The Role of a Proprotein Convertase Inhibitor in Reactivation of Tumor-Associated Macrophages and Inhibition of Glioma Growth.

Tumors are characterized by the presence of malignant and non-malignant cells, such as immune cells including macrophages, which are preponderant. Macrophages impact the efficacy of chemotherapy and may lead to drug resistance. In this context and based on our previous work, we investigated the ability to reactivate macrophages by using a proprotein convertases inhibitor. Proprotein convertases process immature proteins into functional proteins, with several of them having a role in immune cell activation and tumorigenesis. Macrophages were treated with a peptidomimetic inhibitor targeting furin, PC1/3, PC4, PACE4, and PC5/6. Their anti-glioma activity was analyzed by mass spectrometry-based proteomics and viability assays in 2D and 3D in vitro cultures. Comparison with temozolomide, the drug used for glioma therapy, established that the inhibitor was more efficient for the reduction of cancer cell density. The inhibitor was also able to reactivate macrophages through the secretion of several immune factors with antitumor properties. Moreover, two proteins considered as good glioma patient survival indicators were also identified in 3D cultures treated with the inhibitor. Finally, we established that the proprotein convertases inhibitor has a dual role as an anti-glioma drug and anti-tumoral macrophage reactivation drug. This strategy could be used together with chemotherapy to increase therapy efficacy in glioma.

Spatially-resolved protein surface microsampling from tissue sections using liquid extraction surface analysis.

Tissue microenvironment characterization presents a challenge for a better understanding of the full complexity of a pathology. Unfortunately, making a precise "picture" of the disease needs an efficient microsampling method coupled to an accurate localization for performing region-dependent proteomics. Here, we present a method that enables rapid and reproducible extraction of proteins from a tissue section to analyze a specific region at a millimeter scale. The method used a liquid-microjunction extraction with conventional detergent solution for proteomics analysis. We successfully performed immunoblotting experiments and showed the possibility to retrieve and identify more than 1400 proteins from a 1-mm diameter spot size on tissue sections with a high degree of reproducibility both qualitatively and quantitatively. Moreover, the small size of the extracted region achieved by this sampling method allows the possibility to perform multiple extractions on different tissue section points. Ten points on a sagittal rat brain tissue section were analyzed and the measured proteins clearly distinguished the different parts of the brain, thus permitting precise functional mapping. We thus demonstrate that with this technology, it is possible to map the tissue microenvironment and gain an understanding of the molecular mechanisms at millimeter resolution.

Lipidomics for clinical diagnosis: Dye-Assisted Laser Desorption/Ionization (DALDI) method for lipids detection in MALDI mass spectrometry imaging.

Lipid-based biomarkers for research and diagnosis are rapidly emerging to unpack the basis of person-to-person and population variations in disease susceptibility, drug and nutritional responses, to name but a few. Hence, with the advent of MALDI Mass Spectrometry Imaging, lipids have begun to be investigated intensively. However, lipids are highly mobile during tissue preparation, and are soluble in the solvent used for matrix preparation or in the fixing fluid such as formalin, resulting in substantial delocalization. In the present article, we investigated as another alternative, the possibility of using specific dyes that can absorb UV wavelengths, in order to desorb the lipids specifically from tissue sections, and are known to immobilize them in tissues. Indeed, after lipid insolubilization with chromate solution or chemical fixation with osmium tetroxide, heterocyclic-based dyes can be directly used without matrix. Taking into account the fact that some dyes have this matrix-free capability, we identified particular dyes dedicated to histological staining of lipids that could be used with MALDI mass spectrometry imaging. We stained tissue sections with either Sudan Black B, Nile Blue A, or Oil Red O. An important advantage of this assay relies on its compatibility with usual practices of histopathological investigation of lipids. As a new method, DALDI stands for Dye-Assisted Laser Desorption Ionization and allows for future clinical and histopathological applications using routine histological protocols. Additionally, this novel methodology was validated in human ovarian cancer biopsies to demonstrate its use as a suitable procedure, for histological diagnosis in lipidomics field.

