"Experimental groups

Rats were grafted and randomly assigned to the sham (0 W/ kg), 0.25 W/kg, 0.5 W/kg or to the cage control (CC, no restraint) groups. RF–EMF exposures were performed in restraint for 45 min per day, 5 days per week, starting on post-surgical day (PSD)7 and ending at death or at PSD65.

RF–EMF exposures

A RF power source (900-64 type, generator RFPA S.A., RFS9001800-25 model, RF Power Amplifier, France) emit- ting a GSM 900 MHz EMF (1/8 duty factor) pulse modu- lated at 217 Hz was connected to a four-way divider. Each output was connected to a loop antenna [31] that consisted of a two-metallic line circuitry printed on a dielectric substrate, which allowed four simultaneous head-exposures in the two exposure Faraday cages and one sham Faraday cage. A RF detector controlled incident and reflected powers. Exposures were performed daily for 45 min between 9:00 a.m. and 1:00 p.m. in a blinded fashion.

Exposure duration aimed to mimic the heavy user’s cat- egory. Based on questionnaires of previous epidemiological studies it was included in the interval between the regular (1 call per week) and the extremely frequent (5 h per day) cell phone user categories [5]. According to our previous studies, repeatedly 45 min restrained rats elicited cage control-like stress and behaviors [26]. The rat body was restrained in cyl- inders (4.5, 5 and 6 cm in diameter to adapt to rat body size growth between PSD7 and PSD65) holed to minimize body temperature elevation. The head was inserted at restrainer end in a truncated cone to allow the animal to breathe. According to the finite difference time domain calculations, numerical dosimetry indicated that input powers of 0.17 and 0.33 W gave BASARs of 0.25 or 0.5 W/kg, respectively.

C6 cells preparation and tumor grafting

The C6 rodent models of GBM was shown to recapitulate fea- tures of human GBM [30, 32]. C6 cells were purchased from ATCC (C6ATCC®CCL-107TM, LGC Standards, Molsheim, France). They were grown in F-12K (Kaighn’s Modification of Ham’s F-12) Medium, supplemented with 2.5% fetal bovine serum and horse serum to a concentration of 15%, at 37 °C in 5% carbon dioxide at 90% relative humidity. They were seeded at a concentration of 500,000 cells/flask. Once arrived at 80% confluence, cells were suspended in complete PBS buffer (with calcium and magnesium) at a concentration of 1.12 × 106 cells/ml and kept on ice prior to the graft. Injec- tion of 5600 C6 cells were performed in 5 μl in anesthetized rats placed in the stereotaxic apparatus. The 26 Gauge needle (ga26/51 mm/pst3, Phymep SARL, France) was inserted in the striatum (+ 0.96 mm antero-posterior to the bregma, − 3 mm lateral, − 6 mm dorso-ventral) [33]. At the end of the injection, a 10 min’ time lapse was awaited before gently raising the syringe to prevent the cells from sticking-out along the needle.

Monitoring and pathology

We explored the outcome survival. Monitoring was per- formed in a blinded fashion according to Higashikubo et al. [9]. Briefly, rats were examined twice daily for the develop- ment of clinical/neurological and were daily weighed. Weight loss, progressive deterioration, and increase in the number of neurological signs that indicate imminent death were used to select animals for euthanasia. All survivors were euthanized at PSD65.

Anatomopathology

We explored the outcomes tumor size and categorized tumor location by dividing the brain into seven structures. Rats were anaesthetized with isoflurane and received intra-cardiac PBS followed by formaldehyde 4% infusions. Brains were extracted, fixed in 4% formaldehyde for 5 days and stored in ethanol. Using a brain matrix, frontal sections were made at the optical chiasma (0 mm), +3 mm, +6 mm and +9 mm. For each animal, the 3–5 sections containing tumorous tis- sue were dehydrated using the Vacuum Infiltration Processor (VIP 5, Sakura) and embedded in paraffin wax. Blocks were sectioned using the Microtome Tissue Tek® Accu-Cut® SRM 200 (Sakura). Slides were stained using the DS 2000 from Sakura or immune-labelled using the DAKO link 48. The sec- tions were mounted on Tissue-Tek Coverslipper SCA 5600 (Sakura) and stained with hematoxylin and eosine (HE). All pathological examinations were performed in a blinded fash- ion. Numerical images of the slides were produced using the Nanozoomer camera from Hamamatsu at X20 magnification. Tumor length (l), wide (w) and height (h) were measured. The tumor volume was calculated using the formula:

4 lwh Vellipse= 3×𝜋×2×2×2.

Immunohistochemistry for CC3, Ki67 and CD31

We explored the following outcomes: apoptosis, cell division and vascularization as measured, respectively by caspase 3 (CC3), Ki67 and CD31 immunoreactivity, immune cell inva- sion and necrosis as measured on a scale from 1 to 5.

