The role of coronary artery disease in lung transplantation: a propensity-matched analysis.

BACKGROUND AND AIMS: Candidate selection for lung transplantation (LuTx) is pivotal to ensure individual patient benefit as well as optimal donor organ allocation. The impact of coronary artery disease (CAD) on post-transplant outcomes remains controversial. We provide comprehensive data on the relevance of CAD for short- and long-term outcomes following LuTx and identify risk factors for mortality. METHODS: We retrospectively analyzed all adult patients (≥ 18 years) undergoing primary and isolated LuTx between January 2000 and August 2021 at the LMU University Hospital transplant center. Using 1:1 propensity score matching, 98 corresponding pairs of LuTx patients with and without relevant CAD were identified. RESULTS: Among 1,003 patients having undergone LuTx, 104 (10.4%) had relevant CAD at baseline. There were no significant differences in in-hospital mortality (8.2% vs. 8.2%, p > 0.999) as well as overall survival (HR 0.90, 95%CI [0.61, 1.32], p = 0.800) between matched CAD and non-CAD patients. Similarly, cardiovascular events such as myocardial infarction (7.1% CAD vs. 2.0% non-CAD, p = 0.170), revascularization by percutaneous coronary intervention (5.1% vs. 1.0%, p = 0.212), and stroke (2.0% vs. 6.1%, p = 0.279), did not differ statistically between both matched groups. 7.1% in the CAD group and 2.0% in the non-CAD group (p = 0.078) died from cardiovascular causes. Cox regression analysis identified age at transplantation (HR 1.02, 95%CI [1.01, 1.04], p < 0.001), elevated bilirubin (HR 1.33, 95%CI [1.15, 1.54], p < 0.001), obstructive lung disease (HR 1.43, 95%CI [1.01, 2.02], p = 0.041), decreased forced vital capacity (HR 0.99, 95%CI [0.99, 1.00], p = 0.042), necessity of reoperation (HR 3.51, 95%CI [2.97, 4.14], p < 0.001) and early transplantation time (HR 0.97, 95%CI [0.95, 0.99], p = 0.001) as risk factors for all-cause mortality, but not relevant CAD (HR 0.96, 95%CI [0.71, 1.29], p = 0.788). Double lung transplant was associated with lower all-cause mortality (HR 0.65, 95%CI [0.52, 0.80], p < 0.001), but higher in-hospital mortality (OR 2.04, 95%CI [1.04, 4.01], p = 0.039). CONCLUSION: In this cohort, relevant CAD was not associated with worse outcomes and should therefore not be considered a contraindication for LuTx. Nonetheless, cardiovascular events in CAD patients highlight the necessity of control of cardiovascular risk factors and a structured cardiac follow-up.

A contemporary training concept in critical care cardiology.

Critical care cardiology (CCC) in the modern era is shaped by a multitude of innovative treatment options and an increasingly complex, ageing patient population. Generating high-quality evidence for novel interventions and devices in an intensive care setting is exceptionally challenging. As a result, formulating the best possible therapeutic approach continues to rely predominantly on expert opinion and local standard operating procedures. Fostering the full potential of CCC and the maturation of the next generation of decision-makers in this field calls for an updated training concept, that encompasses the extensive knowledge and skills required to care for critically ill cardiac patients while remaining adaptable to the trainee's individual career planning and existing educational programs. In the present manuscript, we suggest a standardized training phase in preparation of the first ICU rotation, propose a modular CCC core curriculum, and outline how training components could be conceptualized within three sub-specialization tracks for aspiring cardiac intensivists.

Luminal breast cancer identity is determined by loss of glucocorticoid receptor activity.

