SUBSTANCE INTERACTING WITH C TERMINAL FRAGMENT OF THE STIM1 FRACTION LOCALIZED TO THE PLASMA MEMBRANE OF THE CELLS, FOR ITS USE IN THE TREATMENT OF CANCERS, AND IN SCREENING AND DIAGNOSTIC METHODS

Abstract

The present invention relates to a substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, for its use in the treatment of cancers. The present invention further relates to a pharmaceutical composition comprising at least one substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of cells, and at least one pharmaceutically acceptable excipient. The invention also refers to the use of isolated C terminal fragment of the STIM1 fraction localized to the plasma membrane of cells in a method for screening candidate molecules for treating cancers, especially pancreatic, colon, breast, Burkitt lymphoma or chronic Lymphocytic Leukaemia. The present invention also relates to a process for predicting the progression and/or monitoring the progression of a cancer, comprising the in vitro detection of the expression of STIM1 in a sample of the tumour of the subject.

Claims

1-16. (canceled)

17. A method of treating cancer comprising the administration of a substance interacting with the C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells to a subject in need of treatment.

18. The method according to claim 17, wherein the peptide sequence of the C terminal fragment of the STIM1 protein is the sequence SEQ ID NO: 2:

19. The method according to claim 17, wherein said cancer is a cancer with cells having a STIM1 fraction localized to the plasma membrane of the cells and with at least a part of its C terminal end being extracellular.

20. The method according to claim 19, wherein said cancer is selected from the group consisting of pancreatic cancer, colon cancer, breast cancer, Burkitt lymphoma and chronic Lymphocytic Leukemia.

21. The method according to claim 20, wherein said pancreatic cancer is pancreatic ductal adenocarcinoma.

22. The method according to claim 17, wherein said substance is selected from the group consisting of an antibody or a fragment thereof, a protein, a peptide, a chemical compound and an aptamer.

23. The method according to claim 17, wherein said substance is administered in combination with at least one other active ingredient.

24. The method according to claim 17, wherein said substance specifically binds to C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells.

25. A pharmaceutical composition comprising at least one substance interacting with C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells, and at least one pharmaceutically acceptable excipient.

26. The pharmaceutical composition according to claim 25, further comprising at least one other active ingredient.

27. A method of screening candidate molecules for treating cancers comprising contacting said candidate molecule with an isolated C terminal fragment of the STIM1 fraction localized to the plasma membrane of the cells and detecting the interaction of said candidate molecule with said C terminal fragment.

28. The method according to claim 27, wherein the method of screening uses a technique selected from the group consisting of biological screening and biophysical screening.

29. The method according to claim 28, wherein screening is a technique selected from the group consisting of immunofluorescence, Western blot, immunoprecipitation, surface plasmon resonance (SPR), flow cytometry, video microscopy, study of calcium flows, enzyme-linked immunosorbent assay (ELISA), fluorescence and luminescence complementation assays and confocal microscopy.

30. A process for predicting the progression and/or monitoring the progression of a cancer, comprising the in vitro detection of the expression of STIM1 in a sample of the tumour of the subject.

31. The process according to claim 30, wherein said cancer is pancreatic cancer.

32. The process according to claim 30, wherein said detection is made with an antibody directed against the C terminal fragment of the STIM1 fraction located at the cell plasma membrane.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0076] FIG. 1 shows that STIM1 expression is reduced in pancreatic cancer cells but its increase is associated with a decreased overall survival in pancreatic cancer. A: Expression of STIM1 protein in pancreatic tissues is detected by immunochemistry and ranked in two grades (low and high STIM1 expression). Representative images of STIM1 staining in pancreatic primary tumors obtained from human pancreas cancer resections (STIM1 antibodies from Sigma-Aldrich clones HPA011088 (1) and N6072 (2)). B: representative Kaplan-Meieroverall survival curves based on the expression of STIM1 in tumor tissues. Patients are split in two groups: high (STIM1(TUM≥2, high grade, dashed line) and low (STIM1(TUM<2, low grade, full line) STIM1 expression (n=48). Data are expressed as t-test of student and Cox analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0077] FIG. 2 shows that STIM1 is implicated in the constitutive calcium entry of Panc-1-Wt cells. Panels A and B: Constitutive entry is evaluated by recording intracellular Ca.sup.2+ concentration variations consequent to changes in extracellular Ca.sup.2+ concentrations. Constitutive Ca.sup.2+ entry is regulated by STIM1 expression as observed by the amplitude of this entry in Panc-1-Wt cells overexpressing (STIM1 OE; n=150; triangles, Panel A1) or under-expressing (SiRNA targeting STIM1; n>40; grey squares, Panel B1) compared to values obtained in cells transfected with an empty vector (squares) or an untargeted SiRNA (SiRNA Ctrl, circles). Representatives recording of constitutive Ca.sup.2+ entry measurements are presented for the different conditions (Panels A2: black line=empty vector, grey line=STIM1OE and B2: black line=siRNA Ctrl, grey line=siRNA STIM1). Data are expressed as mean +/−SEM of n observations and analyzed with a non parametric Mann Whitney test, *P<0.05, **P<0.01 and ***P<0.001.

