Single-cell RNA sequencing of cystic fibrosis liver disease explants reveals endothelial complement activation.

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.

Distinct immunometabolic signatures in circulating immune cells define disease outcome in acute-on-chronic liver failure.

BACKGROUND AIMS: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and non-recovery in ACLF. APPROACH RESULTS: Here we mapped the single-cell transcriptome of circulating immune cells from ACLF-, acute decompensated (AD) cirrhosis patients and healthy individuals. We further interrogate how these findings as well as immunometabolic- and functional profiles associate with ACLF recovery (ACLF-R) or non-recovery (ACLF-NR). Our analysis unveiled two distinct states of classical monocytes (cMon). Hereto, ACLF-R cMons were characterized by transcripts associated with immune- and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1 . Additional metabolomic- and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program and blunted activation among lymphoid populations in ACLF-R patients. Conversely, ACLF-NR cMon featured elevated expression of inflammatory- and stress response genes such as VIM , LGALS2 , and TREM1 along with blunted metabolic activity and increased functionality. CONCLUSIONS: This study identifies distinct immuno-metabolic cellular states that contribute to disease outcome in ACLF patients. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or non-recovery phenotypes which may be harnessed as potential therapeutic targets in the future.

A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Transcriptomic characterization of the histopathological growth patterns in breast cancer liver metastases.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM.

CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Comparison of the single-cell and single-nucleus hepatic myeloid landscape within decompensated cirrhosis patients.

Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

PD-1- CD45RA+ effector-memory CD8 T cells and CXCL10+ macrophages are associated with response to atezolizumab plus bevacizumab in advanced hepatocellular carcinoma.

The combination of atezolizumab plus bevacizumab (atezo/bev) has dramatically changed the treatment landscape of advanced HCC (aHCC), achieving durable responses in some patients. Using single-cell transcriptomics, we characterize the intra-tumoural and peripheral immune context of patients with aHCC treated with atezo/bev. Tumours from patients with durable responses are enriched for PDL1+ CXCL10+ macrophages and, based on cell-cell interaction analysis, express high levels of CXCL9/10/11 and are predicted to attract peripheral CXCR3+ CD8+ effector-memory T cells (CD8 TEM) into the tumour. Based on T cell receptor sharing and pseudotime trajectory analysis, we propose that CD8 TEM preferentially differentiate into clonally-expanded PD1- CD45RA+ effector-memory CD8+ T cells (CD8 TEMRA) with pronounced cytotoxicity. In contrast, in non-responders, CD8 TEM remain frozen in their effector-memory state. Finally, in responders, CD8 TEMRA display a high degree of T cell receptor sharing with blood, consistent with their patrolling activity. These findings may help understand the possible mechanisms underlying response to atezo/bev in aHCC.

Single-cell profiling and zebrafish avatars reveal LGALS1 as immunomodulating target in glioblastoma.

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

Differences in the Tumor Molecular and Microenvironmental Landscape between Early (Non-Metastatic) and De Novo Metastatic Primary Luminal Breast Tumors.

Background: The molecular mechanisms underlying the de novo metastasis of luminal breast cancer (dnMBC) remain largely unknown. Materials and Methods: Newly diagnosed dnMBC patients (grade 2/3, ER+, PR+/-, HER2-), with available core needle biopsy (CNB), collected from the primary tumor, were selected from our clinical-pathological database. Tumors from dnMBC patients were 1:1 pairwise matched (n = 32) to tumors from newly diagnosed patients who had no distant metastases at baseline (eBC group). RNA was extracted from 5 × 10 µm sections of FFPE CNBs. RNA sequencing was performed using the Illumina platform. Differentially expressed genes (DEG)s were assessed using EdgeR; deconvolution was performed using CIBERSORTx to assess immune cell fractions. A paired Wilcoxon test was used to compare dnMBC and eBC groups and corrected for the false discovery rate. Results: Many regulatory DEGs were significantly downregulated in dnMBC compared to eBC. Also, immune-related and hypoxia-related signatures were significantly upregulated. Paired Wilcoxon analysis showed that the CCL17 and neutrophils fraction were significantly upregulated, whereas the memory B-cell fraction was significantly downregulated in the dnMBC group. Conclusions: Primary luminal tumors of dnMBC patients display significant transcriptomic and immunological differences compared to comparable tumors from eBC patients.

