The transcriptional profile of circulating myeloid derived suppressor cells correlates with tumor development and progression in mouse.

Myeloid derived suppressor cells (MDSCs) play key roles in cancer development. Accumulation of peripheral-blood MDSCs (PB-MDSCs) corresponds to the progression of various cancers, but provides only a crude indicator. We aimed toward identifying changes in the transcriptional profile of PB-MDSCs in response to tumor growth. CT26 colon cancer cells and B16 melanoma cells (106) were inoculated into peritoneal cavities of BALB/c mice and subcutaneously to C57-black mice, respectively. The circulating levels and global transcriptional patterns of PB CD11b+Ly6g+ MDSCs were assessed in control mice, and 4, 8, and 11 days following tumor cell inoculation. Although a significant accumulation of PB-MDSCs was demonstrated only 11 days following tumor induction, a pronounced transcriptional response was identified already on day 4 while the tumor was ~1 mm in size. Further transcriptional changes correlated with different stages of tumor growth. Key MDSC genes and canonical signaling pathways were activated along tumor progression. This phenomenon was demonstrated in both cancer models, and a consensus set of 817 genes, involved in myeloid cell recruitment and angiogenesis, was identified. The data suggest that the transcriptional signatures of PB-MDSC may serve as markers for tumor progression, as well as providing potential targets for future therapies.

[THE PROLIFERATIVE EFFECT OF DENDRITIC CELLS IN OVARIAN CANCER AND THE RELATIONSHIP WITH THE IGF SIGNALING PATHWAY].

INTRODUCTION: Epithelial ovarian cancer (EOC) is the principal cause of death from gynecologic cancer in developed countries. While surgery and chemotherapy can improve survival, the mortality and morbidity rates remain significantly high. The insulin-like growth factor (IGF) axis has been shown to play an important part in carcinogenesis of several human malignancies. Preclinical studies reported a significant anti-proliferative activity of IGF1 receptor (IGF1R) inhibitors in ovarian malignancies, however, clinical studies have shown variable response rates. Recent data indicate that immunotherapy could hold promise in improving EOC treatment. Dendritic cells (DCs) which are antigen presenting cells evoke a positive immune response. Moreover, a recent study shows that IGF treatment can inhibit DC maturation. AIMS: To investigate the involvement of IGF1R signaling in DCs and the effect of combined DCs and IGF1R inhibitor treatment on EOC cells growth. METHODS: HL-60 leukemic cells were differentiated to DCs and ligand induced phosphorylated IGF1R levels were measured by Western blotting. Next, inhibition of IGF1R in DCs was applied and the effect of this inhibition on EOC cell lines ES2 and SKOV3 was examined using the migration assay method. RESULTS: The differentiation of HL-60 into DCs was associated with decreased levels of both IGF1R phosphorylation and total IGF1R protein. In addition, in-vitro growth assays (scratch assay) demonstrated an increased growth of both ES2 and SKOV3 cells into the scratch zone when co-cultured with DCs which were not pre-treated with IGF1R inhibitor as compared to treated DCs. CONCLUSIONS: Preliminary data suggest that DC differentiation is associated with IGF1R signaling downregulation. Moreover, inhibition of IGF1R signaling in DCs might decrease EOC growth.

Runx3 in Immunity, Inflammation and Cancer.

In this chapter we summarize the pros and cons of the notion that Runx3 is a major tumor suppressor gene (TSG). Inactivation of TSGs in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago it was suggested that RUNX3 is involved in gastric cancer development, a postulate extended later to other epithelial cancers portraying RUNX3 as a major TSG. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. In contrast, RUNX3 is overexpressed in a significant fraction of tumor cells in various human epithelial cancers and its overexpression in pancreatic cancer cells promotes their migration, anchorage-independent growth and metastatic potential. Moreover, recent high-throughput quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models have unequivocally demonstrated that RUNX3 is not a bona fide cell-autonomous TSG. Importantly, accumulating data demonstrated that RUNX3 functions in control of immunity and inflammation, thereby indirectly influencing epithelial tumor development.

Runx3 at the interface of immunity, inflammation and cancer.

Inactivation of tumor suppressor genes (TSG) in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago claims arose that the RUNX3 member of the RUNX transcription factor family is a major TSG inactivated in gastric cancer, a postulate extended later to other cancers. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. Here we critically re-appraise this paradigm in light of recent high-throughput, quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models. Collectively, these studies unequivocally demonstrate that RUNX3 is not a bona fide cell-autonomous TSG. Accordingly, RUNX3 is not recognized as a TSG and is not included among the 2000 cancer genes listed in the "Cancer Gene Census" or "Network for Cancer Genes" repositories. In contrast, RUNX3 does play important functions in immunity and inflammation and may thereby indirectly influence epithelial tumor development.

The development of endometriosis in a murine model is dependent on the presence of dendritic cells.

Endometriosis is a common condition associated with pelvic pain and infertility. This study group has previously shown that supplementation of dendritic cells led to enhancement of endometriosis lesion growth and angiogenesis. This study determined whether endometriosis is dependent on the presence of endogenous dendritic cells. Surgical induction of endometriosis was performed in CD11c⁺ DTR/GFP transgenic (Tg) female mice in which dendritic cells were ablated upon injection of diphtheria toxin (DT). Mice were allocated into four groups (n=5 each): group I, wild-type mice treated with vehicle; group II, wild-type mice treated with DT; group III, Tg mice treated with DT; and group IV, Tg mice treated with vehicle. After 10 days, mice were killed and endometriosis lesions were analysed by flow cytometry. DT treatment led to ablation of dendritic cells in spleens and endometriosis lesions in Tg mice while no ablation was observed in controls. Corresponding to dendritic cell ablation, endometriosis lesions in group III were ∼5-fold smaller than in the control groups (ANOVA P<0.0001). This study suggests that endometriosis development is dependent on the presence of endogenous dendritic cells. Therapies designed to inhibit dendritic cell infiltration as possible treatments for endometriosis warrant further study.

Myeloid cell alterations in the mouse placenta precede the onset of labor and delivery.

OBJECTIVE: Immature myeloid cells (IMCs) are bone marrow-derived cells that normally differentiate into granulocytes, macrophages, and dendritic cells (DCs) but expand in pathological conditions such as malignancy. DCs are antigen-presenting cells that regulate the immune response. Both IMCs and DCs were shown to take part in angiogenesis; however, little is known of their function in the placenta. We sought to determine whether alterations in DC and IMC populations in the placenta precede the onset of delivery. STUDY DESIGN: Experiments were performed on 6-8 week old C57Bl/6 female mice. Placentas from pregnant mice that were killed on designated days, immunostained using fluorescently labeled anti-CD11b, Gr-1, CD11c, major histocompatibility II (MHCII), and CD45, and analyzed by flow cytometry and immunofluorescent microscopy. RESULTS: Throughout the latter part of pregnancy toward labor and delivery, the CD45(+)CD11b(+)Gr1(+)-IMC population decreased 29 ± 9.1% (day 12) and 30 ± 9.9% (day 15), vs 21 ± 8.1% (day18, n = 21, 15, and 27; P = .006 and P = .004, respectively), whereas the CD45(+)CD11c(+)MHCII(+)-DC population increased 0.87 ± 0.3% (day 12) and 0.70 ± 0.3% (day 15) vs 1.81 ± 1.3% (day 18, n = 21, 15, and 27, P = .002 and P = .001, respectively). Both myeloid cell populations were localized adjacent to CD31(+) endothelial cells in sites of placental angiogenesis. CONCLUSION: Labor and delivery are preceded by proangiogenic-myeloid cell alterations, reflected by a decrease in IMCs and an increase in DCs populating the mouse placenta. The intriguing possibility that delivery is preceded by the maturation of IMCs in part into DCs warrants further studies.

IGF1R inhibition and PD-1 blockade improve anti-tumor immune response in epithelial ovarian cancer.

Introduction: The insulin-like growth factor (IGF) system plays a key role in regulating growth and invasiveness in epithelial ovarian cancer (EOC) and is considered a promising therapeutic target. EOC is an immunosuppressive disease, although there are limited data about the involvement of the IGF1R system in the anti-tumor immune response in the EOC microenvironment. Methods: In the current study, we hypothesized that IGF 1 receptor (IGF1R) involvement in the maturation of dendritic cells (DC) with the co-inhibition of IGF1R and PD-1 would affect the EOC microenvironment. Results: We found that DC pretreated with IGF1R inhibitor resulted in fewer EOC cells. Moreover, in vivo experiments conducted with an EOC mouse model, with anti-PD-1/IGF1R combined, resulted in lower tumor weight compared to individual treatments. Additionally, anti-PD-1/IGF1R treatment increased DC by 34% compared with AEW-541 and 40% with anti-PD-1. The combined treatment increased CD8+ T-cell levels compared to AEW-541 alone. RNA-seq data analysis indicated that anti-PD-1/IGF1R led to a more potent immune response, as reflected by altered gene expression levels related to anti-tumor immune response, compared with either treatment alone. Discussion: These findings provide novel evidence that IGF1R axis inhibition combined with PD-1 blockade may be an effective therapeutic strategy for selected EOC patient populations.

