The HOPX and BLBP landscape and gliogenic regions in developing human brain.

Outer radial glial cells (oRGs) give rise to neurons and glial cells and contribute to cell migration and expansion in developing neocortex. HOPX has been described as a marker of oRGs and possible actor in glioblastomas. Recent years' evidence points to spatiotemporal differences in brain development which may have implications for the classification of cell types in the central nervous system and understanding of a range of neurological diseases. Using the Human Embryonic/Fetal Biobank, Institute of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, HOPX and BLBP immunoexpression was investigated in developing frontal, parietal, temporal and occipital human neocortex, other cortical areas and brain stem regions to interrogate oRG and HOPX regional heterogeneity. Furthermore, usage of high-plex spatial profiling (Nanostring GeoMx® DSP) was tested on the same material. HOPX marked oRGs in several human developing brain regions as well as cells in known gliogenic areas but did not completely overlap with BLBP or GFAP. Interestingly, limbic structures (e.g. olfactory bulb, indusium griseum, entorhinal cortex, fimbria) showed more intense HOPX immunoreactivity than adjacent neocortex and in cerebellum and brain stem, HOPX and BLBP seemed to stain different cell populations in cerebellar cortex and corpus pontobulbare. DSP screening of corresponding regions indicated differences in cell type composition, vessel density and presence of apolipoproteins within and across regions and thereby confirming the importance of acknowledging time and place in developmental neuroscience.

Loss of H3K27me3 in WHO grade 3 meningioma.

Immunohistochemical quantification of H3K27me3 was reported to distinguish meningioma patients with an unfavorable prognosis but is not yet established as a prognostic biomarker within WHO grade 3 meningiomas. We studied H3K27me3 loss in a series of biopsies from primary and secondary malignant meningioma to validate its prognostic performance and describe if loss of H3K27me3 occurs during malignant transformation. Two observers quantified H3K27me3 status as "complete loss", < 50% and > 50% stained cells in 110 tumor samples from a population-based consecutive cohort of 40 WHO grade 3 meningioma patients. We found no difference in overall survival (OS) in patients with > 50% H3K27me3 retention compared to < 50% in the cohort of patients with WHO grade 3 meningioma (Wald test p = 0.5). H3K27me3 staining showed heterogeneity in full section tumor slides while staining of the Barr body and peri-necrotic cells complicated quantification further. H3K27me3 expression differed without a discernible pattern between biopsies from repeated surgeries of meningioma recurrences. In conclusion, our results were not compatible with a systematic pattern of immunohistochemical H3K27me3 loss being associated with OS or malignant transformation of meningiomas and did not support H3K27me3 loss as a useful immunohistochemical biomarker within grade 3 meningiomas due to staining-specific challenges in quantification.

Chitooligosaccharides Improve the Efficacy of Checkpoint Inhibitors in a Mouse Model of Lung Cancer.

YKL-40 (also named chitinase 3 like-1 protein [CHI3L1]) is a secreted chitinase-like protein which is upregulated in cancers and suggested to have pro-tumorigenic activity. YKL-40 lacks enzymatic function, but it can bind carbohydrates such as chitin. Chitooligosaccharides (COS) derived from deacetylation and hydrolysis of chitin might be used for the blockade of YKL-40 function. Here, public single-cell RNA sequencing datasets were used to elucidate the cellular source of YKL-40 gene expression in human tumors. Fibroblasts and myeloid cells were the primary sources of YKL-40. Screening of YKL-40 gene expression in syngeneic mouse cancer models showed the highest expression in the Lewis lung carcinoma (LL2) model. LL2 was used to investigate COS monotherapy and combinations with immune checkpoint inhibitors (anti-PD-L1 and anti-CTLA-4) (ICIs) and radiotherapy (8 Gy × 3) (RT). COS tended to reduce plasma YKL-40 levels, but it did not affect tumor growth. LL2 showed minimal responses to ICIs, or to RT alone. Interestingly, ICIs combined with COS led to delayed tumor growth. RT also enhanced the efficacy of ICIs; however, the addition of COS did not further delay the tumor growth. COS may exert their anti-tumorigenic effects through the inhibition of YKL-40, but additional functions of COS should be investigated.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

Mitotic and Proliferative Indices in WHO Grade III Meningioma.

