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The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence.Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300–14. ©2018 AACR.

Supplementary Figures and Table Legends to support primary figures

Supplemental Figure 1 shows the tumor masses of the subcutaneous tumors shown in Figure 1, as well as the flow cytometry gating strategy of M/OCP populations and the osteoclast differentiation assay experimental schematic. Supplemental Figure 2 shows representative images and the flow cytometry gating strategy for the in vitro osteoclast differentiation assay demonstrated in Figure 2. Supplemental Figure 3 shows that ZA inhibits breast cancer metastasis in a manner that is counteracted by G-CSF in intratibial injections, subcutaneous bone marrow functional assays, and with intracardiac injections of the B1 cell line following pre-treatment with Ctl or ZA and/or recombinant G-CSF. Supplemental Figure 4 shows the tumor masses of the subcutaneous tumors shown in Figure 4, as well as the plasma ALP and NTX concentrations in nude mice with and without ZA treatment. Supplemental Figure 5 shows the representative images and quantifications of the in vitro osteoclast differentiation assay with whole bone marrow from Ctl- or ZA- donors. Supplemental Figure 6 shows the volcano plots, heatmaps of differentially expressed genes, and gene ontology categories for the RNA-sequencing analysis of whole bone marrow from mice treated with Ctl, ZA, G-CSF, or ZA+G-CSF. Supplemental Figure 7 shows the determination of cut point analyses for the plasma G-CSF concentrations and patient outcome data shown in Figure 7.

Functionally enriched gene ontology (GO) terms and differentially enriched genes for the RNA-sequencing tables and heatmaps included in the manuscript text.

Supplementary Table 1 from Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth

Activation of the β-catenin and receptor kinase pathways occurs often in medulloblastoma, the most common pediatric malignant brain tumor. In this study, we show that molecular cross-talk between the β-catenin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways is crucial to sustain medulloblastoma pathophysiology. Constitutive activation of phosphoinositide-dependent protein kinase 1 (PDK1), Akt, and glycogen synthase kinase 3β (GSK-3β) was detected by immunohistochemistry in all primary medulloblastomas examined (n = 41). Small-molecule inhibitors targeting the PI3K/Akt signaling pathway affected β-catenin signaling by inhibition of GSK-3β activity, resulting in cytoplasmic retention of β-catenin and reduced expression of its target genes cyclin D1 and c-Myc. The PDK1 inhibitor OSU03012 induced mitochondrial-dependent apoptosis of medulloblastoma cells and enhanced the cytotoxic effects of chemotherapeutic drugs in a synergistic or additive manner. In vivo, OSU03012 inhibited the growth of established medulloblastoma xenograft tumors in a dose-dependent manner and augmented the antitumor effects of mammalian target of rapamycin inhibitor CCI-779. These findings demonstrate the importance of cross-talk between the PI3K/Akt and β-catenin pathways in medulloblastoma and rationalize the PI3K/Akt signaling pathway as a therapeutic target in treatment of this disease. Cancer Res; 70(1); 266–76

Supplementary Figure Legends 1-8, Tables 1-3 from Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth

Supplementary Figure 7 from Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth

Supplementary Table 2 from Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth

Supplementary Figure 8 from Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth

Thymic atrophy and the progressive immune decline that accompanies it is a major health problem, chronically with age and acutely with immune injury. No solution has been defined. Here we demonstrate that one of the three mesenchymal cell subsets identified by single-cell analysis of human and mouse thymic stroma is a critical niche component for T lymphopoiesis. The Postn+ subset is located perivascularly in the cortical-medullary junction, medulla and subcapsular regions. Cell depletion demonstrated that it recruits T competent cells to the thymus and initiates T lymphopoiesisin vivo. This subset distinctively expresses the chemokineCcl19necessary for niche functions. It markedly declines with age and in the acute setting of hematopoietic stem cell transplant conditioning. When isolated and adoptively transferred, these cells durably engrafted the atrophic thymus, recruited early T progenitors, increased T cell neogenesis, expanded TCR complexity and enhanced T cell response to vaccination. These data define a thymus lymphopoietic niche cell type that may be manipulated therapeutically to regenerate T lymphopoiesis.

