Functional Biological Activity of Sorafenib as a Tumor-Treating Field Sensitizer for Glioblastoma Therapy.

Glioblastoma, the most common primary brain tumor in adults, is an incurable malignancy with poor short-term survival and is typically treated with radiotherapy along with temozolomide. While the development of tumor-treating fields (TTFields), electric fields with alternating low and intermediate intensity has facilitated glioblastoma treatment, clinical outcomes of TTFields are reportedly inconsistent. However, combinatorial administration of chemotherapy with TTFields has proven effective for glioblastoma patients. Sorafenib, an anti-proliferative and apoptogenic agent, is used as first-line treatment for glioblastoma. This study aimed to investigate the effect of sorafenib on TTFields-induced anti-tumor and anti-angiogenesis responses in glioblastoma cells in vitro and in vivo. Sorafenib sensitized glioblastoma cells to TTFields, as evident from significantly decreased post-TTFields cell viability (p < 0.05), and combinatorial treatment with sorafenib and TTFields accelerated apoptosis via reactive oxygen species (ROS) generation, as evident from Poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, use of sorafenib plus TTFields increased autophagy, as evident from LC3 upregulation and autophagic vacuole formation. Cell cycle markers accumulated, and cells underwent a G2/M arrest, with an increased G0/G1 cell ratio. In addition, the combinatorial treatment significantly inhibited tumor cell motility and invasiveness, and angiogenesis. Our results suggest that combination therapy with sorafenib and TTFields is slightly better than each individual therapy and could potentially be used to treat glioblastoma in clinic, which requires further studies.

Study of multistep Dense U-Net-based automatic segmentation for head MRI scans.

BACKGROUND: Despite extensive efforts to obtain accurate segmentation of magnetic resonance imaging (MRI) scans of a head, it remains challenging primarily due to variations in intensity distribution, which depend on the equipment and parameters used. PURPOSE: The goal of this study is to evaluate the effectiveness of an automatic segmentation method for head MRI scans using a multistep Dense U-Net (MDU-Net) architecture. METHODS: The MDU-Net-based method comprises two steps. The first step is to segment the scalp, skull, and whole brain from head MRI scans using a convolutional neural network (CNN). In the first step, a hybrid network is used to combine 2.5D Dense U-Net and 3D Dense U-Net structure. This hybrid network acquires logits in three orthogonal planes (axial, coronal, and sagittal) using 2.5D Dense U-Nets and fuses them by averaging. The resultant fused probability map with head MRI scans then serves as the input to a 3D Dense U-Net. In this process, different ratios of active contour loss and focal loss are applied. The second step is to segment the cerebrospinal fluid (CSF), white matter, and gray matter from extracted brain MRI scans using CNNs. In the second step, the histogram of the extracted brain MRI scans is standardized and then a 2.5D Dense U-Net is used to further segment the brain's specific tissues using the focal loss. A dataset of 100 head MRI scans from an OASIS-3 dataset was used for training, internal validation, and testing, with ratios of 80%, 10%, and 10%, respectively. Using the proposed approach, we segmented the head MRI scans into five areas (scalp, skull, CSF, white matter, and gray matter) and evaluated the segmentation results using the Dice similarity coefficient (DSC) score, Hausdorff distance (HD), and the average symmetric surface distance (ASSD) as evaluation metrics. We compared these results with those obtained using the Res-U-Net, Dense U-Net, U-Net++, Swin-Unet, and H-Dense U-Net models. RESULTS: The MDU-Net model showed DSC values of 0.933, 0.830, 0.833, 0.953, and 0.917 in the scalp, skull, CSF, white matter, and gray matter, respectively. The corresponding HD values were 2.37, 2.89, 2.13, 1.52, and 1.53 mm, respectively. The ASSD values were 0.50, 1.63, 1.28, 0.26, and 0.27 mm, respectively. Comparing these results with other models revealed that the MDU-Net model demonstrated the best performance in terms of the DSC values for the scalp, CSF, white matter, and gray matter. When compared with the H-Dense U-Net model, which showed the highest performance among the other models, the MDU-Net model showed substantial improvements in the HD view, particularly in the gray matter region, with a difference of approximately 9%. In addition, in terms of the ASSD, the MDU-Net model outperformed the H-Dense U-Net model, showing an approximately 7% improvements in the white matter and approximately 9% improvements in the gray matter. CONCLUSION: Compared with existing models in terms of DSC, HD, and ASSD, the proposed MDU-Net model demonstrated the best performance on average and showed its potential to enhance the accuracy of automatic segmentation for head MRI scans.