HFIP extraction followed by 2D CTAB/SDS-PAGE separation: a new methodology for protein identification from tissue sections after MALDI mass spectrometry profiling for personalized medicine research.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and profiling technology have become the easiest methods for quickly accessing the protein composition of a tissue area. Unfortunately, the demand for the identification of these proteins remains unmet. To overcome this bottleneck, we combined several strategies to identify the proteins detected via MALDI profiling including on-tissue protein extraction using hexafluoroIsopropanol (1,1,1,3,3,3-hexafluoro-2-propanol, HFIP) coupled with two-dimensional cetyl trimethylammonium bromide/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D CTAB/SDS-PAGE) for separation followed by trypsin digestion and MALDI-MS analyses for identification. This strategy was compared with an on-tissue bottom-up strategy that we previously developed. The data reflect the complementarity of the approaches. An increase in the number of specific proteins identified has been established. This approach demonstrates the potential of adapted extraction procedures and the combination of parallel identification approaches for personalized medicine applications. The anatomical context provides important insight into identifying biomarkers and may be considered a first step for tissue-based biomarker research, as well as the extemporaneous examination of biopsies during surgery.

Spectroimmunohistochemistry: a novel form of MALDI mass spectrometry imaging coupled to immunohistochemistry for tracking antibodies.

MALDI mass spectrometry imaging (MALDI-MSI) is currently used for clinical applications, such as biomarker identification, particularly for the study of solid tumors. The ability to map specific compounds that have been determined to be biomarkers and therapeutic targets is relevant for the evaluation of the efficacy of targeted therapies. This article describes a new method called Spectro-ImmunoHistoChemistry (SIHC), which combines the use of specific antibodies against markers and mass spectrometric imaging in the MS/MS mode. SIHC is based on direct primary antibody-antigen recognition, trypsin digestion of the antibody overlaying the markers of interest in the tissue section, and MALDI-MSI of the tryptic peptides generated from the antibody. This approach has both clinical and pharmacological applications. First, it can be used as a cross-validation method to monitor the presence specifically of a marker in a tissue section. Second, SIHC could potentially be used as a novel technology for tracking specific antibodies after in vivo injection for anti-cancer treatments. Additionally, SIHC could enable novel clinical applications of MSI, such as monitoring the efficacy of cytotoxic antibody treatments.

Proteomic analyses of serous and endometrioid epithelial ovarian cancers - cases studies - molecular insights of a possible histological etiology of serous ovarian cancer.

PURPOSE: Epithelial ovarian carcinogenesis may occur de novo on the surface of ovarian mesothelial epithelial cells or from cells originating in other organs. Foreign Müllerian cell intrusion into the ovarian environment has been hypothesized to explain the latter scenario. In this study, MALDI MS profiling technology was used to provide molecular insights regarding these potentially different mechanisms. EXPERIMENTAL DESIGN: Using MALDI MS profiling, the molecular disease signatures were established in their anatomical context. MALDI MS profiling was used on serous and endometrioid cancer biopsies to investigate cases of epithelial ovarian cancer. We then applied bioinformatic methods and identification strategies on the LC-MS/MS analyses of extracts from digested formalin-fixed, paraffin-embedded tissues. Extracts from selected regions (i.e. serous ovarian adenocarcinoma, fallopian tube serous adenocarcinoma, endometrioid ovarian cancer, benign endometrium, and benign ovarian tissues) were performed, and peptide digests were subjected to LC-MS/MS analysis. RESULTS: Comparison of the proteins identified from benign endometrium or three ovarian cancer types (i.e. serous ovarian adenocarcinoma, endometrioid ovarian adenocarcinoma, and serous fallopian tube adenocarcinoma) provided new evidence of a possible correlation between the fallopian tubes and serous ovarian adenocarcinoma. Here, we propose a workflow consisting of the comparison of multiple tissues in their anatomical context in an individual patient. CONCLUSION AND CLINICAL RELEVANCE: The present study provides new insights into the molecular similarities between these two tissues and an assessment of highly specific markers for an individualized patient diagnosis and care.