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.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we take advantage of fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors revealed a distinct transcriptional signature that was predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrated vascular remodeling within the endothelial cells and a proliferative signature within the fibroblasts that was associated with poor survival. An in silico cell-to-cell interaction analysis highlighted the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

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.

SummaryThe treatment of primary prostate cancer delicately balances an active surveillance approach for low-risk disease with multimodal treatment including surgery, radiation therapy, and hormonal therapy for high-risk disease. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we sought to comprehensively describe the tumor microenvironment of localized prostate cancer contrasting this with adjacent normal samples and healthy controls. We performed single-cell RNA sequencing and high-resolution spatial transcriptomic analysis. This revealed tumor context dependent changes in gene expression. Our data point towards an immune suppressive tumor microenvironment associated with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We inferred cell-to-cell relationships at an unprecedented scale for ligand-receptor interactions within undissociated tissue sections. Our work provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.HighlightsCharacterization of prostate cancer by combined scRNA-seq and spatial transcriptomic analysisPrimary prostate cancer establishes a suppressive immune microenvironmentThe prostate tumor microenvironment exhibits a high angiogenic gene expression patternA new computational analysis pipeline to deconvolute context-specific differential gene expression

Single cell sequencing methods facilitate the study of tissues at high resolution, revealing rare cell types with varying transcriptomes or genomes, but so far have been lacking the capacity to investigate cell-cell interactions. Here, we introduce CIM-seq, an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between every cell type in a given tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution of these into their constituent cell types using machine learning. CIM-seq is broadly applicable to studies that aim to simultaneously investigate the constituent cell types and the global interaction profile in a specific tissue.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a pan-cancer, multi-omic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. By combining metabolomics, CRISPR screen and gene expression data from more than 700 solid tumor cell lines, we identified DHODH, a critical enzyme in pyrimidine synthesis, as a potential novel treatment target in neuroblastoma. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of high-risk neuroblastoma tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In neuroblastoma cell lines, DHODH gene dependency was found to correlate with MYCN dependency, rendering these cell lines highly sensitive to DHODH inhibition in vitro. In xenograft and transgenic neuroblastoma mouse models, tumor growth was dramatically reduced, and survival extended following treatment with the DHODH inhibitor brequinar. A combination of brequinar and temozolomide cured the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy with curative potential. Overall, DHODH inhibition combined with temozolomde has clear therapeutic potential in neuroblastoma and we propose this combination as a candidate for clinical testing.

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo perturbation of niche signals interferon, granulocyte-colony stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. Niche perturbations induce novel and rapid transitions between these HSC states. Differential expression analysis within each state revealed HSC- and LSK-specific molecular signatures for each perturbation. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq revealed HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and progenitor-specific chromatin and transcriptional features that represent important determinants of regenerative potential during stress hematopoiesis.

