METHOD AND KIT FOR DIAGNOSING EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) OF THE PERITONEUM

Abstract

The invention relates to the field of diagnosis of epithelial-to-mesenchymal transition (EMT), such as mesothelial-to-mesenchymal transition (MMT), in particular EMT of the peritoneum, which often occurs upon peritoneal dialysis. The invention provides a method and kit based on markers comprising at least one extracellular matrix protein, e.g., collagen 13, collagen 6, and/or keratin 34, at least one protein involved in building and/or restructuring of extracellular matrix, e.g., matrix metalloproteinase 1, at least one protein involved in cell-cell and/or cell-matrix contacts, e.g., cadherin 13 or thrombospondin 1, at least one growth factor, e.g., VEGF, and, optionally, at least one BMP antagonist, e.g., Gremlin 1.

Claims

1. A kit for the diagnosis of epithelial-to-mesenchymal transition (EMT), comprising agents for the detection of markers in a sample, the markers comprising: an extracellular matrix protein; a protein involved in building and/or restructuring of extracellular matrix; a protein involved in cell-cell and/or cell-matrix contacts; a growth factor; and optionally, a BMP antagonist.

2. A method for the diagnosis of epithelial-to-mesenchymal transition (EMT), comprising detecting the absence and/or amount of a plurality of markers in a sample, the markers comprising: an extracellular matrix protein; a protein involved in building and/or restructuring of extracellular matrix; a protein involved in cell-cell and/or cell-matrix contacts; a growth factor; and optionally, a BMP antagonist.

3. The kit of claim 1, wherein the agents for the detection of the markers in the sample are antibodies or fragments thereof.

4. The kit of claim 1, wherein the agents for the detection of the markers in the sample are linked to a solid support, wherein the kit preferably comprises an antibody chip.

5. The kit of claim 1, wherein the extracellular matrix protein is a keratin selected from the group comprising keratin 34, and/or a collagen selected from the group comprising collagen 13 and collagen 6, wherein the markers preferably comprise the extracellular matrix proteins keratin 34, collagen 13 and collagen 6.

6. The kit of claim 1, wherein the protein involved in building and/or restructuring of extracellular matrix is a matrix metalloproteinase selected from the group comprising matrix metalloproteinase 1.

7. The kit of claim 1, wherein the protein involved in cell-cell or cell-matrix contacts is a cadherin selected from the group comprising cadherin 13 and/or a thrombospondin selected from the group comprising thrombospondin 1, wherein the markers preferably comprise cadherin 13 and thrombospondin 1.

8. The kit of claim 1, wherein the growth factor is VEGF.

9. The kit of claim 1, wherein the BMP antagonist is gremlin 1.

10. The kit of claim 1, wherein the markers are cadherin 13, collagen 13, collagen 6, keratin 34, matrix metalloproteinase 1, thrombospondin 1, VEGF and Gremlin 1.

11. The method of claim 2, wherein an increased amount of the markers indicates an epithelial-to-mesenchymal transition (EMT).

12. The kit of claim 1, wherein epithelial-to-mesenchymal transition is epithelial-to-mesenchymal transition of the peritoneum, wherein, preferably, the sample is derived from a peritoneal dialysis patient.

13. The kit of claim 1, wherein the sample is selected from the group comprising: a sample from a patient comprising peritoneal effluent, peritoneal fluid, serum, peritoneal tissue; and culture medium or cell lysate from a cell or tissue culture useful as a model for peritoneal dialysis.

14. A use of the kit of claim 1 for diagnosis of epithelial-to-mesenchymal transition, preferably, for diagnosis of epithelial-to-mesenchymal transition of the peritoneum of a peritoneal dialysis patient.

15. A method for predicting the progression of epithelial-to-mesenchymal transition of a tissue, preferably, the peritoneum, of a patient, comprising analyzing the status of the tissue of the patient with a kit of claim 1.

16. A method for optimizing the therapy of a peritoneal dialysis patient, comprising analyzing the status of the peritoneum of the patient with a kit of claim 1, wherein the therapy is adapted to the status of the patient's peritoneum.

17. A method for testing a peritoneal dialysis solution, comprising: contacting a peritoneum or a cell or tissue culture serving as a model of a peritoneum with the solution; and analyzing the status of the peritoneum or the cell or tissue culture with a kit of claim 1.

