Patent Application
20090130693
This invention is generally applicable in the field of biomedical engineering, and specifically relates to a method for diagnosis and/or prognosis of neoplasias in animals.
The invention further relates to a kit for diagnosis and/or prognosis of neoplasias in animals, a reagent for the preparation of such kit and the use of particular biomarkers in such method and/or kit.
Tumor cells modify and interact with their microenvironment by secreting a variety of proteins, including growth factors, extracellular matrix-degrading proteinases involved in tumor invasion, and cell motility factors that support cell migration and metastasis.
Furthermore, a number of additional factors were found to be involved in immunological escape, tumor invasion and angiogenesis, such as immunoregulatory cytokineses and molecules regulating both cell-to-cell and cell-to-substrate interactions (§ 1-3).
Pancreatic adenocarcinoma is a lethal disease, with an expected survival rate of less than 5% at 24 months after diagnosis. The identifications of proteins released by tumor cells may be useful both to understand the interaction between the tumor and the host organ and to find new methods for diagnosis, prognosis or treatment.
In recent years molecular analysis of cancer has been performed using DNA-microarrays, providing global profiles of transcription that reflect the origins (§ 4-6), stage of development (§ 7) and drug sensitivity (§ 7) of tumor cells. This approach has been also used to identify putative secreted proteins by the analysis of mRNAs bound to membrane-associated polysomes (§ 8). Nevertheless, mRNA-based analysis is an indirect approach to molecular analysis of cancer and necessitates validation at the protein level.
One step forward has been made through direct approaches based on proteome analysis (§ 9-11), although several technical problems arise when analyzing of complex protein mixtures (§ 11). The classical proteomic approach is based on two-dimensional gel electrophopresis, known as “2DG”, in which the proteins of interest are isolated and identified by mass spectrometry.
Thanks to the above mentioned method, and to the other approaches described above, various studies have been conducted (§ 12-§ 24) to identify pancreatic tumor “detecting” proteins. These include MMP-1, MMP-7, TIMP1, SERPINE2, TGFBI, MAC-2BP, clusterine, glycerol-3-phosphate dehydrogenase, syndecan-1, TSP-1 and uPA.
Many of these proteins were found to be involved in cell-cell and cell-extracellular matrix adhesion, as well as in extracellular matrix degradation and remodeling, and thus promote invasion and metastasis.
Nevertheless, this solution still has a few apparent drawbacks. While the 2DG approach has a relatively high resolution, this is limited by the difficulty to detect certain classes of proteins.
These include the cell membrane proteins, due to their low solubility in the gel-electrophoresis buffer, proteins of excessively low or high molecular weight, i.e. of less than 10 kDa or more than 200 kDa respectively, and proteins with an extreme isoelectric point, i.e. less than 4 or more than 9. Furthermore, the 2DG analysis cannot detect proteins in small amounts (§ 25).
The Applicant has been engaged in cancer-related research and has surprisingly found novel biomarkers for use in the diagnosis and/or prognosis of neoplasias in animals.
Therefore, the present invention is aimed at providing a method for diagnosis and/or prognosis of neoplasias in animals, as defined in claim 1, which comprises at least the steps of drawing at least one sample from the patient and determining the amount of a biomarker in said at least one sample drawn from the patient, wherein said biomarker is a protein released from pancreatic cells. Advantageously, the biomarker may be selected from the group consisting of: CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclofilin B, beta2 microglobulin, ICA69, whereas the neoplasia may be a tumor of the pancreas.
Thanks to these proteins the reliability of diagnosis and/or prognosis of neoplasias, particularly the carcinoma of the pancreas will be greatly enhanced.
Suitably, the neoplasia may be selected from the group consisting of tumors of the breast, esophagus, head and neck, liver, lung, gastrointestinal tract, prostate, skin, kidney and/of urogenital system, metastases, micrometastases or a combination thereof.
Preferably, the animal mentioned above is a mammal, and more preferably a human.
Suitably, the sample may be a body fluid, preferably selected from the group consisting of blood, plasma, serum, urine, sperm, interstitial fluid, spinal fluid or a combination thereof.
