Patent Application
20090163414
The present invention refers to an in-vitro method for screening specific disease biological markers accessible from the extra cellular space in pathologic tissues.
The discovery of biological markers clinically suited for the accurate detection and selective treatment of diseases represents a major medical challenge. Targeting pathologic tissues without affecting their normal counterparts is one of the most promising approaches for improving treatment efficiency and safety (Wu, A. M. & Senter, P. D. Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23, 1137-1146 (2005); Adams, G. P. & Weiner, L. M. Monoclonal antibody therapy of cancer Nat Biotechnol 23, 1147-1157 (2005); Carter, P. Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 1, 118-129 (2001)). Indisputably, such advances would represent a breakthrough for the therapy of cancer and other diseases. Hence, the identification of valid biological markers including proteins specifically expressed in diseased tissues, has become of utmost importance (Hortobagyi, G. N. Opportunities and challenges in the development of targeted therapies. Semin Oncol 31, 21-27 (2004); Neri, D. & Bicknell, R. Tumour vascular targeting. Nat Rev Cancer 5, 436-446 (2005)). Yet, even the most recent target discovery strategies based on state-of-the-art, high-throughput technologies have not addressed the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human monoclonal antibodies bound to bioactive molecules (Adams, G. P. & Weiner, L. M. Monoclonal antibody therapy of cancer. Nat Biotechnol 23, 1147-1157 (2005); Neri, D. & Bicknell, R. Tumour vascular targeting. Nat Rev Cancer 5, 436-446 (2005).
The identification of biological markers unique to specific pathologic processes would be most valuable for the accurate detection (e.g. by imaging studies) and selective therapy of diseases including cancer. For instance, patients suffering from cancer would certainly benefit from such developments. Indeed, chemotherapeutic agents, usually designed to take advantage of tumor cell characteristics such as high proliferation rates, unfortunately also target normal cycling cells including hematopoietic progenitors. The targeted delivery of these drugs and other bioactive molecules (e.g. radioisotopes, cytokines) to the tumor microenvironment (e.g. proteins specifically expressed in the stromal or vascular compartment of the tumor) by means of binding molecules such as recombinant human antibodies, would represent a considerable therapeutic improvement. This selective strategy would increase the amount of drugs reaching the tumor with little or no toxicity to the host's healthy tissues. The recent development of high-throughput proteomic technologies such as mass spectrometry have facilitated the rapid and accurate identification of small, but complex biological sample mixtures, overcoming many limitations of two-dimensional gel electrophoresis for proteome analysis (Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J Mass Spectrom 36, 1083-91 (2001)) and making target identification easier than ever. The recent development of this high-throughput proteomic technology holds great promise for speeding up the discovery of novel targets, notably in cancer research. For example, gel-free shotgun tandem mass spectrometry has been recently used to compare global protein expression profiles in human mammary epithelial normal and cancer cell lines (Sandhu, C. et al., Global protein shotgun expression profiling of proliferating mcf-7 breast cancer cells. J Proteome Res 4, 674-89 (2005)). Unfortunately, a significant pitfall associated with such an approach is that it provides no clue as to whether proteins of interest are accessible to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies, in human tissues. Indeed, specific, yet poorly accessible proteins expressed in pathologic tissues are expected to be of little value for the development of antibody-based anti-cancer therapies. Strategies that would unveil disease biological markers not only specifically expressed in pathologic tissues but also accessible from the extracellular fluid would help overcome this limitation.
Further, methods are known allowing protein labelling either by in vivo terminal perfusion or by ex vivo perfusion of human pathologic organs. However, the perfusion technique is restricted to experimental animals (e.g. rodents) or surgically resected organs vascularized by a catheterizable artery (e.g. kidney), which excludes many types of pathologic tissues from investigation, namely those which can not be perfused.
The object of the present invention therefore was to provide a new, simple, quick and efficient method to identify, in human tissues originating from biopsies and non-perfusable organs (e.g. cancer lesions present in mastectomy or radical prostatectomy specimens), specific disease biological markers accessible from the extracellular space.
