Practical Considerations for Complex Tissue Dissociation for Single-Cell Transcriptomics

Pohl, Stephanie; Prada, Maria Llamazares; Espinet, Elisa; Jurkowska, Renata Z.

doi:10.1007/978-1-0716-2756-3_19

Cited by 8 publications

(5 citation statements)

References 36 publications

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“…We performed an integrative analysis of LOY-cells in tumor, normal tissue (unchanged large bowel mucosa) surrounding tumor and blood, using experimental tissue dissociation and publicly available scRNA-seq datasets, aiming to identify and characterize LOY-affected leukocytes. Experiments using scRNA-seq methods has recently revolutionized the medical field 53 and carry a promise of extending the research into personalized diagnostics 34 . scRNA-seq allows to overcome limitations regarding the investigation of the LOY frequency and LOY-associated transcriptomic effects, especially when the number of cells is not sufficient for the bulk analysis, as is often the case of immune cells isolated from solid tissues, or when the data from homogenous cell populations is required 13 .…”

Section: Discussionmentioning

confidence: 99%

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Wójcik,

Juhas,

Mohammadi

et al. 2024

Sci Rep

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Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

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Section: Discussionmentioning

confidence: 99%

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Wójcik,

Juhas,

Mohammadi

et al. 2024

Sci Rep

View full text Add to dashboard Cite

show abstract

“…We performed an integrative analysis of LOY-cells in tumor, normal tissue (unchanged large bowel mucosa) surrounding tumor and blood, using experimental tissue dissociation and publicly available scRNA-seq datasets, aiming to identify and characterize LOY-affected leukocytes. Experiments using scRNA-seq methods has recently revolutionized the medical field [50] and carry a promise of extending the research into personalized diagnostics [34]. scRNA-seq allows to overcome limitations regarding the investigation of the LOY frequency and LOY-associated transcriptomic effects, especially when the number of cells is not sufficient for the bulk analysis, as is often the case of immune cells isolated from solid tissues, or when the data from homogenous cell populations is required [13].…”

Section: Discussionmentioning

confidence: 99%

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Wójcik,

Juhas,

Mohammadi

et al. 2023

Preprint

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Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4+ T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY-cells (22%) in comparison to CD4+ T-lymphocytes and cytotoxic CD8+ T-lymphocytes. Using scRNA-seq LOY was also linked to higher expression ofPDCD1, TIGITandIKZF2in Tregs.PDCD1andTIGITencode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

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“…The lung tissues from all donors were cryopreserved on arrival, enabling cell isolation from multiple donors in parallel and minimizing technical bias introduced by multiple batch processing of fresh samples, as documented previously (Llamazares‐Prada et al , 2021 ; Pohl et al , 2023 ). Briefly, specimens were transported in CO 2 ‐independent medium (Thermo Fisher Scientific) supplemented with 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific), and 1% Amphotericin B (Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…Cryopreserved lung tissues were thawed for 2 min in a 37°C water‐bath, collected in 50 ml Falcon tubes, and washed with wash buffer: HBSS supplemented with 2 mM EDTA (Thermo Fisher Scientific), 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific) and 1% Amphotericin B (Fisher Scientific). Tissue was minced into smaller pieces prior to mechanical and enzymatic dissociation as indicated previously (Llamazares‐Prada et al , 2021 ; Pohl et al , 2023 ). Briefly, the minced tissue (1 g) was introduced in GentleMACS C‐tubes (Miltenyi Biotec) containing 10 μM ROCK inhibitor (Y‐27632, Adooq Bioscience), 10 μg DNase I (ProSpec‐Tany TechnoGene), the enzyme mix from the human tumor tissue dissociation kit (Miltenyi Biotec) and 4.5 ml of CO 2 ‐independent media (Thermo Fisher Scientific) supplemented with 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific) and 1% Amphotericin B (Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

High‐resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

et al. 2023

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Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

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Practical Considerations for Complex Tissue Dissociation for Single-Cell Transcriptomics

Cited by 8 publications

References 36 publications

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases

High‐resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

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