[0078] FIG. 3 shows that mSTIM1 expression is correlated to constitutive calcium entry in different pancreatic cancer cell lines BxPC3, Panc-1-Wt and MiaPaca. Panel A1: Constitutive entry is evaluated by recording intracellular Ca.sup.2+ concentration variations consequent to changes in extracellular Ca2+ concentrations. Histograms of constitutive Ca2+ entry amplitudes measured in BxPC3 (diamonds), Panc-1-Wt (squares) and MiaPaca (triangles) cell lines are presented in Panel A1. Representatives recordings of constitutive Ca2+ entry measurements are presented for the different Pancreatic cell lines in Panel A2 (BxPC3: black line, MiaPaca: black bold dashed line, Panc: black dashed line). Data values are plotted as individual experimental points and the mean +/−SEM of n observations is also reported. Data are analyzed by non parametric Mann Whitney analysis, ***P<0.001. Panel B: Amount of mSTIM1 observed in the different pancreatic cell lines (Panc-1-Wt>MiaPaca>BxPC3) is proportional to the amplitude of constitutive Ca.sup.2+ entry measured in these cell lines. Representative overlays of mSTIM1 level detected by flow cytometry in BxPC3 (Panel B1) Panc-1-Wt (Panel B2) and MiaPaca (Panel B3) cells. STIM1 labeling in non permeabilized cells was realized with an anti-STIM1 antibody clone GOK (targeting the STIM1 N-terminal part) coupled to PE (black) and compared to the labeling observed with an isotype (white).

[0079] FIG. 4 shows that the fraction of STIM1 located at the plasma membrane (mSTIM1) has a dual topology in pancreatic cells Panel A: mSTIM1 dual topology on Panc-1-Wt cells plasma membrane was identified by flow cytometry. Line graphs shows representative overlays of mSTIM1 expression (black) detected in non permeabilized cells with a STIM1 antibody targeting the STIM1 N-terminal region (GOK Ab coupled to PE—Panel A1) or with a STIM1 antibody targeting the STIM1 C-terminal region of STIM1 (CDN3H4 clone coupled to PE—Panel A2). White: isotype control. Histograms represent the Mean Fluorescence Intensity values (MFI) ±SEM of mSTIM1 expressing cells (n=6). Panel B: mSTIM1 dual topology on Panc-1-Wt cells plasma membrane was confirmed by an Elisa approach. Histogram display values of optical densities (OD) ±SEM measured in intact Panc-1-Wt cells using antibodies targeting either the N-terminal region (GOK Ab, n=12−Panel B1) or the C-terminal region (CDN3H4 clone, n=4−Panel B2) of STIM1. Panel C: schematic representation of mSTIM double orientation. Data are expressed as mean +/−SEM of n observations and analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0080] FIG. 5 shows that changing total STIM1 expression confirmed mSTIM1 dual topology in Panc-1-Wt cell line. STIM1 mAb reactivity in PANC cells as assessed by direct immunofluorescence staining with flow cytometry analysis in non permeabilized Panc-1-Wt cells transfected with a STIM1 expression vector (V5STIM1Flag: Panel A and B) or a SiRNA targeting STIM1 (SiSTIM1: Panel C and D). Intact cells are labeled with a STIM1 antibody targeting the STIM1 N-terminal region (GOK Ab coupled to PE-Panel A and C) or with a STIM1 antibody targeting the C-terminal region of STIM1 (CDN3H4 clone coupled to PE—Panel B and D). Representative plots of fluorescence of Panc-1-Wt cells stained with STIM1 antibody (black) or isotype-matched control (white) mAbs are shown on a four-decade log scale for each condition. Histograms of Mean Fluorescence intensities (MFI) values are provided for each experimental conditions. MFI values are normalized to control conditions (transfection with an empty vector or a non targeting SIRNA (SiRNA Ctrl). Data are expressed as mean +/−SEM of n observations and analyzed by non parametric Mann Whitney analysis, *P<0.05 and **P<0.01.

[0081] FIG. 6 represents the analysis of mSTIM1 topology by Elisa in pancreatic cancer cell expressing various amount of STIM1. Quantification by Elisa of the amount of STIM1 located at the plasma membrane (Panels A and C) or of total STIM1 expression (Panels B and D) using an antibody targeting a C-Terminal STIM1 epitope (CDN3H4 clone). Histogram display values of optical densities (OD) ±SEM measured in Panc-1-Wt cells transfected with V5 STIM1 Flag normalized and compared to empty vector (Panel A and B; n=8) or transfected with an SiRNA targeting STIM1 (Panel C and D; n>6). Mean values ±SEM are reported for each experimental condition. Data are normalized to control values (transfection with an empty vector or a non targeting SIRNA) and analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0082] FIG. 7 shows that STIM1 located at the plasma membrane has a dual topology in different pancreatic cancer cells. Representative Flow cytometry plots of plasma membrane STIM1 expression in BxPC3 (Panel 1) and MiaPaca (Panel 2) cell lines. Intact cells were labelled with an antibody recognizing an epitope located in the C-terminus of STIM1 (CDN3H4 clone coupled to PE in light grey PE or isotype in dark grey).

