The chick embryo chorioallantoic membrane (CAM) model is gaining increasing attention from cancer researchers worldwide. Its affordability, short experimental duration, robustness, and ease of tumor xenograft visualization make it a valuable tool in cancer research. This review explores recent advancements and potential applications of the avian CAM model, including the following: (1) studying tumor growth and metastasis, (2) investigating mechanisms of tumor chemoresistance, (3) optimizing drug delivery methods, (4) improving bioimaging techniques, (5) evaluating immuno-oncology drug efficacy, (6) examining tumor-extracellular matrix interactions, (7) analyzing tumor angiogenesis, and (8) exploring the roles of microRNAs in cancer. Additionally, we compare the in ovo CAM model with other in vivo animal models and in vitro cell culture systems. Positioned between in vitro and in vivo models in terms of cost-effectiveness and accuracy in cancer recapitulation, the CAM model enhances both preclinical and translational research. Its expanding use in cancer studies and therapy development is expected to continue growing.

Background: The implementation of proton beam irradiation (PBI) for breast cancer (BC) treatment is rapidly advancing due to its enhanced target coverage and reduced toxicities to organs at risk. However, the effects of PBI can vary depending on the cell type. This study aimed to explore the effects of PBI on two BC cell lines, MCF7 and MDA-MB-231. Methods: The relative biological effectiveness (RBE) of PBI was assessed using a clonogenic assay. DNA double-strand break (DSB) repair, epithelial–mesenchymal transition (EMT), and filamentous actin (F-actin) were evaluated using immunofluorescence analysis. The extent of entosis and the senescence-associated β-galactosidase (SA-β-gal) activity were estimated by cytochemistry analysis. The influence of the extracellular matrix was evaluated by cultivating cells in both adherent two-dimensional (2D) environments and within 3D fibrin gels of varying stiffness. The metastatic propensity of cells was investigated using migration tests and the cell encapsulation of carboxylate-modified fluorescent nanoparticles. The comparative tumorigenic potential of cells was investigated using an in vivo model of the chick embryo chorioallantoic membrane (CAM). Results: PBI demonstrated superior efficacy in eliminating MCF7 and MDA-MB-231 cells with RBE 1.7 and 1.75, respectively. Following PBI, MDA-MB-231 cells exhibited significantly lower clonogenic survival compared to MCF7, which was accompanied by the accumulation of phosphorylated histone H2AX (γH2AX), p53-binding protein 1 (53BP1) and Rad51 foci of DNA DSBs repair proteins. After surviving 7 days post-PBI, MCF7 cells exhibited 2.5-fold higher levels of the senescence phenotype and entosis compared to the MDA-MB-231 offspring. Both PBI-survived cell lines had greater capability for 2D collective migration, but their metastatic potential was significantly reduced. A significant influence of extracellular matrix stiffness on the correlation between F-actin expression in PBI-survived cells—an indicator of cell stiffness—and their ability to uptake nanoparticles, a trait associated with metastatic potential, was observed. PBI-survived MDA-MB-231RP subline exhibited a hybrid EMT phenotype and a 70% reduction in tumor growth in the in vivo model of the chick embryo CAM. In contrast, PBI-survived MCF7RP cells exhibit mesenchymal-to-epithelial transition (MET)-like features, and their in vivo tumor growth increased by 66% compared to parental cells. Conclusions: PBI triggers various cellular responses in different BC cell lines, influencing tumor growth through mechanisms like DNA damage repair, stress-induced premature senescence (SIPS), and alterations in the stiffness of tumor cell membranes. Our insights into entosis and the effect of extracellular matrix stiffness on metastatic propensity (nanoparticle uptake) enhance the understanding of the role of PBI in BC cells, emphasizing the need for more research to optimize its therapeutic application.

Idiopathic toe walking (ITW) occurs in about 5% of children. Orthopedic treatment of ITW is complicated by the lack of a known etiology. Only half of the conservative and surgical methods of treatment give a stable positive result of normalizing gait. Available data indicate that the disease is heterogeneous and multifactorial. Recently, some children with ITW have been found to have genetic variants of mutations that can lead to the development of toe walking. At the same time, some children show sensorimotor impairment, but these studies are very limited. Sensorimotor dysfunction could potentially arise from an imbalanced production of neurotransmitters that play a crucial role in motor control. Using the data obtained in the studies of several pathologies manifested by the association of sensory–motor dysfunction and intestinal dysbiosis, we attempt to substantiate the notion that malfunction of neurotransmitter production is caused by the imbalance of gut microbiota metabolites as a result of dysbiosis. This review delves into the exciting possibility of a connection between variations in the microbiome and ITW. The purpose of this review is to establish a strong theoretical foundation and highlight the benefits of further exploring the possible connection between alterations in the microbiome and TW for further studies of ITW etiology.

