Spotfire software

Data analysis of the screening was performed using a customized Hiscreen application (Discngine) connected to Spotfire software (Tibco Software Inc.). The robustness of the assay was evaluated by calculating for each plate the Z’ factor on the percentage of mCherry/α-SG positive cells parameter as follows Z’ = 1 − [3(SDP + SDN)/(MP − MN)] where MP and MN correspond to the means of the positive (30 nM BTZ) and negative (0.1% DMSO) controls, respectively, and SDP and SDN correspond to their S.D. Raw data related to the percentage of mCherry/α-SG positive cells were normalized to the mean of positive and negative controls, which are defined as 100% and 0%, respectively. Raw data related to cell number per field were normalized to the mean of negative controls. Hit selection was performed using in parallel the number of standard deviations from the mean for each readout value (Z-score) calculated per plate individually or per run where all plate data were pooled. Only hits whose Z-score plate and/or Z-score run was ≥2 and that did not decrease cell number by more than 55% compared to 0.1% DMSO condition were selected for subsequent validation step. Hits were then tested at gradual concentrations (10 nM–10 μm) for parallel exploration of their efficacy and toxicity.

Data analysis of the screening was performed using a customized Hiscreen application (Discngine) connected to Spotfire software (Tibco Software Inc.). The robustness of the assay was evaluated by calculating for each plate the Zʹ factor on the percentage of mCherry/α-SG positive cells parameter as follows Zʹ = 1 − [3(SDP + SDN)/(MP − MN)] where MP and MN correspond to the means of the positive (30 nM bortezomib) and negative (0.1% DMSO) controls, respectively, and SDP and SDN correspond to their S.D. Raw data related to the percentage of mCherry/α-SG positive cells were normalized to the mean of positive and negative controls, which are defined as 100% and 0%, respectively. Raw data related to cell number per field were normalized to the mean of negative controls. Hit selection was performed using in parallel the number of standard deviations from the mean for each readout value (Z-score) calculated per plate individually or per run where all plate data were pooled. Only hits whose Z-score plate and/or Z-score run was $⩾3$ and that did not decrease cell number by more than 55% compared to 0.1% DMSO condition were selected for subsequent validation step. Hits were then tested at gradual concentrations (1 nM–100 µM) for parallel exploration of their efficacy and toxicity.

To assess metabolic activity of OEC grown in different conditions, cells were plated at 10000 cells/cm² in 24-well plates and cultured in respective media. At days: 0 (minimum 4 h upon seeding), 1, 3 and 5 cell metabolic activity was evaluated by Resazurin assay as described before^{57 (link)}. All assays were performed in triplicates. To evaluate cell proliferation, OEC were seeded in 24-well plates in FBS-supplemented and XF media and grown for 0, 1, 2, 3, 4 and 5 days. They were then fixed with 4% v/v paraformaldehyde (PFA, 15 min), permeabilized in 0.1% Triton X-100 in PBS (15 min) and blocked with 5% FBS in PBS (1 h) at room temperature (RT). Nuclei were counterstained with DAPI (10 min). Samples were washed with PBS and brought under high content analysis. Plates were scanned and images were collected with the IN Cell Analyzer 2000 (GE Healthcare) at 10x magnification with 5000 events/well. The collected data was processed with Spotfire software (TIBCO Software Inc.) and OEC proliferation rate in different conditions was assessed by quantification of the average number of cells per defined field.

Optimized fluorescent multiplexed immunohistochemistry was performed using tyramide signal amplification in the Leica Bond Rx Automated Stainer (Leica Biosystems, Newcastle, UK) as previously described^{42 (link)}. Cells were stained with antibodies against CD68 (M0876, DAKO, Glostrup, Denmark), CD206 (NBP1-90020, Novus Biological, Littleton, CO, USA), PD-L1 (13684S, Abcam, Cambridge, UK) and cytokeratin (NBP2-29429, Novus, Littleton, CO, USA), and the fluorescence signals were captured with the following fluorophores: Opal 570, Opal 620, Opal 690, and Opal 780. Multiplex-stained slides were obtained using the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, USA). Regions of interest (ROIs) focusing on the invasive tumor margin or the active tumor-stromal interface were carefully chosen by an experienced pathologist (JK) based on the hematoxylin and eosin slides and cytokeratin expression. The images were analyzed using inForm 2.4.11 image analysis software (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, US) and Spotfire software (TIBCO Software Inc., Palo Alto, CA). The data were expressed as the mean number of cells/mm² for each cell population.

Multiplex stained slides were acquired using Vectra® Polaris Quantitative Pathology Imaging System (Perkin Elmer, Boston, MA, USA). Each ×200 multispectral image cube was created by combining images obtained at 10-nm intervals of the emission light spectrum across the range of each emission filter cube. Filter cubes used for multispectral imaging were DAPI (440–680 nm), FITC (520–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (670–720 nm). In each slide, eight to 11 region of interests (ROIs) were selected, and images were spectrally unmixed and segmented (inForm 2.4.1 image analysis software; Perkin Elmer, Wellesley, MA, USA). Data obtained from inForm were sent to Spotfire™ software (TIBCO Software Inc., Palo Alto, CA, USA). Threshold for the positivity of each marker is determined by the pathologist using immunohistochemistry scoring; >0.2 for DOG, >3.0 for CD3, >1.5 for CD8, >1.2 for FoxP3, >1.7 for PD-L1, >0.5 for PD-1, >1.1 for LAG3, >5.0 for Ki-67, >0.7 for CD68, >0.5 for TIM3, and >0.7 for CD204. For each specimen, mean value of the number of cells per mm² in the analysed ROIs was used for further analyses.

The Vectra^® 3.0 Automated Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, USA), was used for the scanning of multiplex-stained slides. The images were visualized in the Phenochart whole slide viewer (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, USA).
The inform 2.3 Image Analysis Software (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, USA) and Spotfire software (TIBCO Software Inc., Palo Alto, CA, USA) were used for analyzing images. The threshold for the positivity of each marker is determined by the pathologist using IHC scoring: >2.8 for CK, >14 for CD8, >10 for PD-L1, and >1.5 for PD-1. The percentage of each immune cell subset was calculated by dividing the absolute number of each subset by the total number of all the cells.