Glucocorticoid receptor (GR) is a transcription factor that plays a crucial role in cancer biology. In this study, we utilized an in silico-designed GR activity signature to demonstrate that GR relates to the proliferative capacity of numerous primary cancer types. In breast cancer, the GR activity status determines luminal subtype identity and has implications for patient outcomes. We reveal that GR engages with estrogen receptor (ER), leading to redistribution of ER on the chromatin. Notably, GR activation leads to upregulation of the ZBTB16 gene, encoding for a transcriptional repressor, which controls growth in ER-positive breast cancer and associates with prognosis in luminal A patients. In relation to ZBTB16's repressive nature, GR activation leads to epigenetic remodeling and loss of histone acetylation at sites proximal to cancer-driving genes. Based on these findings, epigenetic inhibitors reduce viability of ER-positive breast cancer cells that display absence of GR activity. Our findings provide insights into how GR controls ER-positive breast cancer growth and may have implications for patients' prognostication and provide novel therapeutic candidates for breast cancer treatment.

Performance of tumour microenvironment deconvolution methods in breast cancer using single-cell simulated bulk mixtures.

Cells within the tumour microenvironment (TME) can impact tumour development and influence treatment response. Computational approaches have been developed to deconvolve the TME from bulk RNA-seq. Using scRNA-seq profiling from breast tumours we simulate thousands of bulk mixtures, representing tumour purities and cell lineages, to compare the performance of nine TME deconvolution methods (BayesPrism, Scaden, CIBERSORTx, MuSiC, DWLS, hspe, CPM, Bisque, and EPIC). Some methods are more robust in deconvolving mixtures with high tumour purity levels. Most methods tend to mis-predict normal epithelial for cancer epithelial as tumour purity increases, a finding that is validated in two independent datasets. The breast cancer molecular subtype influences this mis-prediction. BayesPrism and DWLS have the lowest combined numbers of false positives and false negatives, and have the best performance when deconvolving granular immune lineages. Our findings highlight the need for more single-cell characterisation of rarer cell types, and suggest that tumour cell compositions should be considered when deconvolving the TME.

TAVI Under Pressure: Intra-balloon Pressure Profiles During Balloon-Expandable TAVR-First Data from a Feasibility Study.

Our study investigated the feasibility to measure pressure profiles inside the inflation balloon during direct implantation of Edwards Sapien 3 ultra-prostheses using an additional syringe with a digital pressure read-out. Pressure profiles of 15 patients for 26 mm valve size were analyzed. Uniform patterns were found for 5 patients similar to those of previously acquired in vitro curves. 10 patients showed strikingly different pressure profiles compared to the above-mentioned group, marked by an earlier pressure increase, single or multiple pressure drops or higher overall pressure. Measuring the percentage of under-expansion of the prostheses, using calibrated angiographic projections revealed a significant difference between both groups. Our data raises the hypothesis that the acquisition of pressure profiles might help to better understand not only the implantation procedure itself but also the highly individual patient-device interaction, offering new information and a new perspective on optimization of TAVR implantation in the future.

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and immunotherapy response.

Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal diversity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.

Dual ProGlide versus ProGlide and FemoSeal for vascular access haemostasis after transcatheter aortic valve implantation.

BACKGROUND: Large-bore arteriotomy for transcatheter aortic valve implantation (TAVI) requires percutaneous vascular closure devices, but real-world data comparing different closure strategies are limited. AIMS: We sought to compare a dual ProGlide strategy vs a combination of one ProGlide and one FemoSeal for vascular closure after TAVI. METHODS: We retrospectively analysed 874 propensity score-matched patients undergoing TAVI at the Munich University Hospital from August 2018 to October 2020. From August 2018 to August 2019, a dual ProGlide strategy was used for vascular closure. From October 2019 to October 2020, a combination of one ProGlide and one FemoSeal was used. The primary endpoint was defined as access-related major vascular complications or bleeding ≥Type 2 according to Valve Academic Research Consortium 3 criteria. RESULTS: Patients in the dual ProGlide group (n=437) had a higher incidence of the primary endpoint than patients treated with one ProGlide and one FemoSeal (n=437; 11.4% vs 3.0%; p<0.001). Furthermore, they had a higher rate of closure device failure (2.7% vs 0.9%; p=0.044) and more often required unplanned surgery or endovascular treatment (3.9% vs 0.9%; p=0.004). The incidence of death did not differ significantly between groups (3.4% vs 1.6%; p=0.08). CONCLUSIONS: A combined ProGlide and FemoSeal strategy might have the potential to reduce access-related vascular complications following TAVI.

