Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer

IntroductionBrain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis.MethodsPeripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation.ResultsPatients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression.ConclusionsThe frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases. Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cells in vitro, suggesting that monitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients

Alternatively spliced tissue factor is not sufficient for embryonic development

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival

Targeting of the TF gene.

<p>The murine TF allele was targeted with a replacement-type vector containing the murine asTF open reading frame flanked by 3 kb 5′ and 5.2 kb 3′ homology arms. Diphtheria toxin (dt) was used for negative selection. After homologous recombination into 129P2/OlaHsd embryonic stem cells, a loxP flanked neomycin resistance cassette was removed by transfection with a Cre expression plasmid.</p

Expression and activity of asTF.

<p>(A) RNA levels of asTF and flTF were measured in E9.5 whole embryos and normalized to S18 expression, N = 5–7, ***P<0.001, **P<0.01 (B) Protein expression of total TF (western blot probed with a rabbit anti-mouse TF polyclonal antibody), asTF (western blot probed with a rabbit anti-mouse asTF polyclonal antibody), and GAPDH in E9.5 whole embryos. Intensity of normalized total TF staining relative to wt (100%) was 74% in asTFKI/wtTF embryos and 19% in asTFKI/asTFKI embryos. Expression of asTF was comparable between homozygous asTFKI, heterozygous asTFKI/wtTF and wt embryos (C) Mouse asTF and rabbit IgG control immunohistochemistry on E9.5 homozygous asTFKI and E10.5 wt yolk sac. Mouse asTF immunohistochemistry on E8.5 to E10.5 extraembryonic (yolk sac) and embryonic (heart) tissue of homozygous asTFKI and wt embryos. (D) TF activity of whole embryo lysates was measured with a plasma clotting assay and normalized to total protein. N = 5, ***P<0.001.</p

Genotype distribution on E9.5 and birth.

<p>Homozygous asTFKI embryos die after developmental day E9.5. Genotype distribution of offspring from heterozygous breeding pairs at birth (black bars) differs from that at embryonic day 9.5 (grey bars). At day 9.5, embryonic genotypes are distributed in a Mendelian manner, while no more homozygous asTFKI pups can be identified at birth (p = 0.0001, χ2-test).</p

Yolk sac histology.

<p>(A) On day E8.5, Haematoxylin and Eosin (H&E) staining and α-SMA immunohistochemistry show comparable integrity, erythrocyte filling, α-SMA staining of the mesodermal layer of homozygous asTFKI and wt yolk sacs. (B) On day E9.5, erythrocyte content, wall thickness, and number of α-SMA expressing cells are reduced in homozygous asTFKI yolk sacs. AsTFKI embryos are found at different stages of growth retardation and wasting. (C) On day E10.5, depletion of erythrocytes, reduced number of α-SMA⁺ cells, thinning and detachment of mesodermal from endodermal layer in homozygous asTFKI yolk sacs. No intact blood filled vessels can be detected anymore in homozygous asTFKI yolk sac en face preparations. (D) Quantification of erythrocyte and α-SMA⁺ cell number in E8.5–E10.5 yolk sacs of homozygous asTFKI and wt embryos ***p<0.001, **p<0.01.</p

Antibody-based targeting of alternatively spliced tissue factor: A new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma

Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to β1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread

Mutant IDH1 and thrombosis in gliomas

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE