Spa-RQ: an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer

Abstract

To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named ‘Spa-RQ’ for ‘Spatial tissue analysis: image Registration & Quantification’. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. To demonstrate Spa-RQ’s applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. This analysis showed histopathology-selective activation of PI3K/AKT and MAPK signalling in Kras mutant murine tumours, as well as high p38MAPK stress signalling in p53 null murine NSCLC. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. Both murine and clinical NSCLC samples could be stratified into ‘MAPK/mTOR’, ‘AKT/mTOR’, and ‘Null’ signature subclasses, suggesting mutually exclusive MAPK and AKT signalling activities. Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes.

Mac-2-binding protein glycan isomer enhances the aggressiveness of hepatocellular carcinoma by activating mTOR signaling

“… 32 , 33 Conversely, MAPK signaling has been reported to be upstream of mTOR in lung cancer cells. 34 Taken together, these reports suggest that M2BPGi-induced galectin-3 enhances mTOR signaling through MAPK signaling. Although mTOR inhibitors have been developed and used in clinical trials for HCC treatment in the past decade, the tumour-suppressive effects of these agents in patients with HCC remain controversial.…”

SOX9 has distinct roles in the formation and progression of different non‐small cell lung cancer histotypes

“…Individual tumours on H&E‐stained images were then outlined and annotated by their histotypes using ImageJ/Fiji [28] (‘Analyze’→‘Tools’→‘ROI manager’) to generate an ROI library for each murine sample. Images of serial sections stained for SOX9, Ki‐67, pAKT, and pERK were registered with the nearby H&E images using our previously reported open‐source software Spa‐R [29]. For quantifying Ki‐67 and SOX9‐positive nuclei, individual tumour images were exported as inverted 8‐bit grayscale images and processed with a CellProfiler pipeline [30] built with nuclei segmentation, intensity measurement, threshold‐based filtration, and percentage calculation modules.…”

The role of SOX9 in non-small cell lung cancer progression is histopathology-selective

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.23.393926 doi: bioRxiv preprint Histopathology-selective roles of SOX9 in lung cancer progression Individual tumours on H&E-stained images were then outlined and annotated by their histotypes using ImageJ/Fiji [28] ('Analyze' -> 'Tools' -> 'ROI manager') to generate a ROI library for each murine sample. Images of serial sections stained with SOX9, Ki67, pAKT and pERK were registered to the nearby H&E images using our previously reported open-source software Spa-R [29], which allows ROI libraries to be applied to marker-stained images (with slight adjustments). For quantifying Ki-67 and SOX9-positive nuclei, individual tumours were exported as inverted 8-bit grayscale images and processed with a CellProfiler pipeline (www.cellprofiler.org) [30] built with nuclei segmentation, intensity measurement, thresholdbased filtration, and percentage calculation modules.…”

“…The parameters in the pipeline were specified for each staining. To measure pERK and pAKT staining in individual tumours, a workflow using Spa-R and ImageJ/Fiji, and thresholds decided by three independent immunohistochemistry experts, were implemented as before [29]. All raw results can be found in the supplementary material, Table S3.…”

“…All raw results can be found in the supplementary material, Table S3. To mark Collagen IV fibres with red pseudo-colour, our in-house open-source Spa-Q software [29] was applied, using identical thresholds for all samples.…”