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While early detection of breast cancer (BC) has improved prognoses, there is an urgent need to improve outcomes for patients with distant metastatic disease. Higher expression of the Notch ligand JAG1 in primary BC tumors is strongly associated with lymph node metastasis and patient mortality, but causality is unclear. We show that JAG1 expression is higher in patients’ metastatic BC cells colonizing lymph nodes than in primary tumors, suggesting that tumor cells with high JAG1 are preferentially able to metastasize to lymph nodes. JAG1 expression is higher in a derivative of BC line MDA-MB-231 selected for tropism to lymph nodes (MDA231-LN) than in the parental line or derivatives with other tropisms. To determine the mechanism(s) of JAG1-mediated metastasis, we generated clonal JAG1 knockout cell lines from MDA231-LN cells with corresponding JAG1 rescue lines. We investigated the role of JAG1 in spontaneous metastasis under clinically relevant conditions by orthotopically implanting JAG1 knockout and expressing cells, resecting the primary tumor, and following long-term metastatic spread in a mouse model. Quantification of tumor cells in blood showed that survival, metastatic burden, and JAG1 expression did not correlate with the number of circulating tumor cells. Conversely, JAG1 expression drove an increase in lymph node and body-wide metastatic burden and a trend toward decreased survival. In this model metastatic cells were abundant throughout lymph vessels, suggesting lymphatics are the primarily route of dissemination. Preliminary transcriptional analysis suggests that JAG1 alters interactions with lymphatic endothelial cells (LEC), potentially via VEGF signaling, leading us to examine downstream events in co-cultures of LEC with lymphatically invasive BC lines. Deciphering tumor-lymphatic endothelial signaling events may open new avenues to target BC metastasis. Citation Format: Benjamin Gordon, Bhairavi Swaminathan, LA Naiche, Jan Kitajewski. Jagged-1 promotes breast cancer metastasis through the lymphatic system [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-10.

Expression of the Notch family of receptors is often upregulated in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on Notch4, which had not been investigated in PDAC. We generated KC (LSL-KrasG12D;p48-Cre), N4−/−KC (Notch4−/−;LSL-KrasG12D;p48-Cre), PKC (p16fl/fl;LSL-KrasG12D;p48-Cre), and N4−/−PKC (Notch4−/−; p16fl/fl;LSL-KrasG12D;p48-Cre) genetically engineered mouse models (GEMM). We performed caerulein treatment in both KC and N4−/−KC mice, and the development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions were significantly diminished in the N4−/−KC than in the KC GEMM (P = 0.01). This in vivo result was validated by in vitro ADM induction of the explant cultures of pancreatic acinar cells from the N4−/−KC and KC mice (P < 0.001), confirming that Notch4 is an important contributor to early pancreatic tumorigenesis. To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the PKC and N4−/−PKC mice. The N4−/−PKC mice had better overall survival (P = 0.012) and significantly reduced tumor burden (PanIN: P = 0.018 at 2 months, PDAC: P = 0.039 at 5 months) compared with the PKC GEMM. RNA-sequencing analysis of pancreatic tumor cell lines derived from the PKC and N4−/−PKC GEMMs revealed that 408 genes were differentially expressed (FDR < 0.05) and Pcsk5 is a potential downstream effector of the Notch4 signaling pathway (P < 0.001). Low expression of Pcsk5 positively correlates with good survival in patients with PDAC (P = 0.028). We have identified a novel role for Notch4 signaling with tumor-promoting function in pancreatic tumorigenesis. Our study also uncovered a novel association between Pcsk5 and Notch4 signaling in PDAC. Significance: We demonstrated that global inactivation of Notch4 significantly improved the survival of an aggressive mouse model for PDAC and provided preclinical evidence that Notch4 and Pcsk5 are novel targets for PDAC therapies.

Supplementary Figure 1. Inactivation of Notch1 had no impact on pancreatic tumorigenesis driven by oncogenic Kras in the context of p16 inactivation.Supplementary Figure 2. Histological analyses of the pancreatic tissues of the PKC, the N1+/- PKC, and the N1fl/flPKC mice revealed no significant differences.Supplementary Figure 3. Representative H&E from the PKC, the N1+/- PKC, and the N1fl/flPKC mice at 2 months of age (PanIN, A, C, E) and 5 months of age (PDAC, B, D, F).Supplementary Figure 4. Comparative histological analyses of PanIN lesions in the PKC, the N1+/- PKC, and the N1fl/flPKC mice revealed no significant differences.Supplementary Figure 5. Inactivation of Notch4 reduced the formation of high-grade PanIN, although not at a statistically significant level.Supplementary Figure 6. Representative Notch4-ICD expression in the pancreases of PKC miceSupplementary Figure 7. The expressions of Notch4 and Endomucin were both detected in the endothelial compartment by IHC.Supplementary Figure 8. Slight differential Endomucin expression was detected between the KC and the N4-/- KC mice by day 21 post the caerulein treatment.Supplementary Figure 9. Inactivation of Notch4 was associated with reduced but not significant Endomucin-positive angiogenesis at 2 months of age.Supplementary Figure 10. Inactivation of Notch4 was associated with significant reduction of Endomucin-positive angiogenesis at 5 months of age.Supplementary Figure 11. Inactivation of Notch4 attenuated desmoplastic reaction in the pancreases treated by Caerulein.Supplementary Figure 12. Reduced desmoplasia was observed in N4-/- PKC mice when compared to PKC mice.Supplementary Figure 13. Inactivation of Notch4 did not alter macrophage infiltration in the pancreases treated by Caerulein.Supplementary Figure 14. Inactivation of Notch4 did not alter macrophage infiltration in the pancreatic tumors induced by oncogenic Kras.Supplementary Figure 15. scRNA-Seq analysis revealed Notch4 expression in the endothelial and CK19+ normal and tumor cells in pancreatic tumorigenesis.Supplementary Figure 16. Increased expression of the NGF gene was observed with Notch4 inactivation, but not with a statistically significant association.Supplementary Figure 17. Notch4 RNA expression level in Panc 08.13 was the highest among candidate human PDAC cell lines.

