Integrator system vis

In order to assess biomarker changes, a pharmacodynamic study was performed for 4 days of treatment with vistusertib, fulvestrant or a combination of the two drugs in the HBCx22 OvaR PDX model. Mice were sacrificed at 4 h after the final treatment and tumours resected. Excised tumours were fixed in 10% neutral buffered formalin and paraffin-embedded, and tissue microarrays (TMA) were built from the blocks. Three xenografts from each treatment group and two tissue cores per tumour were included in the TMA. Sections from the TMA were cut and stained for the expression of biomarkers, as previously described [12 (link)]. The immunohistochemically stained TMA sections were digitally scanned at ×20 with a Hamamatsu NanoZoomer-XR whole-slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan). The quality of the images was checked manually, and the images were analysed with Visiopharm integrator system (VIS) version 2018.9.3.5303 (Visiopharm A/S) using VIS ready to use automated image analysis algorithms (APPs).

To determine the area and volume of spared tissue, we followed a modified method of eriochrome cyanine (EC) staining from Rabchevsky and colleagues [38 (link)] and described in detail by Reigada, 2022 [39 (link)]. Briefly, sections were sequentially stained with EC followed by counter-staining with 5% iron alum and borax-ferricyanide solutions. After dehydration with increasing concentrations of ethanol and Histochoice Cleaning Agent (Sigma-Aldrich), the samples were mounted using DPX (Sigma-Aldrich). EC stained white matter myelin and allowed us to differentiate spared white matter from grey matter or damaged tissue. The area and volume of spared white matter were estimated through the stereological analysis of sections comprising 1 cm around the epicentre (200 µm between sections), using Cavalliery’s method in an Olympus BX61microscope (Olympus) equipped with a motorised stage coupled to a computer running the stereology software Olympus VIS system composed by the Visiopharm Integrator System (VIS; Visiopharm, Horsholm, Denmark) software and the NewCast module for stereology acquisition and image analysis. We randomly overlayed a 2D grid of crosses on top of each section image (20,000 µm² per cross) and the number of hitting crosses was used to obtain an unbiased estimate of the areas. The white matter area was relativised to the total area of each section.

Scanning of ISH stains was done on the Nanozoomer 2.0 (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Using an original magnification of 40× the images were digitally analysed using the software Visiopharm Integrator System (VIS, Visiopharm, Hoersholm, Denmark), as we have described previously [35 (link)]. We performed a threshold analysis using an intensity interval of 0–180.