Study of a plastic scintillating plate-based quality assurance system for pencil beam scanning proton beams.

INTRODUCTION: The purpose of this study was to evaluate a plastic scintillating plate-based beam monitoring system to perform quality assurance (QA) measurements in pencil beam scanning proton beam. METHODS: Single spots and scanned fields were measured with the high-resolution dosimetry system, consisting of a plastic scintillation plate coupled to a camera in a dark box at the isocenter. The measurements were taken at 110-190 MeV beam energies with 30° gantry angle intervals at each energy. Spot positions were determined using the plastic scintillating plate-based dosimetry system at the isocenter for 70-230 MeV beam energies with 30° gantry angle intervals. The effect of gantry angle on dose distribution was also assessed by determining the scanning pattern for daily QA and 25 fields treated with intensity-modulated proton therapy. RESULTS: Spot size, field flatness, and field symmetry of plastic scintillating plate-based dosimetry system were consistent with EBT3 at all investigated energies and angles. In all investigated energies and angles, the spot size measured was ±10% of the average size of each energy, the spot position measured was within ±2 mm, field flatness was within ±2%, and field symmetry was within ±1%. The mean gamma passing rates with the 3%/3 mm gamma criterion of the scanning pattern and 25 fields were 99.2% and 99.8%, respectively. CONCLUSIONS: This system can be effective for QA determinations of spot size, spot position, field flatness, and field symmetry over 360° of gantry rotation in a time- and cost-effective manner, with spatial resolution comparable to that of EBT3 film.

Synergistic effect of TTF and 5-FU combination treatment on pancreatic cancer cells.

The present study investigated the therapeutic potential of combining tumor-treating fields (TTF), a novel cancer treatment modality that employs low-intensity, alternating electric fields, with 5-fluorouracil (5-FU), a standard chemotherapy drug used for treating pancreatic cancer. The HPAF-II and Mia-Paca II pancreatic cancer cell lines were treated with TTF, 5-FU, or their combination. Combination treatment produced a significantly greater inhibitory effect on cancer cell proliferation than each single modality. Furthermore, combination therapy induced a substantially higher rate of pancreatic cancer cell apoptosis and exhibited a synergistic effect in clonogenic assays. Additionally, combination treatment showed a greater inhibition of cancer cell migration and invasion than either TTF or 5-FU alone. In conclusion, these findings suggest that the synergistic properties of TTF and 5-FU result in greater therapeutic efficacy against pancreatic cancer cells than either modality alone and may improve survival rates in patients with pancreatic cancer.

Inter-fractional entrance dose monitoring as quality assurance using Gafchromic EBT3 film.

Introduction: This study describes a simple method of inter-fractional photon beam monitoring to measure the entrance dose of radiation treatment using Gafchromic EBT3 film. Materials and Methods: The film was placed at the center of a 1-cm thick phantom shaped like a block tray and fixed on the accessory tray of the gantry. The entrance dose was measured following the placement of the film in the accessory tray. The dose distribution calculated with the treatment planning system was compared with the dose distribution on the irradiated EBT3 films. The effectiveness of this methodology, as determined by gamma passing rates, was evaluated for the 22 fields of eight three-dimensional conformal radiotherapy (3D-CRT) plans and the 41 fields of nine intensity-modulated radiotherapy (RT) plans. The plans for three-dimensional conformal RT included treatments of the rectum, liver, breast, and head and neck, whereas the plans for intensity-modulated RT included treatments of the liver, brain, and lung. Results: The gamma passing rates for 3D-CRT ranged from 96.4% to 99.5%, with the mean gamma passing rate for 22 fields being 98.0%. The gamma passing rate for intensity-modulated RT ranged from 96.1% to 98.9%, with the mean gamma passing rate for 41 fields being 97.7%. All gamma indices were over the 95% tolerance level. Conclusions: The methodology described in this study, based on Gafchromic EBT3 film, can be utilized for inter-fractional entrance dose monitoring as quality assurance during RT. Clinical application of this method to patients can verify the accuracy of beam delivery in the treatment room.

Technical note: Evaluation of methods for reducing edge current density under electrode arrays for tumor-treating fields therapy.

