Distinct Niches Research Articles

Stem-like CD8+ T cells in cancer.

Stem-like CD8+ T cells (TSL) are a subset of immune cells with superior persistence and antitumor immunity. They are TCF1+ PD-1+ and important for the expansion of tumor specific CD8+ T cells in response to checkpoint blockade immunotherapy. In acute infections, naïve CD8+ T cells differentiate into effector and memory CD8+ T cells; in cancer and chronic infections, persistent antigen stimulation can lead to T cell exhaustion. Recent studies have highlighted the dichotomy between late dysfunctional (or exhausted) T cells (TLD) that are TCF1- PD-1+ and self-renewing TCF1+ PD-1+ TSL from which they derive. TCF1+ TSL cells are considered to have stem cell-like properties akin to memory T cell populations and can give rise to cytotoxic effector and transitory T cell phenotypes (TTE) which mediate tumor control. In this review, we will discuss recent advances made in research on the formation and expansion of TSL, as well as distinct niches required for their differentiation and maintenance in the setting of cancer. We will also discuss potential strategies to generate these cells, with clinical implications for stemness enhancement in vaccine design, immune checkpoint blockade (ICB), and adoptive T cell therapies.

Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells

Current immunotherapies provide limited benefits against Tcell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking Tcell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ Tcells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ Tcells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory Tcells. Importantly, clinical DC vaccines also potentiate Tcell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome Tcell-depleted tumors.

Methane flux at the water-gas interface is influenced by complex interactions among phytoplankton, phosphorus inputs and methane-functional bacteria: A microcosm systems study

Phytoplankton affect carbon cycling and emissions in eutrophic reservoirs dramatically, but our knowledge about carbon emissions response to phytoplankton bloom and phosphorus enrichment is rather limited. Here we performed a microcosm experiment with five treatments to investigate how phytoplankton blooms and phosphorus addition will impact the carbon emissions and the methane-functional bacterial community. During the 43-day incubation, the CH4 and CO2 flux at the water-air interface in the five water columns fluctuated between 7.536 and 16.689 μmol and between 2788.501 and 4142.726 μmol, respectively. The flux of CH4 and CO2 during phytoplankton decay was 1.542 to 10.397 times and 4.203 to 8.622 times higher, respectively, compared to that during phytoplankton growth. Furthermore, exogenous phosphorus increases bloom biomass of phytoplankton and subsequent CH4 production, even with low nitrogen concentrations. The addition of 1 mg KH2PO4 resulted in a conservative increase of 0.0715 μmol in CH4 emission and 11.911 μmol in CO2 emission in the water column, respectively, compared to the in-situ water column. High throughput sequencing determined that hydrogenotrophic Methanoregula dominated methanogens (MPB) and Methylocystaceae dominated methanotrophs (MOB) in the sediment. Phosphorus inhibited the relative abundance of Methanoregula after incubation, resulting in a significant decrease. Real-time quantitative polymerase chain reaction indicated that the absolute abundance of MPB and MOB (i.e., the mcrA gene and the pmoA gene) in the sediments ranged from 5.1354E+06 to 6.3176E+07 copies·g−1 and 1.1656E+06 to 9.5056E+06 copies·g−1, respectively. The mcrA gene showed a preference for sediments with high organic carbon content. The effect of eutrophication on CH4 emissions is closely related to nutrient load and distinct niche of methane-functional bacteria.

First trimester "clean catch" urine and vaginal swab sample distinct microbiological niches.

Untreated asymptomatic bacteriuria (ASB) has been associated with adverse pregnancy outcomes, including pyelonephritis, preterm labor, and low birth weight infants. Thus, routine screening by standard urine culture (SUC) and treatment of ASB are currently recommended for all pregnant women. For this purpose, some researchers claim that vaginal swabs and urine samples can be used as proxies for each other. Because SUC often misses microbes, we used two more sensitive, recently validated detection methods to compare the composition of the urinary and vaginal microbiomes of pregnant females in their first trimester. Both methods yielded similar results. Vaginal and urinary microbial compositions for the same individual were significantly correlated; however, they were not equivalent. We argue that first trimester urinary and vaginal microbiomes are distinct enough to preclude their use as proxies for each other.

Tissue niches and immunopathology through the lens of spatial tissue profiling techniques.

