TAMI-36. TAMEP ARE BRAIN TUMOR PARENCHYMAL CELLS CONTROLLING NEOPLASTIC ANGIOGENESIS AND PROGRESSION

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor-parenchymal cells may promote specific phases of disease-progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Strikingly, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell-population, by conditional Sox2-knockout, drastically reduced glioblastoma-vascularization and -size. TAMEP manipulation profoundly altered vessel function and strongly attenuated the blood-tumor barrier. Hence, our data indicate TAMEP and their progenitors as new targets for glioblastoma therapy.

EPCO-11. SINGLE-NUCLEI TRANSCRIPTOMICS RELATES GLIOBLASTOMA INFILTRATION TO DISTINCT GLIAL PROGENITOR STATES

Our understanding of glioblastoma (GBM) intratumoral heterogeneity, particularly in the context of neurodevelopment, has thus far been primarily focused on the more surgically accessible tumor core niche. In contrast, the biology of GBM cells at the infiltrative edge, which evade surgical resection and drive tumor recurrence, remains poorly characterized. To this end, we microdissected and performed single-nuclei RNA sequencing (snRNA-seq) on approximately 62,000 nuclei taken from the tumor core and from the infiltrative edge of six GBM tumors with diverse genomic drivers, including IDH1, EGFR, PDGFRA, FGFR3, and NF1. Unbiased clustering reveals distinct neoplastic and non-neoplastic populations, further distinguished using copy number variation analysis. After projecting previously defined signatures taken from snRNA-seq analysis of human adult neocortex/subventricular zone and prenatal germinal matrix, we find that approximately 90% of tumor cells recapitulate a neurodevelopment-like molecular phenotype, reprising gene expression signatures of prenatal astrocytes and of a distinct glial intermediate progenitor cell population (g-IPC) that precedes both astrocyte and oligodendrocyte lineage differentiation. Examining the infiltrative edge of samples with the most confident microdissection (n=4), we see that while distinct populations of tumor cells in this niche express proneural and classical signatures, these cells are overall enriched for a g-IPC-like phenotype, relative to the tumor core, irrespective of the tumors’ genomic alterations. A subset of cells at the infiltrative edge, in particular, recapitulates the signature of an uncommitted g-IPC subtype, expressing both astroglial and oligodendroglial markers. Trajectory analyses also reveal distinct branches of core and edge tumor cells, which are predominantly astrocyte- and g-IPC-like, respectively. Differential gene expression analysis of GBM cells at the infiltrative edge vs. tumor core reveals a migration signature, dominated by EGFR, ERBB4, PCDH9, and PCDH15. Ultimately, this high resolution analysis of heterogeneity at the infiltrative edge allows us to uncover potentially targetable drivers of invasion in GBM.

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of notch via ROBO1/CTNNB1/JAG1

ABSTRACT In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating β-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.

Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

OTME-1. TAMEP are brain tumor parenchymal cells controlling neoplastic angiogenesis and progression

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression.

A dendritic-like microtubule network is organized from swellings of the basal fiber in neural progenitors

Neurons of the neocortex are generated by neural progenitors called radial glial cells. These polarized cells extend a short apical process towards the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport in unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in swellings of the basal fiber. We characterize their distribution and demonstrate that they accumulate the minus end stabilizing factor CAMSAP3 and TGN-related membranes, from which the majority of microtubules grow. Finally, using live imaging of human fetal cortex, we show that this organization is conserved in basal radial glial (bRG) cells, a highly abundant progenitor cell population associated with human brain size expansion.

Notch effector Hes1 marks an early perichondrial population of skeletal progenitor cells at the onset of endochondral bone development

SummaryThe perichondrium, a fibrous tissue surrounding the fetal cartilage, is an essential component of developing endochondral bones that provides a source of skeletal progenitor cells. However, perichondrial cells remain poorly characterized due to lack of knowledge on their cellular diversity and subset-specific mouse genetics tools. Single cell RNA-seq analyses reveal a contiguous nature of the fetal chondrocyte-perichondrial cell lineage that shares an overlapping set of marker genes. Subsequent cell-lineage analyses using multiple creER lines active in fetal perichondrial cells – Hes1-creER, Dlx5-creER – and chondrocytes – Fgfr3-creER – illustrate their distinctive contribution to endochondral bone development; postnatally, these cells contribute to the functionally distinct bone marrow stromal compartments. Particularly, Notch effector Hes1-creER marks an early skeletal progenitor cell population in the primordium that robustly populates multiple skeletal compartments. These findings support the concept that perichondrial cells participate in endochondral bone development through a distinct route, by providing a complementary source of skeletal progenitor cells.

PSI-25 Effects of restricted maternal nutrition and realimentation during gestation on the fetal progenitor cell population in semitendinosus muscle of sheep

Satellite cells are muscle stem cells that contribute to postnatal growth. The satellite cell population is established during fetal muscle development, through the retention of Pax7-expressing myogenic progenitor cells. We hypothesized that realimentation during late gestation would ameliorate the negative effect of poor maternal nutrition during mid-gestation on the fetal myogenic progenitor cell population. To test this hypothesis, 47 ewes pregnant with singletons were fed a control diet of 100% of National Research Council (NRC) requirements (CON) starting at day 25 of gestation. At day 50 of gestation, six ewes were euthanized and the remainder were randomly assigned to one of two diets: CON or 60% of CON (RES). On day 90 of gestation, a subset of ewes were euthanized (n = 7 per treatment) and fetal semitendinosus samples were collected. The remaining ewes were maintained on the current diet (CON-CON, RES-RES) or switched to the alternative diet (CON-RES, RES-CON). On day 130 of gestation, all ewes were euthanized for fetal sample collection (n = 6–7 per treatment). Fetal semitendinosus was cryosectioned and immunostained for detection of Pax7(+) cells followed by image analysis. Data were analyzed using the MIXED procedure in SAS. Semitendinosus from RES lambs had a greater number of Pax7(+) cells but similar total cell numbers to CON offspring, resulting in a greater percentage of Pax7(+) cells at d90 of gestation (CON: 13.22 ± 0.74%; RES: 16.01 ± 0.74%, P = 0.01). At day 130, there was no difference in the percentage of Pax7(+) cells between dietary treatment groups (CON-CON: 7.88 ± 0.80%; CON-RES: 6.34 ± 0.74%; RES-RES: 7.82 ± 0.74%; RES-CON: 6.87 ± 0.74%; P > 0.17). The percentage of Pax7(+) cells decreased from day 90 to day 130, regardless of dietary treatment (P < 0.0001). In summary, restricted maternal nutrition may delay progenitor cell differentiation at mid-gestation.