Valerii E. Orel

Background/Objectives: Sarcomas exhibit marked intratumoural heterogeneity at the molecular, cellular and tissue levels, thereby limiting drug delivery and treatment response. Herein, we evaluated the effects of free doxorubicin (FDOX) and liposomal doxorubicin (LDOX) combined with inductive moderate hyperthermia (IMH) on intratumoural heterogeneity in an experimental sarcoma model using magnetic resonance imaging (MRI), histology and immunohistochemistry image analysis. Methods: Female non-inbred rats bearing sarcoma-45 were divided into six groups: control (no treatment), IMH, FDOX, LDOX, FDOX + IMH and LDOX + IMH. FDOX or LDOX was administered every other day for a total of five times, beginning from day 2 after inoculation. IMH was applied locally following drug administration using a 42 MHz radiofrequency electromagnetic field. Treatment-induced changes were assessed by T₁- and T₂-weighted MRI, haematoxylin–eosin–orange (H&E) staining, and Ki-67 and p53 immunohistochemistry. Moran’s spatial autocorrelation index was used as a measure of tumour phenotypic heterogeneity in medical images. Results: Combination treatment with different doxorubicin formulations resulted in distinct patterns of intratumoural heterogeneity in MRI and in H&E-, Ki-67- and p53-stained images. FDOX or LDOX combined with IMH produced greater deviations from the control group in multimodal spatial organisation of sarcoma-45 than the corresponding drug treatments alone. LDOX + IMH reduced tumour growth by 34% relative to the control group and was associated with distinct histological and immunohistochemical remodelling, including connective tissue replacement and the lowest Ki-67 staining level. The lowest p53 staining levels were observed in the LDOX and LDOX + IMH groups. Conclusions: FDOX and LDOX combined with IMH induced distinct tumour remodelling patterns in sarcoma-45. Multimodal analysis of intratumoural heterogeneity in sarcomas may provide additional quantitative information for assessing treatment response.

Background: Visualising magnetic nanoparticle (MNP) clusters is important for inductive moderate hyperthermia (IMH) as their formation influences mechanical force transduction and heat generation in malignant tumours. An applied inhomogeneous stationary magnetic field (ISMF) can direct the arrangement of MNP clusters and the force exerted on cancer cells. Herein, we analysed MNP cluster formation and changes in mechanical properties using digital breast tomosynthesis (DBT) and ultrasound shear wave elastography (SWE) for a breast phantom containing MCF-7 breast cancer cells under the influence of IMH with ISMF. Results: Texture analysis of the tumour-mimicking region revealed that ISMF induced a 13% increase in fractal dimension and a twofold decrease in lacunarity of MNP clusters on DBT images, as well as a 20% decrease in lacunarity of apparent stiffness within fixed regions of interest on SWE images, as compared with non-targeted MNPs (p < 0.05). While the addition of MNPs increased the maximum temperature in the tumour-mimicking region only by 1.3 °C, it led to a 3.8-fold decrease in lacunarity and a 10% increase in fractal dimension on thermal images, as well as an 87% lower fraction of viable MCF-7 cells than IMH with ISMF (p < 0.05). We also considered the clinical relevance for breast cancer patients, given that the heat-pain threshold (~ 42 °C) is close to the temperatures observed during IMH, whereas the forces generated by ISMF remain below typical pressure-pain thresholds. Conclusions: ISMF initiated a more uniform spatial distribution of MNP clusters, altering the SWE-derived apparent stiffness and temperature patterns in the tumour-mimicking region. While causing only a moderate temperature increase (< 42 °C), IMH combination with ISMF and MNPs significantly reduced MCF-7 viability, indicating the additional role of magneto-mechanical effects.