Expression of the Notch family of receptors is often upregulated in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on Notch4, which had not been investigated in PDAC.We generated KC (LSL-KrasG12D;p48-Cre), N4−/−KC (Notch4−/−;LSL-KrasG12D;p48-Cre), PKC (p16fl/fl;LSL-KrasG12D;p48-Cre), and N4−/−PKC (Notch4−/−; p16fl/fl;LSL-KrasG12D;p48-Cre) genetically engineered mouse models (GEMM). We performed caerulein treatment in both KC and N4−/−KC mice, and the development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions were significantly diminished in the N4−/−KC than in the KC GEMM (P = 0.01). This in vivo result was validated by in vitro ADM induction of the explant cultures of pancreatic acinar cells from the N4−/−KC and KC mice (P < 0.001), confirming that Notch4 is an important contributor to early pancreatic tumorigenesis.To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the PKC and N4−/−PKC mice. The N4−/−PKC mice had better overall survival (P = 0.012) and significantly reduced tumor burden (PanIN: P = 0.018 at 2 months, PDAC: P = 0.039 at 5 months) compared with the PKC GEMM. RNA-sequencing analysis of pancreatic tumor cell lines derived from the PKC and N4−/−PKC GEMMs revealed that 408 genes were differentially expressed (FDR < 0.05) and Pcsk5 is a potential downstream effector of the Notch4 signaling pathway (P < 0.001). Low expression of Pcsk5 positively correlates with good survival in patients with PDAC (P = 0.028).We have identified a novel role for Notch4 signaling with tumor-promoting function in pancreatic tumorigenesis. Our study also uncovered a novel association between Pcsk5 and Notch4 signaling in PDAC.Significance:We demonstrated that global inactivation of Notch4 significantly improved the survival of an aggressive mouse model for PDAC and provided preclinical evidence that Notch4 and Pcsk5 are novel targets for PDAC therapies.

Figure S1 shows that N11-13 decoy blocks activation of Notch1 by DLL1 but not JAG2 whereas N110-24 decoy blocks activation of Notch1 by JAG2 but not DLL1. Figure S2 shows that purified N11-13 decoy preferentially inhibited DLL4-mediated Notch1 activation, while purified N11-24 decoy inhibited both DLL4 and JAG1-mediated Notch1 activation. Figure S3 shows using co-immunoprecipitation of N1 decoys and soluble versions of DLL4 and JAG1 lacking transmembrane domains that similar ligand specificity of the decoys was observed as when using membrane bound DLL4 or JAG1. Figure S4 shows serum levels of Notch1 decoys in P5 neonatal mice. Figure S5 shows N1 decoys that block JAG disrupted vascular smooth muscle cell association in the actively remodeling neonatal retinal vasculature, but not the mature retinal vasculature. Figure S6 shows that N11-13, N110-24, and N11-24 decoys block soft agar growth of Mm5MT-FGF4 tumor cells but do not affect growth of KP1-VEGF, LLC or B16-F10 tumor lines. Figure S7 shows N1 decoys detected in mouse serum after adenovirus infection and quantification of these levels using ELISA. Figure S8 shows that JAG- inhibiting decoys disrupt vascular smooth muscle coverage in tumors. Figure S9 shows that N1 decoys expressed in mice cause a modest increase of intestinal goblet cells but no significant change in murine weight. Figure S10 shows N1 decoys had no effect on serum markers of liver damage. Figure S11 shows N1 decoys did not affect liver and kidney histopathology.

Supplementary Figure 4 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 3 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 1 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 2 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

SUMMARYGlioma cells hijack developmental transcriptional programs to control cell state. During neural development, lineage trajectories rely on specialized metabolic pathways. However, the link between tumor cell state and metabolic programs is poorly understood in glioma. Here we uncover a glioma cell state-specific metabolic liability that can be leveraged therapeutically. To model cell state diversity, we generated genetically engineered murine gliomas, induced by deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling cellular fate. N1IC tumors harbored quiescent astrocyte-like transformed cell states while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. N1IC cells exhibit distinct metabolic alterations, with mitochondrial uncoupling and increased ROS production rendering them more sensitive to inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Importantly, treating patient-derived organotypic slices with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles.

In the first trimester of human pregnancy, low oxygen tension or hypoxia is essential for proper placentation and placenta function. Low oxygen levels and activation of signaling pathways have been implicated as critical mediators in the promotion of trophoblast differentiation, migration, and invasion with inappropriate changes in oxygen tension and aberrant Notch signaling both individually reported as causative to abnormal placentation. Despite crosstalk between hypoxia and Notch signaling in multiple cell types, the relationship between hypoxia and Notch in first trimester trophoblast function is not understood. To determine how a low oxygen environment impacts Notch signaling and cellular motility, we utilized the human first trimester trophoblast cell line, HTR-8/SVneo. Gene set enrichment and ontology analyses identified pathways involved in angiogenesis, Notch and cellular migration as upregulated in HTR-8/SVneo cells exposed to hypoxic conditions. DAPT, a γ-secretase inhibitor that inhibits Notch activation, was used to interrogate the crosstalk between Notch and hypoxia pathways in HTR-8/SVneo cells. We found that hypoxia requires Notch activation to mediate HTR-8/SVneo cell migration, but not invasion. To determine if our in vitro findings were associated with preeclampsia, we analyzed the second trimester chorionic villous sampling (CVS) samples and third trimester placentas. We found a significant decrease in expression of migration and invasion genes in CVS from preeclamptic pregnancies and significantly lower levels of JAG1 in placentas from pregnancies with early-onset preeclampsia with severe features. Our data support a role for Notch in mediating hypoxia-induced trophoblast migration, which may contribute to preeclampsia development.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09791-9.

