SCIENCE ADVANCES | RESEARCH ARTICLE HEALTH AND MEDICINE Copyright © 2019 The Authors, some Reduction of intratumoral brain perfusion by rights reserved; exclusive licensee noninvasive transcranial electrical stimulation American Association for the Advancement of Science. No claim to G. Sprugnoli1*, L. Monti2*, L. Lippa3, F. Neri1, L. Mencarelli1, G. Ruffini4, R. Salvador4, G. Oliveri3, original U.S. Government B. Batani3, D. Momi1, A. Cerase2, A. Pascual-Leone5,6, A. Rossi1,7, S. Rossi1,7, E. Santarnecchi1,5† Works. Distributed under a Creative Malignant brain neoplasms have a poor prognosis despite aggressive treatments. Animal models and evidence Commons Attribution from human bodily tumors reveal that sustained reduction in tumor perfusion via electrical stimulation pro- NonCommercial motes tumor necrosis, therefore possibly representing a therapeutic option for patients with brain tumors. Here, License 4.0 (CC BY-NC). we demonstrate that transcranial electrical stimulation (tES) allows to safely and noninvasively reduce intratu- moral perfusion in humans. Selected patients with glioblastoma or metastasis underwent tES, while perfusion was assessed using magnetic resonance imaging. Multichannel tES was applied according to personalized bio- physical modeling, to maximize the induced electrical field over the solid tumor mass. All patients completed the study and tolerated the procedure without adverse effects, with tES selectively reducing the perfusion of the solid tumor. Results potentially open the door to noninvasive therapeutic interventions in brain tumors Downloaded from http://advances.sciencemag.org/ on March 26, 2021 based on stand-alone tES or its combination with other available therapies. INTRODUCTION traction technique, and noninvasive near-infrared spectroscopy) was Glioblastoma (GBM) is the most aggressive and frequent malignant reduced by more than 50%, starting after 15 min of DC stimulation brain tumor. GBM accounts for more than 60% of adult brain neo- and lasting for almost 24 hours. Moreover, reduction of perfusion was plasms and affects approximately 10 per 100,000 people worldwide followed by a decrease in tumor size 1 day after the intervention. The (1). The prevalence of brain metastases (MTX) is estimated to be at least decrease in blood flow caused a decrease in tumoral partial oxygen around 9%, with an incidence between 8.3 and 14.3 per 100,000 (2). pressure (pO2), supposedly promoting tumor necrosis (6). Several, MTX originate from primary tumors, with the most common ones be- not mutually exclusive, mechanisms for ET effects on tumors have ing the lung (19.9%), melanoma (6.9%), kidney (6.5%), breast (5.1%), been proposed: (i) pH alterations near the electrodes due to electrolysis and colorectal (1.8%) cancers (2). Despite aggressive treatment reaction, (ii) apoptosis, (iii) microembolism, (iv) immunomodula- combining neurosurgery, radiation therapy, and chemotherapy (2), tion, (v) toxic derivate of electrochemical reactions, and (vi) vaso- patients with GBM or MTX have a median postdiagnosis survival constriction (3, 6). window of about 12 months (1). Noninvasive application of ET to brain tumors requires technologi- Electrotherapy (ET) involves the application of a low-intensity direct cal developments. In the current study, we investigated the possibility of electric current into the tumor mass by means of two or more platinum affecting brain tumor perfusion via noninvasive electrical DC brain electrodes or indirectly, by electrodes placed in the surrounding tissue stimulation. To assess brain stimulation–induced cerebral blood flow (3). ET offers some promise in the treatment of bodily tumors and can (CBF) changes, we combined magnetic resonance imaging (MRI) be combined with chemotherapy (i.e., electrochemotherapy), leveraging and noninvasive brain stimulation in patients with GBM and MTX. its effect by acting on tumor transmembrane permeability and conse- In the last two decades, novel forms of noninvasive, transcranial electri- quently increasing drug concentration in tumor cells (4). Following the cal stimulations (tES) have been tested on healthy participants as well as first clinical application in patients with lung cancer, ET has been tested in patients with various neurological and psychiatric diseases, with the on other malignant visceral tumors, including hepatocellular carci- aim of modulating or restoring cognitive, motor, and psychological noma, advanced breast cancer, breast hemangioma, and pancreas car- functions (7, 8). In particular, literature on transcranial DC stimulation cinoma (3). In vivo results have confirmed in vitro findings, showing a (tDCS) suggests that low-intensity electrical stimulation (i.e., <2 mA) reduction of the tumor mass or even prevention of tumor recurrence, delivered transcranially affects cortical neurons’ membrane excitability, with minimal side effects after ET (3). In a pioneering experiment, Jarm with effects outlasting the stimulation period (7). However, only a few and colleagues (5) administered low-intensity DC stimulation (0.6 mA) studies have explored the application of tDCS to modulate brain perfu- for 1 hour to subcutaneous fibrosarcoma tumors in mice via two needle sion in humans (9, 10), with no attempts in patients with brain tumors. electrodes located 5 mm from the tumor’s margins. Tumor perfusion Given the ET findings in bodily tumors, we hypothesized that tDCS (assessed by tissue staining with patent blue violet dye, rubidium ex- would similarly decrease brain tumor perfusion. T1-weighted (T1w) and T2-weighted (T2w) MRI sequences were used to manually segment tu- 1 Brain Investigation and Neuromodulation Laboratory, Department of Medicine, mors in patients with GBM and MTX, tracing the solid tumor mass, its Surgery and Neuroscience, Unit of Neurology and Clinical Neurophysiology, Siena necrotic core, and the surrounding tissue affected by edema (Figs. 1 and 2). Medical School, Siena, Italy. 2Unit of Neuroimaging and Neurointervention, “Santa Biophysical modeling applied to individual anatomical models ob- Maria alle Scotte” Medical Center, Siena, Italy. 3Unit of Neurosurgery, “Santa Maria alle Scotte” Medical Center, Siena, Italy. 4Neuroelectrics, Barcelona, Spain. 5Berenson- tained from T1w images (11) was used to compare the electrical field Allen Center for Noninvasive Brain Stimulation, Beth Israel Medical Center, Harvard generated by a large set of tDCS electrode montages in each patient Medical School, Boston, MA, USA. 6Institut Guttmann, Universitat Autonoma Barcelona, (Fig. 2). Specifically, a genetic algorithm accounting for individual an- Barcelona, Spain. 7Department of Medicine, Surgery and Neuroscience, Human Physi- atomical variability and the conductivity of different tumor tissues was ology Section, Siena Medical School, Siena, Italy. *These authors contributed equally to this work. used to identify a stimulation solution inducing (i) the highest electric †Corresponding author. Email: esantarn@bidmc.harvard.edu field over the solid tumor while (ii) minimally affecting the rest of the Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 1 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on March 26, 2021 Fig. 1. Experimental design. (A) Patients underwent a clinical MRI to define and characterize the brain tumor, including standard and gadolinium-enhanced T1w [CET1w (contrast-enhanced T1w)], T2w, fluid-attenuated inversion recovery (FLAIR), ASL, and resting-state T2-BOLD (blood oxygenation level–dependent) MRI [rs-fMRI (resting-state functional MRI)] images. Regions of interest (ROIs) were defined by parcellating the solid component and the necrotic core of the tumor (MTX in this example) using CE T1w sequences and the edema surrounding the tumor by using FLAIR images. (B) Conductivity values were assigned to each ROI as well as to healthy brain tissue according to existing literature (bottom), and then the E-field distribution of current was calculated (top). (C) The personalized