Nanozoomer s360 digital slide scanner

The tissue samples were processed by the Pathology Core at City of Hope, which included embedding, sectioning, and immunohistochemical staining. The HO-1 IHC analysis was performed using the Ventana Discovery Ultra (Ventana Medical Systems, Roche Diagnostics, Indianapolis, USA) automated stainer. Briefly, 5 μm sections of tissue were mounted on positively charged glass slides. The slides were deparaffinized and rehydrated, then treated with an endogenous peroxidase activity inhibitor and antigen retrieval reagent. They were then incubated with an anti-human HO-1 mouse monoclonal primary antibody (1:500; Novus Biologicals), followed by an anti-mouse IgG1 + IgG2a-IgG3 rabbit monoclonal secondary antibody (1:500; Abcam, Cambridge, UK). The staining was visualized using the ChromoMap DAB Kit (CRB DISCOVERY) and counterstained with hematoxylin (Ventana) and cover slips. After IHC staining, whole slide images were acquired using the NanoZoomer S360 Digital Slide Scanner (Hamamatsu Photonics, Las Vegas, NV, USA) and viewed using the NDP.view image viewer software (Hamamatsu Photonics).

Histological examinations were processed by the Pathology Core at the City of Hope. Heart and kidneys from mice were collected and fixed in 4% paraformaldehyde overnight. The fixed tissues were later dehydrated, sectioned into 4 µm paraffin slides, and subjected to Hematoxylin and Eosin (HE), Periodic acid-Schiff (PAS), or Masson’s trichrome staining. For immunohistochemistry (IHC), the slides were deparaffinized, rehydrated and incubated with endogenous peroxidase activity inhibitor and antigen retrieval reagents. IHC was performed on the Ventana Discovery Ultra IHC automated Stainer (Ventana Medical Systems, Roche Diagnostics, Indianapolis, IN, USA). Anti-mouse CD31 (Lot# 4, Cat#77699, Cell Signaling Technology, Danvers, MA, USA) was used at 1:200 dilution, followed by DISCOVERY anti-rabbit HQ (Cat#760-4815, Roche, Indianapolis, IN, USA) and DISCOVERY anti-HQ-HRP (Cat#760-4820, Roche, Indianapolis, IN, USA) incubation. The stains were visualized with DISCOVERY ChromoMap DAB Kit (Cat# 760-159, Ventana) and counterstained with hematoxylin. The IHC stained slides were digitalized and documented by NanoZoomer S360 Digital Slide Scanner (Hamamatsu, Bridgewater, NJ, USA) and viewed by NDP.view2 image viewer software (U12388-01, Bridgewater, NJ, USA).

Organoids were washed and centrifuged at 2 °C at 300 rcf for 5 min. The pellet was resuspended in formalin for 2 h, after which it was embedded in 2.5% low-melting agarose and followed by paraffin embedding. Hematoxylin and eosin (H&E)-stained FFPE sections of both primary tumors and organoids were analyzed by a board-certified veterinary pathologist (M.D.). Primary tumors were classified following the histological classification of Zapulli et al.²⁹ and graded according to the grading system from Peña et al.^{30 (link)}. Immunohistochemistry was performed on FFPE sections using antibodies against different molecular markers (detailed in Supplementary Table S7). Slides were subsequently scanned on NanoZoomer S360 Digital slide scanner (C13220-01, Hamamatsu) and analyzed with QuPath software^{55 (link)}. Scoring of the different immunohistochemical markers was performed following classical veterinary guidelines of Peña et al.^{33 (link)}.