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Triple-negative breast cancer (TNBC), a highly aggressive and metastatic subtype of breast cancer, lacks expression of the estrogen and progesterone receptors and amplification of HER2 and comprises 15-20% of all breast cancer. Incidence and mortality of TNBC are higher in Black Americans than in other racial/ethnic groups. Conversely, Black patients are underrepresented in clinical breast cancer drug trials, limiting our understanding of differential treatment responses. Leukocyte infiltration is a key modulator of TNBC outcome, and Black patients show immunological differences that may contribute to differential outcomes in TNBC. About 60-80% of Black Americans are homozygous for a polymorphism in the Atypical Chemokine Receptor 1 (ACKR1) promoter region that causes loss of ACKR1 expression on red blood cells, also known as the “Duffy-null” serotype. ACKR1 is expressed on tumor cells, red blood cells, and endothelial cells, and can act as a chemokine sink or chemokine concentrator, depending on the context. Whole-tumor RNA analysis shows that overall loss of ACKR1 expression in TNBC tumors correlates strongly with decreased survival, but it is unclear which ACKR1-expressing cell type(s) contribute to this effect. Under inflammatory conditions, ACKR1 expression in endothelial cells localizes chemotactic cytokines at the endothelial junction, and loss of endothelial ACKR1 blocks the ability of leukocytes to extravasate through the endothelial barrier into surrounding tissues. We hypothesized that loss of endothelial/stromal ACKR1 reduces immune infiltration and activation in TNBC and racial/ethnic differences in ACKR1 expression may contribute to health disparities. To determine the role of ACKR1 in tumor immune infiltration, we chose a syngeneic immunocompetent mouse model to incorporate species-matched stromal-tumor interactions. We orthotopically inoculated E0771.LMB cells, a C57BL6/J syngeneic TNBC cell line, into ACKR1null and ACKR1het female mice. Tumor growth was measured triweekly, and tumor mass was measured at the 28-day endpoint. To measure immune infiltration, we fluorescently stained representative tumor cryosections with CD45 to detect all classes of infiltrating hematopoietic cells involved in immune response. There was no significant difference in tumor volume and mass among the ACKR1null and ACKR1het controls. Tumors implanted in ACKR1het controls showed broadly infiltrated leukocytes clusters, while tumors in ACKR1nullhosts showed sparse and infrequent leukocyte infiltration. Conclusion: Stromal ACKR1 does not have a strong effect on primary tumor growth. Loss of stromal ACKR1 reduces infiltration of leukocytes into the tumor microenvironment even when ACKR1 is not manipulated in the tumor cells, suggesting that ACKR1 in endothelial or other stromal cells regulates immune response in TNBC. Impact: Understanding the immunogenic functions of ACKR1 allelic variation will elucidate mechanisms of racially distinct therapeutic response in TNBC, enhance precision oncology, and promote cancer health equity. Citation Format: Chinwe Obianuju Ewenighi-Amankwah, Tanner Roach, Joseph Dufraine, Naiche Adler, Jan Kitajewski. ACKR1 expression in stromal cells regulates immune infiltration in triple negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C035.

Spontaneous preterm birth (sPTB), a major cause of infant morbidity and mortality, must involve premature cervical softening/dilation for a preterm vaginal delivery to occur. Yet, the mechanism behind premature cervical softening/dilation in humans remains unclear. We previously reported the non-pregnant human cervix contains considerably more cervical smooth muscle cells (CSMC) than historically appreciated and the CSMC organization resembles a sphincter. We hypothesize that premature cervical dilation leading to sPTB may be due to (1) an inherent CSMC contractility defect resulting in sphincter failure and/or (2) altered cervical extracellular matrix (ECM) rigidity which influences CSMC contractility. To test these hypotheses, we utilized immunohistochemistry to confirm this CSMC phenotype persists in the human pregnant cervix and then assessed in vitro arrays of contractility (F:G actin ratios, PDMS pillar arrays) using primary CSMC from pregnant women with and without premature cervical failure (PCF). We show that CSMC from pregnant women with PCF do not have an inherent CSMC contractility defect but that CSMC exhibit decreased contractility when exposed to soft ECM. Given this finding, we used UPLC-ESI-MS/MS to evaluate collagen cross-link profiles in the cervical tissue from non-pregnant women with and without PCF and found that women with PCF have decreased collagen cross-link maturity ratios, which correlates to softer cervical tissue. These findings suggest having soft cervical ECM may lead to decreased CSMC contractile tone and a predisposition to sphincter laxity that contributes to sPTB. Further studies are needed to explore the interaction between cervical ECM properties and CSMC cellular behavior when investigating the pathophysiology of sPTB.

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G-protein-coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine-1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of the S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

Aberrant activation of the Wnt/β-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/β-catenin signaling suppressed apoptosis by inhibiting c-Myc–induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc–induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/β-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis.

