One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment ...modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity.
Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc
mice exposed to bilateral thorax irradiation as well as human lung cells grown
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studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc
mice harboring critically short telomeres and lack of telomerase activity.
The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.
Neuroblastoma displays important clinical and genetic heterogeneity, with emergence of new mutations at tumor progression.
To study clonal evolution during treatment and follow-up, an innovative ...method based on circulating cell-free DNA (cfDNA) analysis by whole-exome sequencing (WES) paired with target sequencing was realized in sequential liquid biopsy samples of 19 neuroblastoma patients.
WES of the primary tumor and cfDNA at diagnosis showed overlap of single-nucleotide variants (SNV) and copy number alterations, with 41% and 93% of all detected alterations common to the primary neuroblastoma and cfDNA. CfDNA WES at a second time point indicated a mean of 22 new SNVs for patients with progressive disease. Relapse-specific alterations included genes of the MAPK pathway and targeted the protein kinase A signaling pathway. Deep coverage target sequencing of intermediate time points during treatment and follow-up identified distinct subclones. For 17 seemingly relapse-specific SNVs detected by cfDNA WES at relapse but not tumor or cfDNA WES at diagnosis, deep coverage target sequencing detected these alterations in minor subclones, with relapse-emerging SNVs targeting genes of neuritogenesis and cell cycle. Furthermore a persisting, resistant clone with concomitant disappearance of other clones was identified by a mutation in the ubiquitin protein ligase
Modelization of mutated allele fractions in cfDNA indicated distinct patterns of clonal evolution, with either a minor, treatment-resistant clone expanding to a major clone at relapse, or minor clones collaborating toward tumor progression. Identification of treatment-resistant clones will enable development of more efficient treatment strategies.
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Presentation of exogenous antigens on MHC-I molecules, termed cross-presentation, is essential for cytotoxic CD8
T cell responses. In mice, dendritic cells (DCs) that arise from monocytes (mo-DCs) ...during inflammation have a key function in these responses by cross-presenting antigens locally in peripheral tissues. Whether human naturally-occurring mo-DCs can cross-present is unknown. Here, we use human mo-DCs and macrophages directly purified from ascites to address this question. Single-cell RNA-seq data show that ascites CD1c
DCs contain exclusively monocyte-derived cells. Both ascites mo-DCs and monocyte-derived macrophages cross-present efficiently, but are inefficient for transferring exogenous proteins into their cytosol. Inhibition of cysteine proteases, but not of proteasome, abolishes cross-presentation in these cells. We conclude that human monocyte-derived cells cross-present exclusively using a vacuolar pathway. Finally, only ascites mo-DCs provide co-stimulatory signals to induce effector cytotoxic CD8
T cells. Our findings thus provide important insights on how to harness cross-presentation for therapeutic purposes.
Mammalian genomes are replicated in a cell type-specific order and in coordination with transcription and chromatin organization. Currently, single-cell replication studies require individual ...processing of sorted cells, yielding a limited number (<100) of cells. Here, we develop Kronos scRT, a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform or cell sorting, which allows investigating large datasets obtained from asynchronous cells. By applying our tool to published data as well as droplet-based single-cell whole-genome sequencing data generated in this study, we exploit scRT from thousands of cells for different mouse and human cell lines. Our results demonstrate that although genomic regions are frequently replicated around their population average RT, replication can occur stochastically throughout S phase. Altogether, Kronos scRT allows fast and comprehensive investigations of the RT programme at the single-cell resolution for both homogeneous and heterogeneous cell populations.
A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform ...whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.
After entering tissues, monocytes differentiate into cells that share functional features with either macrophages or dendritic cells (DCs). How monocyte fate is directed toward monocyte-derived ...macrophages (mo-Macs) or monocyte-derived DCs (mo-DCs) and which transcription factors control these differentiation pathways remains unknown. Using an in vitro culture model yielding human mo-DCs and mo-Macs closely resembling those found in vivo in ascites, we show that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DCs and mo-Macs, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Macs. AhR deficiency also impaired the in vivo differentiation of mouse mo-DCs. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DCs and mo-Macs are controlled by distinct transcription factors and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors.
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•IRF4 and MAFB are essential for human mo-DC and mo-Mac differentiation, respectively•AHR activation promotes mo-DC and inhibits mo-Mac differentiation through BLIMP-1•AhR is involved in the in vivo differentiation of mo-DCs in the mouse
How monocytes differentiate into dendritic cells versus macrophages is poorly understood. Goudot et al. show that IRF4 and MAFB are essential for dendritic cell or macrophage differentiation, respectively, and identify aryl hydrocarbon receptor as a major regulator of monocyte fate.
Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, ...are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.
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•FOLR2+ macrophages are tissue-resident cells found in healthy and malignant breasts•FOLR2+ macrophages reside in a perivascular niche in the tumor stroma•FOLR2+ macrophages interact with tumor-infiltrating CD8+ T cells•FOLR2+ macrophages positively correlate with T cell infiltration and better prognosis
A subset of macrophages marked by FOLR2 that interact with tumor-infiltrating CD8+ T cells are associated with favorable prognosis.
The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among ...breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.
Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, ...centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, and this can be extremely costly. To bypass these issues, we have developed a technique, named CenRICH, to enrich for centromeric DNA from human cells based on selective restriction digestion and size fractionation. Combining restriction enzymes cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of fragments >20 kb, resulted in over 25-fold enrichment in centromeric DNA. High-throughput sequencing revealed that up to 60% of the DNA in the enriched samples is made of centromeric repeats. We show that this method can be used in combination with long-read sequencing to investigate the DNA methylation status of certain centromeres and, with a specific enzyme combination, also of their surrounding regions (mainly HSATII). Finally, we show that CenRICH facilitates single-molecule analysis of replicating centromeric fibers by DNA combing. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of ...the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.
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•Genomically simple RTs are infiltrated by T cell and myeloid populations•Clonally expanded T cell phenotypes suggest a tumor-specific response•Checkpoint blockade induces tumor regression and immune memory in vivo•Endogenous retrovirus expression is linked to the immunogenicity of RTs
Leruste et al. find that, despite their low mutation burden, rhabdoid tumors have a high rate of infiltration by T cells and myeloid cells, and the immunogenicity is linked to endogenous retrovirus expression. Immune checkpoint blockade induces tumor regression in a rhabdoid tumor mouse model.
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