Immune checkpoint regulators such as PD-L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In the present paper, we provide evidence that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch(®) system and found PD-L1((+)) CTCs in 11 patients (68.8%). The fraction of PD-L1((+)) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD-L1 on CTCs. The established CTC/PD-L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.

Because the availability of fish genomic data, the number of reported sequences for fish type II helical cytokines is rapidly growing, featuring different IFNs including virus-induced IFNs (IFNφ) and IFN-γ, and IL-10 with its related cytokines (IL-20, IL-22, and IL-26). Many candidate receptors exist for these cytokines and various authors have postulated which receptor chain would be involved in which functional receptor in fish. To date, only the receptor for zebrafish IFNφ1 has been identified functionally. Three genes encoding virus-induced IFNφs have been reported in zebrafish. In addition to these genes clustered on chromosome 3, we have identified a fourth IFNφ gene on chromosome 12. All these genes possess the intron-exon organization of mammalian λ IFNs. In the zebrafish larva, all induce the expression of reporter antiviral genes; protection in a viral challenge assay was observed for IFNφ1 and IFNφ2. Using a combination of gain- and loss-of-function experiments, we also show that all zebrafish IFNφs do not bind to the same receptor. Two subgroups of fish virus-induced IFNs have been defined based on conserved cysteines, and we find that this subdivision correlates with receptor usage. Both receptor complexes include a common short chain receptor (CRFB5) and a specific long chain receptor (CRFB1 or CRFB2).

We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptorindependent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide±lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process.To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.Keywords: antennapedia; cellular uptake; dithionite; lipidbinding; structure-activity relationship.The pAntp peptide, a 16 amino-acid segment corresponding to the third helix of the homeodomain of Antennapedia protein is known to be internalized by different cell lines [1±4]. Following conjugation, pAntp has been used as a vector for intracellular delivery of molecules such as peptides [5±8] and oligonucleotides [9,10]. The pAntp peptide is taken up into the cytoplasm and the nucleus of cells both at 37 8C and 4 8C indicating that this internalization does not occur by endocytosis [1]. In addition, the reverso, enantio, and retro-inverso forms of pAntp are also imported into cells demonstrating that the internalization is a receptor-independent process [11,12]. Phosphorus NMR studies show that the interaction between pAntp peptide and a dispersion of brain phospholipid induces the lamellar to inverted hexagonal phase transition [13]. It was therefore proposed that the pAntp peptide interacts with phospholipids and translocates through the bilayer by transient formation of inverted micelles in the membrane. This model implies that the peptide remains in an aqueous environment and may explain why hydrophilic compounds linked to the peptide are tr...

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system, an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points, CTCs were detected in 37%, 54.9% and 58.7% of patients using CellSearch, CellCollector and EPISPOT, respectively. The cumulative positivity rate of the three CTC assays was 81.3% (87/107) with 21.5% (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66% before RP to 34% after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p < 0.0001) and clinical tumor stage (p = 0.04), while the other assays showed no significant correlations. In conclusion, CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer.

A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein (P-gp), which is capable of lowering intracellular drug concentrations. To overcome this problem, we tested the capability of two peptide vectors that are able to cross cellular membranes to deliver doxorubicin in P-gp-expressing cells. The antitumor effect of peptide-conjugated doxorubicin was tested in human erythroleukemic (K562/ ADR) resistant cells. The conjugate showed potent dose-dependent inhibition of cell growth against K562/ADR cells as compared with doxorubicin alone. Doxorubicin exhibited IC50 concentrations of 65 microM in the resistant cells, whereas vectorized doxorubicin was more effective with IC50 concentrations of 3 microM. After treatment of the resistant cells with verapamil, the intracellular levels of doxorubicin were markedly increased and consequent cytotoxicity was improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement in the cell uptake nor in the cytotoxic effect of the conjugated doxorubicin, indicating that the conjugate bypasses the P-gp. Finally, we show by the in situ brain perfusion method in P-gp-deficient and competent mice that vectorized doxorubicin bypasses the P-gp present at the luminal site of the blood-brain barrier. These results indicate that vectorization of doxorubicin with peptide vectors is effective in overcoming multidrug resistance.

The zebrafish genome contains a large number of genes encoding potential cytokine receptor genes as judged by homology to mammalian receptors. The sequences are too divergent to allow unambiguous assignments of all receptors to specific cytokines, and only a few have been assigned functions by functional studies. Among receptors for class II helical cytokines—i.e., IFNs that include virus-induced Ifns (Ifn-ϕ) and type II Ifns (Ifn-γ), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)—only the Ifn-ϕ–specific complexes have been functionally identified, whereas the receptors for the two Ifn-γ (Ifn-γ1 and Ifn-γ2) are unknown. In this work, we identify conditions in which Ifn-γ1 and Ifn-γ2 (also called IFNG or IFN-γ and IFN-gammarel) are induced in fish larvae and adults. We use morpholino-mediated loss-of-function analysis to screen candidate receptors and identify the components of their receptor complexes. We find that Ifn-γ1 and Ifn-γ2 bind to different receptor complexes. The receptor complex for Ifn-γ2 includes cytokine receptor family B (Crfb)6 together with Crfb13 and Crfb17, whereas the receptor complex for Ifn-γ1 does not include Crfb6 or Crfb13 but includes Crfb17. We also show that of the two Jak2 paralogues present in the zebrafish Jak2a but not Jak2b is involved in the intracellular transmission of the Ifn-γ signal. These results shed new light on the evolution of the Ifn-γ signaling in fish and tetrapods and contribute toward an integrated view of the innate immune regulation in vertebrates.

A cDNA clone of 6.449 kb ch-TC)G (for colonic and hepatic tumor over-expressed gene) initially selected from various human libraries rind completed by 5' nipid amplification of cDNA ends (RACE) PCR is described. The original cDNA clone was extracted frotn an cxpression library constructed from a hurnan tumoral brain. This library WBS screened with an antibody rxised against the cytochrome P450tu that was shown to he ovcr-expressed in chemically induced mouse hepatic tumors. IJsing this D N A as ii probe, a full-length cDNA was characterized. Its nucleolide sequence shows no significant siiiiilarity with any of the gene sequences collected in the various DNA data bases. The translation of the larger open reading frame leads to a putative protein of 1972 amino acids (molecular mass = 218453 Da). Hybridization analyses 011 Southern blot and on inetaphase chromosomes indicate that this gene is present as a single copy in the genome and is localized on the shori arm of chromosome 11. ch-TOG transcripts are present in several human tissues. Over-expression of &TOG in neoplastic livcr and colon compared with the corresponding normal corresponding tissues is denionstrated. The level of the expression of ch-TOG transcripts was also studied in the various differentiation stages of the human colonic adenocarcinoma cell line Caco-2.