Spatial multi-omics analyses of the tumor immune microenvironment.

In the past decade, single-cell technologies have revealed the heterogeneity of the tumor-immune microenvironment at the genomic, transcriptomic, and proteomic levels and have furthered our understanding of the mechanisms of tumor development. Single-cell technologies have also been used to identify potential biomarkers. However, spatial information about the tumor-immune microenvironment such as cell locations and cell-cell interactomes is lost in these approaches. Recently, spatial multi-omics technologies have been used to study transcriptomes, proteomes, and metabolomes of tumor-immune microenvironments in several types of cancer, and the data obtained from these methods has been combined with immunohistochemistry and multiparameter analysis to yield markers of cancer progression. Here, we review numerous cutting-edge spatial 'omics techniques, their application to study of the tumor-immune microenvironment, and remaining technical challenges.

NK cell receptor and ligand composition influences the clearance of SARS-CoV-2.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Imbalanced Osteogenesis and Adipogenesis in Mice Deficient in the Chemokine Cxcl12/Sdf1 in the Bone Mesenchymal Stem/Progenitor Cells.

Bone and bone marrow serve as an imperative ecosystem to various types of cells participating in critical body functions. The chemokine Cxcl12, also known as stromal cell-derived factor 1 (Sdf1), is one of the communication factors in the marrow microenvironment that regulates hematopoietic stem/progenitor cell homeostasis. However, the function of Cxcl12 in other bone marrow cells in vivo is yet to be discovered. Here we report a novel function of Cxcl12 in postnatal bone development and homeostasis. Targeted deletion of Cxcl12 in Paired related homeobox 1 (Prx1)-expressing or osterix (Osx)-expressing mesenchymal stem/progenitor cells (MSPCs), but not in mature osteoblasts, resulted in marrow adiposity and reduced trabecular bone content. In vivo lineage tracing analysis revealed biased differentiation of MSPCs toward adipocytes. In contrast, adult-stage deletion of Cxcl12 in Osx-expressing cells led to reduced bone content but not adiposity. Targeting the receptor Cxcr4 in the Prx1-expressing cells also resulted in reduced trabecular bone content but not adiposity. Our study reveals a previously unidentified role of the MSPC-secreting Cxcl12 that regulates its osteogenesis and adipogenesis through the cell-autonomous and non-autonomous mechanism, respectively; which could further influence the homeostatic control of the hematopoietic system. © 2017 American Society for Bone and Mineral Research.

The distinct biological implications of Asxl1 mutation and its roles in leukemogenesis revealed by a knock-in mouse model.

BACKGROUND: Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a "physiological" dose of mutant ASXL1 remain unexplored. METHODS: We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system. RESULTS: Heterozygotes (Asxl1 tm/+ ) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1 tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors. CONCLUSIONS: We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies.

Higher HOPX expression is associated with distinct clinical and biological features and predicts poor prognosis in de novo acute myeloid leukemia.

Homeodomain-only protein homeobox (HOPX) is the smallest homeodomain protein. It was regarded as a stem cell marker in several non-hematopoietic systems. While the prototypic homeobox genes such as the HOX family have been well characterized in acute myeloid leukemia (AML), the clinical and biological implications of HOPX in the disease remain unknown. Thus we analyzed HOPX and global gene expression patterns in 347 newly diagnosed de novo AML patients in our institute. We found that higher HOPX expression was closely associated with older age, higher platelet counts, lower white blood cell counts, lower lactate dehydrogenase levels, and mutations in RUNX1, IDH2, ASXL1, and DNMT3A, but negatively associated with acute promyelocytic leukemia, favorable karyotypes, CEBPA double mutations and NPM1 mutation. Patients with higher HOPX expression had a lower complete remission rate and shorter survival. The finding was validated in two independent cohorts. Multivariate analysis revealed that higher HOPX expression was an independent unfavorable prognostic factor irrespective of other known prognostic parameters and gene signatures derived from multiple cohorts. Gene set enrichment analysis showed higher HOPX expression was associated with both hematopoietic and leukemia stem cell signatures. While HOPX and HOX family genes showed concordant expression patterns in normal hematopoietic stem/progenitor cells, their expression patterns and associated clinical and biological features were distinctive in AML settings, demonstrating HOPX to be a unique homeobox gene. Therefore, HOPX is a distinctive homeobox gene with characteristic clinical and biological implications and its expression is a powerful predictor of prognosis in AML patients.

Intermediate Progenitors Facilitate Intracortical Progression of Thalamocortical Axons and Interneurons through CXCL12 Chemokine Signaling.

Glutamatergic principal neurons, GABAergic interneurons and thalamocortical axons (TCAs) are essential elements of the cerebrocortical network. Principal neurons originate locally from radial glia and intermediate progenitors (IPCs), whereas interneurons and TCAs are of extrinsic origin. Little is known how the assembly of these elements is coordinated. C-X-C motif chemokine 12 (CXCL12), which is known to guide axons outside the neural tube and interneurons in the cortex, is expressed in the meninges and IPCs. Using mouse genetics, we dissected the influence of IPC-derived CXCL12 on TCAs and interneurons by showing that Cxcl12 ablation in IPCs, leaving meningeal Cxcl12 intact, attenuates intracortical TCA growth and disrupts tangential interneuron migration in the subventricular zone. In accordance with strong CXCR4 expression in the forming thalamus and TCAs, we identified a CXCR4-dependent growth-promoting effect of CXCL12 on TCAs in thalamus explants. Together, our findings indicate a cell-autonomous role of CXCR4 in promoting TCA growth. We propose that CXCL12 signals from IPCs link cortical neurogenesis to the progression of TCAs and interneurons spatially and temporally. Significance statement: The cerebral cortex exerts higher brain functions including perceptual and emotional processing. Evolutionary expansion of the mammalian cortex is mediated by intermediate progenitors, transient amplifying cells generating cortical excitatory neurons. During the peak period of cortical neurogenesis, migrating precursors of inhibitory interneurons originating in subcortical areas and thalamic axons invade the cortex. Although defects in the assembly of cortical network elements cause neurological and mental disorders, little is known how neurogenesis, interneuron recruitment, and axonal ingrowth are coordinated. We demonstrate that intermediate progenitors release the chemotactic cytokine CXCL12 to promote intracortical interneuron migration and growth of thalamic axons via the cognate receptor CXCR4. This paracrine signal may ensure thalamocortical connectivity and dispersion of inhibitory neurons in the rapidly growing cortex.

Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin(+) mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin(+) MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1ß produced by mutant HSCs. In turn, in vivo depletion of nestin(+) cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with ß3-adrenergic agonists that restored the sympathetic regulation of nestin(+) MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.

Mesenchymal progenitors and the osteoblast lineage in bone marrow hematopoietic niches.

The bone marrow cavity is essential for the proper development of the hematopoietic system. In the last few decades, it has become clear that mesenchymal stem/progenitor cells as well as cells of the osteoblast lineage, besides maintaining bone homeostasis, are also fundamental regulators of bone marrow hematopoiesis. Several studies have demonstrated the direct involvement of mesenchymal and osteoblast lineage cells in the maintenance and regulation of supportive microenvironments necessary for quiescence, self-renewal and differentiation of hematopoietic stem cells. In addition, specific niches have also been identified within the bone marrow for maturing hematopoietic cells. Here we will review recent findings that have highlighted the roles of mesenchymal progenitors and cells of the osteoblast lineage in regulating distinct stages of hematopoiesis.

Baicalin induces apoptosis in SW620 human colorectal carcinoma cells in vitro and suppresses tumor growth in vivo.

In the United States, colorectal cancer (CRC) is the second most frequent malignancy and the fourth most common cause of cancer death. Baicalin, a flavone derivative isolated and purified from the dry root of Scutellaria, was assessed for its antitumor effects in human SW620 CRC cells. Baicalin (200 µM) inhibited proliferation of SW620 cells. Baicalin (200 µM) increased activities of caspase-3, -8, and -9 in SW620 cells. Furthermore, flow cytometric analysis of baicalin-treated SW620 cells showed an increase in sub-G1 cells, and the dihydroethidium assay showed significant enhancement of intracellular peroxide production in baicalin-treated cells. Addition of N-acetylcysteine prevented most of the baicalin-induced apoptosis, which in turn mediated cytotoxicity in human SW620 cells. In vivo, baicalin (50 mg/kg/day, i.p.) treatment inhibited 55% of tumor growth in xenografted nude mice by 4 weeks, compared to that of the vehicle control (p < 0.05). Baicalin had no noteworthy influence on body weight. Thus, we suggest the development of baicalin as a potential leading antitumor agent in CRC.

Loss of Cxcl12/Sdf-1 in adult mice decreases the quiescent state of hematopoietic stem/progenitor cells and alters the pattern of hematopoietic regeneration after myelosuppression.

The C-X-C-type chemokine Cxcl12, also known as stromal cell-derived factor-1, plays a critical role in hematopoiesis during fetal development. However, the functional requirement of Cxcl12 in the adult hematopoietic stem/progenitor cell (HSPC) regulation was still unclear. In this report, we developed a murine Cxcl12 conditional deletion model in which the target gene can be deleted at the adult stage. We found that loss of stroma-secreted Cxcl12 in the adult led to expansion of the HSPC population as well as a reduction in long-term quiescent stem cells. In Cxcl12-deficient bone marrow, HSPCs were absent along the endosteal surface, and blood cell regeneration occurred predominantly in the perisinusoidal space after 5-fluorouracil myelosuppression challenge. Our results indicate that Cxcl12 is required for HSPC homeostasis regulation and is an important factor for osteoblastic niche organization in adult stage bone marrow.

Lung defects in neonatal and adult stromal-derived factor-1 conditional knockout mice.