The object is solved by an in vitro method for screening specific disease biological markers which are accessible from the extra cellular space in pathologic tissues for high-affinity ligands comprising the steps of:
It is understood that the in the step of immersing a native pathologic tissue sample in a solution containing a labelling reagent for labelling proteins, only accessible proteins are labelled by the labelling reagent; while non-accessible proteins are not or essentially not labelled. In a preferred embodiment of the method it is judged that said labelled protein(s) is/are accessible for high-affinity ligands from the extra cellular space in the native tissue, most preferred is/are accessible for high-affinity ligands from the extra cellular space in vivo.
The present invention therefore provides a new chemical proteomic method for the reliable identification of target proteins from diseased tissues, which in vivo are accessible from the extra cellular fluid and thus from the bloodstream. By applying this procedure, for example, to small, surgically obtained samples of normal and cancerous human breast tissues, a series of accessible proteins selectively expressed in breast cancer are identified. Some of these breast cancer-associated antigens correspond to extracellular proteins including extra cellular matrix and secreted proteins, and appear to be interesting targets for antibody-based anti-cancer treatments. This powerful technology can theoretically be applied to virtually any tissue and pathologic condition and may become the basis of custom-made target therapies.
According to the present invention at least the pathologic tissue sample is native until the immersion step, preferably the normal tissue sample is also native until the immersion step. As used herein the term “native tissue sample” means that the tissue sample is not denatured and not fixed. As used herein the term “native tissue” comprises native tissue samples as well as the corresponding tissues in vivo.
According to the present invention biological markers for pathologic diseases which are not accessible from the extracellular space in a native tissue sample (and also are not accessible from the extracellular space in vivo) will not or will essentially not be labelled.
An important feature of the present invention is that the native tissue samples are immersed in a solution comprising a reactive compound which is marking and labelling proteins which are accessible from the extra cellular space, by simple diffusion through the native tissue. This means that once the tissue sample is immersed into the solution the reactive compound comprised therein will diffuse through the extracellular space of the native tissue sample and thereby brought into contact with accessible proteins and react with them. The labelled proteins can be easily purified and analysed and identified by proteomic methods. Identification of the labelled proteins is, for example, achieved by liquid chromatography and subsequent mass spectrometry. One major advantage is that the present method does not depend on the possibility that the (native) tissue samples used can be perfused or not. Therefore, according to the method of the present invention any tissue can be investigated and explored in order to seek for accessible biological markers.
As used herein the term “biological marker” represents proteins or polypeptides (which may be modified for example by glycosylation), which are expressed in a given pathologic tissue and which are essentially not expressed in normal tissues, or are expressed on a higher level in the given pathologic tissue than in normal tissues, wherein the biological marker indicates a pathologic condition compared to the normal physiologic condition of normal tissues.
As used herein the term “normal tissue” represents either normal tissue corresponding to the pathologic tissue from the same individual or normal tissue corresponding to the pathologic tissue from other individuals or normal tissue that is not related (in extenso either from a different location in the body, or with a different histologic type) to the pathologic tissue either from the same individual or form other individuals.
In a preferred embodiment the term “normal tissue” refers to the normal tissue corresponding the pathologic tissue from the same individual.
According to the present invention the step of determination of the “differential expression pattern” of the labelled proteins in pathologic tissue samples compared to normal tissue samples means that it is examined, for example, if a given labelled protein is at all expressed in a tissue or not, either pathologic or normal. This analysis may also comprise the assessment of the relation of expression level in pathologic tissue versus normal tissue. Further, this analysis may also comprise the assessment of in how many samples (eventually from different individuals) of a given type of pathologic tissue expression of a given protein is found compared to in how many samples (eventually from different individuals) of the given type of the corresponding or unrelated normal tissue expression of a given protein is found.
According to the present invention there is a step of judging that the labelled protein(s) having higher expression in the native pathologic tissue samples compared to normal tissue samples or being expressed more frequently in respective native pathologic tissue samples compared to normal tissue samples is/are biological marker(s) for pathologic tissue, which are accessible for high-affinity ligands from the extra cellular space in the native tissue. Most preferred, it is judged that the labelled protein(s) having expression in the native pathologic tissue sample but which is not or essentially not expressed in the corresponding and/or unrelated normal tissue is/are biological marker(s) for pathologic tissue, which are accessible for high-affinity ligands from the extra cellular space in the native tissue.