Spatial organization plays a fundamental role in biology, influencing the function of biological structures at various levels. The immune system, in particular, relies on the orchestrated interactions of immune cells with their microenvironment to mount protective or pathogenic immune responses. The COVID-19 pandemic has underscored the significance of studying immunity within target organs to understand disease progression and severity. To achieve this, multiplex histology and spatial transcriptomics have proven indispensable in providing a spatial context to protein and gene expression patterns. By combining these techniques, researchers gain a more comprehensive understanding of the complex interactions at the cellular and molecular level in distinct tissue niches, key functional units modulating health and disease. In this review, we discuss recent advances in spatial tissue profiling techniques, highlighting their advantages over traditional histopathology studies. The insights gained from these approaches have the potential to revolutionize the diagnosis and treatment of various diseases including cancer, autoimmune disorders, and infectious diseases. However, we also acknowledge their challenges and limitations. Despite these, spatial tissue profiling offers promising opportunities to improve our understanding of how tissue niches direct regional immunity, and their relevance in tissue immunopathology, as a basis for novel therapeutic strategies and personalized medicine.

Marine heatwaves modulate the genotypic and physiological responses of reef-building corals to subsequent heat stress.

Back-to-back marine heatwaves in 2016 and 2017 resulted in severe coral bleaching and mortality across the Great Barrier Reef (GBR). Encouragingly, some corals that survived these events exhibit increased bleaching resistance and may represent thermally tolerant populations that can better cope with ocean warming. Using the GBR as a natural laboratory, we investigated whether a history of minimal (Heron Island) or severe (Lizard Island) coral bleaching in 2016 and 2017 equates to stress tolerance in a successive heatwave (2020). We examined the genetic diversity, physiological performance, and trophic plasticity of juvenile (<10 cm) and adult (>25 cm) corals of two common genera (Pocillopora and Stylophora). Despite enduring greater cumulative heat stress (6.3°C week-1 vs. 5.6°C week-1), corals that experienced the third marine heatwave in 5 years (Lizard) exhibited twice as high survival and visual bleaching thresholds compared to corals that had not experienced significant bleaching in >10 years (Heron). Surprisingly, only one shared host-Symbiodiniaceae association was uncovered between locations (Stylophora pistillata-Cladocopium "C8 group") and there was no genetic overlap in Pocillopora-Cladocopium partnerships, suggesting turnover in species composition from recent marine heatwaves. Corals within the species complex Pocillopora that survived the 2016 and 2017 marine heatwaves at Lizard Island were the most resilient, exhibiting three times greater calcification rates than conspecifics at Heron Island. Further, surviving corals (Lizard) had distinct isotopic niches, lower host carbon, and greater host protein, while conspecifics that had not experienced recent bleaching (Heron) had two times greater symbiont carbon content, suggesting divergent trophic strategies that influenced survival (i.e., greater reliance on heterotrophy vs. symbiont autotrophy, respectively). Ultimately, while corals may experience less bleaching and survive repeated thermal stress events, species-specific trade-offs do occur, leaving open many questions related to the long-term health and recovery of coral reef ecosystems in the face of intensifying marine heatwaves.

Habitat and climate influence hybridization among three genetically distinct Canada jay (Perisoreus canadensis) morphotypes in an avian hybrid zone complex.

Examining the frequency and distribution of hybrids across contact zones provide insights into the factors mediating hybridization. In this study, we examined the effect of habitat and climate on hybridization patterns for three phenotypically, genetically, and ecologically distinct groups of the Canada jay (Perisoreus canadensis) in a secondary contact zone in western North America. Additionally, we tested whether the frequency of hybridization involving the three groups (referred to as Boreal, Pacific and Rocky Mountain morphotypes) is similar across the hybrid zones or whether some pairs have hybridized more frequently than others. We reanalyzed microsatellite, mtDNA and plumage data, and new microsatellite and plumage data for 526 individuals to identify putative genetic and phenotypic hybrids. The genetically and phenotypically distinct groups are associated with different habitats and occupy distinct climate niches across the contact zone. Most putative genetic hybrids (86%) had Rocky Mountain ancestry. Hybrids were observed most commonly in intermediate climate niches and in habitats where Engelmann spruce (Picea engelmannii) overlaps broadly with boreal and subalpine tree species. Our finding that hybrids occupy intermediate climate niches relative to parental morphotypes matches patterns for other plant and animal species found in this region. This study demonstrates how habitat and climate influence hybridization patterns in areas of secondary contact and adds to the growing body of research on tri-species hybrid zones.