Advantageously, the concentration of the above mentioned biomarker may be compared with known concentrations of the same biomarker, detected on samples of the same nature from different animals not suffering from neoplasia, preferably animals having a benign tumor.
Conveniently, the prognosis and/or diagnosis of the neoplasia may be determined by comparing the concentration of said biomarker detected on samples drawn from the same patient.
According to another aspect of the invention, a kit is provided for diagnosis and/or prognosis of neoplasias in animals, as defined in claim 11, which comprises a detectable agent linked to a biomarker, wherein said biomarker is a protein released by pancreatic cells.
Advantageously, the biomarker will be selected from the group consisting of: CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclofilin B, beta2 microglobulin, ICA69.
Conveniently, the detectable agent may be selected from the group consisting of an anti-biomarker antibody, preferably of the monoclonal or polyclonal type, a receptor for said biomarker, or functional fragments, or a combination thereof.
Advantageously, the agent may be detectable by measuring chromatography, electrical capacitance, fluorescence, luminescence, mass, molecular weight, radioactivity or a combination thereof.
According to another aspect of the invention, a reagent is provided for diagnosis and/or prognosis of neoplasias, as defined in claim 16, which comprises a detectable agent linked to a biomarker, wherein said biomarker is a protein released by pancreatic cells.
Conveniently, the biomarker may be selected from the group consisting of: CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclofilin B, beta2 microglobulin, ICA69.
In accordance with a further aspect of the invention, there is provided the use of proteins released by pancreatic cells as biomarkers for diagnosis and/or prognosis of neoplasias, as defined in claim 18.
Conveniently, the biomarker may be selected from the group consisting of: CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclofilin B, beta2 microglobulin, ICA69.
Further features and advantages of the invention will be more readily apparent from the detailed description of a few preferred non exclusive embodiments of a method according to the invention, which are shown as a non limiting example with the help of the annexed figures, in which:
CSPG2Nersican, frame A: Immunohistochemistry of primary pancreatic adenocarcinoma shows strongly positive peritumoral stroma, while tumor cells are immunonegative (arrow).
Frame B: the same pattern of reactivity detected in Suit-2 cells implanted in nu/nu mice in a Matrigel® matrix. This shows strong immunostrain with anti-versican antibody at the cell-matrix interface, while tumor cells expression is undetectable. This demonstrates that cancer cells produce and immediately release the protein.
Frame C: RT-PCR analysis shows the presence of versican transcripts in five of the six cell lines, T3M4 being negative. Actin expression is shown to demonstrate equal amounts of starting RNA.
Mac25/Angiomodulin, frame D: tumor cells in primary pancreatic adenocarcinoma show strong cytoplasmic immunostain.
Frame E: Immunohistochemistry of Suit-2 cells implanted in nu/nu mice in Matrigel® matrix shows immunostain with anti-mac25 antibody in both the cytoplasm of cells and the matrix.
Frame F: Western blot analysis of supernatants shows that angiomodulin is released from five of the six tumor cell lines; anti-cdc42 is the negative control antibody, and Ponceau staining indicates the relative amount of proteins loaded on each line.
yellow <20, blue from 20 to 60 and red >60. The top right inset shows a plot limited to proteins having a molecular weight of less than 100 kDa.
As shown in the sections below, the Applicant surprisingly identified 46 proteins (see Table 1 and 2) which may be related to relevant tumor cell features, such as angiogenesis and modification of the extracellular environment.
Twenty-one of these were classified as secreted proteins according to Human Protein reference and GeneCards™ databases, while the remaining ones were classified as cytoplasmic proteins.
Eleven of the twenty-one proteins released in the medium (MMP-1, MMP-7, TIMP1, SERPINE2, TGFBI, MAC-2BP, clusterine, glycerol-3-phosphate dehydrogenase, syndecan-1, TSP-1 and uPA) have already been associated with pancreatic tumor, indicating the validity of the selected approach.
The remaining ten proteins have never been associated to pancreatic tumor heretofore. These include: CSPG2/versican, Mac25/angiomodulin, IGFBP-1, HSPG2/perlecan, syndecan 4, FAM3C, APLP2, cyclofilin B, beta2 microglobulin and ICA69.
The demonstration that CSPG2/versican is produced and immediately released by pancreatic tumor cells challenges a technical-scientific prejudice. A number of studies allege that fibroblasts of stroma cancer are the sole or predominant source of these molecules (§ 35-38).
Mac25/angiomodulin is a member of the insulin-like growth factor binding protein (IGFBP), and has never been associated with pancreatic tumor heretofore.
The finding that Mac25/angiomodulin is produced in vivo by endothelial or tumor cells, even though it is associated to a number of other phenomena (§ 39-41) fulfils a long-felt need in the technical-scientific community.
IGFBP-1 is another member of the IGFBP family and has been recently associated with increased risk of hematological malignancy (§ 42) but never with pancreatic carcinoma heretofore.
HSPG2/perlecan is a major heparan sulfate proteoglycan component of basement membranes and connective tissues. Suppression of its expression is known to inhibit tumor growth and neovascularization in human colon carcinoma xenografts and mouse melanoma allografts (§ 43) but has never been associated with pancreatic tumor heretofore.
Cells transfected with syndecan-4 are further known to be able to bind collagen and show reduced invasive capability (§44) but such protein has been never associated to pancreatic carcinoma heretofore.
FAM3C is a member of a recently cloned cytokine-like gene family (§ 45), and has never been associated to pancreatic tumor heretofore.
The remaining proteins (APLP2, cyclophilin B, beta2 microglobulin and ICA69) have been reported to be expressed by pancreatic tumor cell lines (§ 46-49), but no one has never shown their direct relationship with pancreatic cancer heretofore.
Those of normal skill in the art may wholly reproduce the invention as described in sections below by way of non limiting example of the invention.
As is known, the invention is susceptible of a many changes and variants within the inventive principle disclosed in the annexed claims. All the steps, features, compounds and compositions may be replaced by equivalents, and several different materials may be used as needed, without departure from the scope of the invention.
While the invention has been described with particular reference to the annexed figures, such description shall be intended to only improve the intelligibility of the invention and not to limit the claimed scope in any manner.
A protein identification method known as “MudPIT”, multidimensional protein identification technology (§ 13) was used, which incorporates two-dimensional capillary chromatography as well as an automated tandem mass spectrometry (2DC-MS/MS).
This technique uses the peptides generated from enzymatic digestion of a complex protein mixture, by first separating them by means of two micro-HPLC columns, and then directly analyzing the eluted peaks by tandem mass spectrometry. The identification of the corresponding proteins is then obtained through an automated database search with appropriate software, such as the SEQUEST algorithm for mass spectra data handling (§ 13-15).
Harvest of supernatants Suit-2, IMIM-PC1, IMIM-PC2, T3M4, BI and MCC1 pancreatic tumor cell lines have been used. The first five cell lines have been described in Moore et al (§ 26), and the last one has been established at the Pathology laboratory of the University of Verona (§ 27). The supernatants were harvested following the protocol of Krachmarova et al (§ 28). Cells were grown to 80% confluency in 150 cm2 flasks with complete medium (RPMI 1640, 2 mM glutamine, 10% FBS), gently washed 6 times with serum-free medium (30 mM) and left in 12 ml of serum-free medium for additional 18 h. The supernatants were ultracentrifuged at 100,000 g for 2 h at 4° C. and subjected to analysis. For cell activation analysis, Suit-2 cells were treated with 100 ng/ml phorbol myristate acetate (PMA) and 100 nM ionomicin.
Enzymatic digestion of protein samples Sequencing grade modified trypsin was added to 50 μl of conditioned medium containing 1 μg protein at a 1:50 enzyme:protein ratio (wt:wt) in 100 mM ammonium bicarbonate, pH 8.0 and incubated at 37° C. overnight. The reaction was stopped by acidification with trifluoroacetic acid. A second aliquot of each sample was digested with pepsin at 1:50 enzyme:protein ratio (wt:wt) in 100 mM ammonium acetate pH 3.0 at room temperature for 4 h and immediately analyzed. 10 μl of the peptide mixture so obtained were injected directly into the 2DC-MS/MS.
Two-dimensional capillary chromatography—tandem mass spectrometry (2DC-MS/MS) analysis 10 μl of the peptide mixture obtained from the digestion of the protein samples, were analyzed by means of two-dimensional microchromatography coupled with an ion trap mass spectrometer, using the ProteomeX system equipped with Bioworks 3.1 as graphical interface for data handling. Particularly, peptide mixtures were first separated by means of ion-exchange chromatography (Biobasic-SCX column, 5 μm, 0.3 i.d.×150 mm) through seven steps of increasing ammonium chloride concentration (0, 50, 100, 150, 200, 300, and 600 mM). Each predetermined salt concentration step was directly loaded onto the reversed phase column (Biobasic-C18, 0.180 i.d.×100 mm, ThermoHypersil, Bellofonte, Pa.) and separated with an acetonitrile gradient. The following were used: eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile; the gradient profile was 5% B for 3 min followed by 5 to 50% B within 40 min.
Peptides eluted from the C18 column were analyzed directly with an ion trap LCQXP mass spectrometer equipped with a metal needle (10 μm i.d.). The heated capillary was held at 160° C., ion spray 3.2 kV and capillary voltage 67 V. The spectrum was acquired in positive mode (in the range of 400-1600 m/z) using dynamic exclusion for MS/MS analysis (collision energy 35%).
Data handling of MS results Using the SEQUEST algorithm, the experimental mass spectrum produced was related to the peptide sequence obtained by comparison with the theoretical mass spectrum in the human protein database downloaded from the NCBI website. For peptide matching, the following limits were used: Xcorr scores of more than 1.5 for simple charge peptide ions, of more than 2.0 and 2.5 for double and triple charge ions respectively. The output data obtained from SEQUEST software were treated with the MAProMA (Multidimensional Algorithm Protein Map) algorithm for comparison of protein lists, evaluation of relative abundances, and plotting of virtual 2D maps (§ 29).
Western blotting. Proteins were precipitated from 10 ml of conditioned, serum-free medium by drop-wise addition of 10% (final concentration) tricloroacetic acid with stirring at 4° C. The sample was then centrifuged at 3000 g for 60 min and washed 3 times with an excess of an acetone: methanol (8:1) mixture.) The pellet was air-dried, resuspended in SDS buffer, subjected to SDS-PAGE and Western blotting. Anti-angiomodulin antibody 88 (§ 30) and an anti-versican antibody (clone 2-B-1) were used at 1 μg/ml. The secondary antibody was a rabbit anti-mouse-HRP, and the signal was detected using a chemiluminescence detection kit.
Immunohistochemistry 5 μm paraffin sections were stained using anti-angiomodulin at 5 μg/ml, and anti-versican at 0.5 μg/ml, as described in Cattaneo et al (§ 31).
Xenografting Suit-2 cells in nude mice 2*106 Suit-2 cells were resuspended in 0.4 ml of Matrigel® and inoculated subcutaneously in the flank of four weeks old nu/nu Swiss mice weighing 18-22 g. After 1 wk, the implant was removed, fixed in 10% buffered formalin, paraffin-embedded, and sectioned for immunohistochemistry.
Reverse Transcription-Poltmerase Chain Reaction (RT-PCR). RNA was prepared using the Trizol® extraction kit. One μg of total RNA was reverse transcribed in 20 μl with 100 ng of random hexamers and 200 U of SuperScript II® at 42° C. for 1 h. Polymerase chain reaction was performed as described in Cattaneo et al (§ 31). Amplification of β-actin mRNA was performed for 25 cycles on cDNA as control. The primers to amplify versican were: 5′-GGC TTT GAC CAG TGC GAT TAC-3′ and 5′-CCA GCC ATA GTC ACA TGT CTC-3′.
