Expression Of Cell Lineage Markers Research Articles

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

Abstract 4228: Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity

Abstract Introduction: B7-H3, a B7 family member, is widely expressed in multiple cancers and is associated with poor clinical prognosis. Enoblituzumab (also known as MGA271 or TJ271) is an investigational, Fc-optimized monoclonal antibody that targets B7-H3 and exhibits enhanced antibody-dependent cell-mediated killing of B7-H3 expressing cancer cells. Here we identified the immuno-modulatory function of TJ271 in different cell subsets and pathways as a new mechanism for cancer treatment. Experiments: The function of B7-H3 and TJ271 in regulating immune response was investigated in human PBMCs stimulated with anti-CD3 antibody with or without plate-coated B7-H3 in the presence or absence of TJ271. Gene expression profiles and immunophenotyping of PBMCs after treatments were evaluated by Nanostring nCounter platform and CyTOF mass cytometry, respectively. IFN-γ production from cell supernatant was measured by ELISA. In vitro tumor killing activity by TJ271 and urelumab, a 4-1BB agonist, was evaluated by co-culture of B7-H3 expressing tumor cells and PBMCs stimulated with anti-CD3. The tumor cell killing activity was evaluated by 7-AAD staining on the Incucyte platform. Results: Gene expression of cell lineage markers in antigen-presenting cells, including macrophages and neutrophils was induced, while those in natural killer (NK) cells was suppressed by B7-H3 stimulation, respectively. Furthermore, expression of activation markers and co-stimulatory receptors, including IFNγ, ULBP2, GITR, and 4-1BB, was down-regulated while expression of exhaustion markers in CD8+ T cells and NK cells, including PD-L1, PD-L2, and IDO1, and of genes associated with M2-like macrophages were up-regulated by B7-H3. All this B7-H3-induced differential gene expression pattern was reversed by TJ271 treatment. As revealed by CyTOF, TJ271 treatment abolished B7-H3-mediated increase in the frequency of M2-like monocytes (B7-H3+CD68+CD206+CD16lo or B7-H3-CD68+CD163+CD16+) and led to increased frequency of 4-1BB-expressing CD4, CD8, and NK cells. TJ271 exhibited strong tumor killing activity against ES-2, a B7-H3-expressing ovarian cancer cell line in vitro. Consistent with the TJ271-associated increased 4-1BB expression in T and NK cells, activation of 4-1BB by urelumab further enhanced TJ271-mediated tumor killing activity. Conclusion: Our data confirm the immunosuppressive function of B7H3 and demonstrate the immunoregulatory function of TJ271 as evidenced by the activation of cytolytic T cell and NK cells, reinvigoration of exhausted cells, and suppression of M2-like myeloid cells. These findings provide rationale for combination therapy with blockade of B7-H3 by TJ271 with other immunotherapies to achieve increased clinical efficacy against cancers. Citation Format: Yanni Zhang, Xuejun Liu, Xinyun Zhu, Zhen Sheng, Yan Liu, Zhengyi Wang, Taylor B. Guo. Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity [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 4228.

Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes.

STUDY QUESTIONDoes experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation?SUMMARY ANSWERInhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries.WHAT IS KNOWN ALREADYSex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4.STUDY DESIGN, SIZE, DURATIONFGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 μM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined.PARTICIPANTS/MATERIALS, SETTING, METHODSGonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography–tandem mass spectrometry (LC-MS/MS).MAIN RESULTS AND THE ROLE OF CHANCETreatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis.LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTION Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered.WIDER IMPLICATIONS OF THE FINDINGSThe findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes.STUDY FUNDING/COMPETING INTEREST(S)This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government’s support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.

Follistatin supplementation during in vitro embryo culture improves developmental competence of bovine embryos produced using sex-sorted semen

Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.

Isogenic blood-brain barrier models based on patient-derived stem cells display inter-individual differences in cell maturation and functionality.

The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes, and neurons. Recently, isogenic invitro models of the BBB based on human pluripotent stem cells have been documented, yet the impact of inter-individual variability on the yield and phenotype of such models remains to be documented. In this study, we investigated the impact of inter-individual variability on the yield and phenotype of isogenic models of the BBB, using patient-derived induced pluripotent stem cells (iPSCs). Astrocytes, BMECs, and neurons were differentiated from four asymptomatic patient-derived iPSCs (two males, two females). We differentiated such cells using existing differentiation protocols and quantified expression of cell lineage markers, as well as BBB phenotype, barrier induction, and formation of neurite processes. iPSC-derived BMECs showed barrier properties better than hCMEC/D3 monolayers; however, we noted differences in the expression and activity among iPSC lines. In addition, we noted differences in the differentiation efficiency of these cells into neural stem cells and progenitor cells (as noted by differences in expression of cell lineage markers). Such differences were reflected later in the terminal differentiation, as seen as ability to induce barrier function and to form neurite processes. Although we demonstrated our ability to obtain an isogenic model of the BBB with different patients' iPSCs, we also noted subtle differences in the expression of cell lineage markers and cell maturation processes, suggesting the presence of inter-individual polymorphisms.

Chronic, highly productive HIV infection in monocytes during supportive culture.