multielectrode solution maximizing the E-field on the solid-edema interface was selected. The experimental session was planned 3 to 5 days preceding the surgical intervention, and multichannel tES was performed inside the MRI scanner by means of an MRI-compatible brain stimulation device. The stimulation session included the acquisition of (i) T1w, FLAIR, ASL, and rs-fMRI sequences before tES; (ii) rs-fMRI and ASL during tES; and (iii) FLAIR images after tES. (D) Roughly 1 week after the presurgery MRI, patients underwent neurosurgery with subsequent histological classification and immediate postsurgery computerized tomography (CT) acquisition. (E) Last, approx- imately 1 month after neurosurgery, selected patients underwent a new MRI acquisition and ROI segmentation to perform a second MRI-tES session (F), aimed at evaluating the safety and feasibility of applying tES after neurosurgery. Additional modeling based on CT scan was performed to account for the effects of skull defects created by craniotomy (see also Fig. 3). This was crucial to ensure safety and to study/quantify changes in electric field distribution in the presence of skull defects. Note: All images are presented in neurological convention (right = right). brain (12). Patients underwent tDCS inside the MRI scanner, while (NVU) caused by gliomas in peritumoral areas [i.e., edema (15)] and sequences sensitive to CBF, i.e., arterial spin labeling (ASL), and the absence of healthy neuronal populations able to respond to electrical resting-state functional MRI (rs-fMRI) were acquired before and dur- stimulation in the tumor region, we hypothesized that tDCS would in- ing stimulation. ASL is a noninvasive technique to quantify CBF (i.e., duce a modulation of perfusion (i.e., CBF) with no changes in BOLD- perfusion) without the injection of contrasting agents, leveraging mag- related indices [i.e., regional homogeneity (ReHo) and amplitude of netically labeled arterial blood water protons as an endogenous tracer low-frequency fluctuations (ALFFs)] during stimulation. In selected (13). fMRI allows to indirectly quantify neuronal activity by detecting cases, a tDCS-MRI session after neurosurgery was also carried out, changes in the amount of deoxygenated hemoglobin [expressed by investigating safety and feasibility of tDCS in the presence of skull “blood oxygenation level–dependent” (BOLD) signal (14)]. rs-fMRI breaches. In those cases, computed tomography (CT) images were used was added as a control MRI acquisition to verify the selectivity of tDCS to carefully model the impact of craniotomy on the electrical field gen- effects on perfusion levels. Considering the neurovascular uncoupling erated in the cortex (Fig. 3). Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 2 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on March 26, 2021 Fig. 2. Tumor tracing, modeling, and optimization. (A) MRI images were manually segmented by two independent investigators. Following the Response Assess- ment in Neuro-Oncology recommendations, the solid part (red) of the tumor (GBM in this example) as well as the necrotic core (blue) were identified on CET1w images. The edema (green) of the tissue surrounding the tumor was segmented using FLAIR images. In the postsurgery phase, T2 Turbo Spin Echo (TSE) scans were used to correctly identify the vacuum created by the surgical intervention. ROIs were manually segmented on the corresponding anatomical scan using MRIcro software and 3D Slicer. ROIs were concentric, with the edema ROI usually including both solid and necrotic parts, while the solid tumor ROI included the necrotic one, such as currently done for planning of radiation therapy. (B) Top: Conductivity values were assigned to each ROIs as well as to healthy brain tissue according to existing literature: gray matter (GM) and white matter (WM), cerebrospinal fluid (CSF), skin, and skull. Bottom: A multielectrode solution maximizing stimulation over the edema-solid tumor interface was identified for each patient, with the corresponding E-field distribution calculated on patients’ head models. Resulting E-field was overlaid onto individual anatomical T1w scans, showing specificity of the tES solution targeting the solid tumor (C). (D) In detail, the T1w MRI of the participant was segmented into multiple tissue classes using MARS, SPM-8 segmentation toolbox, and FreeSurfer (left). Models of PITRODE electrodes (cylinders, 1-cm radius) were placed on scalp positions corresponding to the international 10/10 electroencephalography (EEG) system (green circles, center). A genetic algorithm [Stimweaver (12)] was then used to estimate the best multielectrode solution among those possible using 32 positions on the scalp (center), with the final personalized tES montage including up to eight electro- des placed over the hemisphere ipsilateral to the tumor (right). Note: All images are presented in neurological convention (right = right). RESULTS MRI before returning to their hospital ward, where standard of care Safety and clinical monitoring were guaranteed. Third, a questionnaire Patients did not experience any clinical or radiological complication investigating potential tES adverse effects was administered at the during or after tDCS in either pre- or postsurgical tDCS-MRI sessions. end of the tDCS-MRI session (7): Only four patients reported a tin- During postsurgery tDCS, a notably higher amount of stimulation was gling (consistent with common subjective experience during tES) at estimated to reach the brain/tumor because of current shunting via the the beginning of stimulation that progressively vanished and did not skull breaches (Fig. 3). By creating personalized stimulation models produce any discomfort. Last, routine standard-of-care MRI follow-ups accounting for shunting, the maximum value of the electric field mag- did not show any adverse effects of the intervention. As for patient nitude was checked against damage threshold levels based on animal survival, two patients (numbers 5 and 8) are currently alive, while the studies, equal to 61 V/m for a current duration of 20 min (7). For all remaining cohort died after a mean average of 10 months after the first patients, the maximum electric field (E-field) value never exceeded clinical MRI, in line with the average overall survival (OS) estimated in 1 of 13 of this threshold level, and no adverse effects were observed. previous studies (16). Patients were clinically and MRI monitored throughout the entire tDCS-MRI session. First, soon after tDCS, an additional sequence [i.e., Perfusion and fMRI changes fluid-attenuated inversion recovery (FLAIR)] was performed to rule Cerebral blood flow out intracranial complications such as enlargement of the peritumoral A pattern of decreased perfusion induced by tDCS was observed when edema. Qualitative evaluation of pre- and post-tES FLAIR images did absolute CBF values were corrected for the CBF of contralateral healthy not reveal any significant changes in the edema extension (Fig. 4). white matter (WM) (Fig. 5A; see Materials and Methods). In line with Second, patients were monitored for about 1 hour after the tDCS- the predicted effect, perfusion in the solid tumor displayed a significant Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 3 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on March 26, 2021 Fig. 3. Postsurgical modeling. (A) Structural MRI and CT images were used to model the impact of tES after surgery (shown: complete resection of a GBM). Ad hoc ROIs and three-dimensional (3D) renderings were created for both skull breaches and metallic clips that could respectively influence current shunting and affect electrode positioning. (B) New tissue conductivity values were derived by assigning skull defects a conductivity equal to that of the CSF (left), and the amount of current (i.e., E-field) shunting through them was estimated (right). (C) A new multielectrode stimulation solution was implemented to maximize stimulation over the