Publications by authors named "Filomena Spada"

9 Publications

  • Page 1 of 1

High-Dimensional Single-Cell Quantitative Profiling of Skeletal Muscle Cell Population Dynamics during Regeneration.

Cells 2020 07 18;9(7). Epub 2020 Jul 18.

Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.

The interstitial space surrounding the skeletal muscle fibers is populated by a variety of mononuclear cell types. Upon acute or chronic insult, these cell populations become activated and initiate finely-orchestrated crosstalk that promotes myofiber repair and regeneration. Mass cytometry is a powerful and highly multiplexed technique for profiling single-cells. Herein, it was used to dissect the dynamics of cell populations in the skeletal muscle in physiological and pathological conditions. Here, we characterized an antibody panel that could be used to identify most of the cell populations in the muscle interstitial space. By exploiting the mass cytometry resolution, we provided a comprehensive picture of the dynamics of the major cell populations that sensed and responded to acute damage in wild type mice and in a mouse model of Duchenne muscular dystrophy. In addition, we revealed the intrinsic heterogeneity of many of these cell populations.
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http://dx.doi.org/10.3390/cells9071723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407527PMC
July 2020

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution.

Blood 2020 03;135(11):834-844

Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; and.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.
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http://dx.doi.org/10.1182/blood.2019002279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195541PMC
March 2020

HiPPO and PANDA: Two Bioinformatics Tools to Support Analysis of Mass Cytometry Data.

J Comput Biol 2020 08 20;27(8):1283-1294. Epub 2019 Dec 20.

Bioinformatics Unit, Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University London, London, United Kingdom.

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http://dx.doi.org/10.1089/cmb.2019.0384DOI Listing
August 2020

Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis.

Life Sci Alliance 2019 06 25;2(3). Epub 2019 Jun 25.

Department of Biology, University of Rome Tor Vergata, Rome, Italy

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mice are insensitive to this control mechanism. An unbiased mass spectrometry-based proteomic analysis of FAPs from muscles of wild-type and mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.
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http://dx.doi.org/10.26508/lsa.201900437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599969PMC
June 2019

The immunosuppressant drug azathioprine restrains adipogenesis of muscle Fibro/Adipogenic Progenitors from dystrophic mice by affecting AKT signaling.

Sci Rep 2019 03 13;9(1):4360. Epub 2019 Mar 13.

Department of Biology, University of Rome "Tor Vergata", 00133, Rome, Italy.

Fibro/Adipogenic Progenitors (FAPs) define a stem cell population playing a pro-regenerative role after muscle damage. When removed from their natural niche, FAPs readily differentiate into adipocytes or fibroblasts. This digressive differentiation potential, which is kept under tight control in the healthy muscle niche, contributes to fat and scar infiltrations in degenerative myopathies, such as in Duchenne Muscular Dystrophy (DMD). Controlling FAP differentiation by means of small molecules may contribute to delay the adverse consequences of the progressive pathological degeneration while offering, at the same time, a wider temporal window for gene therapy and cell-based strategies. In a high content phenotypic screening, we identified the immunosuppressant, azathioprine (AZA) as a negative modulator of FAP adipogenesis. We show here that AZA negatively affects the adipogenic propensity of FAPs purified from wild type and mdx mice by impairing the expression of the master adipogenic regulator, peroxisome proliferator-activated receptor γ (PPARγ). We show that this inhibition correlates with a decline in the activation of the AKT-mTOR axis, the main pathway that transduces the pro-adipogenic stimulus triggered by insulin. In addition, AZA exerts a cytostatic effect that has a negative impact on the mitotic clonal process that is required for the terminal differentiation of the preadipocyte-committed cells.
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http://dx.doi.org/10.1038/s41598-019-39538-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416262PMC
March 2019

Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells.

Nat Commun 2017 10 24;8(1):1109. Epub 2017 Oct 24.