Transcatheter Aortic Valve Replacement with the Self-Expandable Core Valve Evolut Prosthesis Using the Cusp-Overlap vs. Tricusp-View.

Despite the rapid increase in experience and technological improvement, the incidence of conduction disturbances in patients undergoing transcatheter aortic valve replacement (TAVR) with the self-expandable CoreValve Evolut valve remains high. Recently, a cusp-overlap view (COP) implantation technique has been proposed for TAVR with self-expandable valves offering an improved visualization during valve expansion compared to the three-cusp view (TCV). This study aims to systematically analyze procedural outcomes of TAVR patients treated with the CoreValve Evolut valve using a COP compared to TCV in a high-volume center. The primary endpoint was technical success according the 2021 VARC-3 criteria. A total of 122 consecutive patients (61 pts. TCV: April 2019 to November 2020; 61 pts. COP: December 2020 to October 2021) that underwent TAVR with the CoreValve Evolut prosthesis were included in this analysis. Although there was no difference in the primary endpoint technical success between TCV and COP patients (93.4% vs. 90.2%, OR 0.65, 95% CI 0.16, 2.4, p = 0.51), we observed a significantly lower risk for permanent pacemaker implantation (PPI) among COP patients (TCV: 27.9% vs. COP: 13.1%, OR 0.39, 95% CI 0.15, 0.97, p = 0.047). Implantation of the CoreValve Evolut prosthesis using the COP might help to reduce the rate of PPI following TAVR.

Spatial deconvolution of HER2-positive breast cancer delineates tumor-associated cell type interactions.

In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra- and interpatient heterogeneity; from which we discover shared gene signatures for immune and tumor processes. By integration with single cell data, we spatially map tumor-associated cell types to find tertiary lymphoid-like structures, and a type I interferon response overlapping with regions of T-cell and macrophage subset colocalization. We construct a predictive model to infer presence of tertiary lymphoid-like structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define a high resolution map of cellular interactions in tumors and provide tools generalizing across tissues and diseases.

A single-cell and spatially resolved atlas of human breast cancers.

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.

Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease.

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis.

BACKGROUND: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. METHODS: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. RESULTS: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. CONCLUSIONS: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Inhibitor of Differentiation 4 (ID4) represses mammary myoepithelial differentiation via inhibition of HEB.

Inhibitor of differentiation (ID) proteins dimerize with basic HLH (bHLH) transcription factors, repressing transcription of lineage-specification genes across diverse cellular lineages. ID4 is a key regulator of mammary stem cells; however, the mechanism by which it achieves this is unclear. Here, we show that ID4 has a cell autonomous role in preventing myoepithelial differentiation of basal cells in mammary organoids and in vivo. ID4 positively regulates proliferative genes and negatively regulates genes involved in myoepithelial function. Mass spectrometry reveals that ID4 interacts with the bHLH protein HEB, which binds to E-box motifs in regulatory elements of basal developmental genes involved in extracellular matrix and the contractile cytoskeleton. We conclude that high ID4 expression in mammary basal stem cells antagonizes HEB transcriptional activity, preventing myoepithelial differentiation and allowing for appropriate tissue morphogenesis. Downregulation of ID4 during pregnancy modulates gene regulated by HEB, promoting specialization of basal cells into myoepithelial cells.

The androgen receptor is a tumor suppressor in estrogen receptor-positive breast cancer.

The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a diverse, clinically relevant panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.

Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy.

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1-Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1-Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Stromal cell diversity associated with immune evasion in human triple-negative breast cancer.

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.