We developed a sensitive bioimaging system for sarcoma by creating a cell line that stably expresses both Katushka2S fluorophore and NanoLuc luciferase, enabling robust dual tracking of tumor growth and metastasis in the chick chorioallantoic membrane (CAM) model. NanoLuc luciferase was notably more effective than Katushka2S for identifying tumor cell metastases in embryo tissues. Pretreating CAM with tumor cell-conditioned medium (TCM) significantly increased neovascularization, Ki67 expression, tumor volumes, and metastasis of the most difficult-to-establish low-tumorigenic and low-metastatic U2OS cells, indicating that tumor cell secretome actively alter the CAM vascular environment to aid tumor progression. The bead-based multiplex profiling of the TCM demonstrated a notable increase in pro-angiogenic factors. Neutralizing VEGFA, the most abundant factor in the TCM, effectively counteracted vascular and pro-metastatic effects of TCM. In contrast, elevating VEGFA levels brought back the pro-tumorigenic effects of TCM. This study reveals the importance of tumor cell secretomes in creating the vascular niche in CAM and points to VEGFA as a target to prevent secretome-induced angiogenesis and sarcoma development. Furthermore, our optimized CAM model permits continuous tumor growth and metastasis monitoring in embryonic development, providing a reliable platform for prognostic studies of sarcoma treatments in ongoing anti-angiogenic and multikinase inhibitors trials.

Resistance to anti-angiogenic therapy remains a major obstacle in non–small cell lung cancer (NSCLC). Although bevacizumab effectively neutralizes VEGFA and suppresses neovascularization, its clinical benefit is often transient. Here, we demonstrate that VEGFA inhibition paradoxically intensifies intratumoral hypoxia, leading to stabilization of HIF1A and activation of a compensatory pro-malignant program. Increased HIF1A not only enhances tumor cell migration, epithelial–mesenchymal transition, and chemoresistance, but also upregulates CD105 (Endoglin), a hypoxia-responsive pro-angiogenic mediator that attenuates the anti-vascular effects of bevacizumab. Thus, anti-VEGFA therapy initiates a hypoxia-driven HIF1A–CD105 axis that sustains tumor aggressiveness and vascular adaptation despite VEGFA blockade. We identify the pre–miR-16-1 duplex as a physiological dual-regulatory system capable of simultaneously targeting this adaptive circuit. The guide strand miR-16-5p directly represses VEGFA, recapitulating the anti-angiogenic action of bevacizumab. In contrast, the passenger strand miR-16-1-3p suppresses HIF1A expression, thereby preventing hypoxia-induced malignant phenotypes and limiting CD105 upregulation. Functional analyses revealed that VEGFA inhibition alone promotes hypoxia-associated migration, cytoskeletal remodeling, and cisplatin resistance, whereas co-modulation of miR-16-1-3p abrogates these effects. In a chick chorioallantoic membrane xenograft model, dual regulation of VEGFA and HIF1A markedly reduced vascular density, tumor growth, and metastatic dissemination compared with single anti-angiogenic intervention. Collectively, our findings uncover a hypoxia-mediated resistance mechanism driven by the HIF1A–CD105 axis following VEGFA inhibition and establish the cooperative function of the miR-16 duplex as a strategy to concurrently suppress angiogenesis and its adaptive hypoxic feedback. Targeting both VEGFA and HIF1A may therefore improve the durability of anti-angiogenic therapy in NSCLC.

Resistance to anti-angiogenic therapy remains a major obstacle in non–small cell lung cancer (NSCLC). Although bevacizumab effectively neutralizes VEGFA and suppresses neovascularization, its clinical benefit is often transient. Here, we demonstrate that VEGFA inhibition paradoxically intensifies intratumoral hypoxia, leading to stabilization of HIF1A and activation of a compensatory pro-malignant program. Increased HIF1A not only enhances tumor cell migration, epithelial–mesenchymal transition, and chemoresistance, but also upregulates CD105 (Endoglin), a hypoxia-responsive pro-angiogenic mediator that attenuates the anti-vascular effects of bevacizumab. Thus, anti-VEGFA therapy initiates a hypoxia-driven HIF1A–CD105 axis that sustains tumor aggressiveness and vascular adaptation despite VEGFA blockade. We identify the pre–miR-16-1 duplex as a physiological dual-regulatory system capable of simultaneously targeting this adaptive circuit. The guide strand miR-16-5p directly represses VEGFA, recapitulating the anti-angiogenic action of bevacizumab. In contrast, the passenger strand miR-16-1-3p suppresses HIF1A expression, thereby preventing hypoxia-induced malignant phenotypes and limiting CD105 upregulation. Functional analyses revealed that VEGFA inhibition was accompanied by HIF1A upregulation, while elevated HIF1A promoted cell migration, mesenchymal-like changes, and cisplatin resistance. Co-expression of miR-16-1-3p suppressed HIF1A and attenuated these adverse phenotypes. In a chick chorioallantoic membrane xenograft model, dual regulation of VEGFA and HIF1A markedly reduced vascular density, tumor growth, and metastatic dissemination compared with single anti-angiogenic intervention. Collectively, our findings uncover a hypoxia-mediated resistance mechanism driven by the HIF1A–CD105 axis following VEGFA inhibition and establish the cooperative function of the miR-16 duplex as a strategy to concurrently suppress angiogenesis and its adaptive hypoxic feedback. Concurrent targeting of VEGFA and HIF1A may therefore provide a strategy to enhance the antitumor efficacy of anti-angiogenic therapy in NSCLC.