Involvement of nitric oxide through endocannabinoids release in microglia activation during the course of CNS regeneration in the medicinal leech.

The medicinal leech is notable for its capacity to regenerate its central nervous system (CNS) following mechanical trauma. Using an electrochemical nitric oxide (NO)-selective electrode to measure NO levels, we found that the time course of NO release in the injured leech CNS is partially under the control of endocannabinoids, namely, N-arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol (2-AG). Relative quantification of these endocannabinoids was performed by stable isotope dilution (2AGd8 and AAEd8) coupled to mass spectrometry in course of regeneration process or adenosine triphosphate (ATP) treatment. Data show that 2-AG levels rose to a maximum about 30 min after injury or ATP treatment, and returned to baseline levels 4 h after injury. In same conditions, AEA levels also rapidly (within 5 min) dropped after injury or ATP treatment to the nerve cord, but did not fully return to baseline levels within 4 h of injury. In correlation with these data, chemoattraction activities of endocannabinoids on isolated leech microglial cells have been shown in vitro and in vivo reflecting that control over NO production is accompanied by the controlled chemoattraction of microglia directed from the periphery to the lesion site for neuronal repair purposes. Taken together, our results show that in the leech, after injury concurrent with ATP production, purinergic receptor activation, NO production, microglia recruitment, and accumulation to lesion site, a fine imbalance occurs in the endocannabinoid system. These events can bring explanations about the ability of the leech CNS to regenerate after a trauma and the key role of endocannabinoids in this phenomenon.

Microproteomics by liquid extraction surface analysis: application to FFPE tissue to study the fimbria region of tubo-ovarian cancer.

PURPOSE: We have developed a new method for rapid analysis of a specific region on formalin fixed and paraffin embedded (FFPE) tissue sections. This method combines advantages of direct tissue MS analysis keeping histological information and conventional proteomics approaches for confident identification of proteins in complex sample. EXPERIMENTAL DESIGN: After histological annotation, heat-induced antigen retrieval is performed on FFPE tissue. Using a chemical inkjet printer, trypsin is deposited on discrete regions of less than 1 mm². After protein digestion, a liquid extraction is performed to retrieve all the peptides. Data coming from identification of proteins in cancer and benign region are compared. RESULTS: In total, 3649 unique peptides were identified (with a peptide strict false discovery rate less than 1%) corresponding to 983 and 792 nonredundant protein groups identified in benign and cancer region, respectively. A total of 123 protein groups are found only in cancer region and 315 are specific to the benign part. From these data, it has been possible to obtain different important signaling pathways involved in cancer processes and some proteins already known as biomarkers. CONCLUSIONS AND CLINICAL RELEVANCE: This new approach using a combination of localized on-tissue protein digestion and liquid microextraction followed by LC-MS/MS analysis is useful for advancing our understanding of cancer biology. It is a rapid and innovative technique that will contribute positively to clinical proteomics.

Ovarian cancer molecular pathology.

Ovarian cancer (OVC) is the fourth leading cause of cancer mortality among women in Europe and the United States. Its early detection is difficult due to the lack of specificity of clinical symptoms. Unfortunately, late diagnosis is a major contributor to the poor survival rates for OVC, which can be attributed to the lack of specific sets of markers. Aside from patients sharing a strong family history of ovarian and breast cancer, including the BRCA1 and BRCA2 tumor suppressor genes mutations, the most used biomarker is the Cancer-antigen 125 (CA-125). CA-125 has a sensitivity of 80 % and a specificity of 97 % in epithelial cancer (stage III or IV). However, its sensitivity is 30 % in stage I cancer, as its increase is linked to several physiological phenomena and benign situations. CA-125 is particularly useful for at-risk population diagnosis and to assess response to treatment. It is clear that alone, CA-125 is inadequate as a biomarker for OVC diagnosis. There is an unmet need to identify additional biomarkers. Novel and more sensitive proteomic strategies such as MALDI mass spectrometry imaging studies are well suited to identify better markers for both diagnosis and prognosis. In the present review, we will focus on such proteomic strategies in regards to OVC signaling pathways, OVC development and escape from the immune response.