Background: Osteosarcoma (OS) is the most common primary malignant bone tumor. Doxorubicin (DOX) is extensively used in OS chemotherapy, yet improving patient outcomes remains challenging. This study investigated the effect of free DOX combined with inductive moderate hyperthermia (IMH) on Saos-2 human OS cells. Methods: Cell viability was assessed by trypan blue exclusion. Flow cytometry analyzed apoptosis, necrosis, and reactive oxygen species (ROS) in cells exposed to control (no treatment), IMH (42 MHz frequency, 500 μT magnetic field induction, 564 V/m electric field strength, 15 W output power, and 30 min duration) alone, DOX (0.06 μg/mL) alone, or DOX combined with IMH. The expression of p14^ARF tumor suppressor and epidermal growth factor receptor (EGFR) was evaluated by immunocytochemistry. Spatial autocorrelation analysis quantified the heterogeneity of p14^ARF and EGFR distributions in acquired images. Results: The half maximal inhibitory concentration (IC₅₀) of DOX in Saos-2 cells had minimal variation between 48 h (0.060 ± 0.01 μg/mL) and 72 h (0.055 ± 0.003 μg/mL). DOX + IMH resulted in a 15% increase in early apoptosis and a 20% elevation in ROS levels compared with DOX alone. Immunocytochemical analysis revealed a 37% increase in p14^ARF and a 32% reduction in EGFR expression following combined treatment in comparison to DOX alone. Image analysis showed that DOX + IMH treatment caused the highest Moran’s index values for p14^ARF and EGFR, reflecting less heterogeneous spatial distributions (p < 0.05). Conclusions: IMH enhanced DOX-induced cytotoxicity in Saos-2 cells by initiating ROS-mediated apoptosis and reducing heterogeneity of cellular responses.

Objective: to examine the combined effect of doxorubicin-loaded superparamagnetic nanoparticles and non-ionising electromagnetic irradiation on Walker-256 carcinosarcoma using magnetochemical technology. Materials and methods: Non-inbred female rats were divided into the control (untreated tumour-bearing) and experimental group receiving superparamagnetic Fe3O4 nanoparticles (3 mg/kg, Sigma-Aldrich, USA) loaded with doxorubicin (1.5 mg/kg, Pfizer, Italy) and exposed to a static magnetic (30 mT) and electromagnetic field (42 MHz) for 15 minutes (5 sessions from day 2 after tumour inoculation). Tumour structural changes were evaluated using magnetic resonance imaging at 1.5 T field strength and 63.9 MHz frequency (Intera, Philips, Netherlands), followed by texture heterogeneity analysis with LifeX software (France). Free iron, ceruloplasmin, superoxide radical and nitric oxide were measured by electron paramagnetic resonance spectroscopy to characterise the tumour and blood redox state. Results. Combined treatment reduced tumour volume by 44–64 % on days 13–18 compared with the control (p < 0.05). Texture parameters (intensity, entropy, dissimilarity, autocorrelation, cluster tendency) in tumour regions of interest on T2-weighted images were 17–53 % lower than in the control (p < 0.05). After treatment, electron paramagnetic resonance spectroscopy revealed lower superoxide radical (4.5-fold) and nitric oxide (1.2-fold) levels in tumour samples, in addition to increased free iron, ceruloplasmin (by 11 %) and reduced superoxide (by 54 %), nitric oxide (by 26 %) in blood samples of tumour-bearing animals, compared with the control (p < 0.05). Conclusion. Doxorubicin-loaded superparamagnetic nanoparticles combined with electromagnetic irradiation inhibit Walker-256 carcinosarcoma growth, reduce intratumour heterogeneity and initiate changes in the tumour and blood redox state. Key words: nanocomplex; doxorubicin; electromagnetic irradiation; magnetochemical technology; magnetic resonance imaging; tumour heterogeneity; redox state; Walker-256 carcinosarcoma.

Evaluating intratumor heterogeneity with image texture analysis offers a more sophisticated understanding of sarcoma response to treatment. We examined the effects of inductive moderate hyperthermia (IMH) on sarcoma-45 growth and intratumor heterogeneity across tissue, cellular and molecular levels using magnetic resonance imaging (MRI), ultrasound and histology image analysis. IMH (42 MHz, 20 W) inhibited sarcoma-45 growth kinetics by 34% compared to the untreated control group. T₂-weighted MRI brightness was increased by 42%, reflecting more extensive tumor necrosis, while Young’s modulus increased by 37% due to more pronounced connective tissue replacement in response to IMH. Whereas calculations of Moran’s spatial autocorrelation index revealed distinctions in heterogeneity between tumor core, periphery and capsule regions of interest (ROIs) on MRI, ultrasound and histological examination in the untreated tumor-bearing animals, there was no significant difference between core and periphery after IMH. Exposure to IMH increased overall tumor ROI heterogeneity by 22% on MRI but reduced heterogeneity in the core and periphery on ultrasound and histology images. Ki-67 protein distribution was 25% less heterogeneous on the tumor periphery after IMH. Therefore, this study provides a quantitative characterization of IMH effects on different manifestations of intratumor sarcoma-45 heterogeneity using experimental imaging data.