Small cell lung cancer: circulating tumor cell lines and expression of mediators of angiogenesis and coagulation.

Aim: Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna. Methods: Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated. Results: The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF. Conclusions: Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer.

PARP inhibitor predictive value of the Leuven HRD test compared with Myriad MyChoice CDx PLUS HRD on 468 ovarian cancer patients from the PAOLA-1/ENGOT-ov25 trial.

BACKGROUND: The PAOLA-1/ENGOT-ov25 trial showed improved progression-free (PFS) and overall survival (OS) in homologous recombination deficient (HRD) positive patients treated with olaparib, but not when HRD negative (HRD tested with MyChoice CDx PLUS [Myriad test]). PATIENTS AND METHODS: The academic Leuven HRD test consists of capture-based targeted sequencing of genome-wide single-nucleotide polymorphisms and coding exons of eight HR genes including BRCA1, BRCA2, and TP53. We compared the predictive value of the Leuven HRD versus Myriad HRD test for PFS and OS in the randomised PAOLA-1 trial. RESULTS: 468 patients had left-over DNA after Myriad testing for Leuven HRD testing. Positive/negative/overall percent agreement for the Leuven versus Myriad HRD status was 95%/86%/91%, respectively. Tumours were HRD+ in 55% and 52%, respectively. In Leuven HRD+ patients, 5years PFS (5yPFS) was 48.6% versus 20.3% (HR 0.431; 95% confidence intervals (CI) 0.312-0.595) for olaparib versus placebo, respectively (Myriad test 0.409; 95% CI 0.292-0.572). In Leuven HRD+/BRCAwt patients 5yPFS was 41.3% versus 12.6% (HR 0.497; 95% CI 0.316-0.783), and 43.6% versus 13.3% (HR 0.435; 95% CI 0.261-0.727) for the Myriad test. 5yOS was prolonged in the HRD+ subgroup with both tests 67.2% versus 54.4% (HR 0.663; 95% CI 0.442-0.995) for the Leuven test, and 68.0% versus 51.8% (HR 0.596 95% CI 0.393-0.904) for the Myriad test. HRD status was undetermined in 10.7% and 9.4% of the samples, respectively. CONCLUSIONS: A robust correlation between the Leuven HRD and Myriad test was observed. For HRD+ tumours, the academic Leuven HRD showed a similar difference in PFS and OS as the Myriad test.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

Metabolomic and transcriptomic response to imatinib treatment of gastrointestinal stromal tumour in xenograft-bearing mice.

BACKGROUND: Although imatinib is a well-established first-line drug for treating a vast majority of gastrointestinal stromal tumours (GIST), GISTs acquire secondary resistance during therapy. Multi-omics approaches provide an integrated perspective to empower the development of personalised therapies through a better understanding of functional biology underlying the disease and molecular-driven selection of the best-targeted individualised therapy. In this study, we applied integrative metabolomic and transcriptomic analyses to elucidate tumour biochemical processes affected by imatinib treatment. MATERIALS AND METHODS: A GIST xenograft mouse model was used in the study, including 10 mice treated with imatinib and 10 non-treated controls. Metabolites in tumour extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). RNA sequencing was also performed on the samples subset (n=6). RESULTS: Metabolomic analysis revealed 21 differentiating metabolites, whereas next-generation RNA sequencing data analysis resulted in 531 differentially expressed genes. Imatinib significantly changed the profile of metabolites associated mainly with purine and pyrimidine metabolism, butanoate metabolism, as well as alanine, aspartate, and glutamate metabolism. The related changes in transcriptomic profiles included genes involved in kinase activity and immune responses, as well as supported its impact on the purine biosynthesis pathway. CONCLUSIONS: Our multi-omics study confirmed previously known pathways involved in imatinib anticancer activity as well as correlated imatinib-relevant downregulation of expression of purine biosynthesis pathway genes with the reduction of respectful metabolites. Furthermore, considering the importance of the purine biosynthesis pathway for cancer proliferation, we identified a potentially novel mechanism for the anti-tumour activity of imatinib. Based on the results, we hypothesise metabolic modulations aiming at the reduction in purine and pyrimidine pool may ensure higher imatinib efficacy or re-sensitise imatinib-resistant tumours.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.