Proangiogenic immature myeloid cells populate the human placenta and their presence correlates with placental and birthweight.

OBJECTIVE: To determine whether proangiogenic immature myeloid cells are present in human placentas. STUDY DESIGN: Biopsies were obtained from 61 placentas of term pregnancies. Percentage of CD45(+)CD33(+)LIN2(-)HLADR(-) immature myeloid cells of total CD45(+) hematopoietic cells was determined by flow cytometry. Location of immature myeloid cells in the placenta was identified using confocal microscopy. The proangiogenic potential of immature myeloid cells was analyzed by endothelial tube formation. RESULTS: Immature myeloid cells comprise ∼25% of human placental CD45(+) hematopoietic cells and infiltrate placentas in proximity of blood vessels. The percentage of immature myeloid cells correlated positively with placental weight (r(2) = 0.108, P = .01) and birthweight (r(2) = 0.087, P = .02). Endothelial tube formation was increased in the presence of immature myeloid cells as compared with the presence of CD45(+)LIN2(+) control cells. CONCLUSION: Human placentas are populated by immature myeloid cells in the proximity of blood vessels. Consistent with their involvement in angiogenesis, immature myeloid cells accelerated endothelial tube formation. The presence of immature myeloid cells in pathologic pregnancies warrants further studies.

The expression of heparanase in term and preterm human placentas.

PURPOSE: Heparanase is an endo-ß-glucuronidase that cleaves side chains of heparan-sulfate proteoglycans, an integral constituent of the extra cellular matrix. The abundance of heparanase in placental trophoblast cells implies its role in the processes of placentation and trophoblast invasion. This study aims to explore the involvement of heparanase in parturition and preterm deliveries (PTD). METHODS: Sixteen human placentas were collected following singleton spontaneous onset term vaginal deliveries (n = 6), spontaneous onset preterm vaginal deliveries (n = 7) and term elective cesarean sections (n = 3). Placentas were excluded in case of any maternal chronic illness, pregnancy or delivery complications apart from PTD. Placental tissue samples were dissected, homogenized and proteins were extracted. Additionally, cryosections were prepared from the placental tissues. Heparanase expression was evaluated utilizing western blot analysis and immunofluorescence staining using heparanase specific antibodies. Heparanase expression was compared between the study groups qualitatively and quantitatively. RESULTS: Western blot analysis results demonstrated higher expression of both pro-heparanase and heparanase in PTD placentas compared to term vaginal placentas. Accordingly, immunofluorescence staining shows elevated heparanase expression in PTD placentas compared to term vaginal placentas (5.1 ± 0.92 vs. 1.2 ± 0.18, p < .005). Expression level of heparanase was higher in term cesarean section placentas as compared to term vaginal deliveries placentas, but did not reach statistical significance (1.8 ± 0.39 vs. 1.2 ± 0.18, p = .06). CONCLUSION: This study demonstrates for the first time that preterm vaginal deliveries are associated with higher expression of heparanase in placental tissue. This may imply a direct effect of heparanase on preterm labor. Further studies should evaluate the functional role by which heparanase influence preterm delivery.

The Impact of Lifestyle on Sperm Function, Telomere Length, and IVF Outcomes.

Many risk factors can potentially influence sperm quality. Telomeres confer stability on the chromosome and their dysfunction has been implicated in conditions such as cancer, aging, and lifestyle. The impact of lifestyle on sperm cell telomeres is unclear. The objectives of this study were to evaluate the impact of lifestyle behaviors on telomere length in sperm and to follow the correlation with pregnancy outcomes in patients undergoing in vitro fertilization (IVF). In this prospective observational study, sperm was analyzed for telomere length (TL). Men were asked to report lifestyle behaviors including occupation (physical or sedentary), smoking duration and amount, physical activity, dietary habits, and where they keep their cellular phone (bag, pants, or shirt pocket). Correlations among semen analysis, TL, men's habits, and embryo quality and pregnancy outcomes were evaluated. Among 34 patients recruited, 12 had longer TL and 13 shorter TL. Sperm motility was negatively correlated with TL (Pearson correlation = -.588, p = .002). Smoking adversely affected native sperm motility (53% motility in nonsmokers vs. 37% in smokers; p = .006). However, there was no significant impact on TL. The group with longer telomeres demonstrated significant association with healthy diet (10/12 vs. 6/13; p = .05) and a trend toward more sports activity, weekly (16/84 vs. 7/91; p = .04) compared with the shorter telomeres group. This study suggests that lifestyle, healthy diet, and sports activity are associated with long telomeres in sperm. Sperm quality is also influenced by patients' habits. The study strongly recommends maintaining a healthy lifestyle to preserve general health and fertility.

