Fig 1: (Ai) Histopathological analysis of the PEC‐exposed tumor sections. Pyknotic nuclei of the PECT samples show its effects. However, there is no observable change in the NECT sample in comparison to the PECT sample. As can be seen, the stroma of all exposed samples remains unaffected, demonstrating the non‐destructive effect of PECT on normal tissues. The scale bars are set to 5 µm. Each group contain 10 mouse models (10 biological replicates for each group). (Aii) Histopathological components of the analyzed PECT and NECT samples. The tumoral tissue has remained unchanged in both NECT and control samples. These include normal regional tissue, vascular channels, normal stromal/connective tissue, and fatty tissue. Each group contain 10 mouse models (10 biological replicates for each group). Figure S7A depicts tissue pathology components in detail. (Bi) Ultrasonography images of a twin‐shaped tumor with PEC exposure to the left lump. Due to the identical structure, physiology, origin, and even location of such tumors, this twin‐shaped tumor provided an excellent model for studying the effects of PECT on exposed and non‐exposed tumor tissues. As can be seen, the non‐exposed part maintained tumor growth, whereas the exposed part suppressed tumor growth. (Bii) The H&E images of the half‐exposed twin tumor demonstrate the presence of apoptotic cells in the exposed region and unaffected cancer cells in the non‐exposed region. IHC images based on P53 and Ki67 indicate apoptosis and proliferation, respectively, only in exposed and non‐exposed regions. The scale bars are set to 5 µm. (Biii) Size alterations timeline in two periods of growth and PECT. Throughout the growth period, the total size of both lumps was calculated. For achieving the effects of PECT, each lump was sized separately following the PECT application. Significant size reduction and complete disappearance of the exposed lump compared to the non‐exposed hump clearly demonstrate PECT's localized effect on cancerous regions. (Biv) Pathological tissue components of the mentioned lumps. It is noteworthy that the components of the non‐exposed lump and the control group are identical. However, there is no evidence of cancerous tissue in the exposed lump. These include normal regional tissue, vascular channels, normal stromal/connective tissue, and fatty tissue. (C) Pathological images of control and exposed (~5 kV) mouse models depict no trace of apoptosis or necrosis in the regions exposed to PECT. The cells maintained their natural morphology and assemblies. The scale bars are set to 5 µm. PEC, positive electrostatic charge
Fig 2: (Ai) H&E and IHC analyses of three mouse model cohorts: control (non‐exposed), NECT, and PECT. Groups with exposure to PECT show pyknotic cancer cells with normal stroma in the H&E sections. HIF‐1α, CD31, and Ki67 IHC sections indicated the downregulation of hypoxia, angiogenesis, and proliferative markers, respectively. Cancerous cells have a high density and an irregular shape and structure in non‐exposed and NECT H&E sections, indicating that NECT does not affect cancerous tissues. Overexpression of HIF‐1α, CD31, and Ki67 IHC markers shows high levels of metabolism, angiogenesis, and proliferation in both non‐exposed and NECT samples. The scale bars are set to 20 µm. Each group contain 10 mouse models (10 biological replicates for each group). (Aii) Quantitative results of the expressions show the downregulation of HIF‐1α, CD31, and Ki67 in PECT samples compared to NECT and non‐exposed ones (independent t‐test). All the data are shown as mean ± SD. Each group contain 10 mouse models (10 biological replicates for each group). (Aiii) Tissue components of the histological analysis of PECT, NECT, and non‐exposed samples. These include normal regional tissue, vascular channels, normal stromal/connective tissue, and fatty tissue. Figure S7B depicts tissue pathology components in detail. (B) RT‐PCR results for the three groups, that is, Control, PECT (~2 kV), and PECT (~5 kV). The overexpression of P21, P27, and P53 near the downregulation of CD33, integrin α5, VEGF, and VEGF‐A in exposed groups indicated that PECT induced detachment, followed by apoptosis, in the malignant tumor (independent t‐test). All the data are shown as mean ± SD. Each group contain 10 mouse models (10 biological replicates for each group). (Ci, Cii) Schematic and comparative histological images between the surface and the deepest part of the tumor post‐exposure (~5 kV) with a primary depth of 1.5 cm indicated complete apoptosis and proliferation for these two regions, respectively. Depth‐dependent histopathological images of the exposed tumor indicated the progressed apoptotic region in the lower depth. It is worth noting that the tumor's deepest part measures 6 mm, up from 3 cm before initiating PECT. Hence, more than 2.4 cm of the tumor (in‐depth) was destructed. The scale bars are set to 5 µm. (Ciii) Confocal microscopy of control and exposed (~5 kV) tumors. Immunofluorescent images of the non‐exposed and exposed surfaces and the depth (4 mm) of the exposed tumor revealed a non‐deformed shape, a severely deformed shape with condensed cytoplasm, a deformed shape with a decreased intensity, respectively. PEC exposure induced apoptosis to a depth of 4 mm in the tumor. The scale bars are set to 5 µm
Fig 3: (I) The histopathological characteristics indicated tumoural tissue remains intact in the control group, whereas no tumour tissue was observed in the NDV+liposomal doxorubicin group. Each group includes five mouse models. The scale bars are set to 5 μm. (II) Immunohistochemistry analysis revealed downregulation and upregulation of Ki67 and P53 as proliferative and apoptotic markers, respectively and Overexpression of Ki67 in the control group showed high levels of proliferation. The scale bars are set to 5 μm.
Fig 4: (I) Used real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) to detect the expression level of P16, P21, P53, CD34, integrin α5, VEGF and VEGF‐R on day 26. NDV induced apoptosis in malignant cells via the overexpression of P21, P27 and P53 and downregulation of CD33, integrin α5, VEGF, and VEGF‐A in exposed groups. In this study, the GAPDH gene was used as a control. All the data are exhibited as means ± SD. (II) Kaplan–Meier survival curves of different mouse models in each group. There is no distinction between the control and placebo groups' survival curves (log‐rank test). NDV‐liposomal doxorubicin group had the most survival rate. Survival rates depended on the dose of NDV and decreased in the lower dose of NDV.
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