In a preferred embodiment the method comprises the steps of:
Further preferred the labelling reagent for labelling proteins is a reactive biotin, preferably a biotin reactive ester derivative.
In yet another preferred embodiment the purification step makes use of the label of the labelled proteins as selective marker. It is preferred that the labelled proteins are purified by affinity purification mediated by the label.
Preferably, the label is a biotin residue and purification is performed using streptavidin bound to a resin, wherein the biotin-labelled proteins are bound to the resin via streptavidin.
According to a further preferred embodiment after the purification step the labelled proteins are cleaved to peptides, preferably by proteolytic digestion.
Further preferred, the analysis step comprises mass spectrometry, preferably microsequencing by tandem mass spectrometry.
Preferably, the native pathologic tissue sample is derived from tissues selected from the group consisting of tumor tissue, inflamed tissue, atheromatotic tissue or resulting from degenerative, metabolic and genetic diseases.
Further preferred, accessibility of the biological markers refers to being accessible for high-affinity ligands from the extra cellular space. This means that substances (high-affinity ligands) can diffuse in vitro through the extra cellular space of a given native tissue to accessible biological markers (which are accessible from the extra cellular space in the native tissue, and therefore also in vivo). In consequence, if a specific substance for labelling is able to diffuse in vitro through the extra cellular space of a given pathologic tissue and reaches the biological marker, which is indicating the pathologic condition, then said biological marker (which can be determined according to the present invention) likely will also be accessible in vivo via the extracellular space (extra cellular liquid) and therefore can be considered to be a candidate target protein (biological marker) for detecting the disease (for detecting the presence (diagnosis), and/or evaluating the spread (staging) and/or assessing the evolution (monitoring) of the disease) and for targeting drugs to the cells and tissues expressing said accessible protein (biological marker). Preferably, the substances (high-affinity ligands) which can diffuse in vitro through the extra cellular space of a given tissue to accessible biological markers are selected from the group consisting of antibodies, antibody fragments, drugs, prodrugs, ligands, biotin, and derivatives and conjugates thereof, preferably conjugates of antibodies or antibody fragments with drugs or prodrugs.
In a further preferred embodiment the biological markers are proteins or polypeptides, which are expressed in the given pathologic tissue and not expressed in the corresponding normal tissue, or are expressed on a higher level in the given pathologic tissue than in the corresponding normal tissue, wherein the biological marker indicates a pathologic condition compared to the normal physiologic condition of the corresponding normal tissue.
The object of the present invention is also solved by the use of the aforementioned method for the manufacture of a medicament for therapeutic and/or preventive treatment of a human or animal disease, wherein a high-affinity ligand directed against a biological marker for pathologic tissue is comprised in the medicament, wherein said biological marker is accessible for high-affinity ligands from the extra cellular space. Preferably, the high-affinity ligand is an antibody, more preferred a monoclonal antibody or a recombinant antibody, directed to the biological marker. Preferably, a method is provided for the manufacture of a medicament for therapeutic and/or preventive treatment of a human or animal disease, wherein a high-affinity ligand directed against a biological marker for pathologic tissue is comprised in the medicament, wherein said biological marker is accessible for high-affinity ligands from the extra cellular space, wherein the method is comprising the steps of:
The present invention also provides the use of the aforementioned method for the development of techniques for the detection of pathologic tissues. Preferably, subsequent to the choice of the biological marker (judgement step) according to the present invention antibodies are raised against the biological marker and conjugated with a detectable label well-known in the art, which is detectable, for example by scintigraphy, PET scan, NMR or X-rays. Preferably, a method is provided for the development of techniques for the detection of pathologic tissues, wherein the method is comprising the steps of:
The present invention further provides the use of the aforementioned method for the development of a medicament based on the use of specific ligands, preferably antibodies and antibody-drug conjugates, for therapeutic and/or preventive treatment of human or animal diseases. Preferably, subsequent to the choice of the biological marker judgement step) according to the present invention antibodies are raised against the biological marker and conjugated with a suitable drug for treatment the pathologic cells and tissues by drug targeting. Preferably, a method is provided for the development of a medicament based on the use of specific ligands, preferably antibodies and antibody-drug conjugates, for therapeutic and/or preventive treatment of human or animal diseases, wherein the method is comprising the steps of:
