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.

Ethanol induces replication fork stalling and membrane stress in immortalized laryngeal cells.

Although ethanol is a class I carcinogen and is linked to more than 700,000 cancer incidences, a clear understanding of the molecular mechanisms underlying ethanol-related carcinogenesis is still lacking. Further understanding of ethanol-related cell damage can contribute to reducing or treating alcohol-related cancers. Here, we investigated the effects of both short- and long-term exposure of human laryngeal epithelial cells to different ethanol concentrations. RNA sequencing shows that ethanol altered gene expression patterns in a time- and concentration-dependent way, affecting genes involved in ribosome biogenesis, cytoskeleton remodeling, Wnt signaling, and transmembrane ion transport. Additionally, ethanol induced a slower cell proliferation, a delayed cell cycle progression, and replication fork stalling. In addition, ethanol exposure resulted in morphological changes, which could be associated with membrane stress. Taken together, our data yields a comprehensive view of molecular changes associated with ethanol stress in epithelial cells of the upper aerodigestive tract.

Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

Lung epithelial and myeloid innate immunity in influenza-associated or COVID-19-associated pulmonary aspergillosis: an observational study.

BACKGROUND: Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) affect about 15% of critically ill patients with influenza or COVID-19, respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality, but data on their pathophysiology are scarce. We aimed to explore the role of lung epithelial and myeloid innate immunity in patients with IAPA or CAPA. METHODS: In this observational study, we retrospectively recruited patients who had been admitted to the intensive care unit (ICU) of University Hospitals Leuven, Belgium, requiring non-invasive or invasive ventilation because of severe influenza or COVID-19, with or without aspergillosis, between Jan 1, 2011, and March 31, 2021, whose bronchoalveolar lavage samples were available at the hospital biobank. Additionally, biobanked in vivo tracheobronchial biopsy samples from patients with IAPA or CAPA and invasive Aspergillus tracheobronchitis admitted to ICUs requiring invasive ventilation between the same dates were collected from University Hospitals Leuven, Hospital Network Antwerp (Belgium), and Amiens-Picardie University Hospital (France). We did nCounter gene expression analysis of 755 genes linked to myeloid innate immunity and protein analysis of 47 cytokines, chemokines, and growth factors on the bronchoalveolar lavage samples. Gene expression data were used to infer cell fractions by use of CIBERSORTx, to perform hypergeometric enrichment pathway analysis and gene set enrichment analysis, and to calculate pathway module scores for the IL-1ß, TNF-α, type I IFN, and type II IFN (IFNγ) pathways. We did RNAScope targeting influenza virus or SARS-CoV-2 RNA and GeoMx spatial transcriptomics on the tracheobronchial biopsy samples. FINDINGS: Biobanked bronchoalveolar lavage samples were retrieved from 166 eligible patients, of whom 40 had IAPA, 52 had influenza without aspergillosis, 33 had CAPA, and 41 had COVID-19 without aspergillosis. We did nCounter gene expression analysis on bronchoalveolar lavage samples from 134 patients, protein analysis on samples from 162 patients, and both types of analysis on samples from 130 patients. We performed RNAScope and spatial transcriptomics on the tracheobronchial biopsy samples from two patients with IAPA plus invasive Aspergillus tracheobronchitis and two patients with CAPA plus invasive Aspergillus tracheobronchitis. We observed a downregulation of genes associated with antifungal effector functions in patients with IAPA and, to a lesser extent, in patients with CAPA. We found a downregulated expression of several genes encoding proteins with functions in the opsonisation, recognition, and killing of conidia in patients with IAPA versus influenza only and in patients with CAPA versus COVID-19 only. Several genes related to LC3-associated phagocytosis, autophagy, or both were differentially expressed. Patients with CAPA had significantly lower neutrophil cell fractions than did patients with COVID-19 only. Patients with IAPA or CAPA had downregulated IFNγ signalling compared with patients with influenza only or COVID-19 only, respectively. The concentrations of several fibrosis-related growth factors were significantly elevated in the bronchoalveolar lavage fluid from patients with IAPA versus influenza only and from patients with CAPA versus COVID-19 only. In one patient with CAPA, we visualised an active or very recent SARS-CoV-2 infection disrupting the epithelial barrier, facilitating tissue-invasive aspergillosis. INTERPRETATION: Our results reveal a three-level breach in antifungal immunity in IAPA and CAPA, affecting the integrity of the epithelial barrier, the capacity to phagocytise and kill Aspergillus spores, and the ability to destroy Aspergillus hyphae, which is mainly mediated by neutrophils. The potential of adjuvant IFNγ in the treatment of IAPA and CAPA should be investigated. FUNDING: Research Foundation Flanders, Coronafonds, the Max Planck Society, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, "la Caixa" Foundation, and Horizon 2020.

Timing evolution of lobular breast cancer through phylogenetic analysis.

BACKGROUND: Late distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood. METHODS: We retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients. FINDINGS: The data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients. INTERPRETATION: Our results provide insights into ILC evolution and offer potential paths for optimised ILC care. FUNDING: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

Molecular Heterogeneity Between Paired Primary and Metastatic Lesions from Clear Cell Renal Cell Carcinoma.

Highly effective systemic treatments have globally improved outcomes in metastatic clear-cell renal cell carcinoma (m-ccRCC). However, despite many efforts, reliable biomarkers predicting individual responses are currently lacking. Moreover, mixed responses are commonly observed. We hypothesized that molecular heterogeneity between primary tumors and their metastases could flaw biomarker research based on features of the primary tumor and explain mixed responses. Therefore, we studied the heterogeneity of the ccrcc1-4 molecular subtypes across patient-matched primary and metastatic lesions over time in 62 patients with m-ccRCC who underwent both nephrectomy and metastasectomy. These subtypes characterize underlying disease biology and are associated with outcomes in both the primary and metastatic settings. We observed a concordance rate of 58% (95% confidence interval 45-71%). This concordance was not affected by the interval between nephrectomy and resection of the metastatic lesion. Across discordant pairs, the metastatic lesions mostly exhibited a less favorable molecular subtype. Moreover, primary tumors with the favorable ccrcc2 molecular subtype were characterized by favorable prognosis and a long interval between nephrectomy and metastasectomy. Conversely, tumors with the unfavorable ccrcc4 molecular subtype relapsed quickly and had poor prognosis. Thus, the considerable molecular heterogeneity between patient-matched m-ccRCC primary and metastatic lesions provides an explanation for mixed responses to systemic therapy and could impact the development of biomarker studies in which the primary tumor is often considered a surrogate for metastatic disease. Patient summary: We studied primary tumors and metastases from patients with kidney cancer and found considerable heterogeneity in their molecular features. This heterogeneity explains mixed responses to systemic therapy and is important to take into account in future biomarker studies for this disease.