BACKGROUND: Tumor-treating fields (TTFields) therapy is increasingly utilized clinically because of its demonstrated efficacy in cancer treatment. However, the risk of skin burns must still be reduced to improve patient safety and posttreatment quality of life. PURPOSE: The purpose of this study was to evaluate the methods of constructing electrode arrays that reduce current density exceeding threshold values, which can cause skin burns during TTFields therapy. METHODS: Electrode and body models were generated using COMSOL software. The body model had the dielectric properties of the scalp. The average current density beneath the central region of the electrode was maintained at ∼31 mA/cm2 RMS. The deviations in current density at the edges of the electrode were reduced by three methods: adjustment of the ceramic thickness ratio of the center to the edge from 1/5 to 4/5, adjustment of the radius of the metal plate from 5.0 to 8.0 mm, and insertion of an insulator of width 0.5 to 2 mm at the edge. RESULTS: While using a single circular electrode, adjustment of the ceramic thickness ratio, adjustment of the metal plate radius, and insertion of an insulator near the edge reduced the deviations of current density by 14.6%, 67.7%, and 75.3%, respectively. Similarly, while using circular electrode arrays, inserting an insulator at the edge of each electrode reduced the deviations of current density significantly, from 8.62 to 2.40 mA/cm2 . CONCLUSIONS: Insertion of an insulator at the edge of each electrode was found to be the most effective method of attaining uniform current density distribution beneath the electrode, thereby lowering the risk of adverse effects of TTFields therapy.

The combination of tumor treating fields and hyperthermia has synergistic therapeutic effects in glioblastoma cells by downregulating STAT3.

Glioblastoma multiforme (GBM), the most common type of brain tumor, is a very aggressive and treatment-refractory cancer, with a 5-year survival rate of approximately 5%. Hyperthermia (HT) and tumor treating fields (TTF) therapy have been used to treat cancer, either alone or in combination with other treatment methods. Both treatments have been reported to increase the efficacy of other treatment techniques and to improve patient prognosis. The present study evaluated the therapeutic effects of combining HT and TTF on GBM cell lines. Cells were subjected to HT, TTF, HT+TTF, or neither treatment, followed by comparisons of cell proliferation, apoptosis, migration and invasiveness. Clonogenic assays showed that the two treatments had a synergistic effect. The levels of cleaved PARP and cleaved caspase-3 were higher and apoptosis was increased in cells treated with HT+TTF than in cells treated with HT or TTF alone. In addition, HT+TTF showed greater inhibition of GBM cell migration and invasiveness and greater downregulation of STAT3 than either HT or TTF alone. The stronger anticancer effect of HT+TTF suggested that this combination treatment can increase the survival rate of patients with difficult-to-treat cancers such as GBM.

Thymidine decreases the DNA damage and apoptosis caused by tumor-treating fields in cancer cell lines.

BACKGROUND: Tumor-treating fields (TTFields) is an emerging non-invasive cancer-treatment modality using alternating electric fields with low intensities and an intermediate range of frequency. TTFields affects an extensive range of charged and polarizable cellular factors known to be involved in cell division. However, it causes side-effects, such as DNA damage and apoptosis, in healthy cells. OBJECTIVE: To investigate whether thymidine can have an effect on the DNA damage and apoptosis, we arrested the cell cycle of human glioblastoma cells (U373) at G1/S phase by using thymidine and then exposed these cells to TTFields. METHODS: Cancer cell lines and normal cell (HaCaT) were arrested by thymidine double block method. Cells were seeded into the gap of between the insulated wires. The exposed in alternative electric fields at 120 kHz, 1.2 V/cm. They were counted the cell numbers and analyzed for cancer malignant such as colony formation, Annexin V/PI staining, γH2AX and RT-PCR. RESULTS: The colony-forming ability and DNA damage of the control cells without thymidine treatment were significantly decreased, and the expression levels of BRCA1, PCNA, CDC25C, and MAD2 were distinctly increased. Interestingly, however, cells treated with thymidine did not change the colony formation, apoptosis, DNA damage, or gene expression pattern. CONCLUSIONS: These results demonstrated that thymidine can inhibit the TTFields-caused DNA damage and apoptosis, suggesting that combining TTFields and conventional treatments, such as chemotherapy, may enhance prognosis and decrease side effects compared with those of TTFields or conventional treatments alone.

Sorafenib increases tumor treating fields-induced cell death in glioblastoma by inhibiting STAT3.

