Feasibility of Next-generation Sequencing of Liquid Biopsy (Circulating Tumor DNA) Samples and Tumor Tissue from Patients with Metastatic Prostate Cancer in a Real-world Clinical Setting in Germany.

BACKGROUND AND OBJECTIVE: With European Medicines Agency approval of PARP inhibitors in metastatic castration-resistant prostate cancer and ongoing trials in metastatic hormone-sensitive prostate cancer, detection of genetic alterations in BRCA1/2 and other homologous recombination repair genes has gained an important role. Our aim was to investigate the feasibility and comparability of comprehensive next-generation sequencing (NGS) of liquid biopsy (LB; circulating tumor DNA) and tumor tissue (TT) samples in a real-world clinical setting. METHODS: The study cohort consisted of 50 patients with metastatic prostate cancer (mPC) who had TT NGS performed for BRCA1/2 alterations and consent for additional LB NGS. The Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for TT NGS. The Guardant360 83-gene assay (Guardant Health, Palo Alto, CA, USA) was used for LB NGS, including all types of somatic alterations, microsatellite instability, and blood tumor mutational burden. We calculated BRCA1/2 alteration rates and the negative percentage agreement (NPA) and positive percentage agreement (PPA) between TT and LB results. KEY FINDINGS AND LIMITATIONS: TT NGS was successful in 44/50 patients (88%), with pathogenic BRCA1/2 alterations detected in four (9%). LB NGS was successful in all 50 patients (100%), with BRCA1/2 alterations detected in ten (20%). In a subgroup analysis for the 44 patients with successful TT NGS, NPA was 85% and PPA was 50%. The median time between TT sample collection and blood sampling for NGS was 132 wk (IQR 94-186). The limited sample size and differences in the time of NGS assessment are limitations. CONCLUSIONS AND CLINICAL IMPLICATIONS: LB NGS resulted in a higher detection rate for BRCA1/2 alterations in comparison to conventional TT NGS (20% vs 9%). Ideally, BRCA1/2 testing should be based on both approaches to identify all patients with mPC eligible for PARP inhibitor therapy. PATIENT SUMMARY: Our study shows that genetic tests for both tumor tissue and blood samples results in higher rates of detection of BRCA1/2 gene alterations in patients with metastatic prostate cancer.

The Brain Pre-Metastatic Niche: Biological and Technical Advancements.

Metastasis, particularly brain metastasis, continues to puzzle researchers to this day, and exploring its molecular basis promises to break ground in developing new strategies for combatting this deadly cancer. In recent years, the research focus has shifted toward the earliest steps in the formation of metastasis. In this regard, significant progress has been achieved in understanding how the primary tumor affects distant organ sites before the arrival of tumor cells. The term pre-metastatic niche was introduced for this concept and encompasses all influences on sites of future metastases, ranging from immunological modulation and ECM remodeling to the softening of the blood-brain barrier. The mechanisms governing the spread of metastasis to the brain remain elusive. However, we begin to understand these processes by looking at the earliest steps in the formation of metastasis. This review aims to present recent findings on the brain pre-metastatic niche and to discuss existing and emerging methods to further explore the field. We begin by giving an overview of the pre-metastatic and metastatic niches in general before focusing on their manifestations in the brain. To conclude, we reflect on the methods usually employed in this field of research and discuss novel approaches in imaging and sequencing.

B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis.

Autonomic nerves control organ function through the sympathetic and parasympathetic branches, which have opposite effects. In the bone marrow, sympathetic (adrenergic) nerves promote hematopoiesis; however, how parasympathetic (cholinergic) signals modulate hematopoiesis is unclear. Here, we show that B lymphocytes are an important source of acetylcholine, a neurotransmitter of the parasympathetic nervous system, which reduced hematopoiesis. Single-cell RNA sequencing identified nine clusters of cells that expressed the cholinergic α7 nicotinic receptor (Chrna7) in the bone marrow stem cell niche, including endothelial and mesenchymal stromal cells (MSCs). Deletion of B cell-derived acetylcholine resulted in the differential expression of various genes, including Cxcl12 in leptin receptor+ (LepR+) stromal cells. Pharmacologic inhibition of acetylcholine signaling increased the systemic supply of inflammatory myeloid cells in mice and humans with cardiovascular disease.

tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations.

The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

Cell interactions in the bone marrow microenvironment affecting myeloid malignancies.

The bone marrow is a complex tissue in which heterogeneous populations of stromal cells interact with hematopoietic cells to dynamically respond to organismal needs in defense, hemostasis, and oxygen delivery. Physiologic challenges modify stromal/hematopoietic cell interactions to generate changes in blood cell production. When either stroma or hematopoietic cells are impaired, the system distorts. The distortions associated with myeloid malignancy are reviewed here and may provide opportunities for therapeutic intervention.

Dissecting the spatial bone marrow microenvironment of hematopoietic stem cells.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) reside in specialized anatomical microenvironments within the bone marrow space, termed HSC niches. Different bone marrow imaging modalities have been utilized to visualize HSCs in situ, and unravel the cellular identity of bone marrow cell types located in their immediate proximity. However, despite extensive research, the exact identity of bone marrow populations that physically associate with HSCs remains controversial. RECENT FINDINGS: Recent advances in volumetric imaging enable precise identification of bone marrow populations and their spatial distribution both at tissue-wide scale and single-cell resolution. In addition, single-cell RNA sequencing and mass-cytometry-based approaches dissect the complexity of the bone marrow microenvironment with unprecedented resolution. Here, we review current concepts regarding bone marrow populations that physically associate with HSCs and recent efforts to localize HSCs and their niche populations. SUMMARY: Defining the bone marrow cell types in the immediate proximity of HSCs in homeostasis and stress is key to determine the cellular and molecular cues driving HSC maintenance and regeneration.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

Publisher Correction: Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells.

