Cancer biomarkers detection using microstructured protein chip: implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins, and immobilization conditions (buffer, pH, concentration). We have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions was evaluated on the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that each antibody displays variable biological activities depending on the surface chemistry and on the immobilization procedure. Under optimized conditions, we can reach a limit of detection in tumor antigen as low as 10 pM. Our microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody on the same chip

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection

Evolutionary Dynamics of the Glycan Shield of the Human Immunodeficiency Virus Envelope during Natural Infection and Implications for Exposure of the 2G12 Epitope

Elucidation of the kinetics of exposure of neutralizing epitopes on the envelope of human immunodeficiency virus type 1 (HIV-1) during the course of infection may provide key information about how HIV escapes the immune system or why its envelope is such a poor immunogen to induce broadly efficient neutralizing antibodies. We analyzed the kinetics of exposure of the epitopes corresponding to the broadly neutralizing human monoclonal antibodies immunoglobulin G1b12 (IgG1b12), 2G12, and 2F5 at the quasispecies level during infection. We studied the antigenicity and sequences of 94 full-length envelope clones present during primary infection and at least 4 years later in four HIV-1 clade B-infected patients. No or only minor exposure differences were observed for the 2F5 and IgG1b12 epitopes between the early and late clones. Conversely, the envelope glycoproteins of the HIV-1 quasispecies present during primary infection did not expose the 2G12 neutralizing epitope, unlike those present after several years in three of the four patients. Sequence analysis revealed major differences at potential N-linked glycosylation sites between early and late clones, particularly at positions known to be important for 2G12 binding. Our study, in natural mutants, confirms that the glycosylation sites N295, N332, and N392 are essential for 2G12 binding. This study demonstrates the relationship between the evolving “glycan shield ” of HIV and the kinetics of exposure of the 2G12 epitope during the course of natural infection

Cancer biomarkers detection using 3D microstructured protein chip: Implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins. These parameters relate to surface chemistry and to immobilization conditions (printing buffers, washing etc.). In this study, we have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions were evaluated for the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that the biological activities of the immobilized antibodies were dependent on the surface chemistry and on the immobilization procedure. Optimal immobilization conditions were different for each antibody. Limit of detection in tumor antigen as low as 10 pM can reach under optimized conditions. Our 3D microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody-antigen system on the same chip

Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein

The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75^NTR and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75^NTR and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion

Evaluation in Rhesus Macaques of Tat and Rev-Targeted Immunization as a Preventive Vaccine against Mucosal Challenge with SHIV-BX08

International audienceRecent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of hu- man immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after path- ogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) ex- pressing HIV-1 tat (SFV- tat ) and HIV-1 rev (SFV- rev ) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev . A second group of monkey was primed with SFV- tat only and boosted with MVA- tat . A third group received a tat and rev DNA/ MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN- g production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, us- ing 10 MID 50 of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/ 18) of vacci- nated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected ani- mals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV pro- teins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies