(M-483) Immune-Senescence Communication Networks in Fibroids

Introduction:: Fibroids are tumors of the uterus characterized by a dense fibrotic extracellular matrix leading to heavy menstrual bleeding, pelvic pain, and infertility. The cumulative incidence of fibroids by age 50 is estimated to be within 70-80% of women with a higher prevalence in the Black/African American population [1]. Despite their prevalence and the immense public health burden, non-surgical treatment options are limited. Senescent cells are characterized by an irreversible growth arrest frequently accompanied by expression of cell cycle inhibitors such as p16ink4a and play a role in dysregulated fibrosis.  This senescent cell phenotype, commonly induced by factors such as age, damage, and stress can arise in a variety of cell types (fibroblast, pericyte, endothelial). These cells have stopped dividing but do not die and begin to produce highly inflammatory cytokines and growth factors called the senescence associated secretory phenotype (SASP). We hypothesize the communication between senescent and immune cells are key drivers of the fibroid tumor microenvironment.

Materials and Methods:: Fresh surgical fibroid (and when possible, patient-matched myometrium) samples were collected through approved IRB protocols. When possible, patient matched fibroids and myometrium were examined. Tissues samples were subsampled for immunofluorescent (IF) staining (paraffin embedding) or single cell suspensions. For flow cytometry and single cell RNA sequencing (scRNAseq), samples were processed with mechanical (cut into 1mm pieces) and enzymatic (Collagenase/DNase) dissociation. To ensure the immune compartment was fully captured for sequencing, we enriched for CD45+ using magnetic bead sorting then mixed CD45+ to CD45- cell fractions at a 1:1 ratio. Cells were sequenced using 10x 3’ and sequencing data was aligned with CellRanger 6.0.1 (10x Genomics). Gene expression analysis was performed with the Seurat package [2] in R, while the projectR package [3] was utilized to apply signatures to the scRNAseq data set.

Results, Conclusions, and Discussions::

Quantification of IF staining of myometrium (Myo) and fibroids (Fib) for p16 (senescence, red), CD68 (myeloid, yellow), CD31 (endothelial, green), and DAPI (nuclear, white) revealed a significant increase of senescent cells within fibroids, Fig 1A-B. Flow cytometry was used to study the immune cells within patient-matched myometrium and fibroids. T cells (CD3+) significantly increased while granulocytes (CD15+) decreased in fibroids as a fold-change proportion relative to patient-matched myometrium, Fig 1C. To study the immune-senescence communication pathways, scRNAseq samples were collected which revealed diverse stromal and immune cell populations, Fig 1D. We predicted cellular senescence by applying SenSig, an in vivo derived senescence signature [4] using the projectR [3] method, Fig 1E. The resulting SenSig scores (projection weight) predict a subset of senescent cells within the fibroblasts, smooth muscle cells, pericytes, lymphatic endothelial and in one of the endothelial populations, Fig 1F. Since senescent cells are characterized by their SASP, we analyzed the secreted ligands between the SenSig score+ and SenSig score- cells within these populations. Several genes related to the extracellular matrix and vasculogenesis were upregulated within the predicted senescent cells, Fig 1G.

There was a significant increase in senescence within uterine fibroids relative to patient-matched myometrium, as well as shifts in proportions of immune populations. Flow cytometry and scRNAseq analysis reveals heterogenous cell populations between fibroids and myometrium. Application of a senescent signature predicted senescent cells within several of these populations. We have identified unique secreted ligands between the predicted senescent cells and multiple other cell types that may represent an avenue for new therapeutic targets. Future work using computational algorithms such as Domino [5] to infer cell communication networks between predicted senescent cells and immune populations in the fibroid and myometrium will advance understanding of senescence – immune crosstalk in uterine fibroids.

Acknowledgements (Optional): : Morton Goldberg Chair, Bloomberg Kimmel Institute for Cancer Immunotherapy, NIH Pioneer (DP1AR076959) to JHE and NICHD grant R01HD094380 to MAB and JS, R01HD111243 to JS, MAB, JHE. JCM is funded by K99AG081564.

References (Optional): :

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