A newly diagnosed or recurrent Glioblastoma multiforme (GBM) can be treated with Tumor-treating fields (TTFields), an emerging type of alternative electric field-based therapy using low-intensity electric fields. TTFields have a penchant to arrest mitosis, eventually leading to apoptosis. Therefore, it is regarded as a potential anticancer therapy. However, in this study, we confirmed the combined efficacy of sorafenib and TTFields to improve the treatment efficiency of malignant GBM. Experimentation revealed the ability of sorafenib to decrease the signal transducer and activator of transcription 3 (STAT3) and this inhibition increased the sensitivity of TTFields in preventing tumor expansion. It was found that both combinatorial as well as monotherapy aimed to inhibit or reduce the level of STAT3, but the extent was different and based upon the reaction conditions. This drug is also capable of arresting multiple kinase pathways along with STAT3-related proteins (Mcl-1 and Survivin). STAT3 silencing can also be accomplished by RNA interference and can increase the TTFields-sensitizing effect of sorafenib. If the effects are reversed and gene regulating STAT3 is expressed more, it annihilates the effects of treatment. Moreover, sorafenib plus TTFields significantly inhibited xenograft tumor growth and combinatorial treatment reduced STAT3 expression more effectively in vivo. These in vitro and in vivo results indicate that sorafenib tends to sensitize GBM cells to TTFields-induced apoptosis by inhibiting STAT3.

Tumor treating fields (TTF) treatment enhances radiation-induced apoptosis in pancreatic cancer cells.

PURPOSE: Tumor treating fields (TTF) therapy is a noninvasive method that uses alternating electric fields to treat various types of cancer. This study demonstrates the combined effect of TTF and radiotherapy (RT) in vitro on pancreatic cancer, which is known to be difficult to treat. MATERIALS AND METHODS: In CFPAC-I and HPAF-II pancreatic cancer cell lines, the combined in vitro effect of TTF and RT was evaluated by measuring cell counts, markers of apoptosis, and clonogenic cell survival. The synergy effects were verified using the Valeriote and Carpentier equations. RESULTS: TTF and RT inhibited cancer cell growth more effectively than did monotherapy with TTF or RT. The combined treatment also enhanced apoptosis more than monotherapy, as shown by assays for cleaved poly (ADP-ribose) polymerase (PARP) and annexin V. In addition, on the survival curve, this treatment method has been shown to work synergistically. CONCLUSION: These results suggest that combined treatment with TTF and RT may be a good alternative treatment for patients with pancreatic cancer.

Effectiveness of a Fractionated Therapy Scheme in Tumor Treating Fields Therapy.

This study aimed to evaluate the biological effectiveness of cancer therapy with tumor treating fields using a fractionated treatment scheme that was originally designed for radiotherapy. Discontinuous fractional tumor treating fields of an intensity of 0.9 to 1.2 V/cm and a frequency of 150 KHz were applied to U373 cancer cells and IEC6 normal cells for 3 days, with durations of 3, 6, 12, or 24 h/d. As the treatment duration of the tumor treating fields increased from 3 to 24 h/d, the relative tumor cell (U373) number (% of control) reduced in proportion to the treatment duration. Compared to a 25% cell number reduction (75% of control) for the group of 6 h/d treatment at 1.2 V/cm, only 5% (70% of control) and 8% (67% of control) of additional reductions were observed for the group of 12 and 24 h/d treatment, respectively. This experimental result indicates that the dependence on treatment duration in tumor cell inhibition was weakened distinctly at treatment duration over 6 h/d. For normal cells (IEC6), the relative cell number corresponding to the treatment time of the tumor treating fields at 1.2 V/cm of electric field strength was not decreased much for the treatment times of 3, 6, and 12 h/d, revealing 93.3%, 90.0%, and 89.3% relative cell numbers, respectively, but it suddenly decreased to ∼73% for the 24 h/d treatment. Our results showed that the effects of tumor treating fields on tumor cells were higher than on normal cells for treatment duration of 3 to 12 h/d, but the difference became minimal for treatment duration of 24 h/d. The fractionated scheme, using tumor treating fields, reduced the treatment time while maintaining efficacy, suggesting that this method may be clinically applicable for cancer treatment.

Tumor-treating fields induce autophagy by blocking the Akt2/miR29b axis in glioblastoma cells.