resection borders, showing high spatial specificity and control of induced E-field. (D) In detail, new geometries of the different head tissues (healthy ones and ROIs) were computed after surgery, leading to a new optimized montage maximizing the current on the surgical borders. Note: All images are presented in neurological convention (right = right). CBF reduction of −36% (before = 1.784; during = 1.312; t = 4.653, sence of healthy neuronal populations able to respond to tDCS (e.g., P = 0.001), ranging from −26% in patients with GBM to −45% in pa- solid and necrotic parts). tients with MTX. CBF changes in the necrotic tumor core were not sig- nificant (−8.9%, before = 1.272; during = 1.168; t = 1.124, P = 0.294, ranging from −15% in patients with GBM to −6% in patients with DISCUSSION MTX). Similarly, CBF changes were not significant in the area of peri- Traditional applications of tES are aimed at modifying cortical excitabil- tumoral edema (−5%, before = 1.365; during = 1.299; t = 1.035, P = 0.328, ity in both the healthy and pathological brains (7). Few studies have in- with −8 and −4% in patients with GBM and MTX, respectively). vestigated the possibility of using tES to modify brain perfusion, and Control regions none to date has reported the impact of any tES application in patients No significant changes in CBF were observed in any of the control with brain tumors. Here, we show that multichannel tDCS in patients regions, with a small increase in CBF both ipsilateral (+5.7%, with malignant primary or metastatic brain tumors is safe and feasible before = 0.964; during = 1.019; t = −1.375, P = 0.194) and contralateral both before and after surgery and also leads to significant tissue-specific (+5.4%, before = 1.064; during = 1.122; t = −0.692, P = 0.502) to the reduction of intratumoral perfusion. tumor (Fig. 5B). Notably, these results are in line with the reported effects of ET on rs-fMRI changes bodily tumors, where a strong reduction in tumor perfusion—followed No significant changes were observed with fMRI, neither for ReHo by a decrease in volume and necrosis—has been shown (3, 5, 6). Given (necrotic core, before = 0.797; during = 0.778; t = 0.663, P = 0.526; solid the safety profile of tDCS, the feasibility of its application in both hos- tumor, before = 0.809; during = 0.784; t = 1.585, P = 0.147; edema, pital and home settings, its relatively low cost, and the possibility of its before = 0.825; during = 0.814; t = 1.729, P = 0.118) nor for fractional combination with other drug-based therapies, the present findings ALFF (fALFF) (necrotic core, before = 0.258; during = 0.274; t = −1.642, might lead to additional noninvasive therapeutic options for patients P = 0.139; solid tumor, before = 0.260; during = 0.270; t = −1.451, P = 0.213; with brain tumors. edema, before = 0.268; during = 0.272; t = −1.141, P = 0.283) (fig. S1), cor- Patients with GBM and MTX were selected, considering their high roborating on the one hand the hypothesized lack of neuronal response prevalence and poor prognosis (1, 2, 17). Gliomas’ perfusion is positively to tES due to NVU (e.g., edema regions) and on the other hand the ab- related with the World Health Organization grade and negatively Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 4 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on March 26, 2021 Fig. 4. Safety. FLAIR images acquired before and immediately after tES did not show significant changes in the edema, as shown for two representative patients with GBM at the presurgery (A) as well as postsurgery stimulation sessions (B). Note: Image constrast was manually adjusted to highlight edema borders. All images are presented in neurological convention (right = right). correlated with survival (18, 19). In particular, for GBM, neoangio- malignant cells. For the newly diagnosed GBMs, the interim analysis of genesis with increase in vascularization and perfusion are the most a randomized phase 3 trial showed that TTF combined with standard distinctive histopathological features, related to extreme invasive- chemotherapy (temozolomide) increased the PFS and the OS by about ness and aggressive growth (20). Moreover, a pattern of hyperperfu- 3 months (24). However, for recurrent GBMs, the phase 3 trial did sion in GBM strongly correlates with the Ki67 index, a well-defined not show any significant improvement of PFS or OS (25). Despite the marker of cell proliferation (21). Last, CBF and cerebral blood volume fact that both TTFs and tES are based on electrical stimulation, funda- represent validated and reliable markers of tumor progression (22), and mental technical and conceptual differences exist: (i) TTFs use alternating CBF increase detected with ASL is a predictor of a shorter progression- currents oscillating at extraphysiological frequencies—differently from free survival (PFS) and OS (19). Therefore, the reported tES-induced the DC stimulation used in the present tDCS approach—to impair the reduction in tumor perfusion suggests that repeated applications of proliferation of malignant cells by reducing mitosis; (ii) tES has a good tES might have a potential disease-modifying impact. Moreover, the in- safety profile (7), with no adverse effects other than occasional scalp dividually tailored brain stimulation solution based on personalized bio- itching and redness, while a variable but considerable percentage of physical modeling fits the precision medicine framework, promoting patients receiving TTFs (ranging from 19 to 43%) report dermatolog- the value of individualized treatments. ical adverse effects such as dermatitis, erosions, ulcers, and infections (26); From a molecular point of view, vascular endothelial growth factor (iii) for TTFs to be effective, patients are required to shave their head and A (VEGF-A) seems to be the principal mediator of the pathological wear the TTF devices more than 18 hours/day during the treatment neoangiogenesis in GBM, correlating also with clinical outcomes (23). cycle (4 weeks), whereas tES offers a light and highly portable alternative Considering the importance of inhibiting this specific GBM feature, that usually produces neurophysiological effects (e.g., modulation of antibodies targeting the VEGF pathway [i.e., bevacizumab (BEV)] were corticospinal excitability and cognitive enhancement) even with single, developed and tested. However, controlled phase 3 trials failed to show relatively short (i.e., 30 min) sessions. Future studies should compare a significant effect of BEV on OS in primary GBM (23). Other new the two techniques, which could be used as complementary interven- therapies for GBM include the Optune device (NovoTTF-100A system; tions possibly leading to additive effects. Novocure Ltd., Haifa, Israel), recently cleared by the Food and Drug The postulated mechanism for the reduction of tumor perfusion caused Administration for the treatment of newly diagnosed and recurrent by ET on bodily lesions (5, 6) seems compatible with results obtained GBM. This noninvasive system delivers low-intensity alternating current with tES applications in brain tumors. Occlusion and vasoconstriction of transcranially to the tumor [tumor-treating fields (TTFs), 200 kHz; field the vessels in proximity to the electrodes have been considered the prin- intensity, >0.7 V/cm at the center of the brain] to inhibit the mitosis of cipal effects of ET, caused by electrolysis occurring around both the Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 5 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on March 26, 2021 Fig. 5. Perfusion changes. (A) Significant reduction in WM-corrected CBF (wmCBF = normalized CBF) was observed in the solid tumor during stimulation for both patients with GBM and MTX (−26 and − 45%, respectively; mean decrease = −36%, *P < 0.001), as compared with no changes in the edema (P < 0.328) and necrotic core (P < 0.294). absCBF, absolute CBF. (B) Control ROIs in the contra- and ipsilateral hemispheres to each tumor did not show