In vivo DNA replication dynamics unveil aging-dependent replication stress.

The genome duplication program is affected by multiple factors in vivo, including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10-50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging.

Released bacterial ATP shapes local and systemic inflammation during abdominal sepsis.

Sepsis causes millions of deaths per year worldwide and is a current global health priority declared by the WHO. Sepsis-related deaths are a result of dysregulated inflammatory immune responses indicating the need to develop strategies to target inflammation. An important mediator of inflammation is extracellular adenosine triphosphate (ATP) that is released by inflamed host cells and tissues, and also by bacteria in a strain-specific and growth-dependent manner. Here, we investigated the mechanisms by which bacteria release ATP. Using genetic mutant strains of Escherichia coli (E. coli), we demonstrate that ATP release is dependent on ATP synthase within the inner bacterial membrane. In addition, impaired integrity of the outer bacterial membrane notably contributes to ATP release and is associated with bacterial death. In a mouse model of abdominal sepsis, local effects of bacterial ATP were analyzed using a transformed E. coli bearing an arabinose-inducible periplasmic apyrase hydrolyzing ATP to be released. Abrogating bacterial ATP release shows that bacterial ATP suppresses local immune responses, resulting in reduced neutrophil counts and impaired survival. In addition, bacterial ATP has systemic effects via its transport in outer membrane vesicles (OMV). ATP-loaded OMV are quickly distributed throughout the body and upregulated expression of genes activating degranulation in neutrophils, potentially contributing to the exacerbation of sepsis severity. This study reveals mechanisms of bacterial ATP release and its local and systemic roles in sepsis pathogenesis.

Sepsis is a severe condition often caused by the body's immune system overreacting to bacterial infections. This can lead to excessive inflammation which damages organs and requires urgent medical care. With sepsis claiming millions of lives each year, new and improved ways to treat this condition are urgently needed. One potential strategy for treating sepsis is to target the underlying mechanisms controlling inflammation. Inflamed and dying cells release molecules called ATP (the energy carrier of all living cells), which strongly influence the immune system, including during sepsis. In the early stages of an infection, ATP acts as a danger signal warning the body that something is wrong. However, over time, it can worsen infections by disturbing the immune response. Similar to human cells, bacteria release their own ATP, which can have different impacts depending on the type of bacteria and where they are located in the body. However, it is not well understood how bacterial ATP influences severe infections like sepsis. To investigate this question, Spari et al analysed how ATP is released from Escherichia coli, a type of bacteria that causes severe infections. This revealed that the bacteria secrete ATP directly in to their environment and via small membrane-bound structures called vesicles. Spari et al. then probed a mouse model of abdominal sepsis which had been infected with E. coli that release either normal or low levels of ATP. They found that the ATP released from E. coli impaired the mice's survival and lowered the number of neutrophils (immune cells which are important for defending against bacteria) at the site of the infection. The ATP secreted via vesicles also altered the role of neutrophils but in more distant regions, and it is possible that these changes may be contributing to the severity of sepsis. These findings provide a better understanding of how ATP released from bacteria impacts the immune system during sepsis. While further investigation is needed, these findings may offer new therapeutic targets for treating sepsis.

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND: The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS: The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION: Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

The Placenta-A New Source of Bile Acids during Healthy Pregnancy? First Results of a Gene Expression Study in Humans and Mice.

Bile acids (BAs) are natural ligands for several receptors modulating cell activities. BAs are synthesized via the classic (neutral) and alternative (acidic) pathways. The classic pathway is initiated by CYP7A1/Cyp7a1, converting cholesterol to 7α-hydroxycholesterol, while the alternative pathway starts with hydroxylation of the cholesterol side chain, producing an oxysterol. In addition to originating from the liver, BAs are reported to be synthesized in the brain. We aimed at determining if the placenta potentially represents an extrahepatic source of BAs. Therefore, the mRNAs coding for selected enzymes involved in the hepatic BA synthesis machinery were screened in human term and CD1 mouse late gestation placentas from healthy pregnancies. Additionally, data from murine placenta and brain tissue were compared to determine whether the BA synthetic machinery is comparable in these organs. We found that CYP7A1, CYP46A1, and BAAT mRNAs are lacking in the human placenta, while corresponding homologs were detected in the murine placenta. Conversely, Cyp8b1 and Hsd17b1 mRNAs were undetected in the murine placenta, but these enzymes were found in the human placenta. CYP39A1/Cyp39a1 and cholesterol 25-hydroxylase (CH25H/Ch25h) mRNA expression were detected in the placentas of both species. When comparing murine placentas and brains, Cyp8b1 and Hsd17b1 mRNAs were only detected in the brain. We conclude that BA synthesis-related genes are placentally expressed in a species-specific manner. The potential placentally synthesized BAs could serve as endocrine and autocrine stimuli, which may play a role in fetoplacental growth and adaptation.