[0083] FIG. 8 shows that STIM1 located at the plasma membrane (mSTIM1) of B lymphocyte cell lines has a dual topology. Panel A: Intact JOK Cells were labeled with anti-STIM1 antibodies recognizing the STIM1 N-terminal region (GOK Ab coupled to PE-Panel A1) or the STIM1 Cterminal region (CDN3H4 clone coupled to PE-Panel A2). Representative cytometry plots of mSTIM1 are representative of 11 experiments with the isotype and anti-STIM1 antibody labeling in white and black, respectively. Values of Mean Fluorescence intensity values (MFI) (n=11) of STIM1 expression are presented in histograms along with the mean value ±SEM. Panel B: Detection by Elisa of mSTIM1 in PLP cells incubated with STIM1 clone GOK (Panel B1) or clone CDN3H4 (Panel B2) antibodies or with secondary antibody only (control). Mean +/−SEM of 4 observations is reported along with single values. Data are analyzed by non parametric Mann Whitney analysis, **P<0.01 and ***P<0.001.

[0084] FIG. 9 shows that STIM1 has a dual topology when located at the plasma membrane of B lymphocytes from Chronic Lymphocytic Patients. mSTIM1 detection in intact B cells was tested by flow cytometry immediately after isolation from the peripheral blood. Representative cytometry plots illustrating the labeling in B cells of STIM1 with a STIM1 antibody targeting either the STIM1 N-terminal region (GOK Ab coupled to PE—Panel A) or with a STIM1 antibody targeting the STIM1 C-terminal region of STIM1 (CDN3H4 clone coupled to PE-Panel B)—Labeling with Isotype control is in dark grey. Histogram of Mean Fluorescence Intensity (MFI) values are presented. Mean +/−SEM of n (at least 50) observations is presented on each histogram and data are analyzed by non parametric Mann Whitney analysis, ***P<0.001.

[0085] FIG. 10 represents the validation by Western Blot of the selectivity of the antibody targeting the C terminus of STIM1 clone CDN3H4. Expression of STIM1 was revealed by Western Blot using an antibody targeting a C-Terminal STIM1 epitope (CDN3H4 clone) in Panc-1-Wt cells (Panel A) or HEK (Panel B) over-expressing STIM1. Panc-1-Wt cells were transfected with an expression vector containing STIM1 (STIM1 OE) or empty vector whereas HEK cells were transduced with a lentivirus containing STIM1 (pSTIM1) or an empty vector.

[0086] FIG. 11 shows that constitutive Ca.sup.2+ entry of pancreatic cancer cells is blocked by an anti-STIM1 antibody targeting the STIM1 C-Terminal region. A: Constitutive Ca.sup.2+ entry is measured by changing external Ca.sup.2+ concentration in pancreatic cancer cells incubated with an anti-STIM1 antibody targeting the C terminal domain of STIM1 (CDN3H4 clone black dashed line) or an isotype (black line). Representative recording of this measurement in Panc-1-Wt cells is presented in Panel A. Histograms display single values of Ca.sup.2+ constitutive entry amplitudes along with the with mean amplitude value +/−SEM of the n observations (n>70 cells) in BxPC3 (Panel B), Panc-1-Wt (Panel C) and MiaPaca (Panel D) cell lines. Data are analyzed by with non parametric Mann Whitney analysis, **P<0.01 and ***P<0.001.

[0087] FIG. 12 shows that Store Operated Ca.sup.2+ entry of pancreatic cancer cells is not modulated by an anti-STIM1 antibody targeting the STIM1 C-Terminal region. A: SOCE is recorded after endoplasmic reticulum Ca.sup.2+ store depletion with Thapsigargin (2 μM) in 0 mM external Ca.sup.2+ and re-addition of 1.8 mM extracellular Ca.sup.2+. Pancreatic cells were incubated with an isotype or with the anti-STIM1 antibody targeting the C terminal end of STIM1 (CDN3H4) as illustrated in Panel A for Panc-1-Wt cells. Histograms display the individual values of SOCE amplitude and speed (slope) and mean values +/−SEM of n observations (n>3) in BxPC3 (Panel B), Panc-1-Wt (Panel C) and MiaPaca (Panel D) cell lines. Data are analyzed by non parametric Mann Whitney analysis, ns: non significant.

[0088] FIG. 13 shows that the presence of STIM1 at the plasma membrane presenting its C-Terminal end exposed to the extracellular environment is independent of STIM1 glycosylation status in pancreatic cells. The amount of mSTIM1 at the plasma membrane is evaluated on Panc-1-Wt cell lines by Elisa using antibodies targeting the N-Terminal (GOK clone, Panel A and B) or C-terminal (CDN3H4 clone, Panel C and D) domains of STIM1. Histogram represent individual values of optical density (OD) and their mean +/−SEM of n observations (n>7) for each experimental conditions. Amount of mSTIM1 (Panel A and C) and total STIM1 (B and D) were evaluated in cells transfected with expression vectors containing wild type STIM1 (V5 STIM1F lag), a STIM1 mutated at the glycosylation sites (V5 STIM1 Flag N131 171) or an empty vector. Values are normalized to the OD measured in cells transfected with the empty vector absorbance. Data are analyzed by non parametric Mann Whitney analysis, **P<0.01 and ***P<0.001.

