Development of novel tools for the diagnosis and prognosis of pheochromocytoma using peptide marker immunoassay and gene expression profiling approaches

Pheochromocytomas (PHEOs) are rare catecholamine-producing neoplasias that arise from chromaffin cells of the adrenal medulla or from extra-adrenal locations. These neuroendocrine tumors are usually benign, but may also present as or develop into a malignancy. There are currently no means to predict or to cure malignant tumors which have a poor prognosis. We have recently validated several assays for the measurement of peptides derived from chromogranin A (CgA) and secretogranin II (SgII) in order to determine whether these secreted neuroendocrine products could provide useful, complementary markers for the diagnosis and prognosis of PHEOs. Both the CgA-derived peptide WE14 and the SgII-derived peptide EM66 proved to be sensitive circulating markers for the diagnosis of PHEO. In addition, much higher EM66 levels were measured in benign than in malignant tumoral tissues, suggesting that this peptide could represent a valuable tool for the prognosis of PHEO. We have also initiated a comparative microarray study of benign and malignant PHEOs, which allowed the identification of a set of about 100 genes that were differentially expressed and best discriminated the two types of tumors. A large majority of these genes were expressed at lower levels in the malignant disease and were associated with various characteristics of chromaffin cells, such as hormone secretion signaling and machinery, peptide maturation, and cellular morphology. Altogether, these studies provide novel tools for the management of PHEO, and new insights for the understanding of tumorigenesis in chromaffin cells, which may offer potential therapeutic strategies

Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival

International audienc

Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Abstract Background Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). Methods Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. Results Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. Conclusion Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target

Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas.

International audienceThe aim of the present study was to compare the expression levels of secretogranin II (SgII), prohormone convertases (PC)1 and PC2, and the proteolytic processing of SgII in benign versus malignant pheochromocytomas. Quantitative (Q)-PCR experiments indicated that SgII, PC1, and PC2 mRNAs were overexpressed in pheochromocytoma compared to non-tumoral chromaffin cells (P<0.001) and in benign compared to malignant tumors (P<0.01). Western blot analysis using a human SgII antiserum revealed the occurrence of a 97-kDa band corresponding to the expected size of SgII, with significantly higher quantities in benign than in malignant tumors (P<0.05). Using antisera directed against sequential regions of SgII (N-terminal, secretoneurin [SN], EM66, internal, and C-terminal sequences), we observed distinct processing profiles between benign and malignant pheochromocytomas. In contrast, using PC1 and PC2 antisera no differences between the two types of tumors were found. RIA measurement showed that EM66 median values between benign and malignant chromaffin cell tumors were significantly different (128.5 vs. 6.3 ng/mg protein, respectively; P<0.001). Taken together, these results indicate that, in pheochromocytoma, malignancy is associated with reduced PC1, PC2, and SgII mRNA expression and decreased levels of processing products of SgII, in line with the low concentrations of EM66 that occur in malignant tumors. These data support the notion that SgII-processing products, such as EM66, could represent prognostic markers of pheochromocytomas

Additional file 1: Table S1. of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Distribution of patients with mCRC according to the tumor molecular subtype. (DOCX 33 kb

Additional file 2: Figure S1. of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

CLDN1 gene (222549_at.) expression. a, in 17 normal colorectal mucosa (NM), 20 primary tumor (PT) samples and 19 hepatic metastases (HM); *** = p < 0.0001 (Kruskall Wallis/Dunn’s test). b, Ratio between CLDN1 expression in PT and CLDN1 expression in NM for the 15 paired NM and PT samples from patients with mCRC. Data from the Affymetrix GeneChip Human Genome U133 Array Set (GSE 62322). Figure S2. The 6F6 mAb is specific for CLDN1. a, Reactivity of the hybridoma supernatant 6F6 against CLDN1. Western blotting of protein extracts from SW480 cells stably transfected with CLDN1 and from SW620 cells transduced with shLUC (control) or ShCLDN1. FACS histograms show the binding of the hybridoma supernatant to CLDN1-positive cell lines (SW480-CLDN1 and SW620shLUC) (■), negative control (-----), CLDN1-negative cell lines (―). b, Immunofluorescence experiments in cells that express CLDN1 (SW480-CLDN1) or transfected with empty vector (SW480-pcDNA) using the 6 F6 mAb as primary antibody (green). Images were recorded using a 63X NA objective on a Leica inverted microscope. c, Surface plasmon resonance measurements of the interaction of 6F6 or of an irrelevant mAb (Irr) with membrane extracts from SW620 cells that express CLDN1. d, Cross-reactivity analysis of the 6F6 mAb towards other CLDN proteins. Top: The expression of the various CLDN proteins (as indicated) in cell lysates from parental or CLDN-transfected SW480 cells was tested by western blotting using the relevant antibodies; Bottom: FACS histograms of 6 F6 binding (10 μg/mL) to parental or CLDN-transfected SW480 cells. Gray, 6 F6 mAb; dotted line, no antibody; black line, irrelevant mAb. Figure S3. CLDN1 is expressed in various cancer cell lines a, FACS histograms of the 6F6 mAb binding (gray histogram) to different cancer cell lines (pancreatic cancer: PANC-1, BXPC-3; ovarian cancer: SKOV-3, IGROV-1; hepatocarcinoma: HUH7). b, Quantification of total CLDN1 expression in the cell lines used in a by western blotting using the anti-CLDN1 polyclonal antibody JAY-8. c, CLDN1 mRNA expression in cell lines from the Cancer Cell Line Encyclopedia ( http://www.broadinstitute.org/ccle ). Figure S4. Detection of apoptosis in Difi spheroids using the Celigo™ imaging system and the NucView™ 488 cell membrane-permeable fluorogenic caspase-3 substrate. Difi cells were seeded at a density of 104/ml in FluoroBrite™ DMEM supplemented with 10% fetal bovine serum and incubated or not (NT) with 100 μg/ml of the 6 F6 mAb, the anti-EGFR cetuximab (cetux) or an irrelevant mAb (IRR). The caspase-3 substrate was added (5 μM) at the same time. Images were acquired at day 5. The bright-field and caspase 3 (green) images were merged (top panels) and the histogram (lower panel) represents the mean fluorescence intensity; * = p < 0.05 (t-test). Figure S5. Effects of the 6F6 mAb on cancer cell migration in vitro. a, Wound healing assay: confluent SW620 cell monolayers were scratched and then grown in the presence or not (NT) of 100 μg/ml of the 6 F6 mAb or irrelevant antibody (IRR). Images were captured at day 0 (D0) and day 5 (D5) after wounding. b, Cell migration assay in Boyden chambers. Caco2 cells were pre-incubated or not (NT) with 100 μg/ml of 6F6 or irrelevant (IRR) mAb. Data used for statistical analysis were from at least three independent experiments; **p < 0.01 (Kruskall Wallis/Dunn’s test). (PPTX 2370 kb

Additional file 3: of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Supplementary methods: Flow cytometry experiments. Immunofluorescence studies. Surface plasmon resonance measurements. Cell migration assays. Apoptosis assay. (DOCX 37 kb