Magnetic nanocomposites (MNC) are promising theranostic platforms with tunable physicochemical properties allowing for remote drug delivery and multimodal imaging. Here, we developed doxorubicin-loaded Fe₃O₄–Au MNC (DOX-MNC) using electron beam physical vapor deposition (EB-PVD) in combination with magneto-mechanochemical synthesis to assess their antitumor effect on Walker-256 carcinosarcoma under the influence of a constant magnetic (CMF) and electromagnetic field (EMF) by comparing tumor growth kinetics, magnetic resonance imaging (MRI) scans and electron spin resonance (ESR) spectra. Transmission (TEM) and scanning electron microscopy (SEM) confirmed the formation of spherical magnetite nanoparticles with a discontinuous gold coating that did not significantly affect the ferromagnetic properties of MNC, as measured by vibrating-sample magnetometry (VSM). Tumor-bearing animals were divided into the control (no treatment), conventional doxorubicin (DOX), DOX-MNC and DOX-MNC + CMF + EMF groups. DOX-MNC + CMF + EMF resulted in 14% and 16% inhibition of tumor growth kinetics as compared with DOX and DOX-MNC, respectively. MRI visualization showed more substantial tumor necrotic changes after the combined treatment. Quantitative analysis of T₂-weighted (T₂W) images revealed the lowest value of skewness and a significant increase in tumor intensity in response to DOX-MNC + CMF + EMF as compared with the control (1.4 times), DOX (1.6 times) and DOX-MNC (1.8 times) groups. In addition, the lowest level of nitric oxide determined by ESR was found in DOX-MNC + CMF + EMF tumors, which was close to that of the muscle tissue in the contralateral limb. We propose that the reason for the relationship between the observed changes in MRI and ESR is the hyperfine interaction of nuclear and electron spins in mitochondria, as a source of free radical production. Therefore, these results point to the use of EB-PVD and magneto-mechanochemically synthesized Fe₃O₄–Au MNC loaded with DOX as a potential candidate for cancer magnetic nanotheranostic applications.

Mechanoluminescence is the emission of light by biological objects in response to mechanical stimuli. Existing devices provide limited capability in mechanoluminescence detection for biological objects in the liquid state under the combined magneto-mechanical influence of magnetic nanoparticles when compared with the control measurements of spontaneous chemiluminescence in investigated samples. This emphasizes the need for technical solutions designed to investigate mechanoluminescence in a broader range of specimens and help understand the relationship between magnetic and mechanical forces exerted by magnetic nanoparticles on cancer cells. For this reason, the current study aims to present the analysis of a biophysical basis and equipment design for detecting mechanoluminescence emitted by cancer cells using magnetic nanoparticles. Biophysical aspects clarifying the origin of mechanoluminescence due to elastic, plastic deformation and destruction of biopolymers, as well as mechanical stress-induced free radical reactions, were discussed here. From the description of the proposed equipment design, it appears that the key technical solution to mechanoluminescence detection in cells with magnetic nanoparticles was to exploit a rotating magnetic field that produced the mechanochemical effect on cells with magnetic nanoparticles, giving rise to mechanoluminescence emission. In addition, the author outlined a sample preparation technology for mechanoluminescence detection in cancer cells with magnetic nanoparticles. Measurements of mechanoluminescence emission can potentially serve as an additional quantitative parameter of the magneto-mechanochemical effect on cancer cells in the course of magnetic nanotheranostics for improved accuracy of medical diagnosis as well as evaluation of tumor response to given treatment. Механолюмінесценція – це явище випромінювання світла біологічними об’єктами у відповідь на механічне подразнення. Відомі апарати для реєстрації механолюмінесценції достатньо обмежені у можливості проведення досліджень у біологічних об’єктах у рідкому стані під комбінованим магніто-механічним впливом магнітних наночастинок по відношенню до контрольних вимірів спонтанної хемілюмінесценції досліджуваних зразків. Актуальною є потреба у розробці технічного рішення для дослідження механолюмінесценції, що дозволяє використовувати більш широкий спектр аналізів для більш повного розуміння взаємозв’язку застосування магнітних і механічних сил магнітних наночастинок на злоякісні клітини. Тому, метою даної роботи було проаналізувати біофізичні основи та описати конструкцію запропонованого пристрою для реєстрації механолюмінесценції злоякісних клітин з використанням магнітних наночастинок. Розглянуто біофізичні аспекти механолюмінесценції при пружних, пластичних деформаціях та руйнуванні, а також ті, що можуть бути ініційовані хімічними вільнорадикальними реакціями, спричиненими механічним стресом. Наведено опис конструкції запропонованого пристрою для реєстрації механолюмінесценції клітин з магнітними наночастинками, принциповим технічним рішенням у якому є застосування ротаційного магнітного поля для механохімічного впливу та реєстрації фотонів механолюмінесценції клітин з магнітними наночастинками. Також, описано технологію підготовки проб та реєстрації механолюмінесценції злоякісних клітин з магнітними наночастинками. Оцінка випромінення механолюмінесценції може потенційно слугувати додатковим кількісним параметром магніто-механохімічного ефекту на злоякісні клітини при магнітній нанотераностиці для покращення точності медичної діагностики та оцінки відповіді пухлини на проведену терапію.