Meningiomas with inherently high mitotic indices and poor prognosis, such as WHO grade III meningiomas, have not been investigated separately to establish interchangeability between conventional mitotic index counted on H&E stained slides (MI) and mitotic index counted on phosphohistone-H3 stained slides (PHH3 MI). This study investigates the agreement of MI and PHH3 MI and to analyze the association of progression-free survival (PFS) and MI, PHH3 MI, and the proliferative index (PI, Ki-67) in WHO grade III meningioma. Tumor specimens from 24 consecutive patients were analyzed for expression of Ki-67, PHH3 MI, and MI. Quantification was performed independently by two observers who made replicate counts in hot spots and overall tumor staining. Repeatability in replicate counts from MI and PHH3 MI was low in both observers. Consequently, we could not report the agreement. MI, PHH3 MI and hot spot counts of Ki-67 were associated with PFS (MI hot spot HR = 1.61, 95% CI 1.12-2.31, p = 0.010; PHH3 MI hot spot HR = 1.59, 95% CI 1.15-2.21, p = 0.006; Ki-67 hot spot HR = 1.06, 95% CI 1.02-1.11. p = 0.004). We found markedly low repeatability of manually counted MI and PHH3 MI in WHO grade III meningioma, and we could not conclude that the two methods agreed. Subsequently, quantification with better repeatability should be sought. All three biomarkers were associated with PFS.

Cancer cell stemness, responses to experimental genotoxic treatments, cytomegalovirus protein expression and DNA replication stress in pediatric medulloblastomas.

Despite recent progress in research on brain tumors, including identification of cancer stem-like cells (CSCs), little is known about the interplay of stemness with the commonly observed infection by the human cytomegalovirus (HCMV) and the widespread features of replication stress in these malignancies. To shed more light on these outstanding issues, here we combine immunohistochemical analysis of archival clinical specimens from a cohort of 25 human pediatric medulloblastomas, complemented by functional experiments and analytical approaches to examine three medulloblastoma cell lines. In the clinical samples, we find consistent, yet individually variable subsets of CSCs expressing the stem-cell markers CD133 and CD15, and a candidate marker VEGFR2, across the spectrum of endogenous DNA damage (γH2AX), expression of HCMV immediate early and late proteins, proliferation rate (Ki67) or molecular class of MB. Contrary to MB cell lines DAOY and D324, the D283 cells showed pronounced phenotypic features of stemness, associated with enhanced endogenous DNA damage, exceptionally high susceptibility to infection with HCMV, unorthodox signaling pathway response to ionizing radiation and hyperactive response to hydroxyurea-induced replication stress. Notably, single-molecule DNA fiber analysis revealed aberrantly slow replication fork progression, pronounced fork asymmetry and inability to timely recover from drug-induced fork stalling in stem-like D283 cells, all hallmarks of pronounced chronic replication stress and propensity to genomic instability. These findings provide insights into human medulloblastoma stemness phenotypes, with various susceptibilities to infection by HCMV and impact on replication fork (mal)function, with implications for better understanding pathogenesis and responses to treatment in pediatric brain malignancies.Abbreviations: CSC: cancer stem-like cells; FBS: fetal bovine serum; HCMV: human cytomegalovirus; MB: medulloblastoma; MBSC: medulloblastoma stem cells; MOI: multiplicity of infection; PBS: phosphate-buffered saline; RPA: replication protein A; RS: replication stress; SHH: sonic hedgehog; VEGFR2: vascular endothelia growth factor receptor 2.

The migration and loss of human primordial germ stem cells from the hind gut epithelium towards the gonadal ridge.

Human primordial germ cells (PGCs) can be recognized in the yolk sac wall, from 3-4 weeks post conception (wpc), in the hind gut epithelium from week 4 and in the gonadal area from early week 5. The objective of this study was to map the migration route of PGCs and elucidate the role of the nervous system in this process. Sixteen human specimens, 5-14 wpc obtained from legal abortions were included. On serial paraffin sections, PGCs were detected immunohistochemically by expression of OCT4 and c-Kit, nerve fibers by ß-III-tubulin and stem cell factor (SCF) as a possible chemoattractive cue for PGC migration. PGCs were present in the hind gut epithelium, in the mesenchyme of the dorsal mesentery and in the developing gonadal ridge of 4-6 wpc embryos, prior to connections between the enteric and the sympathetic nervous system. From 6 wpc onwards, the PGCs travelled along the developing nerve fibers from the wall of the hind gut via the dorsal mesentery to the midline of the dorsal wall and laterally into the gonads. Numerous PGCs were still present in the nervous system by 14 wpc. PGCs in 4-5 wpc embryos are suggested to leave the gut epithelium by EMT-like transition. SCF may facilitate further migration, but after establishment of connections between the enteric and sympathetic nervous systems. PGCs follow sympathetic nerve fibers towards the gonads. PGCs failing to exit the nerve branches at the gonadal site, may continue along the sympathetic trunk ending up in other organs where they may form germ cell tumors if not eliminated by apoptosis.