Morpho-functional analysis of patient-derived xenografts reveals differential impact of gastric cancer and chemotherapy on the tumor ecosystem, affecting immune check point, metabolism, and sarcopenia.

OBJECTIVES: Gastric cancer (GC) is an aggressive disease due to late diagnosis resulting from the lack of easy diagnostic tools, resistances toward immunotherapy (due to low PD-L1 expression), or chemotherapies (due to p53 mutations), and comorbidity factors, notably muscle atrophy. To improve our understanding of this complex pathology, we established patient-derived xenograft (PDX) models and characterized the tumor ecosystem using a morpho-functional approach combining high-resolution imaging with molecular analyses, regarding the expression of relevant therapeutic biomarkers and the presence of muscle atrophy. MATERIALS AND METHODS: GC tissues samples were implanted in nude mice. Established PDX, treated with cisplatin or not, were imaged by magnetic resonance imaging (MRI) and analyzed for the expression of relevant biomarkers (p53, PD-L1, PD-1, HER-2, CDX2, CAIX, CD31, a-SAM) and by transcriptomics. RESULTS: Three well-differentiated, one moderately and one poorly differentiated adenocarcinomas were established. All retained the architectural and histological features of their primary tumors. MRI allowed in-real-time evaluation of differences between PDX, in terms of substructure, post-therapeutic changes, and muscle atrophy. Immunohistochemistry showed differential expression of p53, HER-2, CDX2, a-SAM, PD-L1, PD-1, CAIX, and CD31 between models and upon cisplatin treatment. Transcriptomics revealed treatment-induced hypoxia and metabolic reprograming in the tumor microenvironment. CONCLUSION: Our PDX models are representative for the heterogeneity and complexity of human tumors, with differences in structure, histology, muscle atrophy, and the different biomarkers making them valuable for the analyses of the impact of platinum drugs or new therapies on the tumor and its microenvironment.

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.

A novel glycine receptor alpha Z1 subunit variant in the zebrafish brain.

Alpha subunits of the inhibitory glycine receptor (GlyR) display genetic heterogeneity in mammals and zebrafish. This diversity is increased in mammals by the alternative splicing mechanism. We report here in zebrafish, the characterization of a new alphaZ1 subunit likely arising from alphaZ1 gene by an alternative splice process (alphaZ1L). This novel cDNA possesses 45 supplementary nucleotides at the putative exon2/exon3 boundary. The corresponding protein contains 15 additional amino acids in the NH2-terminal domain. Heterologous expression of homomeric GlyRalphaZ1L in human embryonic kidney-293 cells generates glycine-gated strychnine-sensitive chloride channels with no obvious discrepancy with pharmacological properties of GlyRalphaZ1. Moreover, zinc modulation of glycine-induced currents is identical in alphaZ1 and alphaZ1L glycine receptors. During ontogenesis, simultaneous alphaZ1 and alphaZ1L mRNA synthesis have been observed. Embryonic and adult alphaZ1 and alphaZ1L mRNA expressions are restricted to the CNS. Embryonic alphaZ1L mRNA anatomical pattern of expression is, however, highly restrained and strictly limited to the rostral part of the brain revealing a highly regionalized function of alphaZ1L in the CNS. This report contributes to the characterization of the diversity of glycine receptor isoforms in zebrafish and emphasizes the common mechanism used among vertebrates for creating GlyR variety and specificity.

Phylogenetic relationships and chromosomal location of five distinct glycine receptor subunit genes in the teleost Danio rerio.

Glycine receptors mediating synaptic inhibition are heteromeric proteins constituted of alpha and beta subunits. The mammalian GlyR subunits constitute a subgroup in the superfamily of ligand-gated ionic channels. To compare the evolutionary events in the mammalian and teleostean lineages for the receptor family, we first undertook systematic cloning of the constitutive subunits of the zebrafish glycine receptor. The isolation of two alpha subunits (alphaZ1 and alphaZ2) and one beta subunit (betaZ) has been reported previously and we report here the characterization of two novel alpha subunits, alphaZ3 and alphaZ4, increasing the known zebrafish subunits number to four alpha and one beta. Establishment of phylogenetic relationships reveals that alphaZ1, alphaZ3 and betaZeta are orthologous to mammalian alpha1, alpha3 and beta subunits. However, two zebrafish GlyRalpha subunit genes are orthologous to the unique avian and mammalian alpha4 subunit revealing a duplication of the alpha4 gene in zebrafish. Whole-mount in situ hybridization in 24-hours post fertilization (hpf) and 52-hpf embryos of the daughter gene products display very different expression patterns indicating distinct functions of the duplicated genes. Gene mapping reveals that the two duplicated genes are localized on two different linkage groups (LG5 and LG22) as would be daughter genes resulting from a large-scale duplication of the ancestral genome. Finally, we report that a linked pair of genes on human chromosome 4 (alpha3 and beta) is also linked on linkage group 1 in zebrafish (alphaZ3 and betaZ) as a consequence of a mosaic conserved syntheny.

Regional distribution of glycine receptor messenger RNA in the central nervous system of zebrafish.