DIVERSITY AND DISTRIBUTION OF TERMITES (BLATTODEA: ISOPTERA) IN BHIWANI, HARYANA, INDIA

A comprehensive study was undertaken by collecting 51 samples of termites between July and October 2021 in three distinct zones within the Bhiwani district to analyze the termite diversity of the Bhiwani, Haryana region. The interplay of aeolian dunes characterizes the Bhiwani district?s ecological landscape. Nestled within the Indo-Gangetic alluvial plain, the Loharu Banger zone, and the ruggedness of the Aravalli Hills provide three distinct niches for termite diversity. The study sheds light on the intricate tapestry of termite life inhabiting these diverse ecological niches. Sample identification exhibited termites? affiliations to two prominent families, Termitidae and Rhinotermitidae and three subfamilies, Amitermitinae, Macrotermitinae, and Coptotermitinae. Further, delving into the taxonomy unveiled a total of ten termite species inhabiting the various locations across the three ecological regions of Bhiwani district including Coptotermes sp., Microtermes mycophagus Desneux, Odontotermes gurdaspurensis Holmgren and Holmgren, Odontotermes redemanni (Wasmann), Odontotermes obesus, Coptotermes kishori, Amitermes belli (Desneux), Microcerotermes sp., Odontotermes guptai Roonwal and Bose, and Odontotermes vaishno Bose. Various diversity indices, including species richness, Simpson?s diversity index, Shannon-Weaver index, and Pielou?s evenness index, were used to determine the relative abundance and distribution of termite species within each zone. These indexes offer a comprehensive view of termite community structure and ecosystem resilience. This pioneering research marks the first-ever documented record of termite biodiversity in Bhiwani district, Haryana. In a region where the study of termites had hitherto been scarce, this endeavor opens doors to a deeper understanding of these soil-dwelling architects of nature. It lays the foundation for future research and pest management strategies in this dynamic ecosystem.. KEYWORDS :Isoptera, Termite diversity, Taxonomy, Pest management, Soil ecology

Spatial Mapping Reveals Distinct Erythroid Niches in Mice and Humans during Development and Stress

Like hematopoiesis, erythropoiesis requires erythroid niche nurturing and support. While extensive studies have revealed various components and functions of the HSC niches, the erythroid niche is less characterized. The most studied erythroid niche is the bone marrow erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. However, most of the reported studies of EBI involve in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. Here, we utilized multiple spatial mapping technologies, including spatial transcriptomics and whole-mount imaging studies, to characterize erythropoiesis in mouse and human hematopoietic tissues during development and under stress. We first utilized the Visium platform from 10x Genomics to study the spatial transcriptomic profiles of the E14.5 mouse fetal liver, which is the major organ for murine fetal definitive erythropoiesis. The Visium platform contains barcoded spots with a diameter of 55 µm, which is suboptimal for single-cell analyses but ideal for the investigation of cell-cell interactions within their microenvironment. We found that each hepatic lobe exhibited different cluster distributions, indicating distinct cell compositions among different anatomic loci. Further deconvolution of the clusters and co-analysis with corresponding single-cell RNA sequencing data revealed that there was a higher positive correlation between erythroid cells and C1q+ macrophages compared to other macrophages, suggesting that C1q+ macrophages are more likely to be EBI macrophages in mouse. C1q+ macrophages exhibited high expression of known genes involved in EBI macrophage function and erythropoiesis. Cyclic immunofluorescence (CyCIF) and whole-mount immunofluorescence analyses further demonstrated the co-localization of C1q+ macrophages with erythroid cells in the E14.5 fetal liver. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed across different stages of fetal liver development, newborn bone marrow, and adult spleen under physiologic and stress conditions. These findings demonstrate that C1q+ macrophages as EBI macrophages are conserved across different hematopoietic organs in mice. We next applied the same technologies to human hematopoietic tissues during development and stress. In contrast to the mouse, we did not observe a strong positive correlation between erythroid cells and C1q+ macrophages or other macrophages in the human fetal liver. Instead, there is a strong association between erythroid progenitor and maturing erythroid cells. This strong association is also found in different developmental stages of the human fetal liver as well as the bone marrow, which becomes even stronger in regenerating human bone marrow after chemotherapy. We also recapitulated these findings in a human bone marrow organoid model. These results demonstrate the erythroid cell self-sustaining capacities in human erythropoiesis. Taken together, our findings reveal the distinct erythroid niches in mice and humans during development and stress, which provides a comprehensive understanding of erythropoiesis during evolution.