Stromal-derived factor (SDF)-1/CXCL12 is a cytokine that is involved in organogenesis, hematopoiesis, chemoattraction, and wound healing. An SDF-1 knockout mouse (SDF-1(-/-)) has provided important insights into the role of SDF-1 in fetal development. Because the SDF-1 knockout is lethal in the perinatal period, we have created a conditional SDF-1 knockout mouse. In the present study, we induced conditionally knocked out SDF-1 in neonatal mice and found that lung development was compromised; neonatal lungs showed increased alveolar airspace and abnormal ultrastructure. Conditional knockout of SDF-1 in adult mice resulted in an emphysemic morphology, with increased alveolar airspace and thickened alveolar septa. Fluorescence angiography showed pulmonary vessel hyperdilation. To determine whether the hyperdilation involved nitric oxide, we inhibited endothelial nitric oxide synthase (eNOS) with N (G)-nitro-L- arginine methyl ester. This resulted in the inhibition of pulmonary vessel hyperdilation. Western blot results showed increased phosphorylation of eNOS in our induced SDF-1 knockout mice, indicating that eNOS is normally repressed in the presence of SDF-1, and that activation of eNOS contributes to pulmonary pathology. Thus, a conditional knockout mouse has been successfully created for SDF-1; initial characterization indicates that SDF-1 is intimately involved in lung development and physiology.

Gene expression in early and progression phases of autosomal dominant polycystic kidney disease.

BACKGROUND: Little is known about the genes involved in the initial cyst formation and disease progression in autosomal dominant polycystic kidney disease (ADPKD); however, such knowledge is necessary to explore therapeutic avenues for this common inherited kidney disease. FINDINGS: To uncover the genetic determinants and molecular mechanisms of ADPKD, we analyzed 4-point time-series DNA microarrays from Pkd1L3/L3 mice to generate high resolution gene expression profiles at different stages of disease progression. We found different characteristic gene expression signatures in the kidneys of Pkd1L3/L3 mice compared to age-matched controls during the initial phase of the disease. By postnatal week 1, the Pkd1L3/L3 kidney already had a distinctive gene expression pattern different from the corresponding normal controls. CONCLUSION: The genes differentially expressed, either induced or repressed, in ADPKD are important in immune defense, cell structure and motility, cellular proliferation, apoptosis and metabolic processes, and include members of three pathways (Wnt, Notch, and BMP) involved in morphogenetic signaling. Further analysis of the gene expression profiles from the early stage of cystogenesis to end stage disease identified a possible gene network involved in the pathogenesis of ADPKD.

Angiopoietin-like protein 1 decreases blood brain barrier damage and edema following focal cerebral ischemia in mice.

Angiopoietin-like protein (Angptl) 1, a member of the angiopoietin-related protein family, modulates angiogenesis but little else is known of its physiological role. We found that angptl1 was upregulated at the 7th day after focal cerebral ischemia in normal mice. In order to understand the role of angptl1 in cerebral infarction, we induced focal cerebral ischemia in normal and glial fibrillary acidic protein promoter-angptl1 transgenic mice. In the transgenic mice without ischemia, overexpression of angptl1 in the whole brain led to a decrease in cortical microvascular density. Following focal cerebral ischemia, edema, but not infarct size, was less in transgenic mice relative to wild type littermates. This effect might be due to a reduction in the blood brain barrier breakdown, as confirmed by a decrease in Evans Blue leakage in the early post-ischemic phase. We conclude that angptl1 may have a beneficial role in the preservation of vascular integrity following focal cerebral ischemia.

Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells.

BACKGROUND: Stroke is a leading cause of death and disability worldwide; however, no effective treatment currently exists. METHODS AND RESULTS: Rats receiving subcutaneous granulocyte colony-stimulating factor (G-CSF) showed less cerebral infarction, as evaluated by MRI, and improved motor performance after right middle cerebral artery ligation than vehicle-treated control rats. Subcutaneous administration of G-CSF enhanced the availability of circulating hematopoietic stem cells to the brain and their capacity for neurogenesis and angiogenesis in rats with cerebral ischemia. CONCLUSIONS: G-CSF induced increases in bone marrow cell mobilization and targeting to the brain, reducing the volume of cerebral infarction and improving neural plasticity and vascularization.

A novel PEPP homeobox gene, TOX, is highly glutamic acid rich and specifically expressed in murine testis and ovary.

The homeobox gene superfamily has been highly conserved throughout evolution. These genes act as transcription factors during several important developmental processes. To explore the functional roles of homeobox genes in spermatogenesis, we performed a degenerate oligonucleotide polymerase chain reaction (PCR) screening of a testis cDNA library and isolated a novel mouse homeobox gene. This gene, which we named Tox, encodes a homeodomain protein distantly related to members of the Paired/Pax (Prd/Pax) family. A phylogenetic analysis revealed Tox to be a member of the recently defined PEPP subfamily of Paired-like homeobox genes. Tox was mapped to chromosome X, with its homeodomain organized into three exons. A special feature of Tox is that the encoded protein sequence contains two poly-glutamic acid (poly E) stretches, which make Tox highly acidic. Tox transcripts were detected predominately in the testis and ovary of mice. Tox expression in testes was initiated soon after birth, mainly in Sertoli cells and spermatogonia; however, in adult mice, Tox expression shifts to the spermatids and spermatozoa. Tox expression in ovaries was detected in somatic cells of follicles, early on in theca cells, and in both granulosa and theca cells at the later stages of follicular development. Based on these results, Tox may play an important role during gametogenesis.