WP2, Genetic, Epigenetic, Morphology studies of 3D Cultures

Our study will be focused on the realization of tumoroids from tissue aliquots taken from patients with histological diagnosis of and BC and LUAD in humans. We will distinguish, in the each tumoroid, with specific markers, the different cells present in it, we will analyze the epigenetic state analyzing by bisulphite-seq the methylome, and by ATAC-seq we will evaluate the gene-promoter chromatin changes in steady-state or during treatment (drug or specific modification or stress) respect to the normal cells. Specific markers of chromatin changes and related gene expression will be identified by using RNA-seq, which will allow us to build a specific map in each tumor analyzed. The epigenetic changes in specific promoter will be confirmed by Chromatin-Immuno-Precipitation (ChIP) in each gene. All the results obtained will be analyzed by bio-informatics platform (to see WP4).

Tissues dedicated to routine diagnosis will be fixed in 10% buffered formalin. After dehydration, they will be paraffin embedded and sections will be cut to a thickness of 3 mm. Organoids will be released from Matrigel and fixed in 4% buffered paraformaldehyde, washed in phosphate-buffered saline (PBS), and resuspended in pre-dissolved 1% agarose. Agarose blocks will be embedded in paraffin and sectioned as human tissues.

For immunofluorescence analysis, organoids will be deparaffinized, rehydrated, and antigen retrieved in a steam pressure cooker with citrate buffer (pH 6.0). The slides will be blocked in 0.1% bovine serum albumin (BSA), 0.2% Triton X-100, and 0.05% Tween 20 in PBS for 1 h at room temperature (RT). Images will be acquired on a Nikon Eclipse C1 confocal microscope and processed using the Fiji software package.

Whole-exome sequencing (WES) will be performed in patient-derived organoids to profile them along with their matched primary tumors and NAT tissue samples in order to identify somatic mutations in tumor and organoid samples.

Genomic DNA isolation from diagnostic tissues (formalin-fixed paraffin-embedded tissues, FFPE) will be performed using the Maxwell® 16 FFPE Tissue LEV DNA Purification Kit (Promega). gDNA will be isolated from organoids using the QIAamp DNA Mini Kit (QIAGEN).

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) will be performed to observe the organoid surface and ultrastructure. Because of its ultrafine resolution, TEM is an excellent tool for imaging the details of organoid structure. For the TEM study, organoids will be fixed in glutaraldehyde, post-fixed in osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sectionswill be double stained with uranyl acetate and lead citrate. Ultrathin sections (90 nm) will be examined under a Philips EM 208 equipped with a digital camera (Centro Universitario di Microscopia Elettronica e a Fluorescenza [CUMEF]—University of Perugia). SEM does not require ultra-thin specimens; the depth of field is many times greater than TEM and the image can show the three- dimensional topography and surface structure of the specimen. For SEM analysis, the organoids will be fixed in glutaraldehyde, dehydrated and coated with gold. The samples will be examined with a ZEISS-LEO 1525 (Laboratorio Universitario di Nanomateriali—University of Perugia).