The present invention further provides the use of the aforementioned method for the screening of accessible biological markers of a pathologic tissue of an individual patient for the development of an individual treatment protocol. Preferably, subsequent to the choice of the biological marker (judgement step) according to the present invention antibodies are raised against the biological marker and conjugated with a suitable drug for treatment the pathologic cells and tissues by drug targeting. Preferably, a method is provided for the development of a medicament based on the use of specific ligands, preferably antibodies and antibody-drug conjugates, for therapeutic and/or preventive treatment of human or animal diseases, wherein the method is comprising the steps of:
The object is also solved by a method for therapeutic and/or preventive treatment of a human or animal disease, wherein a high-affinity ligand directed against a biological marker for pathologic tissue is used, wherein said biological marker is accessible for high-affinity ligands from the extra cellular space, wherein the method is comprising the aforementioned procedure of the present invention. Preferably, a method is provided for therapeutic and/or preventive treatment of a human or animal disease, wherein a high-affinity ligand directed against an biological marker for pathologic tissue is used, wherein said biological marker is accessible for high-affinity ligands from the extra cellular space, wherein the method is comprising the steps of:
In the present patent application a new, simple, quick and efficient method is provided to identify, in human tissue biopsies, specific disease biological markers accessible from the extra cellular space. The method of the present invention preferably makes use of covalent linking of biotin onto primary amines of accessible proteins brought into contact with a solution of reactive biotin ester derivatives. The method according to the present invention permits the ex vivo biotinylation of human tissues, which originate either from biopsies or from non-perfusable organs (e.g. cancer lesions present in mastectomy or prostatectomy samples), by simple immersion in the biotinylation solution, without prior lysis or homogenisation, and preferably also without denaturation or fixation. Biotinylated proteins are easily purified thanks to the extremely high and specific interaction of biotin with streptavidin, even in lysis buffers containing strong detergents, thus minimizing the non-specific binding during the affinity purification step. Proteolytic digestion of the purified biotinylated proteins preferably is performed directly on-resin, before shotgun mass spectrometry analysis.
In summary the method of the present invention allows the identification of several accessible proteins differentially expressed in pathologic and healthy tissue samples. This method is applicable to virtually any tissue, including tumor tissue samples as well as other pathologic tissues resulting from inflammatory, degenerative metabolic and even genetic diseases. The present invention sought for specific biological markers of human breast cancer, the most frequent form of cancer and the second leading cause of cancer death in American women. Among a list of candidate markers, two of them, versican and periostin, were identified in breast cancers with the chemical proteomic-based approach of the present invention, and were further validated by immunohistochemistry. The accessibility of these extra cellular matrix proteins may be particularly suited for targeting strategies using an intravenous route. It was demonstrated that the method is easy to perform and reliable. Of note, results can be obtained in less than one week. The method is versatile enough to be exploited in the future for complete mapping of primary tumors and associated metastases. It would enable the development of custom-made treatments, targeting only accessible proteins effectively expressed in the diseased tissues. It may be anticipated that once the most relevant, accessible biological markers specific for a patient's disease are identified, high affinity ligands, such as recombinant human antibodies and their fragments, can be prepared to assess the precise localization of the pathologic lesions and to selectively destroy them. As an example, the human antibody L19, a specific ligand of the EDB domain of fibronectin, a biological marker of several cancer types (Zardi, L. et al. Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. Embo J 6, 2337-2342 (1987); Pini, A. et al. Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J. Biol Chem 273, 21769-21776 (1998); Castellani, P. et al. Differentiation between high and low-grade astrocytoma using a recombinant antibody to the extra domain-B of fibronectin. Am J Pathol 161, 1695-1700 (2002)), is currently tested in clinical trials, both as a imaging tool (conjugated to radioactive iodine (Berndorff, D. et al. Radioimmunotherapy of solid tumors by targeting extra domain B fibronectin: identification of the best-suited radioimmunoconjugate. Clin Cancer Res 11, 7053s-7063s (2005); Borsi, L. et al. Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19- to the ED-B domain of fibronectin. Int J Cancer 102, 75-85 (2002)) and as a therapeutic agent (fused with human interleukin-2 (Ebbinghaus, C. et al. Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy. Int J Cancer 116, 304-313 (2005); Menrad, A. & Menssen, H. D. ED-B fibronectin as a target for antibody-based cancer treatments. Expert Opin Ther Targets 9, 491-500 (2005); Carnemolla, B. et al. Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix. Blood 99, 1659-1665 (2002))). The new technology of the present invention will promote the development of selective target therapies and therefore may represent an unprecedent step toward a clean and effective war against diseases and particularly cancer.