SUMMARY Lung alveolar type I cells (AT1) and alveolar type II cells (AT2) regulate the structural integrity and function of alveoli. AT1, covering ~95% of the surface area, are responsible for gas exchange, whereas AT2 serve multiple functions, including alveolar repair through proliferation and differentiation into AT1. However, the signaling mechanisms for alveolar repair remain unclear. Here, we demonstrate, in Pseudomonas aeruginosa-induced acute lung injury in mice, that non-canonical Notch ligand Dlk1 (delta-like 1 homolog) is essential for AT2-to-AT1 differentiation. Notch signaling was activated in AT2 at the onset of repair but later suppressed by Dlk1. Deletion of Dlk1 in AT2 induced persistent Notch activation, resulting in stalled transition to AT1 and accumulation of an intermediate cell population that expressed low levels of both AT1 and AT2 markers. Thus, Dlk1 expression leads to precisely timed inhibition of Notch signaling and activates AT2-to-AT1 differentiation, leading to alveolar repair., Graphical Abstract, In Brief Finn et al. show that Notch signaling is activated in type II cells after alveolar injury but that subsequent Dlk1-mediated inhibition of Notch is required for complete type II-to-type I cell transition and alveolar repair. Thus, Dlk1 and Notch are potential therapeutic targets for treatment of lung injury.

Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibition of Notch1 or its ligand, Dll4, can induce tumor vessel hypersprouting, which interferes with perfusion and reduces tumor development. Unfortunately, blocking Notch1 in endothelial cells of normal tissue can lead to tissue hypoxia and vascular neoplasms. Notch4, another member of the Notch family, is upregulated and increased in the endothelium of certain tumor types compared to normal vasculature. Reducing Notch4, unlike Notch1, causes reduction in developmental and tumor angiogenesis. Using a newly developed anti-Notch4 antibody, E7011, we assessed the effects on Notch4 inhibition on developmental retinal angiogenesis. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, but did decrease radial outgrowth and vein diameter, supporting the hypothesis that Notch4 functions as a pro-angiogenic signal. These findings suggest that the targeting of Notch4 could interfere with tumor angiogenesis and offer a novel therapeutic avenue. To establish the role of Notch4 within the tumor microenvironment, we analyzed its expression in both human and murine tumor samples. Immunofluorescent staining revealed a range of Notch4 expression in both tumor cells and endothelium in human triple negative breast cancers. Several murine syngeneic tumors examined expressed Notch4 primarily in the endothelium with only rare tumor expression. We treated orthotopically implanted Py8119 murine breast carcinomas and B16F10 melanomas with E7011, which significantly reduced tumor size compared to IgG treated controls, indicating that suppression of Notch4 is anti-tumorigenic. To determine mechanistically how E7011 delays tumor growth, we performed single cell RNA sequencing on E7011 and control treated Py8119 tumors. Surprisingly, we found that while Notch4 transcripts were present only in endothelial cells, E7011 administration dramatically altered macrophage and monocyte populations within the tumor. Taken together, these findings indicate that inhibition of tumor endothelial Notch4 by E7011 can regulate the presence of immune cells within the tumor microenvironment and improve tumor control. Citation Format: Jason Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Rahul Vadakath, James Herts, Rajyasree Emmadi, Junji Matsui, Jan Kitajewski. Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment [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 247.

Notch signaling is activated by ligands Delta-like 4 (Dll4) and Jagged1 in the tumor microenvironment to promote tumor angiogenesis and perfusion. Notch activation is associated with poor outcomes in several cancers, particularly triple negative breast cancer (TNBC), and affects both tumor angiogenesis and metastasis. The development of therapeutics targeting angiogenesis, such as the Notch pathway, has again attracted attention. Previous approaches to globally inhibit the Notch pathway or block Dll4/Notch1 activation, such as γ-secretase inhibitors (GSI), have raised safety concerns due to gastrointestinal toxicity due to accumulation of secretory goblet cells in the intestine. Similarly, anti-Dll4 therapy resulted in pathological changes in the liver as well as severe vascular neoplasms when evaluated using preclinical animal models. Development of new approaches for targeting the Notch pathway remains a critical clinical problem currently unaddressed. Our lab has previously developed ligand-specific inhibitors of Notch signaling, called Notch decoys, which are comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain. These Notch decoys bind ligand non-productively and interfere with ligand function. Jagged-specific Notch1 decoys inhibit angiogenesis in vitro and significantly impair tumor growth, tumor angiogenesis, and perfusion without apparent toxicity in mouse models of TNBC. However, this previous work utilized Notch decoys produced via viral expression vectors, which preclude dosage control and limit clinical applicability. We have developed a new generation of Notch “mini” decoys that contain fewer EGF-like repeats. These mini decoys show improved secretion characteristics and can be purified as active proteins in clinically relevant quantities. Using a variety of binding assays, we observed that these Notch mini decoys demonstrate strong but distinct binding to Notch ligands Dll4 and Jagged1 and block Notch signaling when evaluated in cultured cells. Our newly generated Notch decoys can now be sufficiently purified for use in a dose-dependent manner to test the therapeutic role of Notch inhibition on tumor angiogenesis using preclinical animal models. Citation Format: Timothy Sargis, Seock-Won Youn, Hyun Lee, L.A. Naiche, Jan Kitajewski. Notch decoys as potential anti-angiogenic biotherapeutics [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 3207.

A dominant mechanism of sudden cardiac death in the young is the progression of maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy. Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. We discuss approaches for personalized treatments that are indicated by recent advanced understanding of the progression.

