Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.

microscopy before nsPEF application (a), 5 min after (b), and at 12 hours (c). Electrodes site were drawn on (a) with dotted rectangles. The circular zone (1) on (b) highlights the clear loss of perfusion of the treated zone (large vessels and capillaries) 5 min after nsPEF treatment.
Circle 2 in (c) shows an example zone where perfusion did not return in capillaries, compared with another zone (c, circle 3) where vascularization recovered after 12 hours. Scale bar in (c) = 2 mm applies to all images.

SCIENTIFIC REPORTS
2 Supplementary figure 2: The dose-response relationship for the vascular effects of nsPEF was investigated with respect to electric field intensity. A total of 18 independent CAM samples were injected with Rhodamine B-dextran 70k and treated with a single nsPEF at a range of electric field intensities including 0 kV/cm (control condition with the same placement of electrodes, n=4), 10.6 kV/cm (n=2), 16.5 kV/cm (n=2), 22.5 kV/cm (n=3), 34 kV/cm (n=3) and 44kV/cm (n=4). Vessel diameter was measured and the peak decrease in vessel diameter followed a sigmoidal trend with increasing electric field intensity, as shown by the doseresponse curve fit (R-Square=0.99).

SCIENTIFIC REPORTS
3 Supplementary figure 3: The measured and simulated reflection coefficient curves of the nsPEF delivery electrodes. To determine the efficiency of the energy transfer between the generator and the delivery system, reflection coefficient (S11) evaluation was carried out through measurements and simulations. Reflection coefficients of less than -13 dB over the 0-500 MHz frequency bandwidth (without biological solution, dotted lines) and less than -10 dB over the 0-200 MHz frequency bandwidth (with biological solution, solid lines) were obtained corresponding to a good impedance matching and energy transfer. It can be noticed that both measured and simulated results are in good agreement with each other.

Supplementary video 1 :
Multiphoton imaging of quail CAM vasculature visualized in a 3D rotating movie showing intravascular Rhodamine dextran labeled capillaries and vessels in a field of 500*500*200 µm (case 1).

Supplementary video 2 :
Multiphoton imaging of quail CAM vasculature visualized in a 3D rotating movie showing intravascular Rhodamine dextran labeled capillaries and vessels in a field of 500*500*400 µm (case 2).

Supplementary video 3 :
Multiphoton imaging of quail CAM vasculature visualized in a 4D movie (3D over time) showing intravascular Rhodamine dextran labeled capillaries and vessels in a field view of 500*500*400 µm (case 2) that was pulsed at t=6 min with a single 10 ns PEF.

Supplementary video 4 :
Multiphoton imaging of quail CAM vasculature visualized in a 3D rotating movie showing intravascular Rhodamine dextran labeled capillaries and vessels in a field of 500*500*350µm (case 3).

Supplementary video 5 :
Multiphoton imaging of quail CAM vasculature visualized in a 4D movie (3D over time) showing intravascular Rhodamine dextran labeled capillaries and vessels in a field view of 500*500*350 µm (case 3) that was pulsed at t=6 min with a single 10 ns PEF and displayed extravascular fluorescence.

Supplementary video 6 :
Multiphoton imaging of quail CAM vasculature visualized in a 3D rotating movie showing intravascular Rhodamine dextran labeled capillaries and vessels in a field of 500*500*500µm (case 4).

Supplementary video 7 :
Multiphoton imaging of quail CAM vasculature visualized in a 4D movie (3D over time) showing intravascular Rhodamine dextran labeled capillaries and vessels in a field view of 500*500*500 µm (case 4) that was pulsed at t=6 min with a single 10 ns PEF and displayed extravascular fluorescence.

Supplementary video 8 :
Intravascular Rhodamine B-dextran and GFP-U87 grafted on CAM were observed with multiphoton imaging in a series of image sections of the tumoral spheroid over 500*500*250 µm (case 5).