Minzhe Guo1,2 and Yan Tang3
1 The Perinatal Institute and Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
2Department of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH, USA
3Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Single cell RNA sequencing (scRNA-seq) is a technology that allows the analysis of transcriptome of individual cells; that is, to measure and analyze the messenger RNA (mRNA) molecules made by each cell in a tissue or in a biological process. Recent advances in this powerful technology provide us with exciting opportunities to study LAM at an unprecedented resolution, enabling the discovery of novel disease-related cells, predicting biomarkers and potential treatment targets, and providing new insights into the development of LAM.
With the support of The LAM foundation, we performed initial scRNA-seq experiments on four LAM lung explants and generated 20,381 single cell transcriptomic profiles, each measuring the mRNA expression levels of ~2,000 genes in a cell. This data allows us to begin to identify the cell types that exist in LAM lung. To achieve this, we grouped various cells into clusters based on their transcriptomic similarity. Cells in the same cluster have similar gene expression patterns, suggesting that they have similar identity. Using this approach, we identified 18 distinct cell types in the LAM lungs. 17 of them were mapped to known pulmonary cells based on our prior knowledge. The remaining cell cluster is unique and unlike any of the known cell types from the normal human lung. Uniquely, this cell cluster expressed previously identified LAM markers, including PMEL, ACTA2, MLANA, and VEGFD; and thus, we termed it LAMCORE cell. The identification of LAMCORE cell enabled us to further characterize the LAM cells. Using the scRNA-seq data, we predicted a LAMCORE specific signature of 777 genes and identified their associated bioprocesses. These results have provided us with many new candidate genes for future biomarkers and treatment targets, and new insight into the development of LAM.
This pulmonary LAMCORE signature also included genes characteristic of normal uterine tissue but rarely expressed in the normal lung, suggesting a connection between LAM cell and uterus. To further investigate this, we performed single nuclei RNA-seq on uterus obtained from a LAM patient and identified the LAMCORE cells in this uterus, suggesting that uterus might be a potential source of origin of LAM cells. In addition to the LAMCORE cell, single cell and single nuclei RNA-seq data also provided important information about a variety of other cells in lung and uterus that will help us better understand the communication between LAM cells and other cell types in the body. Details of our current scRNA-seq findings were recently published in the American Journal of Respiratory and Critical Care Medicine. Our journey to mining knowledge from this precious data has just started. All scRNA-seq data are freely available to the LAM research community and to all interested researchers.
Single cell analysis further revealed the link between TSC2-deficiency in the lung and COVID-19. We have little information so far about the risks of COVID-19 in women with LAM. We recently performed scRNA-seq on the lungs of five women with LAM. This work was recently published in the European Respiratory Journal. We compared our results with existing single cell data from eight healthy lungs. We focused on the mRNA for ACE2 (Angiotensin-Converting Enzyme 2), which is known to be required for SARS-CoV-2 (the virus that causes COVID-19) particles to enter host cells. We found that ACE2 is primarily expressed in a specialized type of alveolar cell, the type II pneumocyte, in both healthy lungs and LAM lungs. Unexpectedly, we discovered that the percentage of type II pneumocytes expressing ACE2 in LAM lungs was about 3-fold higher than in healthy lungs. We also found that ACE2 expression in LAM was correlated with increased expression of genes whose protein products physically interact with the SARS-CoV-2 virus, and are thought to help the virus replicate.
Interferon molecules have been previously shown to stimulate ACE2 expression. We observed that increased expression of ACE2 in LAM was associated with upregulation of interferon pathways in natural killer (NK) cells. NK cells are specialized immune cells that enable killing of virus-infected cells and tumor cells. These data suggest that NK cells in LAM are hyperactivated, and may be secreting interferon gamma, leading to increased expression of ACE2 in pneumocytes.
Activated mTORC1 signaling, the central defect in LAM, is believed to be useful in helping SARS-CoV-2 multiply. Using cultured primary cells from a LAM-related kidney tumor, we discovered that sirolimus treatment downregulated the interferon pathways and also downregulated a set of previously identified proteins that are believed to interact with SARS-CoV2 viral particles. From a molecular perspective, these data provide the first evidence of a link between mTORC1-dependent remodeling of the LAM microenvironment and SARS-CoV-2/COVID-19, and also suggest a potential beneficial role of sirolimus in COVID-19.
In addition to scRNA-seq, other single cell “omics” technologies are rapidly becoming available, including single cell ATAC-sequencing (scATAC-seq) that allows the analysis of open regions in the chromosomes of individual cells. These open regions allow the binding of regulators to control gene expression, providing information about regulatory mechanisms that is complimentary to that gained with the use of scRNA-seq. We expect the integration of scRNA-seq and scATAC-seq will further advance our understanding of the gene regulation in LAM.
About Minzhe Guo, PhD: Dr. Guo is an Assistant Professor in the Division of Pulmonary Biology at Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine. Dr. Guo’s research focus is on developing computational methods for single cell analysis and using bulk and single cell multiomic approaches to study normal lung development and lung diseases, such as lymphangioleiomyomatosis, idiopathic pulmonary fibrosis, alveolar capillary dysplasia, and cystic fibrosis.
About Yan Tang, PhD: Dr. Tang is an instructor of medicine in the Division of Pulmonary and Critical Care Medicine at Brigham and Women’s Hospital, Harvard Medical School. Dr. Tang’s research focuses on better understanding of tumor and tumor microenvironment in Tuberous Sclerosis Complex using multiple single cell omics profiling, and development of transformative therapy based on RNA medicine and nanomedicine.