[0089] FIG. 14 shows that the localization of STIM1 at the plasma membrane with its C-Terminal outside the cell is independent of STIM1 glycosylation status in pancreatic cells. Amount of mSTIM1 was evaluated by Elisa in MiaPaca cells using an antibody targeting the N terminal (GOK clone Panel A) or the C-terminal (CDN3H4 clone, Panel B) domains of STIM1. Histogram represent singles values of optical densities and the mean +/−SEM of n observations (n>4) detected in MiaPaca cells transfected with expression vectors containing wild type STIM1 (V5STIM1Flag), a STIM1 mutated at the glycosylation sites (V5STIM1Flag N131 171) or an empty vector. Values are normalized to the OD measured in cells transfected with the empty vector absorbance. Data are analyzed with non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0090] FIG. 15 shows the regulation of the presence of STIM1 at the plasma membrane with its C-Terminal out by the TGFβ1 signaling pathway in pancreatic cancer cells. An ELISA experimental approach was used to detect mSTIM 1 (intact cells) and total STIM1 expression (permeabilized cells) using an antibody targeting the C-terminal domain of STIM1 in Panc-1-Wt cells treated with TGFβ1 for different periods of time. Histogram shows single values of optical densities (OD) and their mean +/−SEM of the n observations for the different experimental conditions. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0091] FIG. 16 shows the in vitro functional effects of an antibody targeting the C terminal domain of STIM1 in pancreatic cancer cells. Migration of Panc-1-Wt cells is evaluated for 48 hours using a transwell migration assay with boyden chambers (Panel A, n=8) or by the analysis of wound healing (Panel B, n=12). Cells were incubated all along the experiences with 10 μg/ml of an anti-STIM1 antibody targeting the C terminal domain of STIM1 (CDN3H4 clone). Histograms (Panel A1 and B2) show single values of the % of migration as well as the mean +/−SEM of n experiments. A representative evolution over time of wound healing is presented in panel B1. Representative experiments in Boyden chambers or in wound healing test are illustrated in panels A2 and B3. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0092] FIG. 17 illustrates the In vitro effects on pancreatic cancer cell survival of an antibody targeting the C terminal domain of STIM1. In each experimental approach, cells were treated for 48 hours with 2 different concentrations (5 and 10 μg/ml) of the antibody (CDN3H4 clone) targeting the C terminal domain of STIM1 or with a control isotype. Panel A: Evaluation of the cytotoxic effect (Cell Tox green assay, Promega) on Panc-1-Wt cells of the antibody (clone CDN3H4, n=16). Panel B: Evaluation of cell proliferation (Cell Titer proliferation assay, Promega) of cells treated with the antibody (CDN3H4 clone; n=18). Panel C: Analysis of Panc-1-Wt cell survival (CCK-8 kit, Sigma) when cells were treated with different concentrations of the antibody (CDN3H4 clone, n=12). Histograms represent for each experimental conditions individual values and the mean +/−SEM of n observations. Data were normalized to the mean values obtained for cells treated with the isotype. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0093] FIG. 18 shows the in vitro effects on cell death of pancreatic cancer cells off an antibody targeting the C terminal domain of STIM1. Panc-1-Wt cells are treated with an antibody targeting the C terminal domain of STIM1 (CDN3H4 clone) for 48 hours of treatment with 3 different concentrations of the anti-STIM1 antibody (1, 5 and 10 μg/ml) or with an isotype. Cell death was analysis by flow cytometry by Anexin/Propidium Iodide labeling. Percentage of live cells (black), necrotic cells (late necrosis cells=white, early necrosis cells=light grey) or apoptotic cells (dark grey) submitted to antibody treatment are normalized to the isotype treatment conditions (Panel A). Histogram from panel B present the amount of apoptotic cells (anexin/PI positive cells) detected in each experimental conditions. Data are expressed as mean +/−SEM of n observations (n=3). Data are analyzed with non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0094] FIG. 19 demonstrates that targeting C-Terminal out mSTIM1 to modulate pancreatic cancer cells proliferation using different STIM1 antibody targeting the C terminal end of STIM1. Panel A and B: mSTIM1 C-Terminal out detected on Panc-1-Wt cells plasma membrane by flow cytometry using anti-STIM1 C-Terminal antibody (HPA011088) in panel A and anti-STIM1 C-Terminal antibody (HAP012123) in Panel B. MFI of STIM1 labeling is normalize to isotype labeling (n>3). Panel C and D: In each experimental approach, cells were treated for 48 hours with 10 μg/ml of the anti-STIM1 antibody (HPA011088 in panel C and HPA012123 in panel D) targeting the C terminal domain of STIM1 or with a control isotype. Cell proliferation (using the Cell Titer assay Promega) and cell survival (using CCK8 assay Sigma) were evaluated in cells treated with the antibody (HPA011088 or HPA012123; n>4). Histograms represent for each experimental conditions individual values and the mean +/−SEM of n observations. Data were normalized to the mean values obtained for cells treated with the isotype. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0095] FIG. 20 shows the benefits In vitro of associating the chemotherapeutic agent Gemcitabine with an antibody targeting the C terminal domain of STIM1 to reduce pancreatic cancer cell survival. In each experimental approach, cells were treated for 48 hours with 5 μg/ml of the antibody targeting the C terminal domain of STIM1 (CDN3H4 clone) alone or associated with Gemcitabine (15 nM). Cells were treated with a control isotype in control conditions. Panel A: Evaluation in Panc-1-Wt of the effects of the treatments on cell survival (CCK8 kit, Panel A), on cell proliferation (Cell Titer assay, Panel B) on cell cytotoxicity (Cell Tox green assay, Panel C). Histograms (n=4 in each condition) represent for each experimental conditions individual values and the mean +/−SEM of n observations. Data were normalized to the mean values obtained for cells treated with the isotype. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0096] FIG. 21 illustrates that in colorectal adenocarcinoma cells the expression gradient of mSTIM1 with its C-Terminal out is correlated with the effects on cell survival of anti-STIM1 mAbs targeting the C terminus of STIM1. Panel A-B: Amount of mSTIM1 C-Terminal out in the plasma membrane of colorectal adenocarcinoma cells was obtained by flow cytometry experiments (panel A) or by an Elisa approach (panel B). Histograms represents the mean Fluorescence Intensity values (MFI) ±SEM of mSTIM1 expressing cells (n>3) compared to isotype (panel A) or mean values of optical densities (OD) ±SEM measured in intact cells using antibodies targeting either the C-terminal region (CDN3H4 clone, N>3) of STIM1 (panel B). Panel C and D: Effects on cell proliferation (panel C) and cell survival (panel D) of a 48 hours treatment with 10 μg/ml of an antibody (CDN3H4 clone) targeting the C terminal domain of STIM1 or with a control isotype. Evaluation in panel C of cell proliferation (Cell Titer assay) and of cell survival (CCK8 kit) in panel D when cells were treated with the anti-STIM1 antibody clone CDN3H4 (n=12 in each conditions). Histograms represent for each experimental conditions individual values and the mean +/−SEM of n observations. Data were normalized to the mean values obtained for cells treated with the isotype. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0097] FIG. 22 shows the in vitro effects on colorectal adenocarcinoma (SW620 and Lovo) cell death of an antibody targeting the C terminal domain of STIM1 (CDN3H4 clone). LOVO cells (panels A-B) and SW-620 cells (panels C-D) are treated with 10 μg/ml of CDN3H4 antibody for 48 hours or with an isotype. Cell death was analysis by flow cytometry using an Anexin/Propidium Iodide labeling protocol. Percentage of live cells, necrotic cells or apoptotic cells submitted to the antibody treatment are normalized to the isotype treatment condition (Panels A-C). Histogram from panels B-D present the amount of necrotic cells (anexin/PI positive cells) detected in each experimental conditions. Data are expressed as mean +/−SEM of n observations (n=1).