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration after radiation-induced injury.

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant immune cell in the salivary glands and are attractive therapeutic targets due to their unrivaled capacity to drive tissue repair. Yet, the nature and role of macrophages in salivary gland homeostasis and how they may contribute to tissue repair after injury are not well understood. Here, we show that at least two phenotypically and transcriptionally distinct CX3CR1+ macrophage populations are present in the adult salivary gland, which occupy anatomically distinct niches. CD11c+CD206-CD163- macrophages typically associate with gland epithelium, whereas CD11c-CD206+CD163+ macrophages associate with blood vessels and nerves. Using a suite of complementary fate mapping systems, we show that there are highly dynamic changes in the ontogeny and composition of salivary gland macrophages with age. Using an in vivo model of radiation-induced salivary gland injury combined with genetic or antibody-mediated depletion of macrophages, we demonstrate an essential role for macrophages in clearance of cells with DNA damage. Furthermore, we show that epithelial-associated macrophages are indispensable for effective tissue repair and gland function after radiation-induced injury, with their depletion resulting in reduced saliva production. Our data, therefore, provide a strong case for exploring the therapeutic potential of manipulating macrophages to promote tissue repair and thus minimize salivary gland dysfunction after radiotherapy.

TMIC-36. SPATIAL ARCHITECTURE OF HIGH-GRADE GLIOMA REVEALS TUMOR HETEROGENEITY WITHIN DISTINCT DOMAINS

Abstract High-grade gliomas are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type high-grade glioma (glioblastoma, GBM), increased intra-tumoral heterogeneity is associated with more aggressive disease. Recently, spatial technologies have emerged to dissect this complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. Here, we construct a high-resolution molecular landscape of GBM and IDH-mutant high-grade glioma patient samples to investigate the cellular subtypes and spatial communities that compose high-grade glioma using digital spatial profiling and spatial molecular imaging. This uncovered striking diversity of the tumor and immune microenvironment, that is embodied by the heterogeneity of the inferred copy-number alterations in the tumor. Reconstructing the tumor architecture revealed two distinct niches, one composed of tumor cell states that most closely resemble normal glial cells, associated with microglia; and the other niche populated by monocytes and mesenchymal tumor cells. We further reveal that communication between tumor and immune cells is underpinned by tumor-specific ligands, such as TGFb signaling in astrocyte-like tumor cells. This primary study reveals high levels of intra-tumoral heterogeneity in high-grade gliomas, associated with a diverse immune landscape within spatially localized regions.

TMIC-01. PERICYTES ORCHESTRATE A TUMOR-SUPPRESSIVE MICROENVIRONMENT BY IMPINGING ON THE CROSSTALK BETWEEN MACROPHAGES AND TUMOR-INITIATING CELLS IN GLIOBLASTOMA MULTIFORME

Abstract Glioblastoma multiforme (GBM) is characterized by fast progression, infiltrative growth pattern and a high rate of relapse. A defining feature of GBM is the existence of spatially and functionally distinct cellular niches, i.e. a hypoxic niche, a leading-edge niche, and a peri-vascular niche, in which malignant cells engage in paracrine crosstalk with cell types comprising the tumor microenvironment, including immune cells, astrocytes, and vascular cells. Here, by analysis of single-cell transcriptomic data of human GBM and transgenic mouse models of GBM, we unexpectedly identified pericytes intimately associated with the endothelium as the most active paracrine signaling hub within the tumor parenchyma. Exclusive signaling axes emanating from pericytes were received by endothelial cells, malignant cells, astrocytes, and immune cells. Depletion of pericytes through genetic engineering in several different transgenic and orthotopic mouse models of GBM demonstrated accelerated tumor progression, a disrupted blood-brain-barrier, and premature death of pericyte-poor mice. Mechanistic studies revealed that pericyte deficiency altered the cellular composition of GBM, remodeled the endothelium, and impacted on the immune cell landscape. Specifically, endothelial cells deprived of pericyte association upregulated Colony Stimulating Factor (CSF)-1, which in turn attracted peri-vascular tumor-associated macrophages polarized towards an immune-suppressive phenotype. In the absence of pericytes, the recruited macrophages expressed Hepatocyte Growth Factor (HGF), which reinforced activation of its receptor tyrosine kinase c-Met on a subset of GBM cells harboring an extreme mesenchymal-like state. Indeed, orthotopic implantation of isolated c-Met-expressing GBM cells corroborated their superior tumor-initiating capability and invasive phenotype, compared to cells negative for c-Met. Taken together, we infer that the pericyte represents a critical modulator of GBM development by orchestrating a tumor-suppressive microenvironment; our findings thus highlight the importance of pericyte preservation in the face of current and future GBM therapies.