In a preferred embodiment the object is solved by an in vitro method for screening specific disease biological markers which are accessible from the extra cellular space in pathologic tissues for high-affinity ligands comprising the steps of:
First, the effect of immersion time in the biotin solution on labelling extent and intensity was investigated by histochemistry. Diffusion extent was dependent not only on soaking time, but also on the thickness and composition of the tissue. Increased soaking times (up to 40 minutes) were tested onto breast tissue specimens of variable thicknesses. It was observed that labelling extent, indicative of biotin penetration in the tissues, increased over time (
Expression profiles of biotinylated, accessible proteins in 7 ductal and 3 lobular human breast carcinomas (Table 2) and their matched non-tumoral counterparts were then determined using the MudPIT (Multidimensional protein identification technology) technique, based on 2-dimensional separation of tryptic peptide digest using nanoflow liquid chromatography coupled to electrospray tandem mass spectrometry. The complete list of proteins includes 670 proteins identified with high confidence (Table 3). Reproducibility of the method was assessed by comparing the protein lists generated by multiple runs of the same sample, and by comparing protein lists generated from 3 different samples from the same tumor (
The comparative analysis of nor-tumoral and cancerous breast tissues identified several proteins known to be preferentially localized in the extra cellular compartment Stromal proteins that are selectively expressed in cancer tissues are prime candidates for tumor targeting strategies because (i) they are expected to be more accessible than intracellular proteins, (ii) they are often present in high quantities, and (iii) tumor cells frequently induce changes in the stromal compartment. This “reactive stroma” creates a permissive and supportive environment contributing to cancer progression (Walker, R. A. The complexities of breast cancer desmoplasia. Breast Cancer Res 3, 143-145 (2001); De Wever, O. & Mareel, M. Role of tissue stroma in cancer cell invasion. J Pathol 200, 429-447 (2003)). For these reasons, identification of new stromal proteins specifically involved in cancer is of high interest. Searching for accessible extra cellular matrix (ECM) proteins, versican has been identified as being systematically expressed only in the breast cancer microenvironment (Table 1). Among other potential extra cellular biological markers, periostin and fibronectin were more frequently detected in the cancerous tissues analyzed. Further, the in situ expression of some of these potential markers was investigated by immunohistochemistry in a series of human breast cancers together with their normal counterparts.
Table 1 shows a selection of 10 accessible proteins identified with high confidence. Several proteins of this list appeared to be cancer- or breast cancer-associated proteins (e.g. cytokeratins, anterior gradient protein homolog 2, periostin and versican, among others). Interestingly, the membrane antigen ErbB2 was also identified by MS in the single tumor (out of the 10 tumors tested) that was considered as ErbB2-positive by routine immunohistochemical assessment. Biotinylated proteins included extracellular and plasma membrane proteins, but also included a non-negligible fraction of intracellular proteins. The reasons for this observation include intracellular protein leakage when tissues are sliced, biotin penetration, and strong interactions between cytoplasmic and membrane proteins. Nevertheless, the comparative analysis of non-tumoral versus cancerous breast tissues identified several proteins known to be preferentially localized in the extracellular compartment.