Description

FIGURES

[0048] FIG. 1 shows expression of VEGF in effluent from patients that have been categorized into E (Epithelioid) and NE (Non-Epithelioid), wherein the protein was quantified with antibodies. Categorization was based on Elliptical Factor. Two photographs were taken of each cell culture. Ten cells were randomly chosen and were measured the major and minor axis of cell and calculated a ratio. The mean of each cultured was calculated and if this mean was equal or higher than 2 (the cell was double of long that wide) the culture was classificated as non-epitelioid, and, otherwise, as epitelioid. Epithelioid=12.7312.81 pg/ml (N=20); Non-Epithelioid=43.706.181 pg/ml (N=20)

[0049] FIG. 2 shows expression of GREM1 in effluent from patients that have been categorized into E (Epithelioid) and NE (Non-Epithelioid) as above, wherein the protein was quantified with antibodies. Epithelioid=0.44050.3598 ng/ml (N=20); Non-Epithelioid=0.35550.3756 ng/ml (N=20)

[0050] FIG. 3 shows expression of Thrombospondin 1 in effluent from patients that have been categorized into E (Epithelioid) and NE (Non-Epithelioid) as above, wherein the protein was quantified with antibodies. Epithelioid=5.7917.669 ng/ml (N=20); Non-Epithelioid=66.6242.18 ng/ml (N=20)

[0051] FIG. 4 shows expression of Thrombospondin 1 in effluent from patients that have been categorized according to Mass Transfer Coefficient of creatinine (Cr-MTC), i.e., a parameter to determinate the transport across the peritoneal membrane, wherein the protein was quantified with antibodies. Cr-MTC<11=19.1926.11 ng/ml (N=26); Cr-MTC>11=67.8150.66 ng/ml (N=14)

[0052] FIG. 5 shows expression of COL13 in effluent from patients that have been categorized according to Elliptical Factor, wherein the protein was quantified with antibodies. Epithelioid=166.4120.1 ng/ml, N=33; Non-Epithelioid=228.0132.4 ng/ml, N=43

[0053] FIG. 6 shows expression of COL13 in effluent from patients that have been categorized according to Mass Transfer Coefficient of creatinine (Cr-MTC), wherein the protein was quantified with antibodies. Cr-MTC<11=189.8132.8 ng/ml, N=51; Cr-MTC>11=243.8108.6 ng/ml, N=23

EXAMPLES

Example 1

[0054] Differential gene expression studies were performed in cultured mesothelial cells derived from effluents of PD patients. 9 samples with Epithelioid and 8 samples with Non-Epithelioid phenotypes derived from PD patients were compared with a control pool (mesothelial cells derived from four different healthy donors' omentum). Different groups of induced or repressed genes were obtained and 40 genes were validated by RT-qPCR. Results for subset of 15 genes are shown in Table 1. Furthermore, results were grouped according to use of biocompatible (e.g. BicaVera) and less biocompatible (bioincompatible) PD fluids (e.g. Stay-safe), as shown in Table II. Table Ill shows that the use of bioincompatible PD fluids leads to a higher rate of EMT that use of biocompatible PD fluids. The results in Tables I and II show the high functional significance of the markers of the invention.

[0055] mRNA of CDH13, COL6A3, COL13A1, KRT34, MMP1, THBS1 (also designated TPS1), VEGFA and CDH1 showed a statistic difference between Epithelioid and Non-Epithelioid phenotypes and between the characteristic bio-compatible and bio-incompatible of PD fluids. CD44, TFI2, KDR and THBD showed a significant difference between Epithelioid and Non-Epithelioid phenotypes, and AQP1 differed between bio-compatible and bio-incompatible PD fluids.

TABLE-US-00001 TABLE I Epithelioid Non-Epithelioid GEN N Mean SD N Mean SD p-value CD44 23 2.88 2.94 28 4.87 3.34 0.001 CDH13 23 15.07 13.96 28 26.16 23.03 0.049 COL6A3 23 8.80 10.07 28 18.03 20.91 0.020 COL13A1 23 1.39 1.34 28 3.16 3.54 0.016 GREM1 23 4.03 4.14 28 7.61 11.97 0.798 IL33 23 2.42 2.26 28 4.90 5.78 0.108 KRT34 23 7.59 8.23 28 27.21 33.15 0.012 MMP1 23 1.37 1.25 28 6.73 9.83 0.002 MMP2 23 3.97 3.90 28 4.64 3.27 0.116 TFPI2 23 5.80 5.35 28 10.57 9.91 0.016 THBS1 23 7.36 8.54 28 19.41 24.98 0.022 VEGFA 23 1.84 1.32 28 3.32 3.29 0.088 CDH1 23 0.61 0.46 28 0.25 0.25 0.001 KDR 23 0.87 0.55 28 0.63 0.52 0.041 THBD 23 0.46 0.53 28 0.19 0.12 0.047

