![]() Although powerful in principle, practical factors limit flexible implementation of multiplexed strategies, including availability of specific antibodies of varying host species or signal unmixing accuracy. Recent advances in multiplexed staining techniques have deepened our understanding of spatial phenotypes at tissue levels and yielded data with clinical relevance 6, 7, 8. Tumour heterogeneity can be interrogated by gene-centric methods, such as multi-regional or single cell spatial sequencing, or by phenotypic assays, such as high-content in vitro imaging or tissue profiling using immunohistochemistry (IHC) analyses. During carcinoma progression, such factors contribute to microenvironmental heterogeneity which, in conjunction with tumour cell-intrinsic genetic and molecular heterogeneity, fosters tumour evolution and compromises treatment success 3, 4, 5. ![]() Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes.Įpithelial cells are locally influenced by neighbouring stromal cells and secreted molecules, as well as tissue gradients in oxygenation and mechanical forces 1, 2. 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. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. ![]() 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. ![]() Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. To demonstrate Spa-RQ’s applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named ‘Spa-RQ’ for ‘Spatial tissue analysis: image Registration & Quantification’. ![]()
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