Multiplex immunofluorescence and single‐cell transcriptomic profiling reveal the spatial cell interaction networks in the non‐small cell lung cancer microenvironment


Peng Haoxin12,Wu Xiangrong12,Liu Shaopeng34,He Miao1,Xie Chao3,Zhong Ran1,Liu Jun1,Tang Chenshuo3,Li Caichen1,Xiong Shan1,Zheng Hongbo5,He Jianxing1,Lu Xu34,Liang Wenhua16


1. Department of Thoracic Oncology and Surgery China State Key Laboratory of Respiratory Disease & National Clinical Research Center for Respiratory Disease the First Affiliated Hospital of Guangzhou Medical University Guangzhou China

2. Department of Clinical Medicine Nanshan School Guangzhou Medical University Guangzhou China

3. Department of Computer Science Guangdong Polytechnic Normal University Guangzhou China

4. Department of Artificial Intelligence Research Pazhou Lab Guangzhou China

5. Medical Department Genecast Biotechnology Co., Ltd Beijing China

6. Department of Medical Oncology The First People's Hospital of Zhaoqing Zhaoqing China


AbstractBackgroundConventional immunohistochemistry technologies were limited by the inability to simultaneously detect multiple markers and the lack of identifying spatial relationships among cells, hindering understanding of the biological processes in cancer immunology.MethodsTissue slices of primary tumours from 553 IA∼IIIB non‐small cell lung cancer (NSCLC) cases were stained by multiplex immunofluorescence (mIF) assay for 10 markers, including CD4, CD38, CD20, FOXP3, CD66b, CD8, CD68, PD‐L1, CD133 and CD163, evaluating the amounts of 26 phenotypes of cells in tumour nest and tumour stroma. StarDist depth learning model was utilised to determine the spatial location of cells based on mIF graphs. Single‐cell RNA sequencing (scRNA‐seq) on four primary NSCLC cases was conducted to investigate the putative cell interaction networks.ResultsSpatial proximity among CD20+ B cells, CD4+ T cells and CD38+ T cells (r2 = 0.41) was observed, whereas the distribution of regulatory T cells was associated with decreased infiltration levels of CD20+ B cells and CD38+ T cells (r2 = −0.45). Univariate Cox analyses identified closer proximity between CD8+ T cells predicted longer disease‐free survival (DFS). In contrast, closer proximity between CD133+ cancer stem cells (CSCs), longer distances between CD4+ T cells and CD20+ B cells, CD4+ T cells and neutrophils, and CD20+ B cells and neutrophils were correlated with dismal DFS. Data from scRNA‐seq further showed that spatially adjacent N1‐like neutrophils could boost the proliferation and activation of T and B lymphocytes, whereas spatially neighbouring M2‐like macrophages showed negative effects. An immune‐related risk score (IRRS) system aggregating robust quantitative and spatial prognosticators showed that high‐IRRS patients had significantly worse DFS than low‐IRRS ones (HR 2.72, 95% CI 1.87–3.94, p < .001).ConclusionsWe developed a framework to analyse the cell interaction networks in tumour microenvironment, revealing the spatial architecture and intricate interplays between immune and tumour cells.




Molecular Medicine,Medicine (miscellaneous)

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