significant changes in CBF. Note: y axis = normalized CBF calculated as: absolute CBF (ml/100 g per minute)/contralateral WM’s CBF (ml/100 g per minute). cathode and the anode (5, 6). However, how ET selectively affects further explored to disentangle the pathophysiological mechanism tumor vessels, leaving the physiological reactivity of vessels of the underlining tES-induced CBF reduction. healthy surrounding tissue unaltered, is not clear. It is possible that Studies on ET on bodily tumors have shown that the smaller the the altered micro- and macrostructure characterizing tumor vessels tumor, the greater the resulting decrease in perfusion. In line with these might make them more susceptible to the effects of electric stimulation. results, we observed the most important CBF decrease in patients with These include, for example, lack of smooth muscle cells in the walls, MTX, representing the smallest lesions in our sample (CBF decrease: −45% impaired shunts, and higher intercapillary distance in the context of an in MTX versus −26% in GBM). On the other hand, the different histologic overall irregular microvascular architecture (3, 27). In addition, the high and vascular features of MTX (30) could explain their higher responsive- conductivity of tumors as compared with healthy brain tissues (28), ness to stimulation. However, this hypothesis needs to be tested on a large partly due to the abundance of vessels (27), might result in a greater sample of patients, also considering different types of brain MTX (i.e., current density inside the tumor. Other mechanisms, such as a possible colon, breast, and melanoma) and their locations in the cortical mantle, tES effect on nitric oxide (NO) synthase, cannot be excluded, consid- which can influence the amount of current delivered in the tumor. ering that electrical stimulation has been shown to modify (i.e., increase) The present tES protocol only included a single session of 20 min of NO synthase activity in animal models with consequent increase in tu- stimulation at a total intensity across all electrodes of <4 mA, in line with mor blood flow (29). In vitro and in vivo brain tumor models need to be current safety guidelines (7). Stimulation parameters were defined on Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 6 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE the basis of animal studies showing the peak in CBF decrease at around with tES in our patients, opens the possibility of integrating tES with 20′ of continuous ET, while extravasated red blood cells were observed chemotherapy in conditions where surgery represents the primary ther- within the lesions—and in many cases, also in the surrounding healthy apeutic option, as well as when chemotherapy is the only viable solution. tissues—after 1 hour of stimulation (6). This phenomenon has been re- lated to leakage of tumor vessels induced by electrolysis and impaired vessel permeability. In addition, the application of single electric pulse to MATERIALS AND METHODS the same tumor model has been shown to lead to a slower recovery to Sample baseline perfusion level after repeated stimulation (31), in line with a During a 9-month period, 50 patients with brain neoplasms, referred to similar finding in healthy volunteers receiving multiple tES sessions the Neurosurgery and Neuroradiology Units of the “Santa Maria alle during ASL imaging (9). Scotte” Medical Center of Siena (Italy), agreed to participate in this The analysis of variation in perfusion levels in ipsilateral and contra- study. Only 18 were considered eligible for the study, which had been lateral regions of interest (ROIs) confirmed the selectivity of tES effects previously approved by Local Ethical Committee. Participants were and the accuracy of personalized modeling of induced electric fields. A screened before the experiment by a multidisciplinary team, including trend for CBF increase was noticed in the healthy brain tissue in our neurologists, radiologists, and neurosurgeons. Inclusion criteria were as patients (as opposed to the decrease observed in the tumor’s ROIs follows: (i) age between 18 and 80 years, (ii) valid written informed con- and compatible with spontaneous brain perfusion fluctuations), sug- sent, (iii) strong clinical and radiological suspicion of primary GBM or gesting that tES effects were limited to the regions predicted by the bio- MTX, and (iv) lesion site presumably reachable by noninvasive stimu- physical modeling. lation (i.e., cortico-subcortical location). The exclusion criteria were as Downloaded from http://advances.sciencemag.org/ on March 26, 2021 BOLD imaging sequences (see fig. S1) were included to verify tES follows: (i) lesions in deep WM, brain stem, cerebellum, or basal ganglia; selectivity on perfusion, given the altered neurovascular response (ii) multiple brain lesions; (iii) concomitant radiochemotherapy due to (NVU) observed in the tumoral as well as the surrounding healthy brain the effect of radiotherapy on tumor perfusion that could alter the assess- tissue (15). BOLD-related indices of amplitude and coherence of neu- ment of tES effects; (iv) severe dilatation of ventricles; and (v) any health rovascular activity (fALFF and ReHo, respectively) did not reveal any problem or other potential contraindications to undergo the MRI and significant change during tES in any of the three tumor-related ROIs. experimental procedures. Patients were carefully informed about the fALFF, measuring the amplitude of spontaneous low-frequency fluctua- safety of tES as well as about commonly reported adverse/side effects tions of BOLD signal, is considered an index of regional spontaneous to ensure informed consent. neuronal activity (SNA), and it had been explored on a variety of psy- After the initial MRI session (Fig. 1A), manual segmentation of the chiatric and neurological conditions (32). ReHo is thought to reflect brain tumor was performed in preparation for the tDCS-MRI session coherence of local brain activity and has been associated to abnormal (Fig. 1B). Three patients developed health complications before the activities in many clinical conditions as well (33). Recently, ReHo and experimental intervention (thrombocytopenia, status epilepticus, and ALFF have been explored in relation to the NVU phenomenon in both severe intracranial hypertension) and were excluded. Rapid worsening patients with brain tumors (34, 35) and in a murine brain tumor model of clinical conditions led to urgent neurosurgical intervention (2 to 3 days (15), with results in line with our findings: The lack of changes in ReHo after clinical MRI) in seven patients who were therefore excluded. Stim- and fALFF measured in the edema ROI can be related to previously ulation sessions were conducted in eight patients (mean age, 62.5; SD, reported peritumoral NVU observed in gliomas, caused by the tumor 13 years; five males and three females; see Table 1): Six were diagnosed neoangiogenesis interfering with normal surrounding vasculature (15). with primary GBM and two with MTX from lung cancer. Participants On the other hand, we expected no variation in the tumor regions (i.e., had concomitant neurological conditions: seizures (n = 2), focal neuro- solid tumor and necrotic core) considering the lack of healthy neuronal logical deficits (n = 3), symptoms and signs of endocranial hypertension population modifications responding to tES. (n = 2); cognitive impairment (n = 1). Some patients received cortico- The study has some noteworthy limitations. Because of the main steroids, levetiracetam, and heparin (n = 3) during the study. Tumors goal of establishing safety and feasibility, the sample was small, and were located in different brain regions: frontal lobe (n = 4; two left- we included only two patients with MTX originating both from lung sided), parietal lobe (n = 2; one left-sided), and temporal lobe (n = 2; cancer, thus preventing generalization of findings. This also limited one left-sided). Histological analyses of