Most systemic therapies for prostate cancer target androgen receptor signaling. Given the ongoing disease burden and mortality from advanced prostate cancer that persist despite available therapies, novel therapeutic targets are clearly needed. To this end, we conducted a high-resolution analysis of primary prostate cancer samples across a spectrum of tumor stage using 10x Chromium scRNAseq. Within each patient, we compared the prostate tumor to its adjacent normal tissue. These samples were also compared to healthy prostate samples collected from patients whose prostates were removed in the setting of bladder cancer surgery. Joint analysis revealed a rich repertoire of immune and non-immune stroma cells. Significant alterations were apparent in the prostate tumor microenvironment compared to adjacent normal and healthy samples. One striking difference was an expansion of T-cell cluster characterized by decreased cytotoxic and an increased exhaustion gene signatures. Another difference was the significantly higher regulatory T cell (Treg) activity. There was a substantial population shift within the myeloid compartment. Specifically, tumor tissue showed a selective enrichment in M2-like macrophages which was validated by multiplex immunohistochemistry. In addition, tumor tissue showed a significant increase in the myeloid-derived suppressor cell scoring in tumor inflammatory monocytes that strongly correlated with Treg activity. The changes in both myeloid and T cell gene expression suggest an immune suppressive environment within the tumor. In addition, we performed Slide-seqV2 to generate high spatial resolution maps of cellular organization of prostate tissues. The highly detailed spatial configuration of the healthy prostate tissue showed well-organized prostate epithelial glands surrounded by immune and non-immune stromal cells. This tissue architecture was severely disrupted within the cancerous prostate. Endothelial and fibroblast populations were disorganized and dispersed throughout the invading tumor. Context-dependent differential expression gene analysis on endothelial cells showed significant upregulation of sprouting angiogenesis and vascular endothelial growth factor pathways in the tumor context. In addition, we developed an approach to characterize the coordination between tumor cells and stromal compartment via spatial ligand-receptor (L/R) interaction. Data revealed 405 significant potential communication channels with many of these interactions known to have a role in prostate tumor angiogenesis and progression. These data provide a high resolution landscape of localized prostate cancer via 10x and Slide-seq. Data showed an immunosuppressive milieu in addition to highly active endothelial angiogenic program. L/R interaction analysis revealed interactions that may serve as potential therapeutic targets. Citation Format: Taghreed Hirz, Shenglin Mei, Hirak Sarkar, Youmna Kfoury, Shulin Wu, Chin-Lee Wu, Fei Chen, Evan Z. Macosko, Douglas M. Dahl, Ninib Baryawno, David T. Scadden, Philip J. Saylor, Peter V. Kharchenko, David B. Sykes. An atlas of single-cell and spatial transcriptomics reveals alterations that correlate with human prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2510.

Medulloblastoma is one of the most common pediatric malignant brain tumors, currently comprising four distinct molecular subgroups: wingless [WNT], sonic hedgehog [SHH], and groups 3-4. Efforts to identify the mechanisms of medulloblastoma development have focused on mapping the extent of the tumor cell heterogeneity within each subgroup. Nevertheless, little is known about the role of the tumor microenvironment (TME) in medulloblastoma progression, particularly in subgroups 3-4, which have the worst prognosis due to metastatic disease. In this study, we performed single-cell transcriptomics on 14 human medulloblastoma samples spanning all molecular subgroups, to uncover novel TME-tumor interactions modulating medulloblastoma progression and metastasis. Unsupervised clustering of all medulloblastoma samples revealed 18 subclusters, including tumor granule neuron progenitors (GNPs) in different stages of differentiation, stromal cells (including oligodendrocyte precursors, mature oligodendrocytes, astrocytes, fibroblasts, endothelial cells and pericytes), myeloid cells (including microglia, monocytes and macrophages) as well as lymphoid cells. To investigate subgroup-specific signatures as well as TME-tumor pathways, we analyzed each subgroup separately. Analysis of supporting stroma cells in groups 3-4 demonstrated the presence of 8 different stroma populations, including a population of tumor-associated endothelial cells. Tumor-associated endothelial and fibroblast populations of groups 3-4 showed the highest expression of genes for vascular remodeling and extracellular matrix degradation, suggesting an active reprogramming of the stroma by tumor GNP cells to support medulloblastoma progression. Epithelial-to-mesenchymal transition-like processes that regulate stem cell, invasion and metastatic properties were upregulated in the tumor GNP populations of groups 3-4 compared to SHH and WNT subgroups. Finally, we highlight the presence of the CXCL1-CXCL5/CXCR2 metastasis-associated axis in groups 3-4 medulloblastomas as a potential therapeutic target. Our findings provide biological insights into TME processes for the different subgroups of medulloblastoma and possible new potential therapeutic avenues. Citation Format: Ioanna Tsea, Yana Ruchiy, Manouk Verhoeven, Indranil Sinha, Klas Blomgren, Lena Maria Carlson, John Inge Johnsen, Cecilia Dyberg, Ninib Baryawno. Transcriptomic landscape of medulloblastoma reveals pathways of tumor-stroma remodelling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1677.