Gelatin/zymography for metalloprotease activity Supernatants were centrifuged for 10 min at 14,000 g to remove cell debris. Five μl of 5×SDS sample buffer (5% SDS, 0.5 M Tris-HCl pH 6.8, 25% glycerol) were added to 20 μl of supernatant. The sample was run on an SDS-PAGE gel containing 1 mg/ml gelatin. The gel was washed twice (20 min/cycle) with 2.5% Triton X-100 at room temperature, incubated in 200 ml of activation buffer (10 mM Tris-HCl, 1.25% Triton X-100, 5 mM CaCl2, 1 μM ZnCl2) overnight at 37° C., stained with Coomassie blue and destained with methanol: acetic acid:water (50:10:40).
MudPIT analysis of serum-free supernatants of resting and phorbol-ester activated Suit-2 cell lines identified 46 proteins (Tables 1 and 2). The results were validated for certain proteins by analyzing a panel of tumor cell lines. Evidence that the latter release these proteins in vivo was obtained by immunohistochemistry on both primary pancreatic tumors and in a model consisting of Suit-2 cells embedded in an amorphous matrix and implanted in athymic mice. MudPIT analysis further proved to reveal changes in the amount of secreted proteins after phorbol-ester activation of cells, as reflected by the SEQUEST software score values.
Determination of optimal conditions and time point for collection of supernatants To determine the optimal treatment and supernatant collection times, the 3DC-MS/MS proteomic approach was applied to several different solutions. Such optimal conditions (e.g. determination of the minimum number of cytosolic proteins susceptible of cell damage) turned out to be the protocol of Kratchmarova et al. with minor modifications, consisting in a gentle wash followed by 18 h incubation in protein-free medium
Identification of proteins released by Suit-2 cells Each 2DC-MS/MS analysis of digested samples produced seven reversed-phase chromatograms, which corresponded to the seven SCX column salt steps of increasing ammonium concentration (0, 50, 100, 150, 200, 300, and 600 mM). The peptides eluted from the columns were immediately directed into the mass spectrometer where they were ionized, mass selected, and fragmented. This procedure allowed to identify several peptides that were then associated with their respective proteins by the SEQUEST software.
MudPIT identified 30 proteins released by resting pancreatic cancer cells The MudPIT analysis of the supernatant from Suit-2 cells reproducibly identified, from 4 independent cell cultures, the 30 proteins listed in Table 1, where their putative cellular location according to public databases is also reported. Some of these proteins have never been associated with pancreatic tumor heretofore, including CSPG2/versican and Mac25/angiomodulin. MudPIT data was validated for two of them, CSPG2/versican e Mac25/angiomodulina, because these were present in larger amounts in samples, with respect to the number of peptides detected in supernatants.
CSPG2/versican is released by Suit-2 and primary pancreatic adenocarcinoma cells CSPG2/versican mRNA was detected by RT-PCR in five of the six cell lines under test (
Mac25/angiomodulin is a major secreted protein in pancreatic tumor and is overexpressed in primary pancreatic tumors. Western blot analysis showed that five of the six pancreatic tumor cell lines released Mac25 in the supernatant (
Additional proteins were identified upon phorbol ester/ionophor activation of tumor cells After phorbol ester-ionomycin activation, the MudPIT analysis of the supernatants from Suit-2 cells reproducibly identified, from 4 independent cell cultures, the 16 proteins listed in Table 2, in addition to those detected in resting conditions (Table 1). Among these, six proteins were classified as secreted in public databases. Moreover, it was noticed that several proteins identified in supernatants of both resting and activated conditions had varied score values assigned thereto by SEQUEST software. This suggests that these score values might be related to changes in the amount of proteins detected in the two conditions.
Virtual 2D map of identified proteins To visualize the SEQUEST output data in a user-friendly format, the MAProMA (multidimensional algorithm protein map) software was developed, which automatically plots molecular weight (MW) vs. isoelectric point (pl) for each identified protein, as shown in
The representation of MudPIT results on a 2D map immediately highlights proteins having a very high molecular weight and/or pl, as is the case for the two proteins in
The following documents, set forth hereunder as references, are designated hereinbefore by symbol §, and a number corresponding to the following number list, both enclosed in brackets.
| Number | Date | Country | Kind |
|---|---|---|---|
| VI2005A00059 | Mar 2005 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB06/00478 | 3/6/2006 | WO | 00 | 7/16/2008 |