Tumour mutations in long noncoding RNAs enhance cell fitness.

Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched for previously-reported cancer genes and a range of clinical and genomic features. A number of these lncRNAs promote tumour cell proliferation when overexpressed in in vitro models. Our results also highlight a dense SNV hotspot in the widely-studied NEAT1 oncogene. To directly evaluate the functional significance of NEAT1 SNVs, we use in cellulo mutagenesis to introduce tumour-like mutations in the gene and observe a significant and reproducible increase in cell fitness, both in vitro and in a mouse model. Mechanistic studies reveal that SNVs remodel the NEAT1 ribonucleoprotein and boost subnuclear paraspeckles. In summary, this work demonstrates the utility of driver analysis for mapping cancer-promoting lncRNAs, and provides experimental evidence that somatic mutations can act through lncRNAs to enhance pathological cancer cell fitness.

A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response.

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.

ILC3s restrict the dissemination of intestinal bacteria to safeguard liver regeneration after surgery.

It is generally believed that environmental or cutaneous bacteria are the main origin of surgical infections. Therefore, measures to prevent postoperative infections focus on optimizing hygiene and improving asepsis and antisepsis. In a large cohort of patients with infections following major surgery, we identified that the causative bacteria are mainly of intestinal origin. Postoperative infections of intestinal origin were also found in mice undergoing partial hepatectomy. CCR6+ group 3 innate lymphoid cells (ILC3s) limited systemic bacterial spread. Such bulwark function against host invasion required the production of interleukin-22 (IL-22), which controlled the expression of antimicrobial peptides in hepatocytes, thereby limiting bacterial spread. Using genetic loss-of-function experiments and punctual depletion of ILCs, we demonstrate that the failure to restrict intestinal commensals by ILC3s results in impaired liver regeneration. Our data emphasize the importance of endogenous intestinal bacteria as a source for postoperative infection and indicate ILC3s as potential new targets.

Heat shock protein 90 (HSP90) inhibitors in gastrointestinal cancer: where do we currently stand?-A systematic review.

PURPOSE: Dysregulated expression of heat shock proteins (HSP) plays a fundamental role in tumor development and progression. Consequently, HSP90 may be an effective tumor target in oncology, including the treatment of gastrointestinal cancers. METHODS: We carried out a systematic review of data extracted from clinicaltrials.gov and pubmed.gov, which included all studies available until January 1st, 2022. The published data was evaluated using primary and secondary endpoints, particularly with focus on overall survival, progression-free survival, and rate of stable disease. RESULTS: Twenty trials used HSP90 inhibitors in GI cancers, ranging from phase I to III clinical trials. Most studies assessed HSP90 inhibitors as a second line treatment. Seventeen of the 20 studies were performed prior to 2015 and only few studies have results pending. Several studies were terminated prematurely, due to insufficient efficacy or toxicity. Thus far, the data suggests that HSP90 inhibitor NVP-AUY922 might improve outcome for colorectal cancer and gastrointestinal stromal tumors. CONCLUSION: It currently remains unclear which subgroup of patients might benefit from HSP90 inhibitors and at what time point these inhibitors may be beneficial. There are only few new or ongoing studies initiated during the last decade.

OMIP-088: Twenty-target imaging mass cytometry panel for major cell populations in mouse formalin fixed paraffin embedded liver.

