Image_4_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_3_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_1_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_2_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_6_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_5_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Image_7_Integrated analyses of single-cell transcriptomics identify metastasis-associated myeloid subpopulations in breast cancer lung metastasis.jpeg

Lung metastasis of breast cancer is closely associated with patient morbidity and mortality, which correlates with myeloid cells in the lung microenvironment. However, the heterogeneity and specificity of metastasis-associated myeloid cells have not been fully established in lung metastasis. Here, by integrating and analyzing single-cell transcriptomics, we found that myeloid subpopulations (Tppp3+ monocytes, Isg15+ macrophages, Ifit3+ neutrophils, and Il12b+ DCs) play critical roles in the formation and development of the metastatic niche. Gene enrichment analyses indicate that several tumor-promoting pathways should be responsible for the process, including angiogenesis (Anxa1 and Anxa2 by Tppp3+ monocytes), immunosuppression (Isg15 and Cxcl10 by Isg15+ macrophages; Il12b and Ccl22 by Il12b+ DCs), and tumor growth and metastasis (Isg15 and Isg20 by Ifit3+ neutrophils). Furthermore, we have validated these subpopulations in lung microenvironment of MMTV-PyVT transgenic mice and verified their association with poor progression of human breast cancer. Also, our results elucidated a crosstalk network among four myeloid subpopulations by cell-cell communication analysis. This study, therefore, highlights the crucial role of myeloid cells in lung metastasis and provides insights into underlying molecular mechanisms, which pave the way for therapeutic interventions in breast cancer metastasis to lung.</p

Table1_Endothelial anthrax toxin receptor 2 plays a protective role in liver fibrosis.docx

Hepatocellular carcinoma is one of the leading cancers worldwide and is a potential consequence of fibrosis. Therefore, the identification of key cellular and molecular mechanisms involved in liver fibrosis is an important goal for the development of new strategies to control liver-related diseases. Here, single-cell RNA sequencing data (GSE136103 and GES181483) of clinical liver non-parenchymal cells were analyzed to identify cellular and molecular mechanisms of liver fibrosis. The proportion of endothelial subpopulations in cirrhotic livers was significantly higher than that in healthy livers. Gene ontology and gene set enrichment analysis of differentially expressed genes in the endothelial subgroups revealed that extracellular matrix (ECM)-related pathways were significantly enriched. Since anthrax toxin receptor 2 (ANTXR2) interacts with the ECM, the expression of ANTXR2 in the liver endothelium was analyzed. ANTXR2 expression in the liver endothelium of wild-type (WT) mice significantly decreased after a 4-time sequential injection of carbon tetrachloride (CCl4) to induce liver fibrosis. Next, conditional knockout mice selectively lacking Antxr2 in endothelial cells were generated. After endothelial-specific Antxr2 knockout mice were subjected to the CCl4 model, the degree of liver fibrosis in the knockout group was significantly more severe than that in the control group. In addition, ANTXR2 in human umbilical vein endothelial cells promoted matrix metalloproteinase 2 (MMP2) activation to degrade the ECM in vitro. Finally, endothelial-specific overexpression of Antxr2 alleviated the development of liver fibrosis following adeno-associated virus treatment. Collectively, these results suggested that endothelial ANTXR2 plays a protective role in liver fibrosis. This function of ANTXR2 may be achieved by promoting MMP2 activation to degrade the ECM.</p

Isolated Prom1⁺ embryonic lateral ventricle and SVZ cells in culture give rise to neurospheres expressing neural cell markers but not vascular markers.

<p>Z-stack confocal images of cultured neurospheres generated from Prom1⁺ cells of E12.5 forebrain of prominin-1^lacZ/+ mice in serum free media and in the presence of bFGF revealed co-immunostaining for ß-galactosidase (green) and both Sox2 (A) and Musashi (B) stem cell markers. Z-stack confocal images of secondary differentiated neurospheres induced by removing bFGF and plating on polyL-ornithine and fibronectin coated dishes from Prom1⁺ cells of E16.5–E17.5 forebrain of prominin-1^lacZ/+ mice. Prom1⁺ cells (ß-galactosidase (green) in culture give rise to 3 different neural lineages (C) Olig 2 oligodendrocytes, (D) GFAP astrocytes and (E) Tuj1 neuron-specific progenitor neuronal marker.</p

Prom1 is expressed by endothelial and tumor cells and both Prom1⁺ and Prom1⁻ cells contribute to glioma growth.

<p>H&E sections of brain tumor of 2 to 4 month old mouse generated by crossing <i>Prom1^lacZ/+</i> and Ntv-a;<i>Ink4a-Arf^−/−</i> mice infected with RCAS-PDGF. Tumors (A) were characterized by pseudopalisading necrosis (C) and microvascular proliferation (E), characteristic of glioblastoma. X-gal staining revealed ß-galactosidase activity throughout the tumor (B), at the periphery of pseudopalisading necrosis (D) and within microvascular proliferation“glomeruloid tuft” (F). (G) Z-stack and confocal optical sections revealed that some tumor cells coimmunostained for ß-galactosidase (green) and GFAP (red) and had astrocytic morphology (inset). (H) Other cells within the “glomeruloid tuft” coimmunostained for ß-galactosidase (green) and CD31 endothelial cells (red). (I) Kaplan-Meier analysis comparing survival of adult wild-type mice implanted with tumor cells dissociated from Prom1⁺ (N = 8) and Prom1⁻ neurospheres (N = 8) from PDGF-induced tumors in Ntv-a;<i>Ink4a-Arf^−/− Prom1^lacZ/+</i> mice. (J) Survival curve of adult (8 weeks) wild-type mice implanted with Prom1⁺ (N = 5) and Prom1⁻ (N = 8) PDGF-induced tumors immediately after their cells were dissociated.</p