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A high-resolution spatial map of cilia-associated proteins based on characterization of the human fallopian tube-specific proteome
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer precision medicine. (Cecilia Lindskog/Human Protein Atlas)ORCID iD: 0000-0002-3750-9308
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer precision medicine. (Cecilia Lindskog/Human Protein Atlas)ORCID iD: 0000-0003-1145-6700
Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 17121 Stockholm, Sweden; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer precision medicine.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Molecular changes in the fallopian tubes (FT) play a crucial role in the development of cancer and reproductive disorders. Here, we aimed to map key FT proteins on the single-cell level utilizing an integrated transcriptomics and proteomics approach. Based on RNA-seq, 310 genes were identified as elevated in FT, out of which a majority were associated with motile cilia function. An in-depth spatial characterization was performed for 133 of these genes in FT and other human tissues with motile cilia, localizing the proteins to different subcellular structures of ciliated cells. The specificity for ciliated cells was validated with single-cell RNA-seq and spatial mass-spectrometry data. Our approach enabled us to identify 44 novel cilia-related proteins lacking previous evidence on the protein level, as well as several other proteins not described in the context of cilia biology. The high-resolution spatial map aids in further disentangling pathways involved in infertility and diseases linked to cilia-specific functions.

Keywords [en]
motile cilia, fallopian tube, transcriptomics, spatial proteomics
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-538405OAI: oai:DiVA.org:uu-538405DiVA, id: diva2:1897770
Available from: 2024-09-15 Created: 2024-09-15 Last updated: 2024-09-15
In thesis
1. Spatial characterization of proteins in reproductive tissues: Insights into health and disease states
Open this publication in new window or tab >>Spatial characterization of proteins in reproductive tissues: Insights into health and disease states
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The molecular building blocks of human cells have been increasingly mapped in large-scale projects by emerging high-throughput antibody profiling and sequencing methods. These transformational efforts have shown remarkable progress in resolving the expression levels and spatial locations of proteins in many human cells. This thesis aimed to characterize the spatial protein expression at the single-cell level in human reproductive tissues, testis and fallopian tube (FT), and additionally study how aberrantly expressed testis-proteins repurposed in non-small cell lung cancer (NSCLC) affect the immune microenvironment.

In Paper I, more than 500 proteins with elevated RNA expression levels in the testis were profiled in eight different cell types with immunohistochemistry (IHC). Several poorly characterized proteins, so-called “missing proteins,” were localized at the cell-type level at various stages of spermatogenesis, providing novel insights into their possible function.

In Paper II, a spatiotemporal map of human spermatogenesis was created by combining single-cell transcriptomics and multiplex IHC. High-throughput image analysis determined the cell state-specific protein expression for almost 500 proteins. By examining RNA and protein correlation dynamics, we highlighted the complex spatiotemporal landscape of the human testis. These proteins serve as targets for functional studies.

In Paper III, protein-coding genes elevated in FT based on RNA levels were profiled by IHC, and most proteins were functionally related to cilia motility, a mechanism necessary for creating the tubal flow essential for fertilization. Of 133 proteins annotated at the cell-type level, most were exclusive to ciliated cells, including several proteins previously not described in motile cilia.

In Paper IV, cancer-testis antigens (CTA) were characterized by IHC on more than 300 immunophenotyped NSCLC patients. CTAs are typically expressed in the testis and harbor immunogenic properties that may be used as treatment targets due to aberrant expression in NSCLC. CTAs were associated with immune profiles, such as macrophage and plasma cell infiltration, possibly demonstrating an in situ immunogenic effect. These associations can be studied and exploited as potential immunotherapy targets.

This thesis defines the spatial proteome of reproductive tissues at the single-cell resolution and identifies many proteins with previously unknown functions in reproduction. The integrative approach to mapping tissue-specific cellular diversity at the molecular level shows the importance of combining RNA and protein detection methods. The thesis developed emerging methods like multiplexed staining and bioimage analysis, which hold promise for large-scale efforts. This work significantly contributes to the cellular atlas of reproductive tissues, which historically have not been well-studied.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2024. p. 78
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2076
Keywords
Spatial analysis, Spatial proteomics, Antibody-based profiling, Immunohistochemistry, Transcriptomics, Cancer-testis antigens, Spermatogenesis, Sperm cells, Testis, Fallopian tube, Ciliated cells, Bioimage analysis
National Category
Medical and Health Sciences
Research subject
Molecular Medicine; Medical Science
Identifiers
urn:nbn:se:uu:diva-536635 (URN)978-91-513-2231-5 (ISBN)
Public defence
2024-11-01, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
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Supervisors
Available from: 2024-10-11 Created: 2024-09-15 Last updated: 2024-10-11

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