The purpose of this 20-target imaging mass cytometry (IMC) panel is to identify the main cell types in formalin fixed paraffin embedded (FFPE) mouse liver tissue with the Hyperion™ mass cytometer from Standard BioTools (formerly Fluidigm). The antibody panel includes markers to identify hepatocytes (E-cadherin, HNF4α (hepatocyte nuclear factor 4 alpha), Arginase-1), liver sinusoidal endothelial cells (LSECs; CD206), Kupffer cells (F4/80, CD206), neutrophils (Ly6G, CD11b), bone marrow derived myeloid cells (BMDMs; CD11b), cholangiocytes (E-cadherin high), endothelial cells (CD31, α-SMA), plasmacytoid dendritic cells (CD317), B cells (CD19), T cells (CD3e, CD4, CD8a), NK cells (CD161) as well markers of cell activation (CD44, CD74), proliferation (Ki-67) and to aid in cell segmentation (Pan-Actin, E-cadherin, histone H3). The panel has been tested in other mouse tissues, namely the spleen, colon and lung, and therefore is likely to work across various mouse FFPE samples of interest. It has not been tested using human samples, frozen samples or in suspension mass cytometry because FFPE treatment profoundly changes epitope conformation. In summary, this panel is a powerful tool for pre-clinical research to determine cellular abundance and spatial distribution within mouse tissues and serves as a scaffold, to which more targets can be added for project specific requirements.

Combination of Sterile Injury and Microbial Contamination to Model Post-surgical Peritoneal Adhesions in Mice.

Abdominal surgeries are frequently associated with the development of post-surgical adhesions. These are irreversible fibrotic scar bands that appear between abdominal organs and the abdominal wall. Patients suffering from adhesions are at risk of severe complications, such as small bowel obstruction, chronic pelvic pain, or infertility. To date, no cure exists, and the understanding of underlying molecular mechanisms of adhesion formation is incomplete. The current paradigm largely relies on sterile injury mouse models. However, abdominal surgeries in human patients are rarely completely sterile procedures. Here, we describe a modular surgical procedure for simultaneous or separate induction of sterile injury and microbial contamination. Combined, these insults synergistically lead to adhesion formation in the mouse peritoneal cavity. Surgical trauma is confined to a localized sterile injury of the peritoneum. Microbial contamination of the peritoneal cavity is induced by a limited perforation of the microbe-rich large intestine or by injection of fecal content. The presented protocol extends previous injury-based adhesion models by an additional insult through microbial contamination, which may more adequately model the clinical context of abdominal surgery. Graphical abstract.

Splenic red pulp macrophages provide a niche for CML stem cells and induce therapy resistance.

Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs), and cure relies on their eradication. The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance. Although leukemic infiltration of the spleen is a hallmark of CML, it is unknown whether spleen cells form a niche that maintains LSCs. Here, we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression. Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model. Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen. Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs). These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state, resulting in disease progression and resistance to therapy.

Primary Infection by E. multilocularis Induces Distinct Patterns of Cross Talk between Hepatic Natural Killer T Cells and Regulatory T Cells in Mice.

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients, the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points, downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice.

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus.

Background & Aims: High-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis. Methods: Using a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry. Results: At 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype. Conclusions: Based on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype. Lay summary: Irradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors.

LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.

The LIM-domain protein Ajuba is associated with cell proliferation, a fundamental process of tissue regeneration and cancer. We report that in the liver, Ajuba expression is increased during regeneration and in tumour cells and tissues. Knockout of Ajuba using CRISPR/Cas9 is embryonic lethal in mice. shRNA targeting of Ajuba reduces cell proliferation, delays cell entry into S-phase, reduces cell survival and tumour growth in vivo and increases expression of the DNA damage marker γH2AX. Ajuba binding partners include proteins involved in DNA replication and damage, such as SKP2, MCM2, MCM7 and RPA70. Taken together, our data support that Ajuba promotes liver cell proliferation associated with development, regeneration and tumour growth and is involved in DNA replication and damage repair.

Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells.