Immature myeloid cells accumulate in mouse placenta and promote angiogenesis.

OBJECTIVE: We sought to determine whether CD11b(+)Gr1(+) immature myeloid cells (IMCs), which have been shown to promote tumor angiogenesis, accumulate in the placenta and similarly contribute to blood vessel formation. STUDY DESIGN: Experiments were performed on 6- to 8-week-old C57Bl/6J female mice. Placentas from pregnant mice or B16F10 tumors that were subcutaneously implanted were analyzed by flow cytometry and confocal microscopy. To determine the proangiogenic potential of IMCs, Matrigel plug assays were performed. RESULTS: IMCs infiltrate the placenta in the proximity of blood vessels, reaching peak concentration at midpregnancy. When isolated from either placentas or B16F10 melanoma tumors, IMCs actively promoted endothelial cell migration into Matrigel plugs in vivo. Furthermore, placental IMCs, similar to tumor-derived IMCs, expressed matrix metalloproteinase-9 and Bv8, 2 pivotal proangiogenic proteins. CONCLUSION: IMCs that express matrix metalloproteinase-9 and Bv8 infiltrate placentas of pregnant mice and actively promote angiogenesis. These cells show striking similarity to IMCs that populate malignant tumors.

Lipid droplets in granulosa cells are correlated with reduced pregnancy rates.

BACKGROUND: Lipids are an important source for energy production during oocyte maturation. The accumulation of intracellular lipids binds to proteins to form lipid droplets. This may lead to cellular lipotoxicity. The impact of lipotoxicity on cumulus and granulosa cells has been reported. This pilot study evaluated their correlation to oocyte and embryo quality. DESIGN: Prospective case-control study. SETTING: Referral IVF unit. PATIENTS: Women younger than age 40, undergoing IVF with intracytoplasmic sperm injection. INTERVENTIONS: 15 women with BMI > 30 (high BMI) and 26 women with BMI < 25 (low BMI) were enrolled. IVF outcomes were compared between groups based on BMI. Lipid content in cumulus and granulosa cells was evaluated using quantitative and descriptive methods. Lipid profile, hormonal profile and C-reactive protein were evaluated in blood and follicular fluid samples. Demographic and treatment data, as well as pregnancy rates were collected from electronic medical records. RESULTS: Higher levels of LDL and CRP, slower cell division rate and lower embryo quality were found in the group with high BMI. There was no difference in pregnancy rates between groups. In light of these findings, treatment outcomes were reanalyzed according to patients who became pregnant and those who did not. We found that patients who conceived had significantly lower fat content in the granulosa cells, reflected by mean fluorescence intensity recorded by flow cytometry analysis (23,404 vs. 9370, P = 0.03). CONCLUSIONS: BMI has no effect on lipid content in cumulus and granulosa cells, and does not affect likelihood of pregnancy. However, women who achieved pregnancy, regardless of their BMI, had lower lipid levels in their granulosa cells. This finding is important and further study is needed to evaluate lipid content in granulosa cells as a potential predictor of IVF treatment success.

IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. The insulin-like growth factor (IGF) system plays a key role in regulating growth and invasiveness in several malignancies, including ovarian cancer. IGF1R targeting showed antiproliferative activity of EOC cells. However, clinical studies failed to show significant benefit. EOC cells suppress antitumor immune responses by inducing dendritic cell (DC) dysfunction. The IGF1 axis can regulate DC maturation. The current study evaluated involvement of the IGF1 axis in DC differentiation in EOC. Studies were conducted on EOC and on a human monocyte cell line. Tissue microarray analysis (TMA) was performed on 36 paraffin blocks from EOC patients. Expression of IGF1R, p53, Ki67, BRCA1, and DC markers was evaluated using immunohistochemistry. Co-culture of EOC cells with DC pretreated with IGF1R inhibitor blocked cancer cell migration. TMA demonstrated higher rate of IGF1R protein expression in patients with advanced (76.9%) as compared to early (40%) EOC. A negative correlation between IGF1R protein expression and the CD1c marker was found. These findings provide evidence that IGF1R axis inhibition could be a therapeutic strategy for ovarian cancer by restoring DC-mediated antitumor immunity.

