Cytonuclear

Tissue sections were stained with hematoxylin & eosin (H&E) (hematoxylin, GHS132-1L; eosin Y solution, HT110132-1L; Sigma-Aldrich, St. Louis, MO) for routine light microscopy evaluation. The Masson’s trichrome stain kit (ab150686, Abcam, Cambridge, UK) to detect cardiac fibrosis was used according to the manufacturer’s instructions. Picro Sirius red staining (Direct red 80, 65548, Sigma-Aldrich, St. Louis, MO) and Picric acid (10015714, Xilong, China) was also applied to evaluate the fibrotic area, appearing as red color. Histopathological scores were graded as 0 “-” (no apparent change), 1 (minimal), 2 (moderate), 3 (marked), and 4 (severe) based on the increasing extent and/or complexity of morphological changes^{124 (link),125 (link)}. More details for grading standards are described in Supplementary Table 2.
The stained tissue sections were captured under an Olympus BX53 digital microscope with the cellSens digital imaging software (Olympus, Tokyo, Japan), scanned on a Leica Aperio AT2 scanner (200x magnification; Leica Biosystems), and analyzed with the Halo software v3.0.311.217 (Indica Labs) with Area Quantification (v2.1.3.0; Indica Labs) and CytoNuclear (v2.0.5.0; Indica Labs) algorithm.

The images were quantified using Halo image analysis software (Indica Labs) using CytoNuclear module to quantify CD8 and CD20 staining. All images were subsequently reviewed manually. For each TMA, all cores from UPS patients were selected as the annotated regions to be quantified, while for each slide, areas of high cellularity containing malignant cells were selected, avoiding fibrotic or pauci-cellular regions. The intensity for each stain was assigned under + 1, + 2, or + 3 category. Once set, the image analysis was completed on the annotated regions. For each annotated region, the image analysis software generated the following results: total cells, positive cells, negative cells, percentage of positive cells, percentage of negative cells, average stain one nuclear OD, average stain one cytoplasmic OD, average cell area, average cytoplasm area, average nucleus area, average nucleus perimeter, and tissue area (μm²). The percentage of positive cells was used as our primary measure of biomarker expression to allow for analysis of the cohort as a whole, including patients with specimens in the form of TMA and into survival analyses. For patients with TMA tumor specimens, the percentage of positive cells from triplicate cores was averaged.