Searching for accessible extracellular matrix (ECM) proteins, versican was identified in this study as being systematically and specifically (10 out of the 10 sample pairs tested) detected in the breast cancer samples, but not in the matched normal counterparts (Table 1). Versican, a large proteoglycan secreted by stromal cells in the ECM, is a recognized cell adhesion and motility modulator that may facilitate tumor cell invasion and metastasis (Yamagata, M., et al., Regulation of cell-substrate adhesion by proteoglycans immobilized on extra cellular substrates. J Biol Chem 264, 8012-8 (1989); Evanko, S. P. et al., Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19, 1004-13 (1999); Ricciardelli, C. et al., Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with node-negative primary breast cancer. Clin Cancer Res 8, 1054-60 (2002)). Anti-versican immunoreactivity is increased in the peritumoral stromal matrices of breast (Suwiwat, S. et al., Expression of extra cellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer. Clin Cancer Res 10, 2491-8 (2004)), lung (Pirinen, R. et al., Versican in nonsmall cell lung cancer: relation to hyaluronan, clinicopathologic factors, and prognosis. Hum Pathol 36, 44-50 (2005)), prostate (Ricciardelli, C. et al., Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer. Clin Cancer Res 4, 963-71 (1998)), and colon (Mukaratirwa, S. et al., Versican and hyaluronan expression in canine colonic adenomas and carcinomas: relation to malignancy and depth of tumour invasion. J Comp Pathol 131, 259-70 (2004)) cancers. In addition, increased accumulation of versican in the stroma surrounding breast cancer cells is associated with a higher risk of relapse in node-negative, primary breast cancer (Ricciardelli, C. et al. Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with node-negative primary breast cancer. Clin Cancer Res 8, 1054-60 (2002); Suwiwat, S. et al., Expression of extra cellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer. Clin Cancer Res 10, 2491-8 (2004)). Versican was identified from several peptides (up to 5), and MS spectra of versican peptides were thoroughly examinated (
Periostin is a soluble, secreted ECM-associated protein (Takeshita, S. et al., Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem J 294 (Pt 1), 271-8 (1993)) that localizes with integrins at sites of focal adhesion, thus suggesting a contribution of this protein to cell adhesion and motility (Gillan, L. et al. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility. Cancer Res 62, 5358-64 (2002)). It has been recently shown that human periostin is overexpressed in several cancer types, including colorectal carcinoma (Bao, S. et al., Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway. Cancer Cell 5, 329-39 (2004); Tai, I. T. et al., Periostin induction in tumor cell line explants and inhibition of in vitro cell growth by anti-periostin antibodies. Carcinogenesis 26, 908-15 (2005)), breast adenocarcinoma (Shao, R. et al., Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression. Mol Cell Biol 24, 3992-4003 (2004)), non-small cell lung carcinoma (Sasaki, H. et al., Expression of Periostin, homologous with an insect cell adhesion molecule, as a prognostic marker in non-small cell lung cancers. Jpn J Cancer Res 92, 869-73 (2001)), glioblastoma (Lal, A. et al., A public database for gene expression in human cancers. Cancer Res 59, 5403-7 (1999)), and epithelial ovarian carcinoma (Ismail, R. S. et al., Differential gene expression between normal and tumor-derived ovarian epithelial cells. Cancer Res 60, 6744-9 (2000)). In addition, periostin overexpression in cancer lesions may be associated with advanced disease (Bao, S. et al., Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway. Cancer Cell 5, 329-39 (2004)) and poor outcome (Sasaki, H. et al., Expression of Periostin, homologous with an insect cell adhesion molecule, as a prognostic marker in non-small cell lung cancers. Jpn J Cancer Res 92, 869-73 (2001)). Periostin may facilitate tumor invasion and metastasis by promoting angiogenesis (Bao, S. et al., Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway. Cancer Cell 5, 329-39 (2004); Shao, R. et al., Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression. Mol Cell Biol 24, 3992-4003 (2004)). Given the suspected role of periostin in tumor metastasis, this secreted protein represents an attractive cancer biological marker and target. In the current study, protein expression profiles obtained in the 10 breast cancer and matched non-tumoral tissues analyzed revealed that periostin was among the most frequently extra cellular proteins identified in the tumor samples (10 out of the 10 breast cancers evaluated). This was further confirmed by immunohistochemical experiments demonstrating an up-regulation of periostin in the stroma associated with the breast cancers analyzed in this study, as compared with the stroma associated with the non-tumoral breast glandular/ductal compartment (data not shown).
