Programmed death ligand 1 gene silencing in murine glioma models reveals cell line-specific modulation of tumor growth in vivo

BACKGROUND Glioblastoma is the most common brain tumor in adults and virtually incurable. Therefore, new therapeutic strategies are urgently needed. Immune checkpoint inhibition has not shown activity in various phase III trials and intra- as well as intertumoral expression of programmed death ligand 1 (PD-L1) varies in glioblastoma. METHODS We abrogated constitutive PD-L1 gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo. RESULTS A heterogeneous expression of Pdl1 mRNA and PD-L1 protein was detected in the glioma cell panel in vitro and in vivo. PD-L1, but not PD-L2, was inducible by interferon β and γ. Co-culture with splenocytes induced PD-L1 expression in GL-261 and SMA-560, but not in CT-2A cells, in an interferon γ-dependent manner. Conversely, Pdl1 gene silencing conferred a survival benefit in CT-2A, but not in the other 2 models. Accordingly, PD-L1 antibody prolonged survival in CT-2A glioma-bearing mice. This activity required PD-L1 expression on tumor rather than host cells, and the survival gain mediated by PD-L1 loss was reproduced in immune-deficient RAG$^{-/-}$ mice. CONCLUSIONS PD-L1 is expressed and interferon-inducible in murine glioma cell lines. PD-L1 has model-specific roles for tumor growth. Future studies need to determine which subset of glioblastoma patients may benefit from PD-L1 antagonism as part of a multimodality therapeutic approach to glioblastoma

Differential roles of type I interferon signaling in tumor versus host cells in experimental glioma models

Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers.Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host

Oxidative Stress Levels and DNA Repair Kinetics in Senescent Primary Human Fibroblasts Exposed to Chronic Low Dose Rate of Ionizing Radiation

International audienc

Doxorubicin-provoked increase of mitotic activity and concomitant drain of G0-pool in therapy-resistant BE(2)-C neuroblastoma.

In this study chemotherapy response in neuroblastoma (NB) was assessed for the first time in a transplantation model comprising non-malignant human embryonic microenvironment of pluripotent stem cell teratoma (PSCT) derived from diploid bona fide hESC. Two NB cell lines with known high-risk phenotypes; the multi-resistant BE(2)-C and the drug sensitive IMR-32, were transplanted to the PSCT model and the tumour growth was exposed to single or repeated treatments with doxorubicin, and thereafter evaluated for cell death, apoptosis, and proliferation. Dose dependent cytotoxic effects were observed, this way corroborating the experimental platform for this type of analysis. Notably, analysis of doxorubicin-resilient BE(2)-C growth in the PSCT model revealed an unexpected 1,5-fold increase in Ki67-index (p3-fold increase in active cell cycle (i.e. cells positive staining for PH3 together with incorporation of EdU) (p<0.01). Considering the clinical challenge for treating high-risk NB, the discovery of a therapy-provoked growth-stimulating effect in the multi-resistant and p53-mutated BE(2)-C cell line, but not in the drug-sensitive p53wt IMR-32 cell line, warrants further studies concerning generality and clinical significance of this new observation

Micro colonisation of IMR-32 tumours in the PSCT model.

<p>(A) Schematic illustration; NB cells were injected into an arbitrary position centrally in the PSCT cellular mass, resulting in multiple micro-colonisations from migrating NB cells. (B) A representative FFPE section of a PSCT with four IMR-32 colonies indicated (red borders). (C) IMR-32 colony surrounded by loose mesenchyme. LM = loose mesenchyme; NE = neural epithelium; C = cartilage; M = muscle; Blue arrows = vessels. Size bars: B:5mm, C:500μm.</p

The effects of doxo on PSCT non-malignant embryonic tissues.

<p>Immunohistochemistry staining of formalin-fixed paraffin-embedded PSCT histological slides, following intra peritoneal injection of the host mouse with 8mg/kg doxo (A-D), or 4+4mg/kg doxo (E-H). High frequencies of positive staining for Ki67 in tissues compatible with neural epithelium, muscle and cartilage can be seen, indicative of extensive proliferation (A,C,E,G). Low frequencies of positive staining for cleaved caspase 3 can be seen in NE, muscle and cartilage, indicative of low frequencies of apoptosis (B,D,F,H). Size bars: 50μm.</p

Data set for IMR-32 <i>in vitro</i>.

<p>Data set for IMR-32 <i>in vitro</i>.</p

Data set for BE(2)-C <i>in vitro</i>.

<p>Data set for BE(2)-C <i>in vitro</i>.</p

The effects of doxo in neural epithelium in the PSCT model.

<p>The effects of doxo in neural epithelium in the PSCT model.</p

The effects of doxo on BE(2)-C tumours growing in the PSCT model.

<p>The effects of doxo on BE(2)-C tumours growing in the PSCT model.</p