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.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.

Lipid availability determines fate of skeletal progenitor cells via SOX9.

The avascular nature of cartilage makes it a unique tissue1-4, but whether and how the absence of nutrient supply regulates chondrogenesis remain unknown. Here we show that obstruction of vascular invasion during bone healing favours chondrogenic over osteogenic differentiation of skeletal progenitor cells. Unexpectedly, this process is driven by a decreased availability of extracellular lipids. When lipids are scarce, skeletal progenitors activate forkhead box O (FOXO) transcription factors, which bind to the Sox9 promoter and increase its expression. Besides initiating chondrogenesis, SOX9 acts as a regulator of cellular metabolism by suppressing oxidation of fatty acids, and thus adapts the cells to an avascular life. Our results define lipid scarcity as an important determinant of chondrogenic commitment, reveal a role for FOXO transcription factors during lipid starvation, and identify SOX9 as a critical metabolic mediator. These data highlight the importance of the nutritional microenvironment in the specification of skeletal cell fate.

Joint single-cell genetic and transcriptomic analysis reveal pre-malignant SCP-like subclones in human neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. It is a complex disease that can result in diverse clinical outcomes. In some children, tumors regress spontaneously. Others respond well to existing treatments. But for the high-risk group, which constitutes approximately 40% of all patients, the prognosis remains dire despite collaborative efforts in basic and clinical research. While its exact cellular origin is still under debate, NB is assumed to arise from the neural crest cell lineage including multipotent Schwann cell precursors (SCPs), which differentiate into sympatho-adrenal cell states eventually producing chromaffin cells and sympathoblasts. METHODS: To investigate clonal development of neuroblastoma cell states, we performed haplotype-specific analysis of human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Samples were also assessed using immunofluorescence stainings and fluorescence in-situ hybridization (FISH). RESULTS: Beyond adrenergic tumor cells, we identify subpopulations of aneuploid SCP-like cells, characterized by clonal expansion, whole-chromosome 17 gains, as well as expression programs of proliferation, apoptosis, and a non-immunomodulatory phenotype. CONCLUSION: Aneuploid pre-malignant SCP-like cells represent a novel feature of NB. Genetic evidence and tumor phylogeny suggest that these clones and malignant adrenergic populations originate from aneuploidy-prone cells of migrating neural crest or SCP origin, before lineage commitment to sympatho-adrenal cell states. Our findings expand the phenotypic spectrum of NB cell states. Considering the multipotency of SCPs in development, we suggest that the transformation of fetal SCPs may represent one possible mechanism of tumor initiation in NB with chromosome 17 aberrations as a characteristic element.

An unsupervised method for physical cell interaction profiling of complex tissues.

Cellular identity in complex multicellular organisms is determined in part by the physical organization of cells. However, large-scale investigation of the cellular interactome remains technically challenging. Here we develop cell interaction by multiplet sequencing (CIM-seq), an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between cell types present in a tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution into constituent cell types. CIM-seq estimates parameters such as number of cells and cell types in each multiplet directly from sequencing data, making it compatible with high-throughput droplet-based methods. When applied to gut epithelium or whole dissociated lung and spleen, CIM-seq correctly identifies known interactions, including those between different cell lineages and immune cells. In the colon, CIM-seq identifies a previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is directly adjacent to colonic stem cells. Our results demonstrate that CIM-seq is broadly applicable to unsupervised profiling of cell-type interactions in different tissue types.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Effects of epigenetic modificators in combination with small molecule inhibitors of receptor tyrosine kinases on medulloblastoma growth.

Epigenetic alterations and aberrant expression of genes controlling epigenetic mechanisms have been identified in several cancers, including medulloblastoma, the most common brain tumor in children. Here we show that combining drugs that inhibit two of the most important epigenetic factors, gene methylation and post-translational modifications of protein histone-associated DNA, with small molecule inhibitors of receptor tyrosine kinases induces apoptosis. The histone deacetylation inhibitor, 4-phenylbutyrate (4-PB) and the demethylation agent, 5-Aza-2'deoxycytidine (5-Aza-dC) had minor effects on medulloblastoma cell cytotoxity in single agent treatment whereas a significant enhancement in cell cytotoxity was seen when these drugs were combined with Gleevec. Triple treatment of medulloblastoma cells with 4-PB, 5-Aza and Gleevec were associated with reduced DNA methyltransferase activity, reduced global methylation and induction of apoptosis. Taken together these results suggest that a combination of these drugs may be beneficial in the treatment of medulloblastoma.

Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma.

Childhood neuroblastoma with MYCN-amplification is classified as high-risk and often relapses after intensive treatments. Immune checkpoint blockade therapy against the PD-1/L1 axis shows limited efficacy in neuroblastoma patients and the cancer intrinsic immune regulatory network is poorly understood. Here, we leverage genome-wide CRISPR/Cas9 screens and identify H2AFY as a resistance gene to the clinically approved PD-1 blocking antibody, nivolumab. Analysis of single-cell RNA sequencing datasets reveals that H2AFY mRNA is enriched in adrenergic cancer cells and is associated with worse patient survival. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverts in vivo resistance to PD-1 blockade by eliciting activation of the adaptive and innate immunity. Mapping of the epigenetic and translational landscape demonstrates that H2afy deletion promotes cell transition to a mesenchymal-like state. With a multi-omics approach, we uncover H2AFY-associated genes that are functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell states and immunogenicity in high-risk neuroblastoma.

Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature.

Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo.

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target?

Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.

Recent Advances in Single-Cell RNA-Sequencing of Primary and Metastatic Clear Cell Renal Cell Carcinoma.

Over the past two decades, significant progress has been made in the treatment of clear cell renal cell carcinoma (ccRCC), with a shift towards adopting new treatment approaches ranging from monotherapy to triple-combination therapy. This progress has been spearheaded by fundamental technological advancements that have allowed a deeper understanding of the various biological components of this cancer. In particular, the rapid commercialization of transcriptomics technologies, such as single-cell RNA-sequencing (scRNA-seq) methodologies, has played a crucial role in accelerating this understanding. Through precise measurements facilitated by these technologies, the research community has successfully identified and characterized diverse tumor, immune, and stromal cell populations, uncovering their interactions and pathways involved in disease progression. In localized ccRCC, patients have shown impressive response rates to treatment. However, despite the emerging findings and new knowledge provided in the field, there are still patients that do not respond to treatment, especially in advanced disease stages. One of the key challenges lies in the limited study of ccRCC metastases compared to localized cases. This knowledge gap may contribute to the relatively low survival rates and response rates observed in patients with metastatic ccRCC. To bridge this gap, we here delve into recent research utilizing scRNA-seq technologies in both primary and metastatic ccRCC. The goal of this review is to shed light on the current state of knowledge in the field, present existing treatment options, and emphasize the crucial steps needed to improve survival rates, particularly in cases of metastatic ccRCC.

Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo.

The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Dissecting the immune suppressive human prostate tumor microenvironment via integrated single-cell and spatial transcriptomic analyses.

The treatment of low-risk primary prostate cancer entails active surveillance only, while high-risk disease requires multimodal treatment including surgery, radiation therapy, and hormonal therapy. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we comprehensively describe the tumor microenvironment of localized prostate cancer in comparison with adjacent normal samples and healthy controls. Single-cell RNA sequencing and high-resolution spatial transcriptomic analyses reveal tumor context dependent changes in gene expression. Our data indicate that an immune suppressive tumor microenvironment associates with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We infer cell-to-cell relationships from high throughput ligand-receptor interaction measurements within undissociated tissue sections. Our work thus provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.

The immune cell atlas of human neuroblastoma.

Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.