Whole-organ analysis of TGF-b-mediated remodelling of the tumour microenvironment

Tissue clearing technique may be one of the most powerful strategies for a comprehensive and unbiased analysis of disease progression. Here, we improved a tissue clearing protocol and explored its application in a mouse tumour model of experimental lung metastasis. We established an integrated pipeline for 3D profiling of the tumour microenvironment in combination with our new clearing protocol and machine learning. This pipeline distinguished each cellular component within the tumour microenvironment, and provided information on their spatial relationships. We further explored the role of TGF-b in the tumour microenvironment using this integrated pipeline. TGF-b-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-b-stimulated cancer cells. Further, 3D profiling of the tumour microenvironments revealed accumulation of platelets or macrophages with TGF-b-stimulated cancer cells, suggesting that TGF-b might promote remodelling of the tumour microenvironment through the activation of these cellular components at the metastatic sites, which further enhances the colonization of cancer cells. Hence, an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning will help further the understanding of the role of the tumour microenvironment. Overall design: Target genes for TGF-b in A549 cells were identified.