CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance.

The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs), defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

Establishment of a predictive model for GVHD-free, relapse-free survival after allogeneic HSCT using ensemble learning.

Graft-versus-host disease-free, relapse-free survival (GRFS) is a useful composite end point that measures survival without relapse or significant morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to develop a novel analytical method that appropriately handles right-censored data and competing risks to understand the risk for GRFS and each component of GRFS. This study was a retrospective data-mining study on a cohort of 2207 adult patients who underwent their first allo-HSCT within the Kyoto Stem Cell Transplantation Group, a multi-institutional joint research group of 17 transplantation centers in Japan. The primary end point was GRFS. A stacked ensemble of Cox Proportional Hazard (Cox-PH) regression and 7 machine-learning algorithms was applied to develop a prediction model. The median age for the patients was 48 years. For GRFS, the stacked ensemble model achieved better predictive accuracy evaluated by C-index than other state-of-the-art competing risk models (ensemble model: 0.670; Cox-PH: 0.668; Random Survival Forest: 0.660; Dynamic DeepHit: 0.646). The probability of GRFS after 2 years was 30.54% for the high-risk group and 40.69% for the low-risk group (hazard ratio compared with the low-risk group: 2.127; 95% CI, 1.19-3.80). We developed a novel predictive model for survival analysis that showed superior risk stratification to existing methods using a stacked ensemble of multiple machine-learning algorithms.

Identification of the minimum requirements for successful haematopoietic stem cell transplantation.

Historically, defining haematopoietic subsets, including self-renewal, differentiation and lineage restriction, has been elucidated by transplanting a small number of candidate cells with many supporting bone marrow (BM) cells. While this approach has been invaluable in characterising numerous distinct subsets in haematopoiesis, this approach is arguably flawed. The haematopoietic stem cell (HSC) has been proposed as the critical haematopoietic subset necessary for transplantation. However, due to the presence of supporting cells, the HSC has never demonstrated sufficiency. Utilising the homeobox B5 (Hoxb5)-reporter system, we found that neither long-term (LT) HSCs nor short-term (ST) HSCs alone were sufficient for long-term haematopoietic reconstitution. Critically, reconstitution can be rescued by transplanting combined LT- and ST-HSCs, without supporting cells; a fraction we term the 'Minimum Subset for Transplantation' (MST). The MST accounts for only 0·005% of nucleated cells within mouse BM, and this MST can be cultured, expanded and genetically modified while preserving its rapid haematopoietic engraftment potential. These results support the consideration of an MST approach for clinical translation, especially for gene therapy approaches that require HSC compartment modification.

Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment.

Self-renewal and multipotency are essential functions of hematopoietic stem cells (HSCs). To maintain homeostatic hematopoiesis, functionally uniform HSCs have been thought to be an ideal cell-of-origin. Recent technological advances in the field have allowed us to analyze HSCs with single cell resolution and implicate that functional heterogeneity may exist even within the highly purified HSC compartment. However, due in part to the technical limitations of analyzing extremely rare populations and our incomplete understanding of HSC biology, neither the biological meaning of why heterogeneity exists nor the precise mechanism of how heterogeneity is determined within the HSC compartment is entirely known. Here we show the first evidence that self-renewal capacity varies with the degree of replication stress dose and results in heterogeneity within the HSC compartment. Using the Hoxb5-reporter mouse line which enables us to distinguish between long-term (LT)-HSCs and short-term (ST)-HSCs, we have found that ST-HSCs quickly lose self-renewal capacity under high stress environments but can maintain self-renewal under low stress environments for long periods of time. Critically, exogeneous Hoxb5 expression confers protection against loss of self-renewal to Hoxb5-negative HSCs and can partially alter the cell fate of ST-HSCs to that of LT-HSCs. Our results demonstrate that Hoxb5 imparts functional heterogeneity in the HSC compartment by regulating self-renewal capacity. Additionally, Hoxb5-positive HSCs may exist as fail-safe system to protect from the exhaustion of HSCs throughout an organism's lifespan.

Low Multiplication Value of Absolute Monocyte Count and Absolute Lymphocyte Count at Diagnosis May Predict Poor Prognosis in Neuroblastoma.