Our current knowledge of the different immune cells in neuroblastoma is based on in vitro and in vivo studies mainly focusing on a single cell type. Different studies have conveyed conflicting results and despite the use of anti-GD2 immunotherapy in the clinic, a comprehensive immune cell overview at the single-cell level is still missing and understanding the complete composition of immune cells neuroblastoma will be crucial for the development of novel immunotherapeutics against the disease. In this study, we performed single-cell RNA-sequencing on human neuroblastoma coupled with multiplex immunohistochemistry and survival analysis to provide a complete cellular and molecular immune cell landscape of human neuroblastoma. Further, we contrasted the neuroblastoma data with single-cell RNA-sequencing data from normal fetal adrenal gland to characterize cell-state changes from normal tissue to neuroblastoma. Our analysis revealed 27 immune cell subtypes including distinct subpopulations of myeloid, NK, B and T cells that were associated with a survival benefit. Multiple subtypes of B and NK cells, not seen in neuroblastoma before, were identified. We propose the presence of tertiary lymphoid structures and showed that Active NK cells correlated with improved survival. In contrast to adult cancers, we detected no difference in cytotoxicity and exhaustion score for cytotoxic T cells, nor Treg activity. However, we demonstrated an increase in inflammatory monocyte signature score from normal to tumor derived myeloid cells. Finally, we performed receptor-ligand interaction analysis between tumor, stroma and immune cells, where we highlight several interactions that we suggest for future studies of how to exploit immune cells as a therapeutic option in neuroblastoma. Our findings significantly broaden the understanding of the immune composition in neuroblastoma and provides a resource for the development of novel immunotherapeutics. Citation Format: Bronte Manouk Verhoeven, Shenglin Mei, Thale K. Olsen, Karin K. Gustafsson, Anders Valind, Axel Lindström, David G. Nord, Shahrzad S. Fard, Catharina Hagerling, Peter V. Kharchenko, Per Kogner, John I. Johnsen, Ninib Baryawno. The immune cell atlas of human neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6215.

Background and aims: Neuroblastoma (NB) is the most common and deadly malignancy of infancy. It is an embryonal neuroendocrine tumor arising from the neural crest lineage. Amplification of MYCN is a frequent genetic abnormality, found in approximately 20% of NB tumors, and represents a critical stratifying prognostic marker. Given that MYCN plays a key role in NB tumorigenesis and aggressiveness, the TH-MYCN transgenic mouse model is widely used in NB research. This model is characterized by the overexpression of MYCN in neuroectodermal cells, driven by tyrosine hydroxylase (TH) promoter. This overexpression gives rise to tumors exclusively in the sympathoadrenal system, reflecting human NB features. We aimed to explore the transcriptional landscape of TH-MYCN tumors by single-cell RNA sequencing (scRNA-seq). Methods: Tumors from three homozygous and two hemizygous TH-MYCN mice were dissociated into single cells, profiled by scRNA-seq (Chromium 3’ v2 kit, 10x Genomics), sequenced on the NextSeq platform (Illumina). A total of 41666 single cells were analyzed using pagoda2 and conos. TH-MYCN tumor single-cell transciptomes were further aligned with previously published transcriptomes from the adrenal gland of three mice at embryonic day (E)13.5. To evaluate malignant tumor cells, copy number variants (CNV) were analyzed using inferCNV. Immunohistochemistry was used to validate the bioinformatically predicted cell types. Results: scRNA-seq of tumors from TH-MYCN mice revealed 16 major cell populations, spanning stromal, immune and tumor compartments. MYCN+ tumor cells predominantly resemble sympathetic neuroblasts (Isl1, Stmn1, Nefl, Nefm), while MYCN+ chromaffin-like tumor cells (Pnmt, Cdkn1c, Th, Dbh) were rare. Immunofluorescence stainings confirmed that tumors predominantly consist of proliferating sympathetic neuroblasts. Tumor cell nests were surrounded by supportive stromal and immune cells. The adrenergic tumor cell composition observed in TH-MYCN tumors is highly reminiscent of human NB. Interestingly, we discovered a cluster of immature adrenergic tumor cells aligning with normal fetal cells transitioning from bridge to chromaffin state (characterized by Sox11, Atrx, Xist, Ptprs, Tcf4). Distinct genomic aberrations, particularly unique chromosomal deletions, were found in the immature adrenergic tumor cells relative to their sympathoblast and chromaffin tumor cell counterparts. Conclusions: Examining the cellular composition of TH-MYCN tumors at a transcriptional level gives a detailed view of tumor cell heterogeneity at an unprecedented resolution. This comprehensive atlas of TH-MYCN tumors provides a resource for unmasking novel therapeutic targets. Citation Format: Bethel Tesfai Embaie, Adele Alchahin, Thale Kristin Olsen, Hirak Sarkar, Shenglin Mei, Ninib Baryawno. Single-cell landscape of the TH-MYCN neuroblastoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1678.