In mice, embryonic dermal lymphatic development is well-understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulatedDll4transcripts, and differentially and temporally regulated the expression ofNotch1andHes/Heygenes. Mice nullizygous forNotch4had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5, and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. UnlikeNotch4nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increased in LEC proliferation, described for the loss of LECNotch1. Moreover, loss ofNotch4did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Gliogenesis in the adult mouse brain Neural stem cells in the adult mouse brain can generate both neurons and glia. Exactly where each stem cell is positioned can determine what type of neurons it generates. Delgado et al. show that neural stem cells are also choosy about what sorts of glia they make and when (see the Perspective by Baldwin and Silver). Injury or selective deletion of platelet-derived growth factor receptor β (PDGFRβ) from the stem cells kicked them into overdrive and revealed their selectivity with respect to gliogenesis. An unusual type of glial progenitor cell, intraventricular oligodendrocyte progenitors, are found nestled between the cilia of ependymal cells derived from tight clusters of PDGFRβ-expressing stem cells. Science , abg8467, this issue p. 1205 ; see also abj1139, p. 1151

High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. We investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in early stages of disease. We interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. We propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased metastasis of HGSC via increased adherence of disseminating cells to new metastatic sites in the peritoneum.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference. Fil: Nowak Sliwinska, Patrycja. Université of Lausanne; Suiza. Univeristé of Geneve; Suiza Fil: Alitalo, Kari. Katholikie Universiteit Leuven; Bélgica Fil: Allen, Elizabeth. Katholikie Universiteit Leuven; Bélgica Fil: Anisimov, Andrey. Katholikie Universiteit Leuven; Bélgica Fil: Aplin, Alfred C.. University of Washington; Estados Unidos Fil: Auerbach, Robert. University of Wisconsin; Estados Unidos Fil: Augustin, Hellmut G.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Bates, David O.. University of Nottingham; Reino Unido Fil: Beijnum, Judy R. van. Cancer Center Amsterdam; Países Bajos Fil: Bender, R. Hugh F.. University of California; Estados Unidos Fil: Bergers, Gabriele. Katholikie Universiteit Leuven; Bélgica Fil: Bikfalvi, Andreas. Universite de Bordeaux; Francia Fil: Bischoff, Joyce. Harvard Medical School; Estados Unidos Fil: Böck, Barbara C.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Brooks, Peter C.. Maine Medical Center Research Institute; Estados Unidos Fil: Bussolino, Federico. Università di Torino; Italia. Candiolo Cancer Institute; Italia Fil: Cakir, Bertan. Harvard Medical School; Estados Unidos Fil: Carmeliet, Peter. Katholikie Universiteit Leuven; Bélgica Fil: Castranova, Daniel. Harvard Medical School; Estados Unidos Fil: Cimpean, Anca M.. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Cleaver, Ondine. University Of Texas At Brownsville; Estados Unidos Fil: Coukos, George. Universida de Lausanne; Suiza Fil: Davis, George E.. University of Missouri; Estados Unidos Fil: De Palma, Michele. Swiss Federal Institute of Technology; Suiza Fil: Dimberg, Anna. Uppsala University; Suiza Fil: Dings, Ruud P. M.. University of Arkansas for Medical Sciences; Estados Unidos Fil: Djonov, Valentin. University of Bern; Suiza Fil: Dudley, Andrew C.. University of Virginia; Estados Unidos Fil: Dufton, Neil P.. Imperial College London; Reino Unido Fil: Fendt, Sarah-Maria. VIB Center for Cancer Biology; Bélgica Fil: Ferrara, Napoleone. University of California at San Diego; Estados Unidos Fil: Fruttiger, Marcus. University College London; Estados Unidos Fil: Fukumura, Dai. Harvard Medical School; Estados Unidos Fil: Ghesquière, Bart. Harvard Medical School; Estados Unidos Fil: Gong, Yan. Harvard Medical School; Estados Unidos Fil: Griffin, Robert J.. VIB Center for Cancer Biology; Bélgica Fil: Harris, Adrian L.. University of Oxford; Reino Unido Fil: Hughes, Christopher C. W.. University of California at Irvine; Estados Unidos Fil: Hultgren, Nan W.. University of California at Irvine; Estados Unidos Fil: Iruela-Arispe, M. Luisa. University of California at Los Angeles; Estados Unidos Fil: Irving, Melita. Universida de Lausanne; Suiza Fil: Maidana, Agostina Jainen. Harvard Medical School; Estados Unidos Fil: Kalluri, Raghu. Texas A&M University; Estados Unidos Fil: Kalucka, Joanna. Katholikie Universiteit Leuven; Bélgica Fil: Kerbel, Robert S.. University of Toronto; Canadá Fil: Kitajewski, Jan. University of Illinois; Estados Unidos Fil: Klaassen, Ingeborg. University of Amsterdam; Países Bajos Fil: Kleinmann, Hynda K.. The George Washington University; Estados Unidos Fil: Koolwijk, Pieter. Fondation Asile des Aveugles; Suiza. Universida de Lausanne; Suiza Fil: Kuczynski, Elisabeth. University of Toronto; Canadá Fil: Kwak, Brenda R.. University of Geneva; Suiza Fil: Koen, Marien. HistoGeneX; Bélgica Fil: Melero Martin, Juan M.. University of Liège; Bélgica Fil: Munn, Lance L.. Harvard Medical School; Estados Unidos Fil: Nicosia, Roberto F.. VA Puget Sound Health Care System; Estados Unidos Fil: Noel, Agnes. University of Liège; Bélgica Fil: Nurro, Jussi. University of Eastern Finland; Finlandia Fil: Olsson, Anna-Karin. Uppsala University; Suiza Fil: Petrova, Tatiana V.. Ludwig Institute for Cancer Research Lausanne; Suiza Fil: Pietras, Kristian. Division of Translational Cancer Research; Suecia Fil: Pili, Roberto. Indiana University Simon Cancer Center; Estados Unidos Fil: Pollard, Jeffrey W.. University of Edinburgh; Reino Unido Fil: Post, Mark J.. Maastricht University; Países Bajos Fil: Quax, Paul H. A.. Einthoven Laboratory for Experimental Vascular Medicine; Países Bajos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Raica, Marius. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Randi, Anna M.. Imperial College London; Reino Unido Fil: Ribatti, Domenico. Università degli Studi di Bari; Italia Fil: Ruegg, Curzio. University of Fribourg; Suiza Fil: Schlingemann, Reinier O.. University of Amsterdam; Países Bajos Fil: Schulte Merker, Stefan. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Smith, Lois E. H.. Harvard Medical School; Estados Unidos Fil: Song, Jonathan W.. Ohio State University; Estados Unidos Fil: Stacker, Steven A.. University of Melbourne; Australia Fil: Stalin, Jimmy. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Stratman, Amber N.. National Institutes of Health; Estados Unidos Fil: Van de Velde, Maureen. University of Liège; Bélgica Fil: van Hinsbergh, Victor W. M.. Universida de Lausanne; Suiza Fil: Vermeulen, Peter B.. HistoGeneX; Bélgica. University of Antwerp; Bélgica Fil: Waltenberger, Johannes. University of Münster; Alemania Fil: Weinstein, Brant M.. National Institutes of Health; Estados Unidos Fil: Xin, Hong. University of California at San Diego; Estados Unidos Fil: Yetkin Arik, Bahar. University of Amsterdam; Países Bajos Fil: Yla Herttuala, Seppo. University of Eastern Finland; Finlandia Fil: Yoder, Mervin C.. Indiana University; Estados Unidos Fil: Griffioen, Arjan W.. VU University Medical Center; Países Bajos

TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.

SUMMARY Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/− mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging., In Brief Kim et al. demonstrate a molecular link between PDIA1 and Drp1 oxidoreduction, which protects against mitochondrial fragmentation and ROS elevation, limiting endothelial senescence. This study provides insights into restoring endothelial PDIA1 function or targeting Drp1 Cys oxidation as potential therapeutic strategies for treating diabetes-associated vascular and metabolic diseases., Graphical Abstract

Expression of the Notch family of receptors are often upregulated in pancreatic ductal adenocarcinoma (PDAC), however, the functional impacts of the Notch signaling network on pancreatic tumorigenesis remain unresolved. In this study, we focused on Notch4, which had not been investigated in PDAC. Leveraging the conventional Notch4 deficient mouse line and previously established genetically engineered mouse models (GEMM) for PDAC, we generated KC (LSL-KrasG12D;p48-Cre), N4−/−KC (Notch4−/−;LSL-KrasG12D;p48-Cre), PKC (p16flox/flox;LSL-KrasG12D;p48-Cre), and N4−/−PKC GEMMs (Notch4−/−; p16flox/lox;LSL-KrasG12D;p48-Cre). We performed caerulein treatment in both KC and N4−/−KC mice, and compared the development of acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) between them. The ADM/PanIN lesions were significantly smaller in the N4−/−KC than in the KC GEMM (p=0.01), suggesting that Notch4 deficiency attenuated early pancreatic tumorigenesis. This in vivo result was confirmed by in vitro ADM induction of the explant cultures of mouse pancreatic acinar cells. The number of ADM structures in the N4−/−KC acinar cultures was significantly lower than the KC acinar cultures (p Citation Format: Kiyoshi Saeki, Wanglong Qiu, Richard Friedman, Carrie Shawber, Jan Kitajewski, Jianhua Hu, Gloria H. Su. Inactivation of Notch4 attenuated pancreatic tumorigenesis in mice [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-073.

Summary Neuroblastoma (NB), the most common cancer in the first year of life, presents almost exclusively in the trunk. To understand why an early-onset cancer would have such a specific localization, we xenotransplanted human NB cells into discrete neural crest (NC) streams in zebrafish embryos. Here, we demonstrate that human NB cells remain in an undifferentiated, tumorigenic state when comigrating posteriorly with NC cells but, upon comigration into the head, differentiate into neurons and exhibit decreased survival. Furthermore, we demonstrate that this in vivo differentiation requires retinoic acid and brain-derived neurotrophic factor signaling from the microenvironment, as well as cell-autonomous intersectin-1-dependent phosphoinositide 3-kinase-mediated signaling, likely via Akt kinase activation. Our findings suggest a microenvironment-driven explanation for NB’s trunk-biased localization and highlight the potential for induced differentiation to promote NB resolution in vivo.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor β promoter: PDGFRβ-P2A-CreER T2 . We evaluated the expression of the PDGFRβ-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRβ+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRβ-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.

Introduction Murine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands. Methods Notch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta. Results Notch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells. Discussion Canonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.

[Image: see text] Robust angiogenesis in the corpus luteum is critical for maintenance of pregnancy and thus mammalian female fertility. During angiogenesis, blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Pericytes are associated with capillaries and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial induction of Notch signaling in adjacent pericytes helps recruit and maintain pericyte coverage in some but not all tissue types. We have employed a Notch decoy, N1(10–24), which blocks Notch signaling in a ligand-specific manner, and determined that pharmacological inhibition of Notch ligand Jagged blocks luteal angiogenesis after normal ovulation, resulting in reduced luteal vasculature. Conversely, after ovarian hyperstimulation, a condition which occurs during fertility treatments, Jagged inhibition causes vascular dilation and hemorrhage. These results indicate that Jagged inhibition has effects in different ovarian angiogenic conditions, promoting vascular growth in the corpus luteum and vascular stability in hyperstimulated ovaries.

