Fantl, W. J. | bioRxiv Science

bioRxiv2024

Coordinated protein modules define DNA damage responses to carboplatin at single cell resolution in human ovarian carcinoma models

Tubo-ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy and frequently responds to platinum-based chemotherapy because of common genetic and somatic impairment of DNA damage repair (DDR) pathways. The mechanisms of clinical platinum resistance are diverse and poorly molecularly defined. Consequently, there are no biomarkers or medicines that improve patient outcomes. Herein we use single cell mass cytometry (CyTOF) to systematically evaluate the phosphorylation and abundance of proteins known to participate in the DNA damage response (DDR). Single cell analyses of highly characterized HGSC cell lines that phenocopy human patients show that cells with comparable levels of intranuclear platinum, a proxy for carboplatin uptake, undergo different cell fates. Unsupervised analyses revealed a continuum of DDR responses. Decompositional methods were used to identify eight distinct protein modules of carboplatin resistance and sensitivity at single cell resolution. CyTOF profiling of primary and secondary platinum-resistance patient models shows that a complex DDR sensitivity module is strongly associated with response, suggesting it as a potential tool to clinically characterize complex drug resistance phenotypes.

cancer biology↗

bioRxiv2022

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration

In aging, skeletal muscle strength and regenerative capacity declines due, in part, to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here we capitalize on mass-cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blocking of CD47 alternative polyadenylation or antibody blockade of CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

cell biology↗

bioRxiv2019

Profiling Myelodysplastic Syndromes by Mass Cytometry Demonstrates Abnormal Progenitor Cell Phenotype and Differentiation

PurposeWe sought to enhance the cytometric analysis of MDS by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS.\n\nExperimental DesignTwenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering.\n\nResultsThis high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance (ICUS). These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells.\n\nConclusionsThese results demonstrate that high-parameter cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS.\n\nStatement of SignificanceO_LIHigh-dimensional mass cytometry enables high-resolution characterization of abnormal maker expression and myeloid development in MDS.\nC_LIO_LIThis technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.\nC_LI\n\nStatement Translational RelevanceIn spite of several studies suggesting the utility of flow cytometry in the diagnosis of myelodysoplastic syndrome (MDS), this technique has not been widely adopted. We sought to enhance the utility of cytometry in MDS by performing the first high-dimensional mass cytometry characterization of a cohort of MDS patients. High-dimensional mass cytometry allowed all bone marrow cell populations to be simultaneously analyzed enabling high-resolution characterization of abnormal maker expression and myeloid development in MDS. This approach could identify almost all previously identified aberrant surface marker expression patterns in MDS while simultaneously enabling analysis by unsupervised clustering. Additionally, this mass cytometry analysis approach enabled the modeling of abnormal differentiation in MDS. This technology could enhance MDS diagnosis and therapeutic monitoring and merits further research.

cancer biology↗