Philippe Kischel

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Versican overexpression in human breast cancer lesions: Known and new isoforms for stromal tumor targeting

Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant stroma, proteoglycans provide a reservoir of potential new targets for anticancer therapies, because they can serve to convey toxic payloads in the close proximity of cancer cells and subsequently destroy them. In this context, versican, a proteoglycan largely overexpressed in several solid cancers, bears the potential to be such an ideal target. As 4 main versican isoforms have been characterized, we sought to determine which isoform could represent the best target in human breast cancer. We used a series of 10 primary breast cancer lesions that were characterized as overexpressing the versican protein, when compared with matched normal breast tissues, using shotgun mass spectrometry and immunohistochemistry experiments. Quantitative polymerase chain reaction and western‐blotting experiments were used to evaluate versican isoform expression in breast cancer/normal tissue pairs for which ARN quality was excellent. All known isoforms were significantly overexpressed in the malignant lesions, both at the mRNA and at the protein levels. In the course of this study, we also identified and cloned a new alternatively spliced versican isoform, referred to as V4, which was also found to be upregulated in human breast cancer. This study provides for the first time a comprehensive mRNA and protein analysis of versican isoforms expression in human breast tissues, and offers insights into which therapeutic strategy would be best suited to target versican in human breast cancer lesions.

Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody‐based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry‐based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty‐seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody‐based imaging and therapeutic biomolecular strategies.

Expression and functional implications of troponin T isoforms in soleus muscle fibers of rat after unloading

Abstract. The expression pattern of troponin T (TnT) isoforms was studied in rat soleus muscle fibers in control and after hindlimb unloading (HU) conditions. To determine the functional consequence of TnT expression, the fibers were also examined for their calcium activation characteristics. With regard to TnT expression, four populations of fibers were distinguished in control muscle. Slow fibers expressing only slow isoforms of TnT (TnT1s, 2s, 3s ) were predominant (54%). Hybrid slow fibers (16%) differed from slow fibers by the additional expression of two TnTf isoforms. Hybrid fast fibers (22%) expressed slow and fast isoforms of TnT while fast fibers (8%) expressed only fast TnT isoforms. The expression of the other regulatory protein isoforms was checked for each population. The contractile experiments revealed steeper slopes of the tension/pCa relationship from hybrid slow fibers expressing fast TnT in a completely slow molecular environment. The expression of TnTs in hybrid fast fibers did not modulate the intrinsic co-operativity. After HU, the fast population was increased and reached 55%. The slow population decreased to 41% and a very small amount of hybrid slow fibers remained (4%). These data demonstrated the implication of TnT isoforms in the calcium activation properties and, more particularly, in the modulation of co-operativity within the myofibrillar lattice. Regulation of TnT expression appeared as a very fast and complete process compared to moderate changes of TnC and TnI.

Identification of accessible human cancer biomarkers using ex vivo chemical proteomic strategies.

One promising avenue towards the development of more selective, better anticancer drugs lies in the targeted delivery of bioactive compounds to the tumor environment by means of binding molecules specific for tumor-associated biomarkers. Eligibility of such markers for therapeutic ideally use three criteria: accessibility from the bloodstream; expression at sufficient level, and no (or much lower) expression in normal tissues. Most current discovery strategies (such as biomarker searching into body fluids) provide no clue as to whether proteins of interest are accessible, in human tissues, to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies. To address this limitation, our group recently developed two methodologies based on chemical proteomic modifications, enabling the discovery of proteins accessible from the bloodstream and the extracellular space in human pathological tissues. In this review, we will discuss the potential benefits of these methodologies for the fast discovery of therapeutically valuable biomarkers.

Identification of stromal proteins overexpressed in nodular sclerosis Hodgkin lymphoma

Hodgkin lymphoma (HL) represents a category of lymphoid neoplasms with unique features, notably the usual scarcity of tumour cells in involved tissues. The most common subtype of classical HL, nodular sclerosis HL, characteristically comprises abundant fibrous tissue stroma. Little information is available about the protein composition of the stromal environment from HL. Moreover, the identification of valid protein targets, specifically and abundantly expressed in HL, would be of utmost importance for targeted therapies and imaging, yet the biomarkers must necessarily be accessible from the bloodstream. To characterize HL stroma and to identify potentially accessible proteins, we used a chemical proteomic approach, consisting in the labelling of accessible proteins and their subsequent purification and identification by mass spectrometry. We performed an analysis of potentially accessible proteins in lymph node biopsies from HL and reactive lymphoid tissues, and in total, more than 1400 proteins were identified in 7 samples. We have identified several extracellular matrix proteins overexpressed in HL, such as versican, fibulin-1, periostin, and other proteins such as S100-A8. These proteins were validated by immunohistochemistry on a larger series of biopsy samples, and bear the potential to become targets for antibody-based anti-cancer therapies.

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.