Quiescent neural stem cells (NSCs) in the adult ventricular-subventricular zone (V-SVZ) have a regional identity and undergo activation to generate neurons. The domains for gliogenesis are less explored. Here we show that Platelet-Derived Growth Factor Receptor beta (PDGFRβ) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia with slow baseline kinetics. Selective deletion of PDGFRβ in adult V-SVZ NSCs leads to their release from quiescence uncovering multiple domains in the septal wall for oligodendrocyte and astrocyte formation. Unexpectedly, we identify a novel intraventricular oligodendrocyte progenitor inside the brain ventricles. Together our findings reveal different NSC spatial domains for gliogenesis in the adult V-SVZ that are largely quiescent under homeostasis and may have key functions for brain plasticity.

SUMMARY The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1PR1 activity in ECs, and thereby, conditional deletion of S1PR1 in ECs (EC-S1pr1−/− mice) impairs tumor vascularization and growth. Mechanistically, we show that S1PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-S1pr1−/− mice. Our findings suggest that blocking S1PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling., Graphical Abstract, In Brief Vijay Avin et al. demonstrate an essential role of endothelial cell (EC)-S1PR1 signaling in amplifying VEGFR2-mediated tumor growth. S1PR1 by Gi and c-Abl1 phosphorylates VEGFR2 at Y951, which retains VEGFR2 at EC plasmalemma, thus enabling EC migration, tumor angiogenesis, and growth.

Proper development and function of the mammalian placenta requires interactions between embryo-derived trophoblasts and uterine endothelial cells to form mosaic vessels that facilitate blood flow to a developing conceptus. Notch signaling utilizes a cell–cell contact dependent mechanism to drive cell behaviors, such as differentiation and invasion. In mice, Notch2 is needed for proper placentation and embryo survival. We used transgenic mice with a dominant-negative form of Mastermind-like1 and Cyp19-Cre and Tpbpa-Cre drivers to inhibit canonical Notch signaling in trophoblasts. Both Cre drivers resulted in robust placental expression of dominant-negative Mastermind-like1. All pregnancies progressed beyond mid-gestation and morphological analyses of placentas revealed no differences between mutants and controls. Our data suggest that mouse placentation occurs normally despite dominant negative inhibition of trophoblast canonical Notch signaling and that Notch2 signaling via the canonical pathway is not necessary for placentation., Summary: Using transgenic mice with a dominant-negative form of Mastermind-like1 and Cyp19-Cre and Tpbpa-Cre drivers, we found that dominant negative inhibition of canonical Notch signaling in trophoblast cells does not disrupt placenta formation.

Fusobacterium nucleatum is an oral anaerobe prevalent in intrauterine infection associated with a wide spectrum of adverse pregnancy outcomes. We demonstrate here that F. nucleatum triggers placental inflammation through maternal, rather than paternal, TLR4-mediated signaling. Elimination of TLR4 from maternal endothelial cells alleviated placental inflammation and reduced fetal and neonatal death, while elimination of TLR4 in the hematopoietic cells had no effect. The placental inflammatory response followed a spatiotemporal pattern, with NF-κB activation observed first in the maternal endothelial cells and then in the decidual cells surrounding the endothelium, followed by induction of inflammatory cytokines and chemokines. Supplementation of pregnant mice with fish oil as a source of omega-3 fatty acids suppressed placental inflammation, reduced F. nucleatum proliferation in the placenta, and increased fetal and neonatal survival. In vitro analysis illustrates that omega-3 fatty acids inhibit bacterial-induced inflammatory responses from human umbilical cord endothelial cells. Our study therefore reveals a mechanism by which microbial infections affect pregnancy and identifies a prophylactic therapy to protect against intrauterine infections.

Though survival is improving for breast cancer patients, clinical outcomes remain poor for patients with aggressive molecular subtypes. Triple negative breast cancer (TNBC) is considered the most aggressive breast cancer subtype, and despite having a 90% five-year relative survival rate for localized disease, that number drops to as low as 11% for patients with distant TNBC metastasis. The poor clinical outcomes for TNBC patients are largely due to lack of clinically actionable molecular targets and pathways compared to other breast cancer subtypes. Given the lack of effective treatment strategies for advanced TNBC, there is an urgent and unmet need to understand the means through which tumor cells disseminate and metastasize to distant tissues in hopes of identifying new targets for therapeutic intervention. For tumor cells to metastasize, they acquire complex series of pathophysiological phenotypes, starting with invasion into surrounding tissue and intravasation into systemic lymphatic or blood vascular systems. Only a small fraction of tumor cells can survive in circulation, with a smaller fraction able to extravasate from vessels and establish distant metastases. While some cancer cells partially coopt the mechanisms that leukocytes use to extravasate, circulating TNBC cells also use distinct mechanisms to breach an endothelial barrier. However, the mechanism(s) of TNBC extravasation remains largely unknown, underlying the need to study further. High expression of Notch ligand JAGGED-1 (JAG1) is associated with increased metastasis and mortality in patients with breast cancer. While JAG1 enhances tumor angiogenesis and tumor growth, the means through which JAG1 promotes tumor cell dissemination is not clear. Our preliminary data supports a role for JAG1 as a key regulator of tumor cell interactions with the endothelium, controlling tumor cell adhesion, transendothelial migration (TEM), and transcription of critical paracrine-acting extravasation proteins, such as SPARC and ICAM1. Using both JAG-specific ligand traps as well as CRISPR/Cas9 mediated deletion of JAG1, we show that JAG1 promotes TEM in static transwell models, promotes primary tumor growth, and directs a tumor cell transcriptional program that upregulates cell surface interaction pathways distinct from global Notch inhibition. It is our central hypothesis that JAG1 promotes TNBC migration across the vascular endothelium, thereby allowing disseminated cancer cells to exit from circulation and enhancing TNBC metastasis. To define the JAG1-mediated elements of extravasation, we will examine five CRISPR/Cas9 JAG1 knockout lines in a cutting-edge flow system that recapitulates the vascular shear stress microenvironments where extravasation is thought to occur in vivo. We will evaluate multiple steps of extravasation including tumor cell binding, rolling, and TEM under various flow parameters. Moreover, we will study in vivo lung extravasation using a murine tail vein injection model and interrogate transcriptional candidates downstream of JAG1 as identified by our RNAseq data. Citation Format: Benjamin Gordon, Reyhaan A. Chaudhri, Tim Sargis, L.A. Naiche, Jan K. Kitajewski. JAGGED-1 as a novel metastatic extravasation promoter of triple negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO015.