[0098] FIG. 23 outlines that expression of mSTIM1 C-Terminal out in breast cancer cells is correlated with the effects on cell survival of anti-STIM1 mAbs targeting the C terminus of STIM1. Amount of mSTIM1 C-Terminal out in the plasma membrane of breast cancer cells was evaluated by an Elisa approach (panel A). Histograms represent the mean values of optical densities (OD) ±SEM measured in intact cells using antibodies targeting the C-terminal region (CDN3H4 clone) of STIM1. Panels B and C: In each experiment, cells were treated for 48 hours with 10 μg/ml of the antibody targeting the C terminal domain of STIM1 (CDN3H4 clone) or with a control isotype. Evaluation of cell proliferation (Cell Titer assay, Panel B) or cell survival (CCK8 kit, Panel C) of two types of breast cancer cell lines treated with the anti-STIM1 antibody (CDN3H4 clone; n=8). Histograms represent for each experimental conditions individual values and the mean +/−SEM of n observations. Data were normalized to the mean values obtained for cells treated with the isotype. Data are analyzed by non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.

[0099] FIG. 24 outlines the effects of a STIM1 C terminal peptide on A: PANC-1 cell survival (%), B: PANC-1 cell proliferation (%) and C: PANC-1 cell cytotoxicity (%), for untreated cells (solid circles) and cells treated with STIM1 fragment peptide C8 (corresponding to amino acids 495-530 of STIM1 sequence) 10 μg/ml (triangles).

EXAMPLES

Example 1: Expression of STIM1 Protein as a Pathology Evolution Marker of Pancreatic Adenocarcinoma

[0100] We have demonstrated that the expression of STIM1 protein, in cancer cells of patients with pancreatic adenocarcinoma (Pancreatic Ductal AdenoCarcinoma: PDAC) constitutes a pathology evolution marker (see FIG. 1).

[0101] The expression of STIM1 (average grade) in healthy pancreatic tissues and pancreatic tumor tissues (PDAC) (n=48) is shown in Table 1.

