Role of senescent cells in uterine fibroid pathogenesis

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Uterine fibroids affect 70-80% of US women, leading to heavy menstrual bleeding, pelvic pain, and infertility. A prominent feature of uterine fibroids is the presence of a structurally dysregulated and fibrotic extracellular matrix. Despite their prevalence and the immense public health burden they cause, non-surgical treatment options are limited. Research from many groups has revealed a role of senescent cells in dysfunctional fibrosis, such as idiopathic pulmonary fibrosis and foreign body fibrosis. In previous research we demonstrated that senescent cells secrete soluble factors that modulate immune cells, resulting in a feed-forward loop that reinforces a Type 3 immune response and cellular senescence in a fibrosis model of the foreign body reaction. In this project we have used cutting-edge single cell RNA sequencing and new computational methods to study senescent and immune cells in uterine fibroids. We observed that extensive numbers of senescent cells were present in human fibroids compared to adjacent uterine tissues which had few, or no, senescent cells. Immunofluorescence staining revealed p16+ cells with a fibroblastic morphology were present in all fibroid tissues evaluated. Furthermore, the senescent cells were associated with macrophages and abnormal vascularity in the fibroids compared to normal tissues. Multi-spectral flow cytometric analysis confirmed changes in macrophage phenotype and increased numbers of T cells in the fibroids, compared to normal myometrium. Notably, preliminary single cell RNA Sequencing (scRNASeq) analysis of fibroid and normal myometrium revealed multiple cell types that exhibited senescent phenotypes, paving the way for further mechanistic analysis of immune-stromal communication associated with fibrosis. Finally, senescent cell numbers in fibroids decreased significantly after fibroids were injected with collagenase in a phase 1 clinical trial, in support of senescent cells as a viable therapeutic target for treatment of fibroids. This research will test the overarching hypothesis that senescent – immune cell interactions regulate and contribute to abnormal vascularity and fibrosis in fibroid development and pathogenesis. We will pursue three specific aims. Aim 1 seeks to characterize the presence, phenotype, and function of senescent cells and their interaction with immune cells in uterine leiomyoma. Experiments in Aim 2 will validate key senescent cell interaction in fibroid development and determine the impact of environmental factors on senescent cells. Aim 3 will evaluate the effect of senolytic drugs on uterine leiomyomas, in vitro and in vivo. Elucidation of key relationships and cell drivers of the dysfunctional fibrosis in fibroids is likely to have a broad impact on the field. Unraveling the cell-cell immune communication networks may explain how myometrial tissues transform to develop into fibroids. Affirmation of the key role of senescent – immune cell interactions in fibroid pathogenesis raises the possibility of new treatments targeting senescent cells as therapy.