TABLE-US-00002 TABLE II Biocompatible Bioincompatible GEN N Mean SD N Mean SD p-value CD44 28 3.53 2.94 23 4.51 3.66 0.135 CDH13 28 12.39 8.67 23 31.84 24.64 0.003 COL6A3 28 8.06 8.19 23 20.93 22.57 0.006 COL13A1 28 1.37 1.27 23 3.57 3.77 0.006 GREM1 28 5.45 9.19 23 6.66 9.80 0.977 IL33 28 3.70 5.34 23 3.88 3.82 0.140 KRT34 28 8.73 10.64 23 30.08 35.17 0.010 MMP1 28 4.27 9.49 23 4.36 5.12 0.066 MMP2 28 3.61 3.02 23 5.23 3.98 0.096 TFPI2 28 8.42 6.66 23 8.42 10.38 0.691 THBS1 28 5.78 5.31 23 23.96 26.41 0.000 VEGFA 28 1.90 1.85 23 3.57 3.24 0.007 CDH1 28 0.38 0.43 23 0.46 0.35 0.061 KDR 28 0.66 0.48 23 0.83 0.60 0.325 THBD 28 0.37 0.50 23 0.24 0.18 1.000

TABLE-US-00003 TABLE III Bioincompatible Biocompatible Total Non- 30 16 46 Epithelioid Epithelioid 15 19 34 Total 45 35 80 p-value = 0.06

Example 2

[0056] By Enzyme-Linked ImmunoSorbent Assay (ELISA), protein was determined in supernatant obtained from cultures of mesothelial cells derived from 26 PD patient effluents, distributed like this: 26 samples taken at PD treatment start, 26 samples taken at 12 months, 20 samples taken at 18 months and 11 samples taken at 24 months (data not shown). Mesothelial cells were cultured in Earle's M199 medium supplemented with 20% of FBS and 50 U/ml penicillin, 50 g/ml streptomycin and 2% of Biogro-2. When cells reached confluency, medium was replaced with fresh medium during 24h. Supernatant was collected and stored at 80 C. until use. For analysis of supernatants, the protein in supernatant was normalized with the total protein in the cell lysate. Selected markers were analyzed with ELISA. TSP1, VEGF, MMP2, CDH13 and GREM1 showed different expression between Epithelioid and Non-Epithelioid phenotypes, with a higher amount of all of these proteins in the Non-Epithelioid group.

Example 3

[0057] 40 effluents derived from PD patients were analyzed by ELISA, and exemplary data for different markers on protein level is included herein.

[0058] VEGF in effluent showed a significant difference between Epithelioid and Non-Epithelioid phenotypes (FIG. 1). This indicates that according to progress of EMT of mesothelial cells, VEGF increases. Mass Transfer Coefficient (MTC) of creatinine (Cr) is a clinical parameter than indicates the peritoneal transport state. MTC higher than 11 is characteristic for a pathological peritoneal transport. When results for VEGF in effluent were grouped by MTC, a statistic difference was observed between patients with MTC higher or lower than 11, and VEGF was higher in MTC>11 group. So, VEGF in effluent may be used to determine the functional state of the peritoneal membrane. VEGF in effluent, when grouped by bio-incompatible (more aggressive fluid) and bio-compatible PD fluids, also showed a significant difference between these groups with more VEGF in bio-incompatible fluid.

[0059] GREM1 in effluent didn't show a significant difference between Epithelioid and Non-Epithelioid phenotypes (FIG. 2), with a higher amount of protein in Epithelioid group, but the difference was significant in supernatant, where Non-Epithelioid phenotype present the higher amount of protein. GREM1 in effluent presented statistic difference when it was classified by MTC, with the higher level of protein in MTC<11 group. GREM1 did not show a significant difference between bio-compatible and bio-incompatible PD fluids.

[0060] TSP1 in effluent showed a significant difference between Epithelioid and Non-Epithelioid phenotypes (FIG. 3) and between MTC<11 or MTC>11 groups (FIG. 4). So, TSP1 is related with the EMT of mesothelial cell/MMT process and the transport in peritoneal membrane. TSP1 protein in effluent didn't show differences between bio-compatible and bio-incompatible PD fluids, but the p-value (p=0.16) indicate a tendency to significance.

[0061] COL13 in effluent has been also analyzed and showed differences depending on phenotypes and creatinine transport.