OBJECTIVE: Mucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory and antimicrobial functions and are implicated in liver fibrogenesis. It is not well understood which liver cells function as antigen (Ag)-presenting cells for MAIT cells, and under which conditions stimulatory Ags reach the circulation. DESIGN: We used different types of primary human liver cells in Ag-presentation assays to blood-derived and liver-derived MAIT cells. We assessed MAIT cell stimulatory potential of serum from healthy subjects and patients with portal hypertension undergoing transjugular intrahepatic portosystemic shunt stent, and patients with inflammatory bowel disease (IBD). RESULTS: MAIT cells were dispersed throughout healthy human liver and all tested liver cell types stimulated MAIT cells, hepatocytes being most efficient. MAIT cell activation by liver cells occurred in response to bacterial lysate and pure Ag, and was prevented by non-activating MR1 ligands. Serum derived from peripheral and portal blood, and from patients with IBD stimulated MAIT cells in MR1-dependent manner. CONCLUSION: Our findings reveal previously unrecognised roles of liver cells in Ag metabolism and activation of MAIT cells, repression of which creates an opportunity to design antifibrotic therapies. The presence of MAIT cell stimulatory Ags in serum rationalises the observed activated MAIT cell phenotype in liver. Increased serum levels of gut-derived MAIT cell stimulatory ligands in patients with impaired intestinal barrier function indicate that intrahepatic Ag-presentation may represent an important step in the development of liver disease.

The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.

The small non-coding VTRNA1-1 (vault RNA 1-1) is known to confer resistance to apoptosis in several malignant cell lines and to also modulate the macroautophagic/autophagic flux in hepatocytes, thus highlighting its pro-survival role. Here we describe a new function of VTRNA1-1 in regulating in vitro and in vivo tumor cell proliferation, tumorigenesis and chemoresistance. Knockout (KO) of VTRNA1-1 in human hepatocellular carcinoma cells reduced nuclear localization of TFEB (transcription factor EB), leading to a downregulation of the coordinated lysosomal expression and regulation (CLEAR) network genes and lysosomal compartment dysfunction. We demonstrate further that impaired lysosome function due to loss of VTRNA1-1 potentiates the anticancer effect of conventional chemotherapeutic drugs. Finally, loss of VTRNA1-1 reduced drug lysosomotropism allowing higher intracellular compound availability and thereby significantly reducing tumor cell proliferation in vitro and in vivo. These findings reveal a so far unknown role of VTRNA1-1 in the intracellular catabolic compartment and describe its contribution to lysosome-mediated chemotherapy resistance.Abbreviations: ATP6V0D2: ATPase H+ transporting V0 subunit d2; BafA: bafilomycin A1; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; DMSO: dimethyl sulfoxide; GST-BHMT: glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HCC: hepatocellular carcinoma; LAMP1: lysosomal associated membrane protein 1; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MITF: melanocyte inducing transcription factor; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ncRNA: non-coding RNA; RNP: ribonucleoprotein; SF: sorafenib; SQSTM1/p62: sequestosome 1; STS: staurosporine; tdRs: tRNA-derived RNAs; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; vtRNA: vault RNA transcript.

Intraperitoneal microbial contamination drives post-surgical peritoneal adhesions by mesothelial EGFR-signaling.

Abdominal surgeries are lifesaving procedures but can be complicated by the formation of peritoneal adhesions, intra-abdominal scars that cause intestinal obstruction, pain, infertility, and significant health costs. Despite this burden, the mechanisms underlying adhesion formation remain unclear and no cure exists. Here, we show that contamination of gut microbes increases post-surgical adhesion formation. Using genetic lineage tracing we show that adhesion myofibroblasts arise from the mesothelium. This transformation is driven by epidermal growth factor receptor (EGFR) signaling. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor, are sufficient to induce these changes. Correspondingly, EGFR inhibition leads to a significant reduction of adhesion formation in mice. Adhesions isolated from human patients are enriched in EGFR positive cells of mesothelial origin and human mesothelium shows an increase of mesothelial EGFR expression during bacterial peritonitis. In conclusion, bacterial contamination drives adhesion formation through mesothelial EGFR signaling. This mechanism may represent a therapeutic target for the prevention of adhesions after intra-abdominal surgery.