Objective— Plasma high-density lipoproteins have several putative antiatherogenic effects, including preservation of endothelial functions. This is thought to be mediated, in part, by the ability of high-density lipoproteins to promote cholesterol efflux from endothelial cells (ECs). The ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) interact with high-density lipoproteins to promote cholesterol efflux from ECs. To determine the impact of endothelial cholesterol efflux pathways on atherogenesis, we prepared mice with endothelium-specific knockout of Abca1 and Abcg1 . Approach and Results— Generation of mice with EC-ABCA1 and ABCG1 deficiency required crossbreeding Abca1 fl/fl Abcg1 fl/fl Ldlr −/− mice with the Tie2Cre strain, followed by irradiation and transplantation of Abca1 fl/fl Abcg1 fl/fl bone marrow to abrogate the effects of macrophage ABCA1 and ABCG1 deficiency induced by Tie2Cre. After 20 to 22 weeks of Western-type diet, both single EC- Abca1 and Abcg1 deficiency increased atherosclerosis in the aortic root and whole aorta. Combined EC- Abca1/g1 deficiency caused a significant further increase in lesion area at both sites. EC- Abca1/g1 deficiency dramatically enhanced macrophage lipid accumulation in the branches of the aorta that are exposed to disturbed blood flow, decreased aortic endothelial NO synthase activity, and increased monocyte infiltration into the atherosclerotic plaque. Abca1/g1 deficiency enhanced lipopolysaccharide-induced inflammatory gene expression in mouse aortic ECs, which was recapitulated by ABCG1 deficiency in human aortic ECs. Conclusions— These studies provide direct evidence that endothelial cholesterol efflux pathways mediated by ABCA1 and ABCG1 are nonredundant and atheroprotective, reflecting preservation of endothelial NO synthase activity and suppression of endothelial inflammation, especially in regions of disturbed arterial blood flow.

Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious against IHs. A selective β2-adrenergic receptor (AR) antagonist mirrored propranolol’s effects on HemSCs. These results show that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation., Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol’s effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2, Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibitors of Notch1 or its ligand Dll4 cause tumor vessel hypersprouting but interfere with vessel perfusion causing reduced tumor growth. Unfortunately, normal tissue vessels respond to these inhibitors causing tissue hypoxia or vascular neoplasms. Mice null for Notch4, unlike Notch1 mutants, exhibit reduced developmental and tumor angiogenesis. Notch4 expression is increased in the endothelium of some tumor types. These results suggest that specifically targeting Notch4 could reduce tumor angiogenesis with fewer effects on normal tissue and represents an unexplored therapeutic opportunity. We use a newly developed anti-Notch4 antibody, E7011, and show that in endothelial cells, inhibition of Notch4 signaling suppresses expression of a subset of canonical Notch target genes. Treatment with E7011 also suppresses expression of a unique set of genes not regulated by canonical Dll4-Notch1 signaling, which appear to be distinct Notch4 targets. We administered E7011 to neonatal mice to examine the effects on developmental retinal angiogenesis. A single dose of E7011 significantly reduced retinal vascular outgrowth, demonstrating that E7011 has anti-angiogenic activity in vivo. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, both of which are typically increased by loss of Notch1 signaling. Because the E7011 phenotype was markedly distinct from the Notch1 loss of function, we combined E7011 treatment with endothelial-specific loss of Notch1 (Notch1ECKO) to compare Notch1 to Notch4 function. E7011-treated Notch1ECKO retina showed a compound phenotype, exhibiting both the loss of vascular outgrowth characteristic of E7011/Notch4 and the hypersprouting characteristic of Notch1 loss of function. These results suggest that Notch1 and Notch4 have distinct roles in developmental angiogenesis, with Notch4 being a pro-angiogenic signaling pathway. We evaluated Notch4 expression in the breast cancer tumor microenvironment to determine if Notch4 functions in tumor vessels. Human triple negative breast cancer (TNBC) shows a range of expression of Notch4 in both tumor cells and tumor endothelium. The anti-angiogenic effects that we observe in the retina lead us to hypothesize that E7011 will show anti-tumor activity against tumors that induce Notch4 expression in neighboring endothelium, and that targeting endothelial Notch4 is a novel anti-angiogenic approach. Citation Format: L. A. Naiche, Bhairavi Swaminathan, Yu Kato, Sarita Das, Jason W. Eng, Qanber Raza, Krishna Thakkar, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. A novel Notch4 neutralizing antibody inhibits angiogenesis and tumor growth via a distinct mechanism from endothelial Notch1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1488.