Despite the growing evidences that immune dysfunction contributes to tumor progression, the prognostic value in patients with neuroblastoma regarding circulating immune blood cell counts has not been well characterized. To answer this, we conducted a retrospective study to evaluate the prognostic value of the circulating immune cell counts at diagnosis in a cohort of 55 patients with neuroblastoma. Based on a novel index by multiplying the absolute monocyte count (AMC)/µl and absolute lymphocyte count (ALC)/µl, we sub-grouped patients with AMC × ALC ≥ 1 × 106 (/µl)2 as high group and patients with AMC × ALC < 1 × 106 (/µl)2 as low group. In the entire cohort, the 4-year progression-free survival (PFS), and overall survival (OS) for high group (n = 38) vs low group (n = 17) was 81.7% (95%CI; 63.6-91.3%) and 90.7% (95%CI; 73.8-96.9%) vs 31.7% (11.6-54.1%) and 56.5% (29.7-76.4%; p < 0.001 for PFS and p = 0.015 for OS), respectively, suggesting that a low AMC × ALC is associated with poor prognosis. In the subgroup analysis for high-risk patients, the 4-year PFS and OS for high group (n = 17) vs low group (n = 13) was 59.8% (31.2-79.7%) and 79.8% (49.4-93.0%) vs 8.5% (0.5-31.7%) and 42.0% (15.4-66.8%; p < 0.001 for PFS and p = 0.089 for OS), respectively. Our data demonstrate that AMC × ALC at diagnosis is a cost-effective and easily measurable biomarker for predicting prognosis in neuroblastoma.

[Significance and purification of long-term hematopoietic stem cells in the hematopoietic system].

The hematopoietic stem cells, defined as blood stem cells with self-replication ability and multipotency, are key to successful hematopoietic stem cell transplantation. With the history of transplantation in the past 60 years and advances in stem cell technologies, our understanding of the hematopoietic system has deepened. However, the molecular mechanisms of self-renewal and pluripotency, which are the essence of the hematopoietic stem cells, remain poorly understood. One reason is that the identification/purification methods of the hematopoietic stem cells, particularly the long-term hematopoietic stem cells capable of lifelong self-renewal, is technically difficult owing to their scarcity in the bone marrow and has not been established to this date. Considering that a long-lasting blood production after hematopoietic stem cell transplantation is crucial, it is essential to understand the biology of the long-term hematopoietic stem cells not only scientifically but also clinically. This review describes the scientific and clinical significance of the long-term hematopoietic stem cells by showing the results of the latest researches in the introduction of hematopoietic stem cell identification/purification history.

Neutrophil and monocyte kinetics play critical roles in mouse peritoneal adhesion formation.

Peritoneal adhesions are pathological fibroses that ensnare organs after abdominal surgery. This dense connective tissue can cause small bowel obstruction, female infertility, and chronic abdominal pain. The pathogenesis of adhesions is a fibrotic response to tissue damage coordinated between mesothelial cells, fibroblasts, and immune cells. We have previously demonstrated that peritoneal adhesions are a consequence of mechanical injury to the mesothelial layer sustained during surgery. Neutrophils are among the first leukocytes involved in the early response to tissue damage. Here, we show that when subjected to mechanical stress, activated mesothelial cells directly recruit neutrophils and monocytes through upregulation of chemokines such as CXCL1 and monocyte chemoattractant protein 1 (MCP-1). We find that neutrophils within the adhesion sites undergo cell death and form neutrophil extracellular traps (NETosis) that contribute to pathogenesis. Conversely, tissue-resident macrophages were profoundly depleted throughout the disease time course. We show that this is distinct from traditional inflammatory kinetics such as after sham surgery or chemically induced peritonitis, and suggest that adhesions result from a primary difference in inflammatory kinetics. We find that transient depletion of circulating neutrophils significantly decreases adhesion burden, and further recruitment of monocytes with thioglycolate or MCP-1 also improves outcomes. Our findings suggest that the combination of neutrophil depletion and monocyte recruitment is sufficient to prevent adhesion formation, thus providing insight for potential clinical interventions.

Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow.

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in ß-1,4-galactosyltransferase-1 (ß4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in ß4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of ß4GalT-1-deficient BM cells, suggesting that ß4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Where Hematopoietic Stem Cells Live: The Bone Marrow Niche.