EPCO-13. CNMF-SNS, A FRAMEWORK FOR UNSUPERVISED INTEGRATION OF MULTIOMICS DATA, IDENTIFIES INVASION AND RECURRENCE ASSOCIATED PROGRAMS IN GBM

Abstract Glioblastoma (GBM) is an aggressive cancer of the brain for which novel therapies are urgently needed. Heterogeneity in GBM underlies tumor evolution and resistance to therapy and involves genetic diversity of tumor cells, spatially distinct niches harbouring stem-like cells, and immune cells spanning anti- and pro-tumor states. No single profiling methodology can capture all these facets of tumor biology in the same space and time, necessitating multi-omics data collection and computational methods suited for multi-omic integration across and within cohorts. We therefore set out to develop a generalizable framework for integration that can bridge across cohorts and data types. Using consensus non-negative matrix factorization (cNMF), we discover gene expression programs (GEPs) corresponding to cell types and/or states in an unsupervised fashion. Rather than choosing a single rank (number of programs), we identify both high and low-resolution programs for each dataset, and then integrate programs from all datasets and ranks into a solution network space (SNS) on which graph algorithms identify communities of highly similar GEPs identified in all or some datasets. These communities are characterized by gene set enrichment analyses and association with sample metadata. SNS communities can be used to transfer sample metadata across datasets, enabling multi-omics and multi-cohort integration, even in the absence of shared samples or cells. We showcase the utility of cNMF-SNS in integrating multiomics datasets of GBM from single-cell to bulk RNA-Seq and mass spectrometry proteomics. We further demonstrate the power of cNMF-SNS to define and cross-annotate programs identified in 5 complementary mass spectrometry datasets of GBM, layering on survival, driver gene mutation status, clinical features, and imaging features. We demonstrate robust mapping of biologically and clinically relevant processes, highlighting how programs for tumor cell invasion and therapy resistance can be mined for novel therapeutic targets. cNMF-SNS is available at https://github.com/MorrissyLab/cNMF-SNS.

CSIG-07. HYPERCELLULAR NODULES IN OLIGODENDROGLIOMA REPRESENT POCKETS OF PROLIFERATIVE STEM-LIKE TUMOR CELLS

Abstract INTRODUCTION Oligodendrogliomas are a progressive, infiltrative glioma. By histology, a subset of oligodendrogliomas demonstrate hypercellular nodules (HCN), which often have increased anaplasia compared to surrounding tumor. However, the signals driving anaplasia are unknown. METHODS We first performed a GeoMX 89-protein Digital Spatial Profiling (DSP, Nanostring) panel on a cohort of ten oligodendrogliomas with HCN., followed by spatial whole RNA transcriptome analysis (10x Genomics Visium) on six oligodendrogliomas. For the latter, following unsupervised clustering, we performed Gene Ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA, Qiagen) on the differentially expressed genes between HCN and adjacent tumor. We performed paired single nucleus RNA sequencing for deconvolution of the spatial transcriptomic data. RESULTS AND CONCLUSIONS By protein DSP, we identified a marker of stemness (OLIG2) and a proliferation marker (Ki-67) as present at increased levels (p&amp;lt; 0.05) within HCN. We subsequently performed spatial whole transcriptome analysis. On unsupervised clustering of differentially expressed genes, the HCN spots cluster away from the remaining tumor spots, supporting a distinct niche. GO and IPA both identified increased activity in several central nervous system developmental pathways, providing further evidence for the “precursor-like” state of cells in HCN. We then annotated paired single nucleus RNA sequencing results to align tumor cells with astrocytic-like, oligodendroglial-like, and stem-like cell states. Using this to deconvolute the spatial transcriptomic data, we found that the vast majority of HCN are enriched for either stem-like or OC-like/stem-like tumor cells. Taken together, the findings support HCN as niches for the most precursor-like, proliferative oligodendroglial cells.

Spatial Transcriptomics Reveals Distinct Hematopoietic Stem Cell Niches in Mouse Fetal Liver

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver (FL) after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the FL-HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge thus far, there have been no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver. As a result, we have uncovered two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. To understand the spatial distribution and interactions between FL-HSCs and niche cells, we introduced 2 spatial transcriptomic methods, Slide-seq (10uM resolution), and 10x Genomics Visium (55uM resolution), in our study on E14.5 and E16.5 mouse fetal liver. By integrating with single-cell RNA sequencing, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelial cells, macrophages, megakaryocytes, and mesenchymal stromal cells (MSCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that MSCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing MSCs are enriched in the portal vessel area. Importantly, the majority of FL-HSCs are in close proximity to N-cadherin-expressing MSCs and endothelial cells, compared with other potential niche cells, indicating a supportive role of N-cadherin-expressing MSCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing MSCs are major niche-signaling senders with an enriched expression of niche factors and stemness pathway-related ligands. This finding is consistent with our previous finding that N-cadherin-expressing MSCs can maintain reserve HSCs in the adult bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting FL-HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Kitl mouse model to conditionally knockout the well-studied niche factors, Cxcl12 and Kitl (Scf), in N-cadherin-expressing cells. Interestingly, deletion of either Cxcl12 or Kitl through N-cad-CreERT induction resulted in the expansion of myeloid-biased FL-HSCs. Slide-seq further showed that depletion of Cxcl12 through N-cad-CreERT induction led to the repositioning of FL-HSCs from the PV region, which is enriched with MSCs, to the hepatic sinusoidal region, which lacks MSCs. These findings suggested the existence of distinct niches in the fetal liver: the PV niche is composed of N-cadherin + MSCs and peri-PV endothelial cells, and it maintains FL-HSCs with multilineage potential. In contrast, the sinusoidal niche preliminary consists of sinusoidal endothelial cells, hepatocytes, and hepatoblasts, which facilitates the proliferation of FL-HSCs but with a bias toward myeloid lineage. Previous studies suggested that the two forms of Kitl, soluble Kitl (sKitl) and membrane-bound Kitl (mKitl), may have different functions in supporting HSCs. Here, we discovered that mKitl is highly enriched in MSCs, whereas hepatoblasts are the major source of the sKitl. This finding suggests that mKitl may serve as the key niche factor that contributes to the homing and maintenance of FL-HSCs. In summary, by using cutting-edge spatial transcriptomics, we uncovered the existence of distinct niches, the portal-vessel (PV) niche and the sinusoidal niche, in mouse fetal liver and the critical role of N-cadherin + MSCs in maintaining FL-HSCs and a role of hepatoblasts in promoting proliferation. The insight learned from this study enhances our understanding of the complex regulation of HSCs and opens new avenues for potential clinical applications.

Single-Cell Longitudinal Characterization of FL Heterogeneity and Residual Disease in the Bone Marrow from Low-Tumor Burden FL Enrolled in the Flirt Clinical Trial