Peripheral blood monocytes of HIV-infected individuals carry virus, constituting one potential reservoir. However, most studies of infection in tissue culture find monocytes refractory to HIV replication, suggesting that culture conditions limit the relative susceptibility of this target cell. We employed a tissue culture system optimized for maintenance of human monocytes without differentiation and compared HIV infection efficiency of monocytes and fully differentiated monocyte derived macrophages (MDM). We tested direct virus-cell fusion, expression of cell lineage markers, and productive HIV infection in fresh monocytes, monocytes after varying periods of supportive culture, and fully differentiated MDM comparing cells from individual donors. Fresh, uncultured monocytes allowed modest HIV fusion, however one week culture was sufficient to allow efficient fusion and an increase in expression of CD14, CD16, CD33, and CD105. Compared to freshly isolated monocytes, monocytes infected after a few days in culture produced p24 more quickly, but the peaks of production were similar. Fresh monocytes were highly susceptible to productive HIV infection in supportive culture, roughly equal to MDM from the same donor in expression of extracellular p24 up to five weeks after infection. Taken together our findings indicate that monocytes are biologically capable of supporting chronic, highly productive HIV infection, a capacity that may reflect their status in HIV-infected persons.

Pilot gene expression analysis of inflamed synovium

Background and objectivesWithin the synovial membrane of an rheumatoid arthritis (RA) joint, tissue architecture varies depending on organisation of the infiltrating inflammatory cells (diffuse, aggregates and germinal centres). It has...

Cell proliferation in human ganglionic eminence and suppression after prematurity-associated haemorrhage

In premature infants, germinal matrix haemorrhage in the brain is a common occurrence. However, cell proliferation and fate determination in the normal human germinal matrix is poorly understood. Human ganglionic eminence samples were collected prospectively from autopsies of premature and term infants with no evidence of pathological process (n=78; dying at post-menstrual age 14-88 weeks). The ganglionic eminence was thickest at 20-26 weeks and involuted by 34-36 weeks. Proliferating cells, detected by Ki67 immunoreactivity, were abundant throughout the ganglionic eminence prior to 18 weeks, after which a sharp boundary between the dorsal and ventral ganglionic eminence appeared with reduced cell proliferation in the dorsal region. Ki67 immunoreactivity persisted in the majority of ventral cells until ∼28 weeks, after which time the proportion of proliferating cells dropped quickly. The expression of cell lineage markers (such as Olig2, SOX2, platelet-derived growth factor receptor alpha) showed partitioning at the microscopic level. The hypothesis that germinal matrix haemorrhage suppresses cell proliferation was then addressed. In comparison to controls, germinal matrix haemorrhage (n=47; born at post-menstrual age 18-34 weeks followed by survival of 0 h to 98 days) was associated with significantly decreased cell proliferation if survival was >12 h. The cell cycle arrest transcription factor p53 was transiently increased and the oligodendroglial lineage markers Olig2 and platelet-derived growth factor receptor alpha were decreased. Cell death was negligible. A low level of microglial activation was detected. Haemorrhage-associated suppression of cell proliferation in premature human infants could partially explain the reduced brain size and clinical effects in children who suffer germinal matrix haemorrhage after premature birth.

Isolation and Characterization of a Putative Intestinal Stem Cell Fraction From Mouse Jejunum

Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342. Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1. Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and beta1-integrin, were cytokeratin positive, and survived in culture for up to 14 days. The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.

Cell lineage markers in human pancreatic cancer

The normal pancreas consists of three major cell types or lineages that share a common embryologic origin from pluripotent endodermal precursors. The type of cell that undergoes neoplastic transformation to form a pancreatic carcinoma is controversial and may influence the phenotype and biologic behavior of the tumor. In this study, immunohistologic techniques were used to determine the cell lineage differentiation expressed in 29 primary exocrine pancreatic adenocarcinomas, five metastatic exocrine pancreatic adenocarcinomas, and five islet cell neoplasma. Specimens of normal pancreas and chronic pancreatitis were used for comparison. The cell lineage markers consisted of monoclonal and polyclonal antibodies against trypsin and lipase (acinar cells); secretory component, carbonic anhydrase II, and pancreatic cancer mucin SPan-1 (ductal cells); and chromogranin-A and somatostatin (islet cells). The expression of carcinoembryonic antigen (CEA) and lysozyme were also determined. This collection of markers allowed the differentiation between acinar, ductal, and islet cells of normal pancreas and chronic pancreatitis specimens. The expression of cell lineage markers in islet cell tumors was homogeneous and restricted to chromogranin-A. In contrast, the expression of these markers in primary and metastatic exocrine pancreatic adenocarcinomas was variable. Reactivity with monoclonal anti-CEA was absent in normal pancreas, and was present in 83% of chronic pancreatitis specimens as well as 90% of exocrine pancreatic adenocarcinomas. In addition, lysozyme reactivity was absent in normal pancreas; however, lysozyme was expressed in one case of chronic pancreatitis, 17 cases of primary carcinoma, and three cases of metastatic carcinoma. These findings support the concept that the original transformed cell type in many pancreatic exocrine carcinomas resemble endodermal "stem cells" that retain the capability of differentiation along more than one cell lineage pathway.