SIGNIFICANCE: Hematopoietic stem cells (HSCs) can sustain the production of blood throughout one's lifetime. However, for proper self-renewal of its own population and differentiation to blood, the HSC requires a specialized microenvironment called the "niche." Recent Advances: Recent studies using novel mouse models have shed new light on the cellular architecture and function of the HSC niche. Here, we review the different cells that constitute the HSC niche and the molecular mechanisms that underlie HSC and niche interaction. We discuss the evidence and potential features that distinguish the HSC niche from other microenvironments in the bone marrow. The relevance of the niche in malignant transformation of the HSCs and harboring cancer metastasis to the bone is also outlined. In addition, we address how the niche may regulate reactive oxygen species levels surrounding the HSCs. Critical Issues and Future Directions: We propose future directions and remaining challenges in investigating the niche of HSCs. We discuss how a better understanding of the HSC niche may help in restoring an aged hematopoietic system, fighting against malignancies, and transplanting purified HSCs safely and effectively into patients. Antioxid. Redox Signal. 00, 000-000.

Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

Do pluripotent stem cells exist in adult mice as very small embryonic stem cells?

Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the "VSEL" flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs.

Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

Synergistic effect of Tim4 and MFG-E8 null mutations on the development of autoimmunity.

Phagocytes, including macrophages, recognize phosphatidylserine exposed on apoptotic cells as an "eat me" signal. Milk Fat Globule EGF Factor VIII (MFG-E8) is secreted by one subset of macrophages, whereas Tim4, a type I membrane protein, is expressed by another. These proteins bind tightly to phosphatidylserine on apoptotic cells and enhance their engulfment by macrophages. To study the contribution of these proteins to the engulfment of apoptotic cells, we established a mouse line that was deficient in the genes encoding MFG-E8 and Tim4. The null mutation of Tim4 impaired the ability of resident peritoneal macrophages, but not thioglycollate-elicited macrophages, to engulf apoptotic cells. Mice deficient in either MFG-E8 or Tim4 on the C57BL/6 background developed hardly any autoantibodies, but aged female mice deficient in both MFG-E8 and Tim4 developed autoantibodies in an age-dependent manner. Tumour necrosis factor (TNF) α is known to protect against systemic lupus erythematosus-type autoimmunity, whereas type I IFN accelerates the disease. Indeed, the administration of an anti-TNFα antibody or a reagent that stimulates the IFN-α production [2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane)] enhanced the production of autoantibodies in the MFG-E8- and Tim4-double-deficient mice. These results suggest that the double deficiency of Tim4 and MFG-E8, phosphatidylserine-binding proteins, can trigger autoimmunity and that TNFα and type I IFN regulate reciprocally the development of autoimmune disease.

Identification of Tim4 as a phosphatidylserine receptor.

In programmed cell death, a large number of cells undergo apoptosis, and are engulfed by macrophages to avoid the release of noxious materials from the dying cells. In definitive erythropoiesis, nuclei are expelled from erythroid precursor cells and are engulfed by macrophages. Phosphatidylserine is exposed on the surface of apoptotic cells and on the nuclei expelled from erythroid precursor cells; it works as an 'eat me' signal for phagocytes. Phosphatidylserine is also expressed on the surface of exosomes involved in intercellular signalling. Here we established a library of hamster monoclonal antibodies against mouse peritoneal macrophages, and found an antibody that strongly inhibited the phosphatidylserine-dependent engulfment of apoptotic cells. The antigen recognized by the antibody was identified by expression cloning as a type I transmembrane protein called Tim4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as Timd4). Tim4 was expressed in Mac1+ cells in various mouse tissues, including spleen, lymph nodes and fetal liver. Tim4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of Tim4 in fibroblasts enhanced their ability to engulf apoptotic cells. When the anti-Tim4 monoclonal antibody was administered into mice, the engulfment of apoptotic cells by thymic macrophages was significantly blocked, and the mice developed autoantibodies. Among the other Tim family members, Tim1, but neither Tim2 nor Tim3, specifically bound phosphatidylserine. Tim1- or Tim4-expressing Ba/F3 B cells were bound by exosomes via phosphatidylserine, and exosomes stimulated the interaction between Tim1 and Tim4. These results indicate that Tim4 and Tim1 are phosphatidylserine receptors for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved.

Neuropilin-1 promotes unlimited growth of ovarian cancer by evading contact inhibition.

