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.

During the cell life cycle, extracellular vesicles (EVs) transport different cargos, including organelles, proteins, RNAs, DNAs, metabolites, etc., that influence cell proliferation and apoptosis in recipient cells. EVs from metastatic cancer cells remodel the extracellular matrix and cells of the tumor microenvironment (TME), promoting tumor invasion and metastatic niche preparation. Although the process is not fully understood, evidence suggests that EVs facilitate genetic material transfer between cells. In the context of NSCLC, EVs can mediate intercellular mitochondrial (Mt) transfer, delivering mitochondria organelle (MtO), mitochondrial DNA (mtDNA), and/or mtRNA/proteinaceous cargo signatures (MtS) through different mechanisms. On the other hand, certain populations of cancer cells can hijack the MtO from TME cells mainly by using tunneling nanotubes (TNTs). This transfer aids in restoring mitochondrial function, benefiting benign cells with impaired metabolism and enabling restoration of their metabolic activity. However, the impact of transferring mitochondria versus transplanting intact mitochondrial organelles in cancer remains uncertain and the subject of debate. Some studies suggest that EV-mediated mitochondria delivery to cancer cells can impact how cancer responds to radiation. It might make the cancer more resistant or more sensitive to radiation. In our review, we aimed to point out the current controversy surrounding experimental data and to highlight new paradigm-shifting modalities in radiation therapy that could potentially overcome cancer resistance mechanisms in NSCLC.

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.

The TGF-β signaling pathway has both tumor-suppressive and metastasis-promoting effects in cancer, yet the molecular determinants governing this switch remain unclear. Here, we investigated the miR-16-1-3p/MDM2/p53 axis as a potential regulator of TGF-β/Smad signaling output in osteosarcoma. miR-16-1-3p overexpression alone markedly reduced the proliferative and clonogenic potential of U2OS cells, while its combination with TGF-β treatment strongly enhanced G1-phase arrest and nearly abolished tumor growth capacity. miR-16-1-3p inhibited TGF-β-induced actin remodeling and epithelial-mesenchymal transition (EMT)-associated changes. While TGF-β promoted both 2D and 3D migration, miR-16-1-3p overexpression, alone or combined with TGF-β, counteracted its pro-migratory effects. Mechanistically, miR-16-1-3p reduced MDM2 expression and stabilized p53, which was associated with enhanced p21 induction and suppression of proliferative responses under TGF-β stimulation. Co-administration of TGF-β and miR-16-1-3p markedly increased cisplatin sensitivity in wild-type U2OS cells and reduced tumor nodule volume, Ki67 expression, and metastasis in the chicken chorioallantoic membrane model. Collectively, these findings suggest that miR-16-1-3p biases TGF-β signaling output toward anti-growth responses, while attenuating pro-migratory effects through MDM2 inhibition and p53 stabilization, providing a mechanistic rationale for improving therapeutic responses in osteosarcoma.

Oncogenesis is inevitably associated with microRNA expression deregulation. Thus, development of both miRNA targeting substances or small RNA for ant-cancer therapy have been reported. Specifically, repression of the miR-16-1-3p and miR-16-2-3p activities play pivotal roles in osteosarcoma and many other cancers. The majority of miRNA sensors use protein degradation to measure miRNA activities. Here we report miRNA sensors that use fluorescent protein synthesis rather than degradation to measure miRNA activity. Specifically, miR-16-1-3p and miR-16-2-3p sensors consist of the bidirectional tet-On system driving the expression of the Katusha2S protein that is regulated by the RNA interference and GFP as a reference. These sensors specifically detect mir16-1-3p and mir16-2-3p small RNA mimics in the osteosarcoma cell line after doxycycline induction. Kinetic measurements of the reporter responses to the miRNA mimics revealed that pre-induced sensors reach significant differences from the control faster, within 2h than the sensors that were induced after mimic transfection. Thus, kinetic measurements of the fluorescent protein synthesis during doxycycline induction of the tet-On system are feasible for determination of the small RNA activity.

Background: Chemotherapy resistance and disease progression remain major causes of mortality in osteosarcoma (OS). The miR-16 family is linked to tumors, but the specific function and treatment potential of the miR-16-1-3p passenger strand in OS remain unclear. Methods: RNA sequencing and clinical data from 82 OS patients in the TARGET-OS cohort were analyzed. Patients were stratified by progression status. Target genes of miR-16-1-3p were predicted using TargetScan and evaluated by gene set enrichment analysis (GSEA). Principal component analysis (PCA) and Kaplan–Meier survival analyses were performed based on cumulative Z-score–derived expression of the target gene set. Functional enrichment was assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The impact of lentivirus-transduced miR-16-1-3p on U2OS cells was examined, focusing on proliferation, cell cycle, migration, colony formation, cisplatin sensitivity, and tumor growth in the CAM in vivo model. A fluorescent miRNA sensor was developed to assess intracellular target-binding functionality of the miR-16-1-3p passenger strand. Direct regulation of candidate target gene expression was validated using luciferase reporter assays and quantitative PCR. Expression of validated targets was further examined in a limited cohort of 15 chemotherapy-treated OS patients. Results: GSEA demonstrated significant enrichment of predicted miR-16-1-3p target genes among genes upregulated in patients with progressive disease. Kaplan–Meier analysis showed that elevated cumulative expression of predicted miR-16-1-3p target genes was associated with poorer overall survival in osteosarcoma patients. GO and KEGG analyses revealed enrichment of pathways related to translational regulation, mitochondrial function, and chemotherapy-associated signaling. A fluorescent miR-16-1-3p sensor system demonstrated sequence-dependent suppression of reporter activity following miR-16-1-3p overexpression. In functional assays performed in both U2OS and HOS cells, miR-16-1-3p overexpression significantly suppressed proliferation and migration and increased cisplatin sensitivity compared with control (scrambled miRNA) virus-transduced cells. Quantitative PCR and luciferase reporter assays further supported the interaction of miR-16-1-3p with SLC38A1 and ABCA13, two candidate genes potentially associated with chemoresistance. Conclusion: The miR-16-1-3p passenger strand exhibited functional activity associated with tumor-suppressive phenotypes and increased cisplatin sensitivity in osteosarcoma cells. Using integrative bioinformatics, fluorescent sensor assays, and functional experiments in U2OS and HOS cells points to miR-16-1-3p possibly regulating cancer characteristics and how osteosarcoma reacts to chemotherapy.