Ultrastructural analysis of breast cancer patient-derived organoids

Background Breast cancer Patient Derived Organoids (PDO) have been demonstrated to be a reliable model to study cancer that promised to replace and reduce the use of animals in pre-clinical research. They displayed concordance with the tissue of origin, resuming its heterogenicity and representing a good platform to develop approaches of personalized medicines. Although obtain PDOs from mammary tumour, was a very challenging process, several ongoing studies evaluated them as a platform to study efficacy, sensitivity and specificity of new drugs and exploited them in personalized medicine. Despite tissue organization represented a crucial point to evaluate in a 3-dimensional model, since it could influence drug penetration, morphology of breast cancer PDOs has not been analysed yet. Here, we proposed a complete ultrastructural analysis of breast PDOs obtained from tumour and healthy tissues to evaluate how typical structures observed in mammary gland were resumed in this model. Methods 81 samples of mammary tissue (healthy or tumour) resulting from surgical resections have been processed to obtain PDO. The resulting PDOs embedded in matrigel drop have been processed for transmission electron microscopy and analysed. A comparison between ones from healthy and ones from cancerous tissue has been performed and PDOs derived from tumour tissue have been stratified according to their histological and molecular subtype. Result The morphological analysis performed on 81 PDO revealed an organized structure rich in Golgi, secretion granules and mitochondria, which was typical of cells with a strong secretory activity and active metabolism. The presence of desmosomes, inter and intracellular lumens and of microvilli and interdigitations signified a precise tissue-organization. Each PDO has been classified based on whether or not it possessed (i) peripheral ridges in mitochondria, (ii) intracellular lumens, (iii) intercellular lumens, (iv) micro-vesicles, (v) open desmosomes, (vi) cell debris, (vii) polylobed nuclei, (viii) lysosomes and (ix) secretion granules, in order to identify features coupled with the cancerous state or with a specific histological or molecular subtype. Conclusion Here we have demonstrated the suitability of breast cancer PDO as 3-dimensional model of mammary tissue. Besides, some structural features characterizing cancerous PDO have been observed, identifying the presence of distinctive traits.

Ultrastructural Analysis of Apoptosis Induced By Zinc Oxide Nanosphere in Human Hepatoma Huh7 Through Mitochondria Dysfunction

The efficiently of hepatocellular carcinoma therapy predominantly depends on advancement of nanomedicine. Zinc oxide nanostructure is promising material in nanomdicine field due to their unique properties. Zinc oxide Nanosphere (ZnO Ns) was fabricated with a size 50 nm diameter by a developed Sol-gel approach, using non-toxic biotemplate yeast extract. The physicochemical properties of zinc oxide nanosphere were estimated by Fourier-transform infrared spectrum (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscope (TEM). Liberated zinc ions released from the zinc oxide nanosphere suspended medium was determined by ICP-AS. The viability was tested by using HuH7 and Vero cells.ZnO nanosphere was more effectively on cell line than released Zn ions. The cell cycle arrested at G1/S. Also, the apoptosis assay by using Annexin-V/PI showed that apoptosis of HuH7 by ZnO nanosphere is concentration and time-dependent. The mechanism of apoptosis was evaluated via using techniques such as RT-PCR and flow cytometry. The results revealed significance up-regulated of Bax, P53, and Cytochrome C, while a Bcl2 results displayed significance down-regulated.Caspase 3 assay results showed that the apoptosis mechanism was intrinsic and extrinsic pathways. Also, ZnO nanosphere and free Zn+2 ions induced oxidative stress via increasing reactive oxygen species (ROS) and lipid peroxidation. The ultrastructural analysis of HuH7 cell was occurred by TEM.Transmission electron microscope analysis of HuH7 after treated with ZnO nanosphere at different times revealed a chromatin condensation of the nuclear periphery fragmentation appearance. Interestingly, the apoptosis of HuH7 cells induced by Zinc oxide nanosphere, showed the canonical ultrastructure features of apoptotic nuclei, and fragmented by budding. Furthermore, There were many vacuoles that filled in the cytoplasm, majority lipid droplets, which resembling those observed in foamy cells, and vesicles with intact membranes, which were recognized as swollen mitochondria.