Despite efforts in osteosarcoma (OS) research, the role of inductive moderate hyperthermia (IMH) in delivering and enhancing the antitumor effect of liposomal doxorubicin formulations (LDOX) remains unresolved. This study investigated the effect of a combination treatment with LDOX and IMH on Saos-2 human OS cells. We compared cell viability using a trypan blue assay, apoptosis and reactive oxygen species (ROS) measured by flow cytometry and pro-apoptotic Bax protein expression examined by immunocytochemistry in response to IMH (42 MHz frequency, 15 W power for 30 min), LDOX (0.4 μg/mL), and LDOX plus IMH. The lower IC₅₀ value of LDOX at 72 h indicated increased accumulation of the drug in the OS cells. LDOX plus IMH resulted in a 61% lower cell viability compared to no treatment. Moreover, IMH potentiated the LDOX action on the Saos-2 cells by promoting ROS production at temperatures of p < 0.05). Therefore, we determined that IMH could enhance LDOX delivery and its antitumor effect via altered membrane permeabilization, ROS generation, and a lower level of visualized Bax heterogeneity in the Saos-2 cells, suggesting the potential translation of these findings into in vivo studies.

Introduction. Induced moderate hyperthermia (IMH) of malignant tumors involves the use of radio frequencies for selective local action of thermal and non-thermal effects in the tumor area. The current work aims to evaluate the effect of local inductive moderate hyperthermia (IMH) on the heterogeneity of different regions of interest (ROIs) delineated on MRI scans of sarcoma-45 bearing animals using texture analysis. Methods. The study of the experimental tumor process was conducted on non-inbred female rats (DNP “NIR”, Kyiv) with sarcoma-45. Computer planning of IMH parameters for different tumor ROIs based on Comsol Multiphysics v. 5.6. (COMSOL AB, Stockholm) software revealed that the average value of the specific absorption rate was higher in the tumor core (3.6 W/kg) than in the periphery (3.3 W/kg) or capsule (2.3 W/kg). Results. A significant tumor volume reduction was observed on day 24 following implantation in response to IMH delivered by a MagTherm (Radmir, Ukraine) apparatus. Conclusions. Comparative analysis of ROI heterogeneity, determined by the Moran's autocorrelation index of T1-weighted MRI scans, showed that IMH led to a 21 % decrease in the value of this texture parameter across all tumor ROIs, on average, as compared with the control group which received no treatment. The reported increase in heterogeneity of sarcoma-45 was most likely associated with a more pronounced formation of the fibrous capsule and tumor destruction in the core and periphery ROIs in response to IMH.

Summary. Chaos theory (nonlinear dynamics) defines cancer as a complex adaptive system in which each cyclic point corresponds to the bifurcation at which changes in signaling pathways emerge. Quantitative assessment of chaos in digital medical images such as electron microscopy, histology and cytology sections collected from patients with malignant cutaneous melanoma employed the following calculation parameters: the irregularity of external contours, internal heterogeneity based on brightness distribution of macromo­lecules, chromosomes, organelles, inclusion bodies, cells and tissues, kurtosis, entropy and the asymmetry coefficient. The present study undertook a nonlinear analysis of the chaotic hierarchy of malignant melanoma. However, considerably more studies will need to be carried out to determine the exact interrelationship between different levels of the hierarchy in the biological system. Multidisciplinary collaborations are therefore essential to find evidence to answer questions that remain open to researchers and oncologists.