patients with MTX classified the possibility to control for possible markers of prediction to tES re- their lesions as MTX of adenocarcinoma (immunohistochemical patterns: sponse and to investigate potential correlations between changes in per- patient 2: TTF1+, CK7+, CKPOOL±, P15+, GATA3−; patient 7: CK7+; fusion and clinical outcome. In addition, future investigations on TTF1+; p63−; CDX2−; CK20−). For histological data of patients with preclinical models are needed to address the molecular changes under- GBM, please refer to table S1. lining the observed modifications in perfusion levels. In summary, the present findings support the safety and feasibility of Tumor segmentation tES in patients with brain tumors, both before and after brain surgery. The ROIs for each patient were created on the basis of the presurgical Electrical stimulation on bodily tumors seems to enhance the effect of MRI assessment (Fig. 1), independently by two investigators (G.S. and chemotherapy treatment (27) by increasing the exposure of malignant L.Mo.). Following the Response Assessment in Neuro-Oncology recom- cells to chemotherapy, as a consequence of reduced blood flow caused mendations, the solid part of the tumor and the necrotic core were iden- by vessels’ vasoconstriction. In addition, a recent study (36) has showed tified on the contrast-enhanced T1w images. One patient with MTX did that tES applied on endothelial monolayers [mimicking the healthy not show manually identifiable necrosis inside the tumor. The edema in human blood-brain barrier (BBB)] can induce a transient, reversible, the tissue surrounding the tumor was segmented using FLAIR images. and polarity-dependent increment in water and solute flux. The possi- In the postsurgery phase, T2 Turbo Spin Echo (TSE) scans were used to bility to increase transport of molecules across the BBB (e.g., relevant for correctly identify the vacuum created by the surgical intervention. ROIs chemotherapy), along with the reduction of tumor perfusion observed were manually segmented on the corresponding anatomical scan using Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 7 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Table 1. Patients’ data. F, female; L, left; M, male; R, right. Patient Age Gender Lesion Location (lobe, side) Clinical presentation tES session Pharmacological treatment 1 46 M GBM Frontal, L Cognitive deficit Pre- and postsurgery Corticosteroids, levetiracetam 2 79 F MTX (lung) Temporal, R Seizures Presurgery Corticosteroids, levetiracetam 3 67 M GBM Frontoparietal, R Focal neurological deficit Presurgery Corticosteroids, levetiracetam 4 77 F MTX (lung) Frontal, R Seizures Presurgery Corticosteroids, levetiracetam 5 54 M GBM Frontal, R Endocranial hypertension Presurgery Corticosteroids, levetiracetam, heparin 6 45 F GBM Temporal, L Endocranial hypertension Presurgery Corticosteroids, levetiracetam, heparin 7 67 M GBM Frontal, L Focal neurological deficit Pre- and postsurgery Corticosteroids, levetiracetam, heparin 8 65 M GBM Parietal, L Focal neurological deficit Postsurgery Corticosteroids, levetiracetam Downloaded from http://advances.sciencemag.org/ on March 26, 2021 MRIcro software (www.mccauslandcenter.sc.edu/crnl/mricro) and 3D (0.138 S/m) and two-thirds higher in the edema nodes (0.458 S/m; Slicer (www.slicer.org/). As presented in Figs. 1 to 3, ROIs were concen- see Figs. 2 and 3 for examples), according to prior similar modeling tric, with the edema ROI usually including both solid and necrotic parts, work (38). In three participants, a reevaluation was performed after while the solid tumor ROI included the necrotic core, reflecting what is neurosurgery. Skull defects created by the surgical intervention were currently done during radiotherapy planning (37). For the postsurgery manually segmented on T1w images with the guide of the skull CT stimulations, CT scans of the skull were used to map the skull defects rendering. Head model regions representing skull defects were as- caused by the craniotomy on the T1w images used for biophysical signed a conductivity equal to that of CSF (Fig. 3). modeling to model the potential shunting of electrical current through the skull defects. T1w images were also used to create a ROI of the me- Stimulation montage optimization tallic clips on the scalp to optimize a tDCS solution with no electrodes tES is usually performed by means of two relatively big (area = 35 cm2) close to the postsurgical scar (Fig. 3). Resulting ROIs were compared, rectangular scalp electrodes (7). However, these so-called “bifocal” and an agreement about the final set of masks to be used for biophysical montages do not allow for fine targeting of specific cortical regions be- modeling and neuroradiological analysis was reached by consensus cause of low spatial resolution of the resulting E-field. In this study, we (G.S. and L.Mo.). optimized a solution based on multiple (up to eight) smaller (area = p cm2) circular electrodes that, when guided by neuroimaging data, Individual head model generation can be systematically placed on the scalp to maximize the induced Individual head models were performed for each participant by using a field in the brain over rather small structures, e.g., a brain tumor (12, 39). combination of freely available software tools and custom MATLAB To achieve such resolution, structural MRI images (T1w) of each pa- scripts (MathWorks, Natick, MA, USA; www.mathworks.com), follow- tient were preprocessed using the ad hoc pipeline described above. ing a pipeline derived from (11). T1w MRI images of each participant Cortical GM surfaces were obtained and then intersected with the ede- were segmented into scalp, skull, air, cerebrospinal fluid (CSF), gray ma as well as solid and necrotic tumor masks, defining a target region matter (GM), and WM masks using a MARS toolbox and an SPM 8 aimed at maximizing the normal component of the electric field (En) (www.fil.ion.ucl.ac.uk/spm/) segmentation toolbox (Figs. 2 and 3). over the intersection of the edema and solid tumor mask (Fig. 2). This Models of PITRODE PISTIM 3.14-cm2 Ag/AgCl electrodes (Neuro- target region was then used as input for a genetic algorithm [Stimweaver electrics, Barcelona, Spain) were placed in the scalp positions correspond- algorithm (12)] comparing multiple multielectrode montages com- ing to the international 10/10 electroencephalography (EEG) system. posed by up to eight stimulating electrodes located on any of the 32 Finite-element meshes of the head and electrodes were created using positions of the international 10/10 scalp EEG system. Solutions were Iso2Mesh, a MATLAB toolbox. The meshes were then imported to found using constrained least-squares minimization to determine the Comsol (Comsol Inc., Burlington, MA, USA; www.comsol.com), where electrode positions and currents that induced a weighted En-field that the E-field calculations were performed. The manually segmented best approximated the weighted target (i.e., the solid tumor). The masks of the edema, necrotic core, and solid tumor were used to define desired En-field was set to 0.25 V/m (the positive value indicates that the mesh nodes that belong to each of these tissues and used to change the target En-field is directed into the cortical surface, thus presumed the electrical conductivity of the tissues to match that of the lesion. All to have an excitatory effect) in the target area. Stimulation was per- healthy brain tissue nodes were assigned electrical conductivity values formed using PITRODE PISTIM electrodes (cylindrical 1-cm radius, appropriate to DC low-frequency electrical currents: 0.330 S/m (scalp), p cm2 area, Ag/AgCl/gel electrode; www.neuroelectrics.com), with a 0.008 S/m (skull), 10−5 S/m (air), 1.790 S/m (CSF), 0.400 S/m (GM), and maximal current of 2.0 mA at any electrode and a maximal total injected 0.150 S/m (WM). The conductivity of the necrotic core nodes was set to current of 4.0 mA across a maximal number of eight electrodes (Fig. 2). that of CSF. The conductivities of the solid tumor and edema nodes Stimulation was applied for 20 min in a single session inside the MRI were defined on the basis of the average WM and GM conductivity scanner. These parameters are within the recommended safety param- values: half of this average conductivity in the solid tumor nodes eters for tES in humans (7). Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 8 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE MRI-tES session Each tag and control pair was subtracted to create 45 perfusion- Before starting the MRI-tES session, patients were comfortably seated weighted images. These images were used to create quantified maps in a chair, and scalp was gently cleaned with an alcohol solution to im- of CBF using the software ASLtbx. Specifically, CBF was quantified prove skin conductivity under the corresponding electrode. An MRI- as ml/100 g per minute (absolute CBF) using the equation recom- compatible brain stimulator was used for the tES session (Starstim 8 mended by the ASL white paper (13) system, with a multichannel MRI kit, Neuroelectrics, Barcelona, Spain), and participants wore the device throughout the entire MRI session. f ¼ DMlR1a2MexpðwR 0a 1a Þ ½1  expðtR1a Þ1 Conductive rubber electrodes were inserted in circular sponge sockets with a radius of 1 cm, having the same area (3.14 cm2) as the PITRODE where f is the CBF, DM is the difference signal between the control PISTIM used for the head model generation. The sockets were dipped and label acquisitions, R1a is the longitudinal relaxation rate of blood, t into a saline solution to ensure electric conductivity. Participants were is the labeling time, w is the postlabeling delay time, a is the labeling then brought into the MRI scanner, and the electrodes were connected efficiency, l is the blood/tissue water partition coefficient, and M0 is ap- to the MRI-compatible cables. Electrode impedance was checked before proximated by the control image intensity. Four-dimensional CBF launching the first MRI sequence; this also served to ensure that parti- images were masked to remove out-of-brain voxels and normalized cipants were familiarized with the tES-induced scalp sensations (e.g., to the Montreal Neurological Institute (MNI) template in SPM12. tingling) before the MRI session. Particular care was taken to minimize CBF images were processed using partial volume correction in the na- head motion via cushions and custom-made padding. Participants were tive ASL spaces and subsequently normalized to MNI space using a provided with earplugs and were instructed to try to relax and not to linear affine transformation. Downloaded from http://advances.sciencemag.org/ on March 26, 2021 move during the MRI acquisition time. CBF-positive values were extracted from the voxels of the three con- centric ROIs (necrotic core, solid tumor, and edema). The edema and MRI acquisition solid tumor ROIs were obtained by subtraction (i.e., edema − solid MRI scans were performed on a Siemens Avanto Syngo VB17 scanner part = real edema defined as “edema” in the manuscript; solid part − with a 12-channel head coil (Siemens Medical Solutions, Erlangen, necrotic core = real solid, defined as “solid tumor”), while the necrotic Germany). Pulsed arterial spin-labeled images were acquired using a ROI was kept the same (“necrotic core”). To verify the selectivity and PICORE Q2T sequence [TR (repetition time) = 3100 ms, TE (echo specificity of the tES effect, we extracted the CBF absolute values from time) = 22 ms, TI (inversion time) 1 = 800 ms, TI2 = 1600 ms, number additional ROIs that did not show any sign of tumor invasion at the of slices = 16, thickness = 6.3 mm, gap = 7.8 mm, imaging matrix = 84 × 84, MRI scans in both the contralateral and ipsilateral hemisphere with re- flip angle = 90°, acquisition duration = 4 min and 45 s]. High-resolution spect to the lesion (“control ROIs”). Last, following recent guidelines for T1w axial images covering the whole brain were acquired using a 3D- CBF analysis, we calculated also the normalized CBF by dividing the MPRAGE sequence (TR = 1880 ms, TE = 3.38 ms, TI = 1100 ms, flip CBF absolute values by the healthy contralateral WM’s CBF (22). angle = 15°, number of slices = 176, thickness = 1 mm, gap = 0 mm, As a control analysis, quantitative evaluation of CBF were also per- imaging matrix = 256 × 256). FLAIR images were acquired with a formed by using the automated Olea Sphere 3.0 postprocessing plat- sagittal orientation (TR = 5000 ms, TE = 333 ms, TI = 1800 ms, flip form integrated in the MRI scan (Olea Medical SA, La Ciotat, France; angle = 120°, thickness = 1 mm, number of slices = 176, imaging www.olea-medical.com). The Bayesian method was applied to motion matrix = 224 × 256). T2 TSE images were acquired with a coronal ori- corrected images to calculate CBF maps. Given that Olea Sphere is de- entation (TR = 4310 ms, TE = 151 ms, TI = 1800 ms, flip angle = 150°, signed for clinical reading of ASL images, the software was not suitable number of slices = 32, thickness = 4 mm, number of averages = 2, imaging for additional custom analyses required by the study design (e.g., cor- matrix = 444 × 448). Functional images were acquired using a standard rection for CBF values of contralateral WM tissue). CBF values obtained echo-planar imaging (EPI) resting state (TR = 2000 ms, TE = 20 ms, flip on this platform were compared with those obtained by the ad hoc angle = 70°, number of slices = 37, thickness = 3.59 mm, gap = 4.64 mm, pipeline developed on MATLAB R2014a, SPM12, and ASLtbx. Raw imaging matrix = 448 × 448, acquisition duration = 8 min and 36 s). CT CBF values were identical for the two pipelines, following the same pre- image acquisition was obtained with a BrightSpeed, CT99, GE Hangwei processing of T1w and ASL data. Medical Systems, GE Healthcare scanner. The x-ray source was composed of a single-bend scanning electron beam (120 kV, 320 mA), and volumetric fMRI preprocessing acquisition was based on 96 images (in-plane resolution, 521 × 512; To control for the selectivity and specificity of stimulation on CBF, ad- thickness = 1 to 1.3 mm) acquired by using a field of view of 25.0 cm. ditional rs-fMRI scans were added to the protocol. ReHo and fALFF indices were extracted with CONN software (www.conn-toolbox.org/). ASL preprocessing ALFF was considered an index of regional SNA, in contrast to the Functional imaging data processing and analyses were performed using more classical functional connectivity analysis that provide insight into MATLAB R2014a (MathWorks, Natick, MA, USA; www.mathworks. interregional interactions between areas. fALFF used the ratio of the com), SPM12 (Wellcome Department of Cognitive Neurology, UK; power spectrum of low frequency (0.01 to 0.08 Hz) to that of the entire www.fil.ion.ucl.ac.uk/spm), and ASLtbx (https://cfn.upenn.edu/~zewang/ frequency range (e.g., 0 to 0.25 Hz for a TR = 2 s) of BOLD signal, ASLtbx.php). The first of 91 ASL volumes was used as the M0 image, with allowing to discriminate the signal from liquor cisterns to cortical re- the remaining 90 volumes used as 45 control-label pairs (labeling first). gions. On the other hand, regional homogeneity (ReHo) was thought A six-parameter rigid body motion spatial transformation was used to reflect neural synchronization of a given voxel with its neighboring to align the raw EPI time series. Then, the functional images were co- voxels, with higher ReHo value indicating high regional synchroniza- registered to the T1w images. The spurious motion component caused tion. In the present analysis, ReHo was calculated according to previous by the systematic label/control alternation was regressed out from the literature calculating the Kendall’s coefficient of concordance of the motion parameters before applying the transformation on the images. time series of a voxel in relation to those of 26 nearest neighbors. Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 9 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE Statistical analysis 10. C. Baeken, J. Remue, M.