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.

Lipid starvation results in skeletal progenitors favouring commitment to chondrogenic over osteogenic fate, a process mediated by FOXO transcription factors and SOX9. The avascular nature of cartilage makes it a unique tissue(1-4), 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.

Neuroblastoma is a heterogeneous embryonal malignancy and the most deadly tumor of childhood, although a minor subset may show spontaneous differentiation. It arises from the multipotent neural crest lineage during development. Some of this multipotency is retained in neuroblastoma, which can give rise to both adrenergic and mesenchymal tumor cells. The mechanisms enabling such dual fates are unknown, but likely help neuroblastoma to evade existing therapies. To understand neuroblastoma plasticity, we analyzed patient tumors using single-cell transcriptomics. In addition to the heterogeneous adrenergic and mesenchymal populations, we identify a subpopulation of malignant cells resembling Schwann cell precursors (SCPs). This SCP-like population connects the adrenergic and mesenchymal compartments through transitions structurally reminiscent of the SCP cell-fate decision fork that occurs during normal development. While the directionality of such transitions in neuroblastoma remains to be established, this finding expands the potential reservoirs of malignant cells, and suggests intratumoral plasticity mechanisms relevant for therapeutic resistance and relapse.

Single cell sequencing methods facilitate the study of tissues at high resolution, revealing rare cell types with varying transcriptomes or genomes, but so far have been lacking the capacity to investigate cell-cell interactions. Here, we introduce CIM-seq, an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between every cell type in a given tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution of these into their constituent cell types using machine learning. CIM-seq is broadly applicable to studies that aim to simultaneously investigate the constituent cell types and the global interaction profile in a specific tissue.

Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer, represent incurable disease and are refractory to immunotherapy. We sought to define distinct features of the bone marrow microenvironment by analyzing single cells from prostate cancer patients’ involved bone, uninvolved bone and distant bone sites as well as bone from cancer-free, orthopedic patients and healthy individuals. Metastatic prostate cancer was associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to prostate cancer bone metastases. The chemokine CCL20 was notably overexpressed by myeloid cells, as was its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic prostate bone metastases restored T cell reactivity and significantly prolonged animal survival. Comparative high resolution analysis of prostate cancer bone metastasis shows a targeted approach for relieving local immunosuppression for therapeutic effect.

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.

Summary Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.

Cancer relapse begins at the moment malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the by-products of massive surrounding cell death. In acute myeloid leukemia (AML), complete remissions are common, but few are cured. We tracked AML cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells and conducted unbiased metabolic analysis, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine synthesis and glutathione generation, but not oxidative phosphorylation. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual cells and improved survival in AML mouse models. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce a metabolic collapse in cancer cells to overcome chemoresistance.

The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence. Significance: Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. Cancer Res; 78(18); 5300–14. ©2018 AACR.