Cancerous and non-tumoral human breast tissue samples were obtained from mastectomy specimens, immediately sliced and soaked into freshly prepared biotinylation solution (1 mg.ml−1 of EZ-link Sulfo NHS-LC biotin (Pierce) dissolved extemporaneously in PBS [pH 7.4]). The time frame from the excision in the operating theatre to the biotinylation step required typically less than 10 minutes. Each biotinylation reaction was stopped by a 5 minute incubation in a primary amine-containing buffer solution (e.g. 50 mM Tris [pH 7.4]). Tissue samples were then snap-frozen in liquid nitrogen, except for a tiny portion of each sample that was directly immersed in formalin and then processed for further histological and histochemical investigations. Additional tissue samples not included in the biotinylation procedure were routinely processed for histopathological diagnosis. Controls included adjacent tissue slices immersed in PBS. The Ethics Committee of the University Hospital of Liege reviewed and approved the specific protocol used in this study, and written informed consent was obtained from all patients.
In order to assess the diffusion depth of reactive biotin ester derivatives in the biotinylated tissues, formalin-fixed, paraffin-embedded breast tissue sections were incubated with avidin-peroxidase conjugates with the use of the Vectastain ABC kit (Vector Labortories, Burlingame, Calif., USA), according to the manufacturer's instructions. Immunohistochemical experiments were performed as previously described by Waltregny et al. (Waltregny, D. et al. Prognostic value of bone sialoprotein expression in clinically localized human prostate cancer. J Natl Cancer Inst 90, 1000-1008 (1998)). For the immunohistochemical detection of versican, antigen retrieval was performed by incubating the slides with chondroitinase. Anti-versican (clone 12C5, Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, Iowa, USA) antibody was applied onto the sections at a dilution of 1:200. Control experiments included omission of the primary antibody in the procedure.
Pulverization of frozen biotinylated biopsies was performed using a Mikro-Dismembrator U (Braun Biotech, Melsungen, Germany) and generated tissue powder that was resuspended first in a PBS buffer containing a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany). Homogenates were sonicated (2×30″) with a 2 mm microprobe and soluble proteins were subjected to a preclearing step consisting in human serum albumin (HSA) and immunoglobulins (IgGs) depletion (Qproteome HSA and IgGs Removal Kit, Quiagen). This step was included to limit the number of HSA and IgGs peptides detected by MS, but did not hinder detection of low abundant proteins (data not shown). Insoluble pellet was resuspended in 2% SDS in PBS, and lysates were sonicated (3×30″). HSA- and IgGs-depleted soluble proteins fraction and detergent-solubilized proteins were pooled and boiled for 5 minutes. Protein concentration was determined using the BCA protein assay reagent kit (Pierce Chemical Co.). Streptavidin-sepharose slurry (Amersham Biosciences, 150 μl per mg of total proteins) was equilibrated by three washes in buffer A (1% NP40 and 0.1% SDS in PBS), and protein binding was allowed for 2 hours at room temperature in a rotating mixer. The resin was then washed twice with buffer A, twice with buffer B (0.1% NP40, 1M NaCl in PBS), twice with buffer C (0.1M sodium carbonate in PBS, pH 11), and once with ammonium hydrogenocarbonate (50 mM, pH 7.8). Binding of the biotinylated proteins onto the resin and washing efficiencies were checked by SDS-PAGE, and further either by Coomassie blue staining or by blotting for subsequent detection of biotin by streptavidin-horseradish peroxidase. On-resin digestion was carried out overnight at 37° C. with agitation, using modified porcine trypsin (Promega) in 100 μl final volume of ammonium hydrogenocarbonate (pH 7.8). The supernatants were collected, protein concentration was determined, and once evaporated, the peptides were resuspended in 0.1% formic acid. Samples were analysed by nanocapillary liquid chromatography-electrospray tandem mass spectrometry (nLC-ESI MS/MS).