-A. Vanderhasselt, A. R. Brunoni, S. de Witte, R. Duprat, E. H. W. Koster, R. de Raedt, G.-R. Wu, Increased left prefrontal brain perfusion after MRI Group-level values introduced to the statistical analyses are reported in compatible tDCS attenuates momentary ruminative self-referential thoughts. Brain Stimul. Fig. 5. Preprocessed, WM-corrected CBF values acquired before and 10, 1088–1095 (2017). during tES were compared using paired t test statistics for the entire 11. P. C. Miranda, A. Mekonnen, R. Salvador, G. Ruffini, The electric field in the cortex during sample (n = 10; 7 before surgery, 3 after surgery), with an a level transcranial current stimulation. Neuroimage 70, 48–58 (2013). of 0.05. The analysis was conducted separately for each ROI, i.e., solid 12. G. Ruffini, M. D. Fox, O. Ripolles, P. C. Miranda, A. Pascual-Leone, Optimization of multifocal transcranial current stimulation for weighted cortical pattern targeting from tumor (surgical border/residual solid tumor for postsurgery analyses), realistic modeling of electric fields. Neuroimage 89, 216–225 (2014). edema, and necrotic core. Separate paired t test analyses were conducted 13. D. C. Alsop, J. A. Detre, X. Golay, M. Günther, J. Hendrikse, L. Hernandez-Garcia, H. Lu, for ipsilateral/contralateral control regions as well. The same model was B. J. MacIntosh, L. M. Parkes, M. Smits, M. J. P. van Osch, D. J. J. Wang, E. C. Wong, used to look at before versus during tES differences in fALFF and ReHo G. Zaharchuk, Recommended implementation of arterial spin-labeled perfusion MRI for clinical applications: A consensus of the ISMRM perfusion study group and the European values extracted from solid tumor, edema, and necrotic core. Given the consortium for ASL in dementia. Magn. Reson. Med. 73, 102–116 (2015). small sample size and independence of each analysis, no correction for 14. M. D. Fox, M. A. Halko, M. C. Eldaief, A. Pascual-Leone, Measuring and manipulating brain multiple comparisons was applied. connectivity with resting state functional connectivity magnetic resonance imaging (fcMRI) and transcranial magnetic stimulation (TMS). Neuroimage 62, 2232–2243 (2012). Brain surgery and anatomopathological analysis 15. R. W. Pak, D. H. Hadjiabadi, J. Senarathna, S. Agarwal, N. V. Thakor, J. J. Pillai, A. P. Pathak, Implications of neurovascular uncoupling in functional magnetic resonance imaging Approximately 1 week after the first MRI evaluation (Fig. 1), patients (fMRI) of brain tumors. J. Cereb. Blood Flow Metab. 37, 3475–3487 (2017). underwent brain neurosurgery. Complete resection (defined as the 16. G. Minniti, G. Lombardi, S. Paolini, Glioblastoma in elderly patients: Current management resection of more than 98% of a tumor (40)) was performed on two and future perspectives. Cancers (Basel) 11, 336 (2019). Downloaded from http://advances.sciencemag.org/ on March 26, 2021 patients, subtotal resection (defined as the resection between the 50 17. N. D. Arvold, D. D. Shi, A. A. Aizer, A. D. Norden, D. A. Reardon, E. Q. Lee, L. Nayak, I. F. Dunn, A. J. Golby, M. D. Johnson, E. B. Claus, E. A. Chiocca, K. L. Ligon, P. Y. Wen, and 98% of a tumor) on three patients, partial resection (resection of B. M. Alexander, Salvage re-irradiation for recurrent high-grade glioma and comparison less the 50% of a tumor) on one patient, and biopsy on the remaining to bevacizumab alone. J. Neurooncol. 135, 581–591 (2017). two patients. There were no intraoperative complications and post- 18. L. S. Hu, J. M. Eschbacher, A. C. Dueck, J. E. Heiserman, S. Liu, J. P. Karis, K. A. Smith, operative CT scans ruled out postoperative intracranial bleeding, ten- W. R. Shapiro, D. S. Pinnaduwage, S. W. Coons, P. Nakaji, J. Debbins, B. G. Feuerstein, sion pneumoencephalon, ischemia, or brain swelling. See table S1 and L. C. Baxter, Correlations between perfusion MR imaging cerebral blood volume, microvessel quantification, and clinical outcome using stereotactic analysis in recurrent the “Sample” section for the immunohistochemical findings. high-grade glioma. AJNR Am. J. Neuroradiol. 33, 69–76 (2012). 19. X. J. Qiao, B. M. Ellingson, H. J. Kim, D. J. J. Wang, N. Salamon, M. Linetsky, A. R. Sepahdari, B. Jiang, J. J. Tian, S. R. Esswein, T. F. Cloughesy, A. Lai, L. Nghiemphu, W. B. Pope, Arterial SUPPLEMENTARY MATERIALS spin-labeling perfusion MRI stratifies progression-free survival and correlates with Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ epidermal growth factor receptor status in glioblastoma. AJNR Am. J. Neuroradiol. 36, content/full/5/8/eaau9309/DC1 672–677 (2015). Fig. S1. rs-fMRI changes. 20. N. N. Gatson, E. A. Chiocca, B. Kaur, Anti-angiogenic gene therapy in the treatment of Table S1. Histological data of patients with GBM. malignant gliomas. Neurosci. Lett. 527, 62–70 (2012). 21. G. A. Alexiou, A. Zikou, S. Tsiouris, A. Goussia, P. Kosta, A. Papadopoulos, S. Voulgaris, A. P. Kyritsis, A. D. Fotopoulos, M. I. Argyropoulou, Correlation of diffusion tensor, dynamic susceptibility contrast MRI and 99mTc-Tetrofosmin brain SPECT with tumour REFERENCES AND NOTES grade and Ki-67 immunohistochemistry in glioma. Clin. Neurol. Neurosurg. 116, 41–45 1. D. A. Reardon, K. L. Ligon, E. A. Chiocca, P. Y. Wen, One size should not fit all: Advancing (2014). toward personalized glioblastoma therapy. Discov. Med. 19, 471–477 (2015). 22. M. Jovanovic, S. Radenkovic, T. Stosic-Opincal, S. Lavrnic, S. Gavrilovic, B. Lazovic-Popovic, 2. M. Valiente, M. S. Ahluwalia, A. Boire, P. K. Brastianos, S. B. Goldberg, E. Q. Lee, E. Le Rhun, I. Soldatovic, R. Maksimovic, Differentiation between progression and pseudoprogresion M. Preusser, F. Winkler, R. Soffietti, The evolving landscape of brain metastasis. by arterial spin labeling MRI in patients with glioblastoma multiforme. J. BUON 22, Trends Cancer 4, 176–196 (2018). 1061–1067 (2017). 3. H. M. C. Ciria, M. M. González, L. O. Zamora, L. E. B. Cabrales, G. V. S. González, 23. J. Jeck, R. Kassubek, J. Coburger, S. Edenhofer, S. S. Schönsteiner, A. C. Ludolph, B. Schmitz, L. O. de Oliveira, R. Zanella, A. C. Buzaid, O. Parise, L. M. Brito, C. A. A. Teixeira, J. Engelke, R. Mayer-Steinacker, J. Lewerenz, L. Bullinger, Bevacizumab in temozolomide M. das Neves Gomes, G. Moreno, V. F. da Veiga, M. Telló, C. Holandino, Antitumor effects refractory high-grade gliomas: Single-centre experience and review of the literature. of electrochemical treatment. Chin. J. Cancer Res. 25, 223–234 (2013). Ther. Adv. Neurol. Disord. 11, 1756285617753597 (2018). 4. A. Hills, J. Stebbing, Electrotherapy: Enlightening modern medicine. Lancet Oncol. 15, 24. R. Stupp, S. Taillibert, A. A. Kanner, S. Kesari, D. M. Steinberg, S. A. Toms, L. P. Taylor, 1060–1061 (2014). F. Lieberman, A. Silvani, K. L. Fink, G. H. Barnett, J.-J. Zhu, J. W. Henson, H. H. Engelhard, 5. T. Jarm, Y. A. B. D. Wickramasinghe, M. Deakin, M. Čemažar, J. Elder, P. Rolfe, G. Sersa, T. C. Chen, D. D. Tran, J. Sroubek, N. D. Tran, A. F. Hottinger, J. Landolfi, R. Desai, M. Caroli, D. Miklavčič, Blood perfusion of subcutaneous tumours in mice following the application Y. Kew, J. Honnorat, A. Idbaih, E. D. Kirson, U. Weinberg, Y. Palti, M. E. Hegi, Z. Ram, of low-level direct electric current. Adv. Exp. Med. Biol. 471, 497–506 (1999). Maintenance therapy with tumor-treating fields plus temozolomide vs temozolomide 6. T. Jarm, M. Cemazar, F. Steinberg, C. Streffer, G. Sersa, D. Miklavcic, Perturbation of blood alone for Glioblastoma. JAMA 314, 2535–2543 (2015). flow as a mechanism of anti-tumour action of direct current electrotherapy. Physiol. Meas. 25. R. Stupp, E. T. Wong, A. A. Kanner, D. Steinberg, H. Engelhard, V. Heidecke, E. D. Kirson, 24, 75–90 (2003). S. Taillibert, F. Liebermann, V. Dbalý, Z. Ram, J. L. Villano, N. Rainov, U. Weinberg, 7. A. Antal, I. Alekseichuk, M. Bikson, J. Brockmöller, A. R. Brunoni, R. Chen, L. G. Cohen, D. Schiff, L. Kunschner, J. Raizer, J. Honnorat, A. Sloan, M. Malkin, J. C. Landolfi, F. Payer, G. Dowthwaite, J. Ellrich, A. Flöel, F. Fregni, M. S. George, R. Hamilton, J. Haueisen, M. Mehdorn, R. J. Weil, S. C. Pannullo, M. Westphal, M. Smrcka, L. Chin, H. Kostron, C. S. Herrmann, F. C. Hummel, J. P. Lefaucheur, D. Liebetanz, C. K. Loo, C. D. McCaig, S. Hofer, J. Bruce, R. Cosgrove, N. Paleologous, Y. Palti, P. H. Gutin, NovoTTF-100A versus C. Miniussi, P. C. Miranda, V. Moliadze, M. A. Nitsche, R. Nowak, F. Padberg, physician’s choice chemotherapy in recurrent glioblastoma: A randomised phase III A. Pascual-Leone, W. Poppendieck, A. Priori, S. Rossi, P. M. Rossini, J. Rothwell, trial of a novel treatment modality. Eur. J. Cancer 48, 2192–2202 (2012). M. A. Rueger, G. Ruffini, K. Schellhorn, H. R. Siebner, Y. Ugawa, A. Wexler, U. Ziemann, 26. M. M. Mrugala, J. Ruzevick, P. Zlomanczuk, R. V. Lukas, Tumor treating fields in M. Hallett, W. Paulus, Low intensity transcranial electric stimulation: Safety, ethical, legal neuro-oncological practice. Curr. Oncol. Rep. 19, 53 (2017). regulatory and application guidelines. Clin. Neurophysiol. 