We report the cloning of the zebrafish beta subunit of the glycine receptor and compare the anatomical distribution of three glycine receptor subunit constituents in adult zebrafish brain (alphaZ1, alphaZ2 and betaZ) to the expression pattern of homologous receptor subunits (alpha1, alpha2 and beta) in the mammalian adult CNS. Non-radioactive hybridization was used to map the distribution of the alphaZ1, alphaZ2 and betaZ glycine receptor subunit messenger RNAs in the adult zebrafish brain. The anterior-posterior expression gradient found in adult zebrafish brain was similar to that reported in mammalian CNS. However, the glycine receptor transcripts, notably the alphaZ1 subunit, were more widely distributed in the anterior regions of the zebrafish than in the adult mammalian brain. The isoform-specific distribution pattern was less regionalized in zebrafish than in the rat mammalian CNS. Nevertheless, there was some regionalization of alphaZ1, alphaZ2 and betaZ transcripts in the diencephalic and mesencephalic nuclei where different sensory and motor centers express either alphaZ1/betaZ or alphaZ2 subunits. In contrast to the widespread distribution of the beta subunit in adult mammalian brain, alphaZ2 messenger RNA presented the widest expression territory of all three glycine receptor subunits tested. alphaZ2 messenger RNA was expressed in the absence of alphaZ1 and betaZ messenger RNA in the outer nuclear layer of the retina, the inferior olive and the raphe of the medulla oblongata, as well as in the nucleus of Cajal of the medulla spinalis. In contrast, an identified central neuron of the reticular formation, the Mauthner cell, expresses all three glycine receptor subunits (alphaZ1, alphaZ2 and betaZ). This report extends the already described glycine receptor expression in the vertebrate CNS and confirms the importance of glycine-mediated inhibition in spinal cord and brainstem.

Cloning, expression and electrophysiological characterization of glycine receptor alpha subunit from zebrafish.

The glycine receptor is a ligand-gated anion channel protein, providing inhibitory drive within the nervous system. We report here the isolation and functional characterization of a novel alpha subunit (alphaZ1) of the glycine receptor from adult zebrafish (Danio rerio) brain. The predicted amino acid sequence is 86%, 81% and 77% identical to mammalian isoforms alpha1, alpha3 and alpha2, respectively. AlphaZ1 exhibits many of the molecular features of mammalian alpha1, but the sequence patterns in the M4 and C-terminal domains are more similar to alpha2/alpha3. Phylogenetic analysis indicates that alphaZ1 is more closely related to the mammalian alpha1 subunits, being positioned, however, on a distinct branch. The alphaZ1 messenger RNA is 9.5 kb, similar to that described previously for alpha1 messenger RNAs. When expressed in Xenopus oocytes or a human cell line (BOSC 23), alphaZ1 forms a homomeric receptor which is activated by glycine and antagonized by strychnine. This receptor demonstrates unexpectedly high sensitivity to taurine and can also be activated by GABA. These results are consistent with physiological findings in lamprey and goldfish, and they suggest that this teleost fish glycine receptor displays a lower selectivity to neurotransmitters than that reported for glycine mammalian receptors.

Enhancement of calcium signaling and proliferation responses in activated human T lymphocytes. Inhibitory effects of K+ channel block by charybdotoxin depend on the T cell activation state.

T cell receptor (TCR) stimulation, leading to T cell activation and ultimately to cell proliferation and differentiation, evokes elevations of [Ca2+]i with a high variability between individual T lymphocytes. We have used Ca(2+)-imaging of Fura-2 loaded cells to study the origin of the variation in Ca2+ signals and its consequences for the final cellular response. We found that, compared to resting cells, the percentage of responding cells and the average amplitude of the Ca2+ signal upon TCR re-stimulation by PHA increases in the first 5 days of T cell activation and declines thereafter, with more pronounced [Ca2+]i oscillations in later stages. In parallel, an enhancement of T cell proliferation is observed. Stronger stimulation of the TCR/CD3 complex by co-crosslinking CD3 with CD4/CD8 molecules evokes oscillating Ca2+ responses irrespective of the activation state, indicating that the basic capacity for Ca2+ signaling is essentially the same in resting and activated cells. Nevertheless, also the amplitude of the CD3+CD4/8 response shows a transient additional increase during the first days of T cell activation. Experiments with the K+ channel blocker charybdotoxin (CTX) indicate that [Ca2+]i oscillations depend critically on K+ channel functioning, but suppression of these oscillations by CTX does not significantly affect the average amplitude of the Ca2+ signal nor PHA-induced proliferation. However, when applied during the first 4-5 days of activation, CTX reduces in addition the average level of the TCR evoked Ca2+ response and inhibits subsequent proliferation.

Maturation and integration of purified foetal neurons transplanted into the adult brain.

Fetal neural transplants presently developed as a therapeutic strategy for neurodegenerative diseases include both the neurons of interest and their cellular environment. These glial and vascular cells may be detrimental by, for instance, expressing foreign MHC antigens. This study was undertaken to determine whether purified neurons would survive transplantation into an adult host brain. Embryonic rat spinal neurons were purified by panning and transplanted into adult hosts' brain. During the first three weeks post-transplantation the grafts contained essentially packed immature neurons. Later transplants contained large, multipolar neurons, demonstrating the ability of transplanted neurons to mature in the adult environment. The adult host appears actively involved in the integration of such a transplant by complementing it with microglial and vascular cells.