Department of Chemistry and Pharmacy University of Sassari, Sassari, 07100, Italy.

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.
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http://dx.doi.org/10.1038/s41467-017-01015-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653675PMC
October 2017

Opposing roles of Nfkb2 gene products p100 and p52 in the regulation of breast cancer stem cells.

Breast Cancer Res Treat 2017 04 11;162(3):465-477. Epub 2017 Feb 11.

European Cancer Stem Cell Research Institute, School of Biosciences, Cardiff University, Hadyn Ellis Building, Maindy Road, Cathays, Cardiff, CF24 4HQ, UK.

Purpose: Nuclear factor-kappa B (NF-κB) signalling has been shown to regulate properties of breast cancer stem cells. However, the specific contribution of the non-canonical NF-κB pathway, components of which are elevated in aggressive breast cancer has not been addressed.

Methods: Through shRNA silencing of the Nfkb2 gene, the role of p100/p52 in 4T1 and N202.1A cell lines were assessed by NF-κB reporter, invasion, tumoursphere and orthotopic transplantation assays. The processing of p100 into p52 was also inhibited with a p97 ATPase inhibitor, NMS-873, and its effects on tumoursphere formation was assessed.

Results: Knockdown of Nfkb2 led to opposing changes in NF-κB-dependent transcription. NF-κB activity was elevated in 4T1 cells and this resulted in increased motility, cancer stem cell (CSC) activity and tumourigenicity in vivo. Conversely, depletion of Nfkb2 in N202.1a cells decreased NF-κB activity, CSC properties and tumourigenicity in vivo. By selectively overexpressing the p52 subunit in Nfkb2 depleted cells, we found that the increased malignancy in 4T1 cells could not be reverted in the presence of p52, whereas the decreased tumourigenicity of N202.1a cells could be rescued by p52. These results indicate that p100 and its subunit p52 have opposing effects on breast CSC activity. Accordingly, inhibition of an upstream regulator of p100 processing was effective in reducing tumoursphere formation of N202.1A and SKBR3 (ErbB2 ) cells without aggravating that of 4T1 and MDA-MB-231 (ErbB2) cells.

Conclusion: These findings indicate that inhibiting the processing of p100 may be a potential therapeutic strategy to suppress CSC activity in a subset of breast tumours.
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http://dx.doi.org/10.1007/s10549-017-4149-0DOI Listing
April 2017

Characterization by mass cytometry of different methods for the preparation of muscle mononuclear cells.

N Biotechnol 2016 Sep 7;33(5 Pt A):514-23. Epub 2016 Jan 7.

Department of Biology, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Biological processes that are mediated by cell-cell interactions in heterogeneous populations are best approached by methods that have single cell resolution. Most of these methods rely on the preparation, from solid tissues, of cell suspensions by enzymatic digestion, followed by analysis of single cell reactivity to an antibody panel that allows the discrimination of cell populations and characterization of their activation state. Thus for any specific biological problem, both efficient and at the same time mild, protocols for cell separation, together with tissue specific panels of antibodies, need to be developed and optimized. Here we characterize an antibody panel that permits the discrimination of mononuclear muscle cell populations by mass cytometry and use it to characterize the cell populations obtained by three different cell extraction procedures from muscle fibers. We show that our panel of antibodies, albeit limited and incomplete, is sufficient to discriminate most of the mononuclear muscle cell populations and that each cell extraction method yields heterogeneous cell populations with a different relative abundance of the distinct cell types.
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http://dx.doi.org/10.1016/j.nbt.2015.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4925466PMC
September 2016

SIGNOR: a database of causal relationships between biological entities.

Nucleic Acids Res 2016 01 13;44(D1):D548-54. Epub 2015 Oct 13.

Department of Biology, University of Rome Tor Vergata, Rome, Italy

Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12,000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.
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http://dx.doi.org/10.1093/nar/gkv1048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702784PMC
January 2016