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Spatial characterization of proteins in reproductive tissues: Insights into health and disease states
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer precision medicine. Human Protein Atlas (HPA). (Cecilia Lindskog/Human Protein Atlas)ORCID iD: 0000-0002-3750-9308
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:uu:diva-536635ISBN: 978-91-513-2231-5 (print)OAI: oai:DiVA.org:uu-536635DiVA, id: diva2:1897774
Public defence
2024-11-01, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2024-10-11 Created: 2024-09-15 Last updated: 2024-10-11
List of papers
1. Cell Type-Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Proteomics
Open this publication in new window or tab >>Cell Type-Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Proteomics
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2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 12, p. 4215-4230Article in journal (Refereed) Published
Abstract [en]

One of the most complex organs in the human body is the testis, where spermatogenesis takes place. This physiological process involves thousands of genes and proteins that are activated and repressed, making testis the organ with the highest number of tissue-specific genes. However, the function of a large proportion of the corresponding proteins remains unknown and testis harbors many missing proteins (MPs), defined as products of protein-coding genes that lack experimental mass spectrometry evidence. Here, an integrated omits approach was used for exploring the cell type-specific protein expression of genes with an elevated expression in testis. By combining genome-wide transcriptomics analysis with immunohistochemistry, more than 500 proteins with distinct testicular protein expression patterns were identified, and these were selected for in-depth characterization of their in situ expression in eight different testicular cell types. The cell type-specific protein expression patterns allowed us to identify six distinct clusters of expression at different stages of spermatogenesis. The analysis highlighted numerous poorly characterized proteins in each of these clusters whose expression overlapped with that of known proteins involved in spermatogenesis, including 85 proteins with an unknown function and 60 proteins that previously have been classified as MPs. Furthermore, we were able to characterize the in situ distribution of several proteins that previously lacked spatial information and cell type specific expression within the testis. The testis elevated expression levels both at the RNA and protein levels suggest that these proteins are related to testis-specific functions. In summary, the study demonstrates the power of combining genome-wide transcriptomics analysis with antibody-based protein profiling to explore the cell type-specific expression of both well-known proteins and MPs. The analyzed proteins constitute important targets for further testis-specific research in male reproductive disorders.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
testis, reproduction, spermatogenesis, antibody-based proteomics, missing proteins, protein evidence, immunohistochemistry, transcriptomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-400735 (URN)10.1021/acs.jproteome.9b00351 (DOI)000502164100015 ()31429579 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation
Available from: 2020-01-17 Created: 2020-01-17 Last updated: 2024-09-15Bibliographically approved
2. A spatiotemporal atlas of human spermatogenesis based on single-cell transcriptomics and multiplex antibody imaging
Open this publication in new window or tab >>A spatiotemporal atlas of human spermatogenesis based on single-cell transcriptomics and multiplex antibody imaging
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(English)Manuscript (preprint) (Other academic)
Keywords
testis, multiplex immunohistochemistry, spermatogonial stem cells, male germ cell differen-tiation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-538406 (URN)
Available from: 2024-09-15 Created: 2024-09-15 Last updated: 2024-09-23
3. A high-resolution spatial map of cilia-associated proteins based on characterization of the human fallopian tube-specific proteome
Open this publication in new window or tab >>A high-resolution spatial map of cilia-associated proteins based on characterization of the human fallopian tube-specific proteome
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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
motile cilia, fallopian tube, transcriptomics, spatial proteomics
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-538405 (URN)
Available from: 2024-09-15 Created: 2024-09-15 Last updated: 2024-09-15
4. Expression of cancer-testis antigens in the immune microenvironment of non-small cell lung cancer
Open this publication in new window or tab >>Expression of cancer-testis antigens in the immune microenvironment of non-small cell lung cancer
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2023 (English)In: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 17, no 12, p. 2603-2617Article in journal (Refereed) Published
Abstract [en]

The antigenic repertoire of tumors is critical for successful anti-cancer immune response and the efficacy of immunotherapy. Cancer-testis antigens (CTAs) are targets of humoral and cellular immune reactions. We aimed to characterize CTA expression in non-small cell lung cancer (NSCLC) in the context of the immune microenvironment. Of 90 CTAs validated by RNA sequencing, eight CTAs (DPEP3, EZHIP, MAGEA4, MAGEB2, MAGEC2, PAGE1, PRAME, and TKTL1) were selected for immunohistochemical profiling in cancer tissues from 328 NSCLC patients. CTA expression was compared with immune cell densities in the tumor environment and with genomic, transcriptomic, and clinical data. Most NSCLC cases (79%) expressed at least one of the analyzed CTAs, and CTA protein expression correlated generally with RNA expression. CTA profiles were associated with immune profiles: high MAGEA4 expression was related to M2 macrophages (CD163) and regulatory T cells (FOXP3), low MAGEA4 was associated with T cells (CD3), and high EZHIP was associated with plasma cell infiltration (adj. P-value < 0.05). None of the CTAs correlated with clinical outcomes. The current study provides a comprehensive evaluation of CTAs and suggests that their association with immune cells may indicate in situ immunogenic effects. The findings support the rationale to harness CTAs as targets for immunotherapy.

Place, publisher, year, edition, pages
John Wiley & Sons, 2023
Keywords
cancer-testis antigens, immune phenotype, immune-oncology, non-small cell lung cancer
National Category
Cancer and Oncology Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-522496 (URN)10.1002/1878-0261.13474 (DOI)001020469000001 ()37341056 (PubMedID)
Funder
Swedish Cancer Society, 21 1790Knut and Alice Wallenberg Foundation, 2015.0344Sjöberg Foundation
Available from: 2024-02-07 Created: 2024-02-07 Last updated: 2024-09-15Bibliographically approved

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