RESUMO
Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-ß (TGF-ß) in cancer metastasis. TGF-ß-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-ß-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-ß-stimulated cancer cells, suggesting that TGF-ß might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.
Assuntos
Adenocarcinoma de Pulmão/secundário , Movimento Celular/efeitos dos fármacos , Técnicas de Preparação Histocitológica , Neoplasias Pulmonares/patologia , Fator de Crescimento Transformador beta/farmacologia , Microambiente Tumoral , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Citocinas/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/metabolismoRESUMO
Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.