Follicular Lymphoma (FL) remains a clinically challenging disease as most patients undergo a succession of clinical response/relapse cycles suggesting that current therapies do not eradicate residual tumor cells. Genetic studies suggest that FL relapses seed from a reservoir of rare Cancer Precursor Cells (CPCs), which likely remain resistant to treatment due to their homing to propitious niches such as the bone marrow (BM) and/or to the acquisition of a specific functional B-cell state. Yet, the rarity of such CPCs and the use of bulk sequencing technologies have so far hampered their direct characterization. Profiling CPC remains thus a major challenge in the FL field. Here, we identify and characterize FL cells before/after rituximab monotherapy within the BM of low tumor burden FL, looking for CPC-like cells supporting minimal residual disease. We harnessed a unique collection of longitudinal BM aspirates from 15 newly diagnosed low-tumor burden FL patients enrolled in the phase III clinical trial FLIRT (NCT02303119) evaluating Rituximab monotherapy and sampled at diagnosis (M0) and at one-year post-treatment start (M12). We generated a multi-modal single-cell dataset combining gene expression, B-cell receptor (BCR) and T-cell receptor (TCR) repertoire (Fig1A). We used BCR sequence analysis as a pivotal method to assign the clonal malignant identity within variably infiltrated BM at diagnosis and track the clonally related residual disease FL cells at M12. We compared FL B cell transcriptional heterogeneity after rituximab therapy. We also integrated BM T cell transcriptomes from both timepoints to study compositional changes of the BM microenvironment linked to a B-cell depletion treatment. We profiled 9,222 BM infiltrating FL cells. Single cell clustering showed a unique transcriptional signature for malignant cells of each FL patient, indicating a major inter-patient heterogeneity. By contrast, the non-malignant B cell clusters were mixed across patients and organized into transcriptional signatures encompassing normal B-cell differentiation steps (Fig1B, left). We then used unsupervised metaclustering of malignant B cells to uncover shared transcriptional states across patients, yielding 6 metaclusters (Mcl) expressing distinct gene expression programs. Mcl gene expression signatures observed in FL BM shared some similarities with the transcriptional programs observed in FL lymph nodes such as non-cycling Germinal Center-like (Mcl.1) or Mem-like (Mcl.3 and Mcl.4) states. This suggests that FL transcriptional heterogeneity is conserved between distinct niches. BM FL cells also upregulated several pathways, including glycolytic metabolism ( GAPDH), NF-κB signaling, negative regulators of cell cycle, or entry into quiescence programs. By analyzing BM samples obtained at M12, we were able to track and profile very rare clonally-related FL cells in 80% of FL patients with a range of 2 to 65 residual FL cells per patient. Those residual FL cells present a heterogeneous cellular phenotype partly redundant with the transcriptional heterogeneity found at diagnosis indicating that residual cells cover a plurality of cell states whose links with clinical outcome is underway. Differential expression analysis in one case also revealed a transcriptional shift in residual FL cells (including overexpression of BCL2) compared to BM tumor cells at diagnosis (Fig1B, right), providing novel insights into putative CPC identity with possible implications for personalized therapies. Finally, exploration of the FL T-cell microenvironment revealed abundant TFH-like cells at diagnosis that were barely detectable in healthy donors. Interestingly, high frequency of the TFH-like subset was maintained at remission suggesting a potential role as a niche for therapy-resistant B cells and later relapses. In summary, our single cell RNAseq analysis in the frame of a clinical trial provide the first longitudinal map of FL heterogeneity in the BM and characterize putative CPCs resistant to Rituximab-therapy. Ongoing efforts are now focusing on the microenvironment drivers of this intra-patient BM diversity and on specific molecular features of BM residual cells that could represent therapeutic vulnerabilities in this particular low-tumor burden FL population.

Microbial biogeography of the eastern Yucatán carbonate aquifer.

The extensive Yucatán carbonate aquifer, located primarily in southeastern Mexico, is pockmarked by numerous sinkholes (cenotes) that lead to a complex web of underwater caves. The aquifer hosts a diverse yet understudied microbiome throughout its highly stratified water column, which is marked by a meteoric lens floating on intruding seawater owing to the coastal proximity and high permeability of the Yucatán carbonate platform. Here, we present a biogeographic survey of bacterial and archaeal communities from the eastern Yucatán aquifer. We apply a novel network analysis software that models ecological niche space from microbial taxonomic abundance data. Our analysis reveals that the aquifer community is composed of several distinct niches that follow broader regional and hydrological patterns. This work lays the groundwork for future investigations to characterize the biogeochemical potential of the entire aquifer with other systems biology approaches.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