128, 1774–1809 (2017). 27. T. Jarm, M. Cemazar, D. Miklavcic, G. Sersa, Antivascular effects of electrochemotherapy: 8. E. Santarnecchi, N. R. Polizzotto, M. Godone, F. Giovannelli, M. Feurra, L. Matzen, A. Rossi, Implications in treatment of bleeding metastases. Expert Rev. Anticancer Ther. 10, S. Rossi, Frequency-dependent enhancement of fluid intelligence induced by transcranial 729–746 (2014). oscillatory potentials. Curr. Biol. 23, 1449–1453 (2013). 28. B. Song, P. Wen, T. Ahfock, Y. Li, Numeric investigation of brain tumor influence on the 9. X. Zheng, D. C. Alsop, G. Schlaug, Effects of transcranial direct current stimulation (tDCS) current distributions during transcranial direct current stimulation. IEEE Trans. Biomed. Eng. on human regional cerebral blood flow. Neuroimage 58, 26–33 (2011). 63, 176–187 (2016). Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 10 of 11 SCIENCE ADVANCES | RESEARCH ARTICLE 29. B. F. Jordan, P. Sonveaux, O. Feron, V. Grégoire, N. Beghein, B. Gallez, Nitric oxide– Acknowledgments: We would like to thank the study participants and their families, as well mediated increase in tumor blood flow and oxygenation of tumors implanted in muscles as the radiology technicians for their support during the MRI acquisitions. Funding: E.S. and stimulated by electric pulses. Int. J. Radiat. Oncol. Biol. Phys. 55, 1066–1073 (2003). A.P.-L. are partially supported by the BROAD Institute at Harvard-MIT (Boston, MA) via 30. R. R. Langley, I. J. Fidler, The biology of brain metastasis. Clin. Chem. 59, 180–189 (2013). 2016P000351. E.S. and A.P.-L. are partially supported by the Defense Advanced Research 31. G. Sersa, M. Cemazar, C. S. Parkins, D. J. Chaplin, Tumour blood flow changes induced by Projects Agency (DARPA) via HR001117S0030. E.S. is supported by the Beth Israel Deaconess application of electric pulses. Eur. J. Cancer 35, 672–677 (1999). Medical Center (BIDMC) via the Chief Academic Officer (CAO) grant 2017 and by the National 32. S. L. Fryer, B. J. Roach, K. Wiley, R. L. Loewy, J. M. Ford, D. H. Mathalon, Reduced amplitude Institute of Aging at NIH via R01-AG060981-01. A.P.-L. is further supported by the Berenson- of low-frequency brain oscillations in the psychosis risk syndrome and early illness Allen Foundation, the Sidney R. Baer Jr. Foundation, grants from the NIH (R01HD069776, schizophrenia. Neuropsychopharmacology 41, 2388–2398 (2016). R01NS073601, R21 MH099196, R21 NS082870, R21 NS085491, and R21 HD07616), and Harvard 33. T. Wu, X. Long, Y. Zang, L. Wang, M. Hallett, K. Li, P. Chan, Regional homogeneity changes Catalyst/The Harvard Clinical and Translational Science Center (NCRR and the NCATS NIH, in patients with Parkinson’s disease. Hum. Brain Mapp. 30, 1502–1510 (2009). UL1 RR025758). The content of this paper is solely the responsibility of the authors and does 34. S. Agarwal, H. Lu, J. J. Pillai, Value of frequency domain resting-state functional magnetic not necessarily represent the official views of Harvard Catalyst, Harvard University and its resonance imaging metrics amplitude of low-frequency fluctuation and fractional affiliated academic health care centers, the National Institutes of Health, and the Sidney amplitude of low-frequency fluctuation in the assessment of brain tumor-induced R. Baer Jr. Foundation. Author contributions: Conceptualization: G.S. and E.S. Methodology: neurovascular uncoupling. Brain Connect. 7, 382–389 (2017). E.S., G.R., and R.S. Investigation: L.Mo., L.L., F.N., L.Me., G.O., B.B., E.S., and A.C. Software: 35. S. Agarwal, H. I. Sair, J. J. Pillai, The resting-state functional magnetic resonance imaging D.M., E.S., G.R., and R.S. Formal analysis: E.S. Writing: Original draft, G.S. and E.S. Review and regional homogeneity metrics–Kendall’s coefficient of concordance-regional editing: G.S., E.S., A.P.-L., S.R., and G.R. Resources: G.R., and E.S. Supervision: A.P.-L., A.R., S.R., homogeneity and coherence-regional homogeneity–are valid indicators of tumor-related and E.S. Competing interests: G.R. and R.S. work for Neuroelectrics. G.R. is a founder of neurovascular uncoupling. Brain Connect. 7, 228–235 (2017). Neuroelectrics. A.P.-L. serves on the medical advisory board for Neuroelectrics. The other authors 36. L. M. Cancel, K. Arias, M. Bikson, J. M. Tarbell, Direct current stimulation of endothelial declare that they have no competing interests. Data and materials availability: All data needed monolayers induces a transient and reversible increase in transport due to the to evaluate the conclusions in the paper are present in the paper and/or the Supplementary electroosmotic effect. Sci. Rep. 8, 9265 (2018). Materials. Additional data related to this paper may be requested from the authors. Downloaded from http://advances.sciencemag.org/ on March 26, 2021 37. S. Fogh, M. Wahl, M. Anwar, D. Haas-Kogan, J. L. Clarke, P. K. Sneed, Standardization and quality assurance of radiation therapy volumes for adults with high-grade gliomas. Submitted 30 July 2018 Semin. Radiat. Oncol. 24, 259–264 (2014). Accepted 10 July 2019 38. P. C. Miranda, A. Mekonnen, R. Salvador, P. J. Basser, Predicting the electric field distribution Published 14 August 2019 in the brain for the treatment of glioblastoma. Phys. Med. Biol. 59, 4137–4147 (2014). 10.1126/sciadv.aau9309 39. G. Ruffini, F. Wendling, R. Sanchez-Todo, E. Santarnecchi, Targeting brain networks with multichannel transcranial current stimulation (tCS). Curr. Opin. Biomed. Eng. 8, 70–77 (2018). Citation: G. Sprugnoli, L. Monti, L. Lippa, F. Neri, L. Mencarelli, G. Ruffini, R. Salvador, G. Oliveri, 40. N. Ahmadloo, A.-A. Kani, M. Mohammadianpanah, H. Nasrolahi, S. Omidvari, A. Mosalaei, B. Batani, D. Momi, A. Cerase, A. Pascual-Leone, A. Rossi, S. Rossi, E. Santarnecchi, Reduction of M. Ansari, Treatment outcome and prognostic factors of adult glioblastoma multiforme. intratumoral brain perfusion by noninvasive transcranial electrical stimulation. Sci. Adv. 5, J. Egypt Natl. Canc. Inst. 25, 21–30 (2013). eaau9309 (2019). Sprugnoli et al., Sci. Adv. 2019; 5 : eaau9309 14 August 2019 11 of 11 Reduction of intratumoral brain perfusion by noninvasive transcranial electrical stimulation G. Sprugnoli, L. Monti, L. Lippa, F. Neri, L. Mencarelli, G. Ruffini, R. Salvador, G. Oliveri, B. Batani, D. Momi, A. Cerase, A. Pascual-Leone, A. Rossi, S. Rossi and E. Santarnecchi Sci Adv 5 (8), eaau9309. DOI: 10.1126/sciadv.aau9309 Downloaded from http://advances.sciencemag.org/ on March 26, 2021 ARTICLE TOOLS http://advances.sciencemag.org/content/5/8/eaau9309 SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/08/12/5.8.eaau9309.DC1 MATERIALS REFERENCES This article cites 40 articles, 3 of which you can access for free http://advances.sciencemag.org/content/5/8/eaau9309#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions Use of this article is subject to the Terms of Service Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).