Tumor-treating fields (TTFs) - a type of electromagnetic field-based therapy using low-intensity electrical fields - has recently been characterized as a potential anticancer therapy for glioblastoma multiforme (GBM). However, the molecular mechanisms involved remain poorly understood. Our results show that the activation of autophagy contributes to the TTF-induced anti-GBM activity in vitro or in vivo and GBM patient stem cells or primary in vivo culture systems. TTF-treatment upregulated several autophagy-related genes (~2-fold) and induced cytomorphological changes. TTF-induced autophagy in GBM was associated with decreased Akt2 expression, not Akt1 or Akt3, via the mTOR/p70S6K pathway. An Affymetrix GeneChip miRNA 4.0 Array analysis revealed that TTFs altered the expression of many microRNAs (miRNAs). TTF-induced autophagy upregulated miR-29b, which subsequently suppressed the Akt signaling pathway. A luciferase reporter assay confirmed that TTFs induced miR-29b to target Akt2, negatively affecting Akt2 expression thereby triggering autophagy. TTF-induced autophagy suppressed tumor growth in GBM mouse models subjected to TTFs as determined by positron emission tomography and computed tomography (PET-CT). GBM patient stem cells and a primary in vivo culture system with high Akt2 levels also showed TTF-induced inhibition. Taken together, our results identified autophagy as a critical cell death pathway triggered by TTFs in GBM and indicate that TTF is a potential treatment option for GBM.

PLOD3 suppression exerts an anti-tumor effect on human lung cancer cells by modulating the PKC-delta signaling pathway.

Current lung cancer treatments are far from satisfactory; thus, finding novel treatment targets is crucial. We recently identified procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), which is involved in fibrosis and tissue remodeling as a radioresistance-related protein in lung cancer cells; however, its mechanism is unclear. In this study, we designed human PLOD3-specific short interfering (si)RNAs and tested their effects on tumor growth inhibition in vitro and in vivo. PLOD3 knockdown overcame chemoresistance and decreased radioresistance by inducing caspase-3-dependent apoptosis in lung cancer cells. Furthermore, PLOD3 interacted with PKCδ to activate caspase-2,4-dependent apoptosis through ER-stress-induced IRE1α activation and the downstream unfolded-protein response pathway. In a mouse xenograft model, PLOD3 knockdown promoted radiation-induced tumor growth inhibition, without side effects. Moreover, lung cancer patients with high PLOD3 expression showed poorer prognosis than those with low PLOD3 expression upon radiotherapy, suggesting that PLOD3 promotes tumor growth. Therefore, PLOD3 siRNA suppresses radioresistance and chemoresistance by inducing apoptosis and renders PLOD3 as a candidate lung cancer biomarker. PLOD3 gene therapy might enhance the efficacy of radiotherapy or chemotherapy in lung cancer patients.

Selective toxicity of tumor treating fields to melanoma: an in vitro and in vivo study.

Tumor treating fields (TTFs) are a newly developed cancer therapy technology using an alternating electric field that may be a possible candidate for overcoming the limitations of conventional treatment methods currently used in cancer treatment. Although clinical results using TTFs appear promising, concerns regarding side effects must be clarified to demonstrate the effectiveness of this treatment method. To investigate the side effects of TTF treatment, the damage to normal cell lines and normal tissue of a mouse model was compared with the damage to tumor cells and tumors in a mouse model after TTF treatment. No serious damage was found in the normal cells and normal tissues of the mouse model, suggesting that the side effects of TTF treatment may not be serious. Our evidence based on in vitro and in vivo experiments suggests that TTF may cause selective damage to cancer cells, further demonstrating the potential of TTF as an attractive alternative to conventional cancer treatment modalities.

Gold nanoparticles as a potent radiosensitizer in neutron therapy.

The purpose of this study was to investigate the potential of gold nanoparticles as radiosensitizer for use in neutron therapy against hepatocellular carcinoma. The hepatocellular carcinoma cells lines Huh7 and HepG2 were irradiated with γ and neutron radiation in the presence or absence of gold nanoparticles. Effects were evaluated by transmission electron microscopy, cell survival, cell cycle, DNA damage, migration, and invasiveness. Gold nanoparticles significantly enhanced the radiosensitivity of Huh7 and HepG2 cells to γ-rays by 1.41- and 1.16-fold, respectively, and by 1.80- and 1.35-fold to neutron radiation, which has high linear energy transfer. Accordingly, exposure to neutron radiation in the presence of gold nanoparticles induced cell cycle arrest, DNA damage, and cell death to a significantly higher extent, and suppressed cell migration and invasiveness more robustly. These effects are presumably due to the ability of gold nanoparticles to amplify the effective dose from neutron radiation more efficiently. The data suggest that gold nanoparticles may be clinically useful in combination therapy against hepatocellular carcinoma by enhancing the toxicity of radiation with high linear energy transfer.