The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling.

This study reports on the C-terminal fragment of the 11S proteasome activator complex (PA28 or Reg alpha), a novel ovarian-specific biomarker of early and late stages of ovarian cancer (OVC) relapse, in patient biopsies after chemotherapy. A total of 179 tissue samples were analyzed: 8 stage I, 55 stage III-IV, 10 relapsed serous carcinomas, 25 mucinous carcinomas and 12 borderline and 68 benign ovarian tissue samples. This fragment was detected by MALDI mass spectrometry profiling in conjunction with a novel extraction method using hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propanol; HFIP) solvents for protein solubilization and by immunohistochemistry using a specific antibody directed against the C-terminal fragment of PA28. Due to its specific cellular localization, this fragment is a suitable candidate for early OVC diagnosis, patient prognosis and follow-up during therapy and discriminating borderline cancers. Statistical analyses performed for this marker at different OVC stages reflect a prevalence of 77.66 ± 8.77 % (with a correlation coefficient value p < 0.001 of 0.601 between OVC and benign tissue). This marker presents a prevalence of 88 % in the case of tumor relapse and is detected at 80.5 % in stage I and 81.25 % ± 1.06 in stage III-IV of OVC. The correlation value for the different OVC stages is p < 0.001 of 0.998. Taken together, this report constitutes the first evidence of a novel OVC-specific marker.

Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech.

BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

MALDI imaging mass spectrometry in ovarian cancer for tracking, identifying, and validating biomarkers.

BACKGROUND: Among biomarkers, cancer-antigen 125 (CA-125) is the most studied. We propose an analytical tool to track ovarian carcinoma biomarkers, that is, the MALDI mass spectrometry imaging. MATERIAL/METHODS: Ovarian carcinomas and benign ovaries were directly analyzed by MALDI-TOF-MS. After automatic profiling and mass spectrometry imaging analyses, hierarchical clustering based on principal component analysis in nonsupervised mode was carried out. On the same samples, preparations were performed to investigate peptides, then proteins, followed by high mass proteins, in an automatic profiling to specific signatures for diagnosis. Using tissue bottom-up strategy on tissue digestion, and mass spectrometry imaging after by shotgun sequencing by nalano-LC-IT-MS in MS/MS mode from washing samples from on tissue digested peptides, several biomarkers were found. RESULTS: A list of specific biomarkers from the ovarian carcinoma regions was obtained and classified as proteins associated with cell proliferation, involved in immune response modulation, signaling to the cytoskeleton, and tumor progression. These specific biomarkers were then validated by immunocytochemistry using Tag-mass technology, cell biology, Western blot, and by PCR (using SKOV-3 ovarian epithelial cancer cells). A link between the immune regulation (innate immunity, tolerance) and virus cause is also discussed. CONCLUSIONS: From the biomarkers identified, proteins involved in immune response modulation and cell proliferation have been pointed out in this study. Two new markers have been identified using such a strategy, that is, fragment C-terminal of the PSME1 (Reg-Alpha) and mucin-9.

Cathepsin L and cystatin B gene expression discriminates immune coelomic cells in the leech Theromyzon tessulatum.

Previous studies evidenced that cystatin B-like gene is specifically expressed and induced in large circulating coelomic cells following bacterial challenge in the leech Theromyzon tessulatum. In order to understand the role of that cysteine proteinase inhibitor during immune response, we investigated the existence of members of cathepsin family. We cloned a cathepsin L-like gene and studied its tissue distribution. Immunohistochemical studies using anti-cathepsin L and anti-cystatin B antibodies and ultrastructural results demonstrated the presence of three distinct coelomic cell populations: (1) the chloragocytes, which were initially defined as large coelomocytes, (2) the granular amoebocytes and (3) small coelomic cells. Among those cells, while chloragocytes contain cystatin B and cathepsin L, granular amoebocytes contain only cathepsin L and the third cell population contains neither cathepsin nor inhibitor. Finally, results evidenced that cathepsin L immunopositive granular amoebocytes are chemoattracted to the site of injury and phagocyte bacteria.