Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas.

STUDY QUESTION: What are the cellular composition and single-cell transcriptomic differences between myometrium and leiomyomas as defined by single-cell RNA sequencing? SUMMARY ANSWER: We discovered cellular heterogeneity in smooth muscle cells (SMCs), fibroblast and endothelial cell populations in both myometrium and leiomyoma tissues. WHAT IS KNOWN ALREADY: Previous studies have shown the presence of SMCs, fibroblasts, endothelial cells and immune cells in myometrium and leiomyomas. However, there is no information on the cellular heterogeneity in these tissues and the transcriptomic differences at the single-cell level between these tissues. STUDY DESIGN, SIZE, DURATION: We collected five leiomyoma and five myometrium samples from a total of eight patients undergoing hysterectomy. We then performed single-cell RNA sequencing to generate a cell atlas for both tissues. We utilized our single-cell sequencing data to define cell types, compare cell types by tissue type (leiomyoma versus myometrium) and determine the transcriptional changes at a single-cell resolution between leiomyomas and myometrium. Additionally, we performed MED12-variant analysis at the single-cell level to determine the genotype heterogeneity within leiomyomas. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected five MED12-variant positive leiomyomas and five myometrium samples from a total of eight patients. We then performed single-cell RNA sequencing on freshly isolated single-cell preparations. Histopathological assessment confirmed the identity of the samples. Sanger sequencing was performed to confirm the presence of the MED12 variant in leiomyomas. MAIN RESULTS AND ROLE OF CHANCE: Our data revealed previously unknown heterogeneity in the SMC, fibroblast cell and endothelial cell populations of myometrium and leiomyomas. We discovered the presence of two different lymphatic endothelial cell populations specific to uterine leiomyomas. We showed that both myometrium and MED12-variant leiomyomas are relatively similar in cellular composition but differ in cellular transcriptomic profiles. We found that fibroblasts influence the leiomyoma microenvironment through their interactions with endothelial cells, immune cells and SMCs. Variant analysis at the single-cell level revealed the presence of both MED12 variants as well as the wild-type MED12 allele in SMCs of leiomyomatous tissue. These results indicate genotype heterogeneity of cellular composition within leiomyomas. LARGE SCALE DATA: The datasets are available in the NCBI Gene Expression Omnibus (GEO) using GSE162122. LIMITATIONS, REASONS FOR CAUTION: Our study focused on MED12-variant positive leiomyomas for single-cell RNA sequencing analyses. Leiomyomas carrying other genetic rearrangements may differ in their cellular composition and transcriptomic profiles. WIDER IMPLICATIONS FOR THE FINDINGS: Our study provides a cellular atlas for myometrium and MED12-variant positive leiomyomas as defined by single-cell RNA sequencing. Our analysis provides significant insight into the differences between myometrium and leiomyomas at the single-cell level and reveals hitherto unknown genetic heterogeneity in multiple cell types within human leiomyomas. Our results will be important for future studies into the origin and growth of human leiomyomas. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the National Institute of Child Health and Human Development (HD098580 and HD088629). The authors declare no competing interests.

Patients with abdominal aortic aneurysms have reduced levels of microRNA 122-5p in circulating exosomes.

OBJECTIVE: There are currently no specific biomarkers to identify patients with abdominal aortic aneurysms (AAAs). Circulating exosomes contain microRNAs (miRNA) that are potential biomarkers for the presence of disease. This study aimed to characterize the exosomal miRNA expression profile of patients with AAAs in order to identify novel biomarkers of disease. METHODS: Patients undergoing duplex ultrasound (US) or computed tomography (CT) for screening or surveillance of an AAA were screened to participate in the study. Cases with AAA were defined as having a max aortic diameter >3 cm. Circulating plasma exosomes were isolated using Cushioned-Density Gradient Ultracentrifugation and total RNA was extracted. Next Generation Sequencing was performed on the Illumina HiSeq4000 SE50. Differential miRNA expression analysis was performed using DESeq2 software with a Benjamini-Hochberg correction. MicroRNA expression profiles were validated by Quantitative Real-Time PCR. RESULTS: A total of 109 patients were screened to participate in the study. Eleven patients with AAA and 15 non-aneurysmal controls met study criteria and were enrolled. Ultrasound measured aortic diameter was significantly larger in the AAA group (mean maximum diameter 4.3 vs 2.0 cm, P = 6.45x10-6). More AAA patients had coronary artery disease (5/11 vs 1/15, P = 0.05) as compared to controls, but the groups did not differ significantly in the rates of peripheral arterial disease and chronic obstructive pulmonary disease. A total of 40 miRNAs were differentially expressed (P<0.05). Of these, 18 miRNAs were downregulated and 22 were upregulated in the AAA group compared to controls. After false discovery rate (FDR) adjustment, only miR-122-5p was expressed at significantly different levels in the AAA group compared to controls (fold change = 5.03 controls vs AAA; raw P = 1.8x10-5; FDR P = 0.02). CONCLUSION: Plasma exosomes from AAA patients have significantly reduced levels of miRNA-122-5p compared to controls. This is a novel exosome-associated miRNA that warrants further investigation to determine its use as a diagnostic biomarker and potential implications in AAA pathogenesis.

TREM1 activation of myeloid cells promotes antitumor immunity.

Myeloid cells in the tumor microenvironment (TME) can exist in immunosuppressive and immunostimulatory states that impede or promote antitumor immunity, respectively. Blocking suppressive myeloid cells or increasing stimulatory cells to enhance antitumor immune responses is an area of interest for therapeutic intervention. Triggering receptor expressed on myeloid cells-1 (TREM1) is a proinflammatory receptor that amplifies immune responses. TREM1 is expressed on neutrophils, subsets of monocytes and tissue macrophages, and suppressive myeloid populations in the TME, including tumor-associated neutrophils, monocytes, and tumor-associated macrophages. Depletion or inhibition of immunosuppressive myeloid cells, or stimulation by TREM1-mediated inflammatory signaling, could be used to promote an immunostimulatory TME. We developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody with enhanced FcγR binding. PY159 is a TREM1 agonist that induces signaling, leading to up-regulation of costimulatory molecules on monocytes and macrophages, production of proinflammatory cytokines and chemokines, and enhancement of T cell activation in vitro. An antibody against mouse TREM1, PY159m, promoted antitumor efficacy in syngeneic mouse tumor models. These results suggest that PY159-mediated agonism of TREM1 on tumoral myeloid cells can promote a proinflammatory TME and offer a promising strategy for immunotherapy.

Targeting TREM2 on tumor-associated macrophages enhances immunotherapy.

Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2-/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2-/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.

Female reproductive tract has low concentration of SARS-CoV2 receptors.

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2.

Female reproductive tract has low concentration of SARS-CoV2 receptors.

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2 , in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2 , Cathepsin B ( CTSB ), and Cathepsin L ( CTSL ) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2. Our findings suggest that COVID-19 is unlikely to contribute to pregnancy-related adverse outcomes such as preterm birth, transmission of COVID-19 through breast milk, oogenesis and female fertility.