Paraffin sections were deparaffinized and antigen retrieval was carried out with the Dako Target Retrieval Solution. The protocol for retrieval included 20 min at 98 °C in EDTA pH9 for CC3, 20 min at 98 °C in citrate pH6 for Ki67, and 10 min at 121 °C in EDTA pH9 for CD31. Rabbit monoclonal antibody to CC3 (Cell signaling, #9664), rab- bit polyclonal antibody to Ki67 (Abcam, ab15580) and to CD31 (Abcam, ab28364) were used as primary antibodies at the concentrations of 1/800, 1/1000 and 1/200, respec- tively. After blocking endogenous peroxydase activity (Dako EnVision®+ System-HRP (DAB), Peroxydase Block) and blocking nonspecific protein labeling (DAKO Protein block serum free), the slides were incubated with diluted primary antibody for 1 h at room temperature. The tissue sections were then washed and incubated with appropriate second- ary antibodies for 30 min (Dako, K4011). Immuno-reactive signals were detected using DAB substrate solution (Dako EnVision®+ System-HRP, DAB + substrate buffer/liquid DAB + chromogen, 5 min’ incubation). Finally, the sections were lightly counterstained with Mayer’s hematoxylin. Neg- ative controls were obtained by substitution of the primary antibodies with isotype control immunoglobulin (rabbit IgG, Dako, X0936) in the immunohistochemical staining proce- dure, with a concentration equivalent to the primary anti- body. Automatic quantification was performed on numeri- cal slides. The number of mitotic figures were counted (five fields at magnification × 400 = 0.11 mm2). The tumor micro- environment was assessed by the semi-quantitative gradation of the severity of immune cell invasion (by counting mac- rophages and lymphocytes) and necrosis. Semi-quantitative gradation included 6 grades: 0 = no lesion, 1 = negligible, 2 = weak, 3 = moderate, 4 = marked, 5 = severe. All histo- logical examinations were performed in a blinded fashion.

Statistics

Survival data and means ± standard error of the mean were analyzed using Graph Pad Prism Statistics software. RF–EMF effects were tested by comparing the sham-, 0.25 W/kg- and 0.5 W/kg-exposed groups together, using the log-Rank test for trend for survival data, the Spearman correlation coefficient and partial coefficient (adjusted on survival) for quantitative data and the chi-squared test of a contingency table analysis of the binary outcomes. Restraint effect was tested by comparing the CC group with the sham-exposed group using the log-Rank, the Mann–Whit- ney test, and the Fisher’s exact test of a contingency table

Immunohistochemistry for CC3, Ki67 and CD31

We explored the following outcomes: apoptosis, cell division and vascularization as measured, respectively by caspase 3 (CC3), Ki67 and CD31 immunoreactivity, immune cell inva- sion and necrosis as measured on a scale from 1 to 5.

Paraffin sections were deparaffinized and antigen retrieval was carried out with the Dako Target Retrieval Solution. The protocol for retrieval included 20 min at 98 °C in EDTA pH9 for CC3, 20 min at 98 °C in citrate pH6 for Ki67, and 10 min at 121 °C in EDTA pH9 for CD31. Rabbit monoclonal antibody to CC3 (Cell signaling, #9664), rab- bit polyclonal antibody to Ki67 (Abcam, ab15580) and to CD31 (Abcam, ab28364) were used as primary antibodies at the concentrations of 1/800, 1/1000 and 1/200, respec- tively. After blocking endogenous peroxydase activity (Dako EnVision®+ System-HRP (DAB), Peroxydase Block) and blocking nonspecific protein labeling (DAKO Protein block serum free), the slides were incubated with diluted primary antibody for 1 h at room temperature. The tissue sections were then washed and incubated with appropriate second- ary antibodies for 30 min (Dako, K4011). Immuno-reactive signals were detected using DAB substrate solution (Dako EnVision®+ System-HRP, DAB + substrate buffer/liquid DAB + chromogen, 5 min’ incubation). Finally, the sections were lightly counterstained with Mayer’s hematoxylin. Neg- ative controls were obtained by substitution of the primary antibodies with isotype control immunoglobulin (rabbit IgG, Dako, X0936) in the immunohistochemical staining proce- dure, with a concentration equivalent to the primary anti- body. Automatic quantification was performed on numeri- cal slides. The number of mitotic figures were counted (five fields at magnification × 400 = 0.11 mm2). The tumor micro- environment was assessed by the semi-quantitative gradation of the severity of immune cell invasion (by counting mac- rophages and lymphocytes) and necrosis. Semi-quantitative gradation included 6 grades: 0 = no lesion, 1 = negligible, 2 = weak, 3 = moderate, 4 = marked, 5 = severe. All histo- logical examinations were performed in a blinded fashion.

Statistics

Survival data and means ± standard error of the mean were analyzed using Graph Pad Prism Statistics software. RF–EMF effects were tested by comparing the sham-, 0.25 W/kg- and 0.5 W/kg-exposed groups together, using the log-Rank test for trend for survival data, the Spearman correlation coefficient and partial coefficient (adjusted on survival) for quantitative data and the chi-squared test of a contingency table analysis of the binary outcomes. Restraint effect was tested by comparing the CC group with the sham-exposed group using the log-Rank, the Mann–Whit- ney test, and the Fisher’s exact test of a contingency table analysis. All hypothesis tests used a criterion level of α = 0.05 excepted for the test of RF–EMF effect on tumor location in seven different areas (tested using an adjusted criterion level of α = 0.006)."

Wistar rats, male, C6 grafted tumors

HAWC