Case center

All tissues were fixed in 10% buffered formalin and stored in a cool and dry environment. For all patient cohorts, a tissue microarray was constructed using the next-generation tissue microarray approach (ngTMA) [17 (link)]. First, diagnostic H&E slides corresponding to each case were re-reviewed. The most representative one to three H&E slides were selected for each cohort and scanned (Pannoramic P250, 3DHistech). Slides were uploaded onto a digital slide management interface (Case Center, 3DHistech) and annotated using a tissue microarray annotation tool of various sizes (0.6mm or 1.0mm) and colors to designate the different histological areas for capturing in the TMA (Supplemental Figure 2). Next, corresponding donor blocks were loaded into the automated tissue arrayer (TMA Grandmaster), aligned with the digital slide and its annotations, and finally cored for TMA construction. Details of the ngTMA core numbers and sizes can be found in Supplemental Table 4. The use of all material in this study was approved by the corresponding ethics committee (Munich, Germany: Klinikum rechts der Isar (no. 1926/7), Bern, Switzerland: Ethics commission of the canton of Bern (200/14)).

All CK-stained slides were scanned using a slide scanner (Pannoramic P250, 3DHistech, Hungary). Scans were uploaded onto a case-managing platform (Case Center, 3DHistech, Hungary). Each scan was then re-reviewed for the presence of TB and the following annotations of 1.0 mm in diameter were made using a digital TMA annotation tool:
The tissue blocks were loaded into a semi-automated tissue microarrayer (TMA Grandmaster, 3DHistech, Hungary). Annotated digital scans were aligned with an image of the corresponding tissue block and punched out in the annotated regions, ensuring the capture of TB. Punched cores were transferred into a recipient paraffin block. This led to the construction of three ngTMAs; two containing human tissue, one with the low-grade budding tumors (75 spots) and one including the high-grade budding tumors (149 spots). The third TMA (84 spots) contained all mouse tissue.