Laurent Ramont

Background: Colorectal cancer (CRC) is the second leading cause of cancer‐related death worldwide. The French CRC screening campaign is based on fecal immunochemical tests (FIT), confirmed by colonoscopy, an invasive procedure with a poor participation rate. This study aimed to compare the expression of circulating exosomal proteins (MMP14, β1‐Integrin subunit, β3‐Integrin subunit, and α1(I) Collagen chain) in patients with CRC or adenomas.Methods: A total of 71 patients were recruited, including 24 controls (normal colonoscopy), 11 patients with adenoma, and 36 with CRC. Plasmatic exosomal protein expression was measured by western blot analysis and reported to either protein or exosome content.Results: The three groups were comparable regarding clinical characteristics. A significant difference was observed for MMP14 relative expression (p = 0.0007), MMP14 expression reported to exosomal protein content (p = 0.0003), and MMP14 expression reported to exosome content (p = 0.0005). These three parameters were significantly higher in patients with adenoma vs. control patients (p = 0.0013, p = 0.0004, and p = 0.0003, respectively). Only MMP14 relative intensity was significantly higher in the CRC group vs. the control group (p = 0.0018).Conclusions: Exosomal MMP14 is a promising early diagnostic biomarker for CRC and adenoma. These preliminary results warrant confirmation in larger studies using quantitative measurements such as ELISA or flow cytometry.

Biochemical and FTIR approaches to discriminate ovarian cancer cell-derived EVs.

The expression of glypicans in different hair follicle (HF) compartments is still poorly understood. Heparan sulfate proteoglycans (HSPGs) distribution in HF is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. Our previous study proposed a novel approach to assess hair histology and glypican-1 (GPC1) distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). We show in the present manuscript for the first time complementary data on the distribution of glypican-4 (GPC4) and glypican-6 (GPC6) in HF at different phases of the hair growth cycle using IR imaging. Findings were supported by Western blot assays focusing on the GPC4 and GPC6 expression in HFs. Like all proteoglycan features, the glypicans are characterized by a core protein to which sulfated and/or unsulfated glycosaminoglycan (GAG) chains are covalently linked. Our study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), GAG, and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western blot. Thus, in one analysis, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a chemical and label-free manner. From a dermatological point of view, IRSI may constitute a promising technique to study alopecia.

Small leucine-rich proteoglycans (SLRPs) are major regulators of extracellular matrix assembly and cell signaling. Lumican, a member of the SLRPs family, and its derived peptides were shown to possess antitumor activity by interacting directly with the catalytic domain of MMP-14 leading to the inhibition of its activity. The aim of the present report was to characterize by in silico three-dimensional (3D) modeling the structure and the dynamics of four SLRPs including their core protein and their specific polysaccharide chains to assess their capacity to bind to MMP-14 and to regulate its activity. Molecular docking experiments were performed to identify the specific amino acids of MMP-14 interacting with each of the four SLRPs. The inhibition of each SLRP (100 nM) on MMP-14 activity was measured and the constants of inhibition ( K_i) were evaluated. The impact of the number of glycan chains, structures, and dynamics of lumican on the interaction with MMP-14 was assessed by molecular dynamics simulations. Molecular docking analysis showed that all SLRPs bind to MMP-14 through their concave face, but in different regions of the catalytic domain of MMP-14. Each SLRPs inhibited significantly the MMP-14 activity. Finally, molecular dynamics showed the role of glycan chains in interaction with MMP-14 and shielding effect of SLRPs. Altogether, the results demonstrated that each SLRP exhibited inhibition of MMP-14 activity. However, the differential targeting of MMP-14 by the SLRPs was shown to be related not only to the core protein conformation but also to the glycan chain structures and dynamics.

Ovarian cancer remains one of the most fatal cancers due to a lack of robust screening methods of detection at early stages. Extracellular matrix (ECM) mediates interactions between cancer cells and their microenvironment via specific molecules. Lumican, a small leucine-rich proteoglycan (SLRP), maintains ECM integrity and inhibits both melanoma primary tumor development, as well as metastatic spread. The aim of this study was to analyze the effect of lumican on tumor growth of murine ovarian epithelial cancer. C57BL/6 wild type mice (n = 12) and lumican-deficient mice (n = 10) were subcutaneously injected with murine ovarian epithelial carcinoma ID8 cells, and then sacrificed after 18 days. Analysis of tumor volumes demonstrated an inhibitory effect of endogenous lumican on ovarian tumor growth. The ovarian primary tumors were subjected to histological and immunohistochemical staining using anti-lumican, anti-αv integrin, anti-CD31 and anti-cyclin D1 antibodies, and then further examined by label-free infrared spectral imaging (IRSI), second harmonic generation (SHG) and Picrosirius red staining. The IR tissue images allowed for the identification of different ECM tissue regions of the skin and the ovarian tumor. Moreover, IRSI showed a good correlation with αv integrin immunostaining and collagen organization within the tumor. Our results demonstrate that lumican inhibits ovarian cancer growth mainly by altering collagen fibrilogenesis.

Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), displays anti-tumor properties through its direct interaction with MMP-14. Lumican-derived peptides, such as lumcorin (17 amino acids) or L9M (10 amino acids), are able to inhibit the proteolytic activity of MMP-14 and melanoma progression. This work aimed to visualize the interactions of lumican-derived peptides and MMP-14. Molecular modeling was used to characterize the interactions between lumican-derived peptides, such as lumcorin, L9M, and cyclic L9M (L9Mc, 12 amino acids), and MMP-14. The interaction of L9Mc with MMP-14 was preferential with the MT-Loop domain while lumcorin interacted more with the catalytic site. Key residues in the MMP-14 amino acid sequence were highlighted for the interaction between the inhibitory SLRP-derived peptides and MMP-14. In order to validate the in silico data, MMP-14 activity and migration assays were performed using murine B16F1 and human HT-144 melanoma cells. In contrast to the HT-144 melanoma cell line, L9Mc significantly inhibited the migration of B16F1 cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. L9Mc significantly inhibited the proliferation of B16F1 but not of HT-144 cells in vitro and primary melanoma tumor growth in vivo. Thus, the site of interaction between the domains of MMP-14 and lumcorin or L9Mc were different, which might explain the differences in the inhibitory effect of MMP-14 activity. Altogether, the biological assays validated the prediction of the in silico study. Possible and feasible improvements include molecular dynamics results.

Procalcitonin (PCT) may be useful for early risk stratification in the emergency department (ED), but the transposition of published data to routine emergency practice is sometimes limited. An observational retrospective study was conducted in the adult ED of the Reims University Hospital (France). Over one year, 852 patients suspected of infection were included, of mean age 61.7 years (SD: 22.6), and 624 (73.2%) were hospitalized following ED visit. Overall, 82 (9.6%) patients died during their hospitalization with an odds ratio (OR) of 5.10 (95% CI: 2.19–11.87) for PCT ≥ 0.5, in multivariate logistic regression analyses. Moreover, 78 (9.2%) patients were admitted to an ICU, 74 (8.7%) had attributable bacteremia, and 56 (6.6%) evolved toward septic shock with an OR of 4.37 (2.08–9.16), 6.38 (2.67–15.24), and 6.38 (2.41–16.86), respectively, for PCT ≥ 0.5. The highest discriminatory values were found for patients with age <65 years, but PCT lost its discrimination power for in-hospital mortality in patients with a bronchopulmonary infection site or a temperature ≥37.8°C and for ICU admission in patients with severe clinical presentations. PCT could be helpful in risk stratification, but several limitations must be considered, including being sometimes outperformed by a simple clinical examination.

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.

Tetrastatin, a 230 amino acid sequence from collagen IV, was previously demonstrated to inhibit melanoma progression. In the present paper, we identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin in vivo and in vitro on melanoma cell proliferation, migration and invasion. We demonstrated that QS-13 binds to SK-MEL-28 melanoma cells through the α_vβ₃ integrin using blocking antibody and β3 integrin subunit siRNAs strategies. Relevant QS-13 conformations were extracted from molecular dynamics simulations and their interactions with α_Vβ₃ integrin were analyzed by docking experiments to determine the binding areas and the QS-13 amino acids crucial for the binding. The in silico results were confirmed by in vitro experiments. Indeed, QS-13 binding to SK-MEL-28 was dependent on the presence of a disulfide-bound as shown by mass spectroscopy and the binding site on α_Vβ₃ was located in close vicinity to the RGD binding site. QS-13 binding inhibits the FAK/PI₃K/Akt pathway, a transduction pathway that is largely involved in tumor cell proliferation and migration. Taken together, our results demonstrate that the QS-13 peptide binds α_vβ₃ integrin in a conformation-dependent manner and is a potent antitumor agent that could target cancer cells through α_Vβ₃.