Cell d software

All relevant organs described above were paraffin-embedded and stained with H&E to perform a complete histopathological analysis and a clinical pathologist supervised all samples for toxicity evaluation. Tumor sections stained with either H&E or DAPI were also used to assess the cytotoxic effect of the administered T22-PE24-H6 nanotoxin in vivo, by counting the number of cell death bodies in 10 high-power fields (magnification 400x). DAPI staining was performed in Triton X-100 (0.5%) permeabilized sections mounted with DAPI mounting media (ProLong Gold antifade reagent, Thermo Fisher). Samples were evaluated under a fluorescence microscope at a wavelength of λex = 334 nm/λem = 465 nm.
In the orthotopic in vivo experiments, the spreading of cells from the primary tumor (colon) to other distant organs was analyzed in H&E-stained samples. We studied those organs in which metastatic dissemination is expected in CRC: liver, lung, mesenteric lymph nodes, and peritoneum. An Olympus microscope with the Cell^D Olympus software was used to count the number and measure the size (expressed as µm²) of all observed metastatic foci in three different slices of each organ.

We histologically examined the primary tumours and all organs with expected metastases, to evaluate the degree of differentiation, necrotic areas, apoptotic and mitotic rate, and tumour invasion and vascularization. Three H&E sections for each metastatic site were examined microscopically to identify micro- and macro-metastases in each organ.
We recorded the number and the area of micro- and macroscopic foci found in the affected organs. Foci areas were quantified using CellD Olympus software (v3.3, Olympus, Japan). Metastatic foci were considered macroscopic when their diameter exceeded 1 mm, meaning that the measured area in their tumour sections was higher than 785,000 μm². All foci with a diameter lower than 1 mm were considered microscopic (Folkman, 1983 (link)). Visible foci were defined as those reaching a diameter of over 3 mm.
To compare the invasive capacity of the ORT and SC+ORT primary tumours, we counted 40 fields (3–5 sections per tumour) at the tumour invasive front for each tumour group. After staining with anti-A1/A3 keratin, we recorded the number of keratin-positive single cells and keratin-positive tumour cell clusters, containing five or less cells (tumour budding), per 100× tumour field.
H&E primary tumour sections of each group were used to quantify the number of apoptotic cells per field. Ten 400× fields per primary tumour were analyzed.