Runx3 prevents spontaneous colitis by directing the differentiation of anti-inflammatory mononuclear phagocytes.

Mice deficient in the transcription factor Runx3 develop a multitude of immune system defects, including early onset colitis. This paper demonstrates that Runx3 is expressed in colonic mononuclear phagocytes (MNP), including resident macrophages (RM) and dendritic cell subsets (cDC2). Runx3 deletion in MNP causes early onset colitis due to their impaired maturation. Mechanistically, the resulting MNP subset imbalance leads to up-regulation of pro-inflammatory genes as occurs in IL10R-deficient RM. In addition, RM and cDC2 display a marked decrease in expression of anti-inflammatory/TGF ß-regulated genes and ß-catenin signaling associated genes, respectively. MNP transcriptome and ChIP-seq data analysis suggest that a significant fraction of genes affected by Runx3 loss are direct Runx3 targets. Collectively, Runx3 imposes intestinal immune tolerance by regulating maturation of colonic anti-inflammatory MNP, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans, including gastrointestinal tract diseases such as Crohn's disease and celiac.

Progesterone treatment enhances the expansion of placental immature myeloid cells in a mouse model of premature labor.

INTRODUCTION: immature-myeloid cells (IMCs) are proangiogenic bone marrow (BM)-derived cells that normally differentiate into inflammatory cells such as neutrophils, monocytes and dendritic cells (DCs). We characterized placental IMCs comparing their gene expression and subpopulations to tumor IMCs, and tested our hypothesis that progesterone that inhibits preterm labor, may affect their abundance and differentiation. METHODS: differences between IMC-subpopulations in subcutaneous tumors versus placentas in C57BL/6 or ICR (CD-1) mice were analyzed by flow cytometry and gene expression was detected by microarrays. BM- and placental cells were incubated with or without progesterone and IMC subpopulations were analyzed. For preterm labor induction pregnant mice pretreated or not with progesterone were or were not treated with Lipopolysaccharide (LPS). RESULTS: we detected enrichment of granulocytic-IMCs in placentas compared to tumors, paralleled by a decrease in monocytic-IMCs. mRNA expression of placenta- versus tumor IMCs revealed profound transcriptional alterations. Progesterone treated BM-CD11b+ cells ex-vivo induced enrichment of granulocytic-IMCs and a decrease in monocytic-IMCs and DCs. LPS treatment in-vivo led to an increase in BM-IMCs in both progesterone pretreated or non-pretreated mice. In the placenta LPS decreased the IMC population while progesterone led to complete abrogation of this effect. DISCUSSION: placental IMCs differ from tumor-IMCs in both subpopulations and gene expression. Progesterone enhances the proliferation of placenta-specific granulocytic IMCs ex-vivo and LPS induced labor is accompanied by a decrease in placental IMCs only in progesterone non-pretreated mice. We thus speculate that the protective effect of progesterone in preventing preterm labor may be explained at least in part by this specific anti-inflammatory effect.

The Role of the Insulin-Like Growth Factor 1 Pathway in Immune Tumor Microenvironment and Its Clinical Ramifications in Gynecologic Malignancies.

Treatment of patients with gynecologic malignancies diagnosed at advanced stages remains a therapeutic challenge. Survival rates of these patients remain significantly low, despite surgery and chemotherapy. Advances in understanding the role of the immune system in the pathogenesis of cancer have led to the rapid evolution of immunotherapeutic approaches. Immunotherapeutic strategies, including targeting specific immune checkpoints, as well as dendritic cell (DC) immunotherapy are being investigated in several malignancies, including gynecological cancers. Another important approach in cancer therapy is to inhibit molecular pathways that are crucial for tumor growth and maintenance, such as the insulin-like growth factor-1 (IGF1) pathway. The IGF axis has been shown to play a significant role in carcinogenesis of several types of tissue, including ovarian cancer. Preclinical studies reported significant anti-proliferative activity of IGF1 receptor (IGF1R) inhibitors in gynecologic malignancies. However, recent clinical studies have shown variable response rates with advanced solid tumors. This study provides an overview on current immunotherapy strategies and on IGF-targeted therapy for gynecologic malignancies. We focus on the involvement of IGF1R signaling in DCs and present our preliminary results which imply that the IGF axis contributes to an immunosuppressive tumor microenvironment (TME). For the long term, we believe that restoring the TME function by IGF1R targeting in combination with immunotherapy can serve as a new clinical approach for gynecological cancers.