Introduction Rare non-hematopoietic cells in the bone marrow make essential contributions to hematopoiesis. Despite tremendous progress in understanding the mouse bone marrow microenvironment, the precise identity of human non-hematopoietic bone marrow cells and their spatial organization remain largely uncharacterized. Here, we performed single cell transcriptomics and spatial proteomics, assembling the first comprehensive atlas of human bone marrow cellular composition and organization. Methods Using fresh femoral head samples obtained from orthopedic hip replacement surgeries, we first performed single-cell RNA sequencing (scRNA-Seq) to create a comprehensive human bone marrow atlas profiling 29,325 enriched non-hematopoietic cells as well as 53,417 hematopoietic cells from enzymatically digested femoral heads from 12 individuals. We next employed Co-Detection by Indexing (CODEX) multiplexed imaging of 18 bone marrow samples, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. Results Our transcriptional analysis revealed nine distinct non-hematopoietic bone marrow cell types including mesenchymal stromal cell (MSC) subsets, osteolineage cells, vascular smooth muscle cells, and both sinusoidal and arterial endothelial cells (Figure 1A). We found that MSCs were heterogeneous and hierarchically organized. One specific cell cluster, which we termed Fibro-MSCs, corresponded to previous descriptions of mesenchymal stem cells and was both computationally and functionally shown to be the most stem/progenitor-like. Next, we performed computational cellular communication analysis which revealed that two MSC subsets, Adipo and THY1+ MSCs, provided the majority of canonical hematopoietic supportive factors such as CXCL12, KITLG, IL7, and PTN. Next, we used CODEX to systematically study spatial relationships between these cells. Consistent with the scRNA-Seq cellular communication analysis, we found that Adipo and THY1+ MSCs were in the bone marrow interacting with hematopoietic cells, while osteolineage cells and Fibro-MSCs were found within the trabecular bone region. We then performed unsupervised neighborhood analysis to analyze the distinct niches of the bone marrow. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, where early myeloid progenitors were characterized by very low levels of HIF1α, in contrast to HIF1α hi neutrophils which were found largely residing near sinusoids. We also employed point pattern statistics and permutation tests to analyze the proximity of cell types to manually identified structures in the bone marrow. This analysis revealed that CD34+ hematopoietic stem and progenitor cells (HSPCs) were frequently contacting adipocytes (Figure 1B), but not sinusoids or bone. Collectively, these results led us to propose a data-driven working model for how human myelopoiesis is spatially organized, with the earliest myeloid progenitors being specified from HSPCs near adipocytes, then migrating to the peri-arteriolar/peri-endosteal early myeloid progenitor niche, and then finally localizing near sinusoids which facilitates bone marrow egress as they mature to neutrophils. To evaluate if our CODEX atlas could be used to study disease states, we mapped new bone marrow images from acute myeloid leukemia (AML) patients and controls to our reference. We used machine learning to identify true mutant cells based on NPM1c expression patterns. We discovered Adipo and THY1+ MSC expansion in AML patients compared to controls, with expanded MSCs spatially associated with leukemic blasts. Our results highlight the potential of our CODEX atlas to contextualize new datasets and study bone marrow organization in disease states, and provide further rationale to study MSC-AML blast interactions. Conclusions Taken together, our scRNA-Seq and CODEX data act in concert, demonstrating the diversity of non-hematopoietic stromal elements, establishing the source of hematopoietic cytokines, and defining the spatial organization in healthy human bone marrow. We envision our multiomic, spatially resolved atlas of human bone marrow will serve as a critical resource for future study of the bone marrow microenvironment in both healthy and disease states.

Responses of Phytoplankton Communities to the Effect of Both River Plume and Coastal Upwelling

AbstractRiver plumes and coastal upwelling systems are both nutrient‐abundant habitats compared to the open ocean, and the mechanisms that structure the phytoplankton community when these occur together are unclear. In this study we investigated the dynamics and drivers of phytoplankton biomass and community composition in the Pearl River plume‐coastal upwelling system on the northern South China Sea shelf during summer. We found that phytoplankton biomass was lower in the plume than in upwelled water. Diatoms were the only dominant group in the upwelled water. In contrast, diatoms and Synechococcus were co‐dominant in the plume, and the negative correlation between the proportions of these two groups indicated that they occupied distinct niches. The lower phytoplankton biomass in plume waters was due to nutrient limitation, and there was a transition from limitation by phosphorus to limitation by nitrogen in the plume along its path. This bottom‐up control limited only diatoms, whereas Synechococcus was limited mainly by top‐down control via microzooplankton grazing. This difference led to niche differentiation of the dominant phytoplankton groups in the plume‐upwelling system. This discovery of niche differentiation enhances understanding of food web structure and will facilitate modeling of marine biogeochemical cycles.

Composition, functional, and niche differentiation of fungal communities in the rhizosphere and root endosphere of Cinnamomum migao in southwestern China.

Roots are key parts of plant material circulation and energy flow, creating two distinct niches for the plant microbiome, such as the rhizosphere and root endosphere, which interact to maintain plant growth and health. In this study, two niches of plant rhizosphere and root endosphere were selected to study the composition and differences of fungi communities in order to better understand how differences in the structure and function of plant fungi communities affect plant health. The community structure, diversity, and ecological function of fungi in the rhizosphere and root endosphere of Cinnamomum migao were studied using high-throughput sequencing techniques, traditional culture methods, and the FUNGuild database. The results of the analysis of alpha diversity showed that the diversity of rhizosphere fungal communities in C. migao was much higher than that of root endosphere communities. Some similarities and differences were noted between the two niche fungal communities, and some fungi had niche preferences. Functional prediction results demonstrated that fungi in the rhizosphere and root endosphere adopt multiple trophic modes, mostly saprophytic fungi. This study provided a basis for an in-depth understanding of the structural variation, niche differentiation, and function of plant root-related fungal microbiota. We believe that it could provide guidance on the subsequent development of beneficial fungi.