatic devices are essential for laparoscopic hepatectomy. However, these devices are not generalized in basic medical organizations. Therefore, a suite of simple and easy hemostatic devices is shown in this article, which can make the laparoscopic hepatectomy easier to perform.

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...