Summary Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer (PCa), represent incurable disease, and are refractory to immunotherapy. We seek to define distinct features of the bone marrow (BM) microenvironment by analyzing single cells from bone metastatic prostate tumors, involved BM, uninvolved BM, and BM from cancer-free, orthopedic patients, and healthy individuals. Metastatic PCa is associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to PCa bone metastases. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic PCa bone metastases restores T cell reactivity and significantly prolongs animal survival. Comparative high-resolution analysis of PCa bone metastases shows a targeted approach for relieving local immunosuppression for therapeutic effect.

Nervous system injury is a frequent result of cancer therapy involving cranial irradiation, leaving patients with marked memory and other neurobehavioral disabilities. Here, we report an unanticipated link between bone marrow and brain in the setting of radiation injury. Specifically, we demonstrate that bone marrow-derived monocytes and macrophages are essential for structural and functional repair mechanisms, including regeneration of cerebral white matter and improvement in neurocognitive function. Using a granulocyte-colony stimulating factor (G-CSF) receptor knockout mouse model in combination with bone marrow cell transplantation, MRI, and neurocognitive functional assessments, we demonstrate that bone marrow-derived G-CSF-responsive cells home to the injured brain and are critical for altering neural progenitor cells and brain repair. Additionally, compared with untreated animals, animals that received G-CSF following radiation injury exhibited enhanced functional brain repair. Together, these results demonstrate that, in addition to its known role in defense and debris removal, the hematopoietic system provides critical regenerative drive to the brain that can be modulated by clinically available agents.

The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.

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.

The niche, as first conceptualized, was in conflict with the prevailing wisdom that stem cells have internal logic. The niche hypothesis has been indisputably confirmed. Yet, recent findings indicate little plasticity of epigenetically scripted hematopoietic stem/progenitors. Reconciling this conflict requires re-envisioning the niche as an enabler, not designer, of cell fate.

Cancer-related mortality due to solid tumor malignancies is overwhelmingly due to the development and progression of metastases. In advanced prostate cancer, metastases most often involve the bone and generally represent incurable disease. It remains unclear what aspects of the bone marrow microenvironment make it hospitable to metastatic dissemination. Similarly, the impact of the metastatic tumor on hematopoiesis and the marrow immune response is poorly understood. We took advantage of rare spinal cord decompression surgeries to profile marrow and metastatic tumors from men with advanced prostate cancer at single-cell resolution. Our analysis contrasts the cellular composition and transcriptional states in matched samples of tumor and liquid bone marrow collected at adjacent vertebral body levels, as well as bone marrow of orthopedic patients without malignancy. Metastatic prostate cancer was associated with hematopoietic suppression and multifaceted immune distortion. There was exhaustion of specific T cell subsets, appearance of inflammatory monocytes and macrophages, and alteration of cytokine profiles. Computational analysis showed association between the presence of specific myeloid subsets and the level of T lymphocyte dysfunction in the tumor fraction. We screened for potential signaling axes that may underlie this interaction. Among them was chemokine CCL20, notably overexpressed by myeloid cells, as was its cognate CCR6 receptor, expressed on T cells. We developed a syngeneic mouse model of bone-metastatic prostate cancer to explore this observation, and demonstrated that disruption of the CCL20-CCR6 axis from either side resulted in significant prolongation of survival. Our results further indicated that this dual overexpression was associated with repressed immune responses. Overall, comparative high-resolution analysis of bone marrow reveals distinct alterations associated with prostate cancer bone metastases that may be amenable to therapeutic targeting with the goal of altering cancer progression. Citation Format: Ninib Baryawno, Youmna Kfoury, Nicolas Severe, Shenglin Mei, Karin Gustafsson, Taghreed Hirz, Thomas Brouse, Elizabeth W. Scadden, Anna A. Igolkina, Bryan D. Choi, Nikolas Barkas, John H. Shin, Philip J. Saylor, David T. Scadden, David B. Sykes, Peter V. Kharchenko, as part of the Boston Bone Metastasis Consortium. Impact of metastatic prostate cancer on human bone marrow [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA23.