Dual-specificity phosphatase Pyst2-L is constitutively highly expressed in myeloid leukemia and other malignant cells.

Northern blotting confirmed previous results indicating that the mitogen-activated protein kinase (MAPK) phosphatase Pyst2-L was highly expressed in leukocytes obtained from acute myeloid leukemia (AML) patients. High levels of Pyst2-L mRNA were expressed in bone marrow (BM) and peripheral leukocytes from nine AML and acute lymphoblastic leukemia (ALL) patients. BM from healthy individuals expressed very low levels of Pyst2-L. Whereas high levels of Pyst2-L mRNA and protein were detected in several leukemia cell lines, Pyst2-L mRNA was detected neither in 33/34 samples of normal peripheral blood mononuclear cells (PBMC) nor in leukocyte fractions enriched with CD34+ cells. Certain solid tumor and lymphoblastoid cell lines expressed high levels of Pyst2-L mRNA. In view of the association of Pyst2-L to MAPK signaling cascades, we tested if cell activation, a process involving MAPK signaling, influences Pyst2-L expression. Indeed, activation of T cells and endothelial cells increased Pyst2-L in these cells. Furthermore, TPA, a known MAPK activator, induces the expression of both Pyst2-L mRNA as well as the Pyst2-L protein in leukemia cells. This induction was partially inhibited by PD098059, an Mek1/2-specific inhibitor. Based on the results of this and previous studies, we hypothesize that the high levels of Pyst2-L detected in the active state of AML and ALL diseases and in other types of cancer reflect an altered MAPK signaling pathway in such malignant processes. This alteration may be the result of a failed attempt to counter the constitutive activation of MAPK in transformed cells or alternatively, may represent the activated state of such cells.

Loss of osteoblast Runx3 produces severe congenital osteopenia.

Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3(fl/fl)/Col1α1-cre), but not chondrocyte-specific (Runx3(fl/fl)/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization.

Carcinogen-induced skin tumor development requires leukocytic expression of the transcription factor Runx3.

Carcinogen-induced skin tumorigenesis depends heavily on proinflammatory tumor-promoting processes. Here, we show that leukocytic Runx3 expression is central to the two-stage DMBA/TPA-induced skin tumorigenesis. Runx3-null mice were highly resistant to this process and concomitant ablation of Runx3 in dendritic and T cells fully recapitulated this resistance. Mechanistically, this resistance was associated with a shift in the skin cytokine milieu toward a tumor nonpermissive microenvironment. Specifically, leukocytic Runx3 loss substantially increased the antitumorigenic cytokine thymic stromal lymphopoietin (TSLP) and profoundly decreased two protumorigenic cytokines, interleukin-17a and osteopontin. Therefore, inflammation-mediated tumor promotion requires leukocytic Runx3 expression, as its loss creates a unique cytokine composition that polarizes the tumor microenvironment to a potent antitumorigenic state.

Immature myeloid cells derived from mouse placentas and malignant tumors demonstrate similar proangiogenic transcriptional signatures.

OBJECTIVE: To determine whether CD11b(+)Gr1(+) immature myeloid cells (IMCs), initially identified to infiltrate tumors and support angiogenesis and recently identified also in mouse and human placentas, are similar in that they share common gene expression. DESIGN: Animal experiment. SETTING: Reproductive immunology laboratory. ANIMAL(S): All 6- to 8-week-old C57Bl/6 female mice. MAIN OUTCOME MEASURE(S): We analyzed gene expression of IMCs isolated from placentas of pregnant mice (n = 3) and Lewis lung carcinoma tumors (n = 3), using flow cytometry. Expression patterns were compared to primary muscle cells (n = 4), using Affymetrix microarrays. Quantitative polymerase chain reaction (PCR) was used to validate microarray data. Similarity of gene expression was evaluated with the mass-distance algorithm. RESULT(S): The IMCs that infiltrate mouse placentas share ∼500 expressed genes with tumor IMCs (set a). This gene set is enriched with proangiogenic and inflammatory genes. Unique gene expression sets for tumor IMCs (set b) and placenta IMCs (set c) were also detected. CONCLUSION(S): The IMCs derived from placentas and tumors express common molecular signatures, suggesting similar origins and functions. This observation lends further support to the notion that the placenta uses a similar angiogenic machinery as tumors for survival and growth. Unique gene-sets, differentially expressed in tumor versus placenta-derived IMCs, may be required for specific IMC-hosting tissue interactions.