Peptide separation by reverse-phase liquid chromatography was performed on an “Ultimate LC” system (LC Packings) completed with a Famos autosampler and a Swichos II Microcolumn switching device for sample clean-up, fractionation and preconcentration. Sample (20 μL at 0.25 μg/μL 0.1% formic acid) was first trapped on a SCX micro pre-column (500 μM internal diameter, 15 mm length, packed with MCA50 bioX-SCX 5 μm; LC Packings) at a flow rate of 200 nL/min followed by a micro pre-column cartridge (300 μM i.d., 5 mm length, packed with 5 μm C18 PepMap100; LC Packings). After 5 min the precolumn was connected with the separating nanocolumn (75 μm i.d., 15 cm length, packed with 3 μm C18 PepMap100; LC Packings) equilibrated in mobile phase A (0.1% formic acid in 2:98 of acetonitrile:degassed milliQ water). A linear elution gradient was applied with mobile phase B (0.1% formic acid in 80:20 of acetonitrile:degassed milliQ water) from 10% to 40% spanning on 95 minutes. The outlet of the LC system was directly connected to the nano electrospray source of an Esquire HCT ion trap mass spectrometer (Bruker Daltonics, Germany). Mass data acquisition was performed in the mass range of 50-2000 m/z using the standard-enhanced mode (8100 m/z per second). For each mass scan, a data dependant scheme picked the 3 most intense doubly or triply charged ions to be selectively isolated and fragmented in the trap. The resulting fragments were analyzed using the Ultra Scan mode (m/z range of 50-3000 at 26000 m/z per second). SCX-trapped peptides were stepwise eluted with 5 salt concentrations (10 mM, 20 mM, 40 mM, 80 mM, and 200 mM), each followed by the same gradient of mobile phase B.
Raw spectra were formatted in DataAnalysis software (Bruker Daltonics). Portion of the chromatogram containing signal (i.e. with base peak chromatogram signal above 50000 arbitrary units) was processed to extract and deconvolute MS/MS spectra, without smoothing or background substraction. A signal/noise ratio of 3 was applied to filtrate irrelevant data in the MS/MS spectra and generate the mass list. Charge deconvolution was performed on both MS and MS/MS spectra. A retention time of 1.5 minutes was allowed for compound elution to minimise detection redundancy of parents of identical masses and charge states. Both deconvoluted and undeconvoluted data were incorporated in the mgf file.
Protein identification was performed using different databases and different softwares featuring different built-in algorithms. Protein were first identified using the NCBI nonredundant database (NCBlnr, release 20060131) through the Phenyx web interface (GeneBio, Geneva, Switzerland). The mass tolerance of precursor ions was set at 0.6; allowed modifications were partial oxidization of methionines and partial lysin-modifications with LC biotin. One misscut was also allowed. For each tumor samples, identification of more than 400 proteins were obtained. Additional searches were performed against the minimally redundant SWISS-PROT human protein database (Nesvizhskii, A. I. & Aebersold, R. Interpretation of Shotgun Proteomic Data: The Protein Inference Problem. Mol Cell Proteomics 4, 1419-1440 (2005)) through the MS/MS ion search algorithm of the Mascot search engine (Mascot and Mascot Daemon v2.1.0) (Perkins, D. N. et al., Electrophoresis 20, 3551-67 (1999)) running on a multi-processor computer cluster. The mass tolerance of precursor and fragmented ions were set at 0.6 and 0.3 respectively; allowed modifications were partial oxidization of methionines. Stringent filtration was deliberately used: the absolute probability (P) was set to 0.01 (i.e. less than 1% probability of a random match), and the MudPIT scoring was used, while discriminating peptides with ion scores inferior to 15. Despite this “aggressive” filtering, proteins hits were manually inspected, particularly for those proteins identified by only one peptide. These precautions were taken to ascertain the accuracy of protein identification reported in Table 1. Proteins of interest were identified by both Phenyx and Mascot search engines, the sequence coverage being usually higher with Phenyx (data not shown).
Medium-density tissue microarrays (TMAs) comprising 0.6 mm cores of various normal human tissues were assembled as previously described (Kononen, J. et al. (1998) Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4, 844-847; Perrone, E. E. et al. (2000) Tissue Microarray Assessment of Prostate Cancer Tumor Proliferation in African-American and White Men. J Natl Cancer Inst 92, 937-939). Formalin-fixed, paraffin-embedded blocks of normal human tissues were retrieved from the Department of Pathology of the University Hospital of Liege. Most normal tissues were from surgical specimens, except for neuronal tissues, which were obtained from autopsies. Initial sections were stained with haematoxylin and eosin to verify histology. Two TMAs were generated with the use of a manual tissue arrayer (Beecham Instruments). Duplicate cores were included in the TMA for each specific tissue or organ analysed. A total of 120 cores were examined for immunohistochemical expression of versican. Immunostaining intensity was scored as absent, weak, moderate or strong by 2 observers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06100141.8 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/069679 | 12/13/2006 | WO | 00 | 7/7/2008 |