Haematopoietic stem cells self-renew and differentiate into all blood lineages throughout life, and can repair damaged blood systems upon transplantation. Asymmetric cell division has previously been suspected to be a regulator of haematopoietic-stem-cell fate, but its existence has not directly been shown1. In asymmetric cell division, asymmetric fates of future daughter cells are prospectively determined by a mechanism that is linked to mitosis. This can be mediated by asymmetric inheritance of cell-extrinsic niche signals by, for example, orienting the divisional plane, or by the asymmetric inheritance of cell-intrinsic fate determinants. Observations of asymmetric inheritance or of asymmetric daughter-cell fates alone are not sufficient to demonstrate asymmetric cell division2. In both cases, sister-cell fates could be controlled by mechanisms that are independent of division. Here we demonstrate that the cellular degradative machinery-including lysosomes, autophagosomes, mitophagosomes and the protein NUMB-can be asymmetrically inherited into haematopoietic-stem-cell daughter cells. This asymmetric inheritance predicts the asymmetric future metabolic and translational activation and fates of haematopoietic-stem-cell daughter cells and their offspring. Therefore, our studies provide evidence for the existence of asymmetric cell division in haematopoietic stem cells.

Fate Distribution and Regulatory Role of Human Mesenchymal Stromal Cells in Engineered Hematopoietic Bone Organs.

The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs' fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments.

Stress-Induced Changes in Bone Marrow Stromal Cell Populations Revealed through Single-Cell Protein Expression Mapping.

Stromal cell populations that maintain hematopoietic stem and progenitor cells (HSPCs) are generally characterized in steady-state conditions. Here, we report a comprehensive atlas of bone marrow stromal cell subpopulations under homeostatic and stress conditions using mass cytometry (CyTOF)-based single-cell protein analysis. We identified 28 subsets of non-hematopoietic cells during homeostasis, 14 of which expressed hematopoietic regulatory factors. Irradiation-based conditioning for HSPC transplantation led to the loss of most of these populations, including the LeptinR+ and Nestin+ subsets. In contrast, a subset expressing Ecto-5'-nucleotidase (CD73) was retained and a specific CD73+NGFRhigh population expresses high levels of cytokines during homeostasis and stress. Genetic ablation of CD73 compromised HSPC transplantation in an acute setting without long-term changes in bone marrow HSPCs. Thus, this protein-based expression mapping reveals distinct sets of stromal cells in the bone marrow and how they change in clinically relevant stress settings to contribute to early stages of hematopoietic regeneration.

A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia.

Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis, and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA sequencing (scRNA-seq) to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes spanning new fibroblastic and osteoblastic subpopulations including distinct osteoblast differentiation trajectories. Emerging acute myeloid leukemia impaired mesenchymal osteogenic differentiation and reduced regulatory molecules necessary for normal hematopoiesis. These data suggest that tissue stroma responds to malignant cells by disadvantaging normal parenchymal cells. Our taxonomy of the stromal compartment provides a comprehensive bone marrow cell census and experimental support for cancer cell crosstalk with specific stromal elements to impair normal tissue function and thereby enable emergent cancer.

Inflammatory signals directly instruct PU.1 in HSCs via TNF.

The molecular mechanisms governing the transition from hematopoietic stem cells (HSCs) to lineage-committed progenitors remain poorly understood. Transcription factors (TFs) are powerful cell intrinsic regulators of differentiation and lineage commitment, while cytokine signaling has been shown to instruct the fate of progenitor cells. However, the direct regulation of differentiation-inducing hematopoietic TFs by cell extrinsic signals remains surprisingly difficult to establish. PU.1 is a master regulator of hematopoiesis and promotes myeloid differentiation. Here we report that tumor necrosis factor (TNF) can directly and rapidly upregulate PU.1 protein in HSCs in vitro and in vivo. We demonstrate that in vivo, niche-derived TNF is the principal PU.1 inducing signal in HSCs and is both sufficient and required to relay signals from inflammatory challenges to HSCs.

Automated Microfluidic System for Dynamic Stimulation and Tracking of Single Cells.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.

Lineage marker synchrony in hematopoietic genealogies refutes the PU.1/GATA1 toggle switch paradigm.

Molecular regulation of cell fate decisions underlies health and disease. To identify molecules that are active or regulated during a decision, and not before or after, the decision time point is crucial. However, cell fate markers are usually delayed and the time of decision therefore unknown. Fortunately, dividing cells induce temporal correlations in their progeny, which allow for retrospective inference of the decision time point. We present a computational method to infer decision time points from correlated marker signals in genealogies and apply it to differentiating hematopoietic stem cells. We find that myeloid lineage decisions happen generations before lineage marker onsets. Inferred decision time points are in agreement with data from colony assay experiments. The levels of the myeloid transcription factor PU.1 do not change during, but long after the predicted lineage decision event, indicating  that the PU.1/GATA1 toggle switch paradigm cannot explain the initiation of early myeloid lineage choice.

In vitro biomimetic engineering of a human hematopoietic niche with functional properties.

In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs' behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis.

Inductive and Selective Effects of GSK3 and MEK Inhibition on Nanog Heterogeneity in Embryonic Stem Cells.

Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.