OBJECTIVE: Neuropilin-1 (NRP-1) is a receptor for both semaphorin and vascular endothelial growth factor and is up-regulated in a variety of human cancers. While there are some reports of NRP-1 expression in ovarian neoplasm, those results differ in pattern of its expression and its role in ovarian cancer is still unclear. We sought to investigate the expression pattern and role of NRP-1 in ovarian cancer. METHODS: NRP-1 expression was analyzed with eighty-seven ovarian tissue samples by immunohistochemistry and four ovarian cell lines by quantitative RT-PCR and Western blotting. To detect its molecular role in ovarian cancer, WST-1 assay, invasion assay and soft agar assay were performed with or without NRP-1 suppression by the introduction of short hairpin RNAs. RESULTS: NRP-1 expression was found to be enhanced in ovarian cancer compared with ovarian surface epithelium (OSE), benign adenoma and tumors of low malignant potential. In vitro, NRP-1 expression was augmented threefold during malignant transformation of OSE cells with oncogene ras, suggesting an association between NRP-1 and oncogenesis. Suppression of NRP-1 reduced cell proliferation in a dense state, indicating that persistently high expression of NRP-1 in ovarian cancer enhances proliferation through evasion of contact inhibition. Suppression of NRP-1 also decreased cell growth in soft agar and invasion to the extracellular matrix in vitro. CONCLUSIONS: These results suggest that NRP-1 is not only associated with oncogenesis, but also with ovarian cancer malignancy, and this molecule is a targeting candidate for the treatment of ovarian malignancies.

Immortalized ovarian surface epithelial cells acquire tumorigenicity by Acrogranin gene overexpression.

Malignant transformation is caused by multi-step genetic mutations, and growth factors are believed to play important roles in developing and maintaining malignant phenotype. However, there is no direct evidence that a specific growth factor contributes to malignant transformation of phenotypically normal cells. In order to assess the function of Acrogranin (also known as granulin epithelial precursor; GEP) in ovarian carcinogenesis, ovarian surface epithelial (OSE) cells, which are supposed to be the origin of primary ovarian epithelial cancer, were transfected with combined genes of hTERT, SV40 LT, and Acrogranin. Introduction of hTERT and SV40 LT was sufficient for immortalizing OSE cells but not enough for tumor formation in nude mice. In contrast, transfection and overexpression of Acrogranin in immortalized OSE cells showed augmented clonogenicity in soft agar and obvious tumorigenicity in nude mice. This is the first study showing evidence that a specific growth factor plays a direct role in malignant transformation in ovarian cancer development.

Oncogenic property of acrogranin in human uterine leiomyosarcoma: direct evidence of genetic contribution in in vivo tumorigenesis.

To identify potential oncogenes that contribute to the development of uterine leiomyosarcoma, we conducted a cDNA microarray analysis between normal uterine smooth muscle and uterine leiomyosarcoma. We found that acrogranin (also named PCDGF or progranulin) is overexpressed in uterine leiomyosarcoma. With immunohistochemical staining of 12 leiomyosarcoma cases, we verified acrogranin expression in tumor cells. Furthermore, the intensity of acrogranin expression correlated with high histologic grade and poor prognosis. To directly analyze the oncogenic properties of acrogranin, we established an immortalized uterine smooth muscle cell line by transfection of human telomerase reverse transcriptase into primary culture. This cell line retained the original characteristics of uterine smooth muscle cells, including spindle-shaped extension as well as expression of vimentin, estrogen receptor alpha, progesterone receptor, and alpha smooth muscle actin. Transfection of acrogranin into the immortalized uterine smooth muscle cells resulted in colony formation in soft agar, but the diameter of the colonies did not exceed 100 mum. Transfection of both acrogranin and SV40 early region (SV40ER) into the immortalized uterine smooth muscle cells resulted in an increased number of colonies and increased colony size in soft agar versus transfection of SV40ER alone. We show that only immortalized uterine smooth muscle cells expressing both acrogranin and SV40ER are capable of tumor formation in nude mice. Thus, acrogranin is overexpressed in uterine leiomyosarcoma cells, particularly in high-grade cases, and forced expression of acrogranin in immortalized uterine smooth muscle cells contributes to malignant transformation, which suggest that acrogranin plays an important role in the pathogenesis of uterine leiomyosarcoma.