TABLE-US-00002 TABLE 1 Variable Average Standard deviation Test Student STIM1 tumor 1.58 0.72 0.002** STIM1 healthy 1.95 0.19 **P < 0.01

[0102] The expression of STIM1 in healthy pancreatic tissues and cancer pancreatic tissues defined by the percentage of low and high grades (n=48) is shown in Table 2.

TABLE-US-00003 TABLE 2 Variable Expression of STIM1 STIM1 tumor Low: 47.92% High: 52.08% STIM1 healthy Low: 8.7% High: 91.3%

[0103] The expression of STIM1 protein is decreased in cancer cells compared to normal peri-tumoral cells. However the increase in STIM1 expression in cancer cells is correlated with poor prognosis.

[0104] STIM1 expression was determined using classical immunohistochemistry protocols. A panel of two different validated antibodies directed against STIM1 was used. Briefly, for IHC analysis of STIM1 expression, formalin-fixed, paraffin-embedded (FFPE) tissue blocks containing both tumor and normal pancreatic tissue were obtained from patients at different pancreatic cancer stages and who had surgery. IHC staining was performed using automated IHC staining system (Roche Diagnostics Ventana Benchmark, N750-BMKU-FS) in accordance with the manufacturer's recommendations. The level of expression of our proteins is scored (1 to 3) corresponding for the score 0 to a weak or nonexistent expression of STIM1 and 3 corresponding to a strong expression of our proteins of interests. Statistical analysis was done using Wilcoxon paired tests.

Example 2: The Level of Expression of STIM1 Modulates the Constitutive Entry of Calcium

[0105] The level of expression of STIM1 modulates the constitutive entry of calcium: (see FIG. 2). The amplitude of this entry is related to the level of expression of STIM1 protein at the plasma membrane of pancreatic cancer lines (see FIG. 3).

[0106] Panc cells were transfected with 5 μg of an expression vector containing the STIM1 gene (pSTIM1 vector) or an empty vector using the transfecting agent lipo293 following commercial recommendations. Cells were transfected with 5 μM of an SiRNA targeting STIM1 (SEQ ID NO: 3: ugagggaagaccucaauua) or a SiCtrl using the transfecting agent lipoRNAimax following commercial recommendations.

[0107] For constitutive Ca.sup.2+ entry (CCE) measurements, Panc cells were loaded with 2 μM of the Fura-2/AM fluorescent dye in the presence of 2 μM Pluronic acid for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 5 mM CaCl.sub.2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl.sub.2) for 100 s and again with the original 5 mM CaCl.sub.2)—containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.

[0108] The amount of mSTIM1 is evaluated by flow cytometry using 5×10.sup.6 cells per condition. Cells were taken off culture plates using an Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and then incubated with 1 μl of an anti-STIM1 antibody coupled with PE (GOK-PE, BD Biosciences; 20 μg/ml) or with 0.5 μl of an isotype control coupled with PE in 50 μl of PBS on cells for 30 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman coulter).

Example 3: Demonstration of the Double Topology of the mSTIM1 Protein

[0109] We demonstrated by cytometry and Elisa approaches using anti-STIM1 antibodies targeting the C terminal end (clone CDN3H4, sc-66173, Santacruz) and the N terminal end (clone GOK, BD transduction Laboratory) of the STIM1 protein that the fraction of this protein STIM1 localized to the plasma membrane (mSTIM1) has a double topology with an orientation in which the N-terminal domain of the protein is extracellular (called N ter out), as previously established, but also an orientation in which the terminal C domain of mSTIM1 is extracellular (Cter out orientation) (see FIG. 4). The dual orientation of mSTIM1 has been demonstrated in pancreatic cells in endogenous expression of STIM1 and using cells over-expressing or under-expressing STIM1 or (see FIGS. 5 and 6).

[0110] Panc cells were transfected with 5 μg of an expression vector containing the STIM1 gene (pSTIM1 vector) or an empty vector using the transfecting agent lipo293 following commercial recommendations. Cells were transfected with 5 μM of an SiRNA targeting STIM1 (sequence: UGAGGGAAGACCUCAAUUA, SEQ ID NO: 3) or a SiCtrl using the transfecting agent lipoRNAimax following commercial recommendations.

[0111] The amount of STIM1 was evaluated using flow cytometry or Elisa approaches.

[0112] For the ELISA measurements, 10.sup.6 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (0.1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (10 μl CDN3H4 sc-66173, Santacruz; 1 μg/ml) for 1 h30 at RT. After 3 washes with PBS, cells were incubated with in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.

[0113] For determination of the amount of STIM1 using flow cytometry, 5×10.sup.6 cells were used per condition. Pancreatic cells were taken off culture plates using Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and incubated with 50 μL of PBS containing anti-STIM1 antibody directed against the N terminus (1 μl GOK-PE, BD Biosciences; 20 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) or 0.5 μL of an isotype control for 30 min on ice. In case of incubation with the uncoupled anti-STIM1, cells were washed and then incubate with a PE conjugated secondary antibody for 20 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman Coulter Life Sciences).

Example 4: Dual Orientation of mSTIM1 in Various Cancer Cell Lines

[0114] The dual orientation of mSTIM1 has been observed in various pancreatic cell lines (see FIG. 7), but also in colon adenocarcinoma cell lines (see FIG. 21), breast adenocarcinoma cells (see FIG. 23), B lymphocytes from hematologic malignancies (B-lymphocytes of patients suffering from Chronic lymphocytic leukemia, Burkitt cell lines) (see FIGS. 8 and 9). Dual topology was identified by flow cytometry or Elisa approaches.

[0115] For the ELISA measurements, 10.sup.6 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (0.1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (10 μl CDN3H4 sc-66173, Santacruz; 1 μg/ml) for 1 h30 at RT. After 3 wash with PBS, cells were incubated in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.

[0116] For determination of the amount of STIM1 using flow cytometry, 5×10.sup.6 cells were used per condition. Pancreatic cells were taken off culture plates using Enzyme free solution for 5 min at 37° C. Cells were then centrifuged for 5 min at 1500 rpm and incubated with 50 uL of PBS containing anti-STIM1 antibody directed against the N terminus (1 μl GOK-PE, BD Biosciences; 20 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) or 0.5 μL of an isotype control for 30 min on ice. In case of incubation with the uncoupled anti-STIM1, cells were washed and then incubate with a PE conjugated secondary antibody for 20 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman Coulter Life Sciences).

Example 5: Demonstration of the Specificity of the Detection of STIM1 by Cter Antibodies

[0117] By western blot approaches (see FIG. 10), it has been shown that an anti-STIM1 antibody targeting the C-terminal domain of the protein (STIM1: clone CDN3H4, sc-66173, Santacruz) well recognizes STIM1 protein. Flow cytometry and Elisa approaches have shown that this antibody recognizes the membrane fraction of STIM1 (mSTIM1). This specificity of the detection of mSTIM1 by Cter antibodies was confirmed by flow cytometry and Elisa by labelling with STIM1 in intact non permeabilized cells expressing different levels of this protein (see FIGS. 5 and 6). This antibody makes it possible to detect specifically in flow cytometry the membrane fraction of STIM1 (mSTIM1) as well as the majority fraction of STIM1 located at the reticulum membrane endoplasmic on permeabilized cells or broken cells.

[0118] Protein extraction was performed by scratching 107 Panc 1-wt cells on ice with a lysis buffer containing: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 2.5 mM Na+ pyrosodium tetraphosphate, 1 mM glycerophosphate, 1 mM Na+ orthovanadate, 1 μg/ml leupeptin and a protease inhibitor cocktail. Protein extracts were sonicated and centrifuged for 12 min at 16,000 g. Protein concentration of cell lysates were determined using the Folin method. 75 μg of proteins were run on SDS-PAGE 7.5% polyacrylamide gels in denaturing conditions, and then transferred onto PVDF (PolyVinyliDene Fluoride) membrane sheets. Unspecific blocking was done by incubation with 5% fat milk in PBS, 0.1% tween 20 for 1 hour. Blots were incubated overnight with 5% fat milk in PBS, 0.1% tween 20, containing mouse monoclonal anti-STIM1 (CDN3H4 clone Santacruz; 1:1,000 dilution) or mouse monoclonal anti-GAPDH antibody (6C5 clone Abcam; 1:10,000 dilution). Blots were incubated with Horseradish Peroxydase (HRP)-conjugated goat anti-mouse after washing with PBS, 0.1% tween 20 and revealed with the Luminata Forte reagent. All results were normalized upon GAPDH quantification.

Example 6: Constitutive Ca.SUP.2+ Entry of Pancreatic Epithelial Cells is Inhibited by the Use of the Anti-STIM1 Antibody

[0119] We demonstrate that constitutive Ca.sup.2+ entry of pancreatic epithelial cells is inhibited by the use of the anti-STIM1 antibody clone CDN3H4 (clone CDN3H4, sc-66173, Santacruz) (see FIG. 11). This antibody does not have effect on activated Ca.sup.2+ entry by release of intracellular calcium stores (SOCE: Store Operated Ca.sup.2+ Entry) (see FIG. 12).

[0120] For SOCE measurement 10.sup.6 cells were seeded in 96 wells. Cells are loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™, Molecular Probes) fluorochrome according to the manufacturer's protocol. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca.sup.2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. Intracellular calcium level variations were monitored by using the FlexStation 3™ (Molecular Devices, Berkshire, UK), Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340 nm (bound to Ca.sup.2+) and 380 nm (unbound to Ca.sup.2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca.sup.2+ concentrations. The SOCE was elicited by releasing the Ca.sup.2+ stores from the endoplasmic reticulum with thapsigargin (2 μM) solution under Ca.sup.2+-free conditions to determine the magnitude of intracellular Ca.sup.2+ release (Hepes-buffered solution). Next, cells were returned to a Ca.sup.2+-containing Hepes-buffered solution to measure SOCE. The magnitude and speed of SOCE were estimated.

[0121] For constitutive Ca.sup.2+ entry (CCE) measurements, Panc-1 cells were loaded with 2 μM of the Fura-2/AM fluorescent dye in the presence of 2 μM Pluronic acid for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 5 mM CaCl.sub.2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl.sub.2) for 100 s and again with the original 5 mM CaCl.sub.2)—containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.

Example 7: The Presence at the Plasma Membrane of mSTIM1 Having a Cter Out Orientation is Independent of Glycosylation of STIM1 and Regulated by TGFβ Signaling

[0122] The presence at the plasma membrane of mSTIM1 having a Cter out orientation is independent of glycosylation of STIM1 (glycosylation at position AA N131 and N171) in contrast to its Nter out orientation (see FIGS. 13 and 14).

[0123] The amount of mSTIM1 having a Cter out orientation is regulated by different cell signalling pathways and especially by the way TGFβ, an over-activated pathway in pancreatic cancer. TGFβ pathway stimulation induces an increased amount of mSTIM1 with Cter orientation in pancreatic cancer cells (see FIG. 15).

[0124] Panc and Miapaca cells were overexpressed with 5 μg of the expression vector pSTIM1 vector containing wild type STIM1 (pSTIM1) or mutated STIM1 (N131Q N171Q STIM1) or an empty vector using the transfecting reagent lipo293 following commercial recommendations. To evaluate the influence of TGFβ signaling cells were treated with 25 ng/ml TGFβ1 for 5 min, 15 min, 30 min, 24 h and 48 h in DMEM medium at 37° C.

[0125] Amount of STIM1 at the plasma membrane was evaluated by ELISA. 10.sup.6 cells were loaded on 96 wells plates in DMEM medium. Cells were fixed using PFA 2% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (1 μl GOK, BD Biosciences; 1 μg/ml) or the C terminus (5 μl CDN3H4 sc-66173, Santacruz; 10 μg/ml) for 1 h30 at RT. After 3 washes with PBS, cells were incubated with in PBS +5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H2504 solution. ELISA plate was read at 392 nm in absorbance and the optical density normalized using absorbance emitted at 495 nm by cells following incubation with Janus green.

Example 8: Anti-STIM1 Antibody Targeting the C Terminal Fraction of STIM1 Reduces In Vitro Migration of Panc-1-Wt Pancreatic Cancer Cells

[0126] Anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) reduces in vitro migration of Panc-1-Wt cells evaluated by transwell migration (Boyden chambers) or by wound healing technique (see FIG. 16). For the migration measurements (wound healing technique), 10.sup.6 cells were loaded on 96 wells plate in DMEM medium. Panc-1-Wt confluent cells were manually scratched, and cells were treated with anti-STIM1 CDN3H4 (10 ug/ml) or an isotype. Acquisition was done during 48 hours with a rate of one picture per hour. Area of a wound was calculated in images representing the time-series.

[0127] For migration assays based on migration through filters, 5×10.sup.6 cells were loaded on the top of a Boyden chamber (transwell Corning 8 μM pores) and incubated with or without anti-STIM1 targeting the C terminus of STIM1 (CDN3H4 clone 10 μg/ml) for 48 hours at 37° C. in DMEM media without SVF. The bottom plate was loaded with 10% SVF DMEM medium. After removing cells from the upper face of the filter, five fields of cells were manually counted after fixation of migrated cells and Dapi nucleus coloration.

Example 9: The Anti-STIM1 Antibody Targeting the C Terminal Fraction of STIM1 Reduces In Vitro the Survival of Panc-1-Wt Cells Pancreatic Cancer Cells, Colon Cancer Cells and Breast Cancer Cells

[0128] The anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) reduces in vitro the survival of pancreatic cancer cells (see FIG. 17), colonic pancreatic cancer cells (see FIG. 21) and breast cancer cells (see FIG. 23). Treatment with Anti-STIM1 antibody targeting the C terminal fraction of STIM1 reduces cell survival mainly by reducing cell proliferation (see FIG. 17 and FIGS. 21 and 23). In vitro, Anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4) let to cell cytotoxicity by inducing apoptosis of these cells (see FIGS. 18 and 22). Different antibody targeting the C terminal end of STIM1 also induces a decrease in cell survival (FIG. 19). The effects of anti-STIM1 antibodies are proportional to the amount of mSTIM1 orientated with its C terminal end out (FIGS. 21 and 23).

[0129] For cell proliferation, cytotoxicity, and cell survival measurements, 10.sup.6 cells were seeded in 96 wells plates in 100 μL of DMEM medium. Cells were incubated for 48H hours at 37° C. with an anti-STIM1 antibody targeting the C terminal of STIM1 (CDN3H4, Santa Cruz; HPA011088 or HPA012123 clones, Sigma). Cell proliferation was evaluated using the Cell Titer kit proliferation assay (Promega). Cytotoxicity was evaluated using the Cell Tox green cytotoxicity assay (Promega) and cell survival measured using the CCK8 kit from Sigma. All these kits were used following manufacturer recommendations and using a plate reader

[0130] For the apoptosis measurements, 5×10.sup.6 cells were seeded in 12 wells plates. Cells were incubated for 48 hours with anti-STIM1 CDN3H4 antibody (10 ug/ml) at 37° C. After wash, cells were taken off plates using an enzyme free solution. Cells were centrifuged for 5 min at 1500 rmp and suspended in 50 μL of PBS containing 1 μL of AnnexinV-FiTC and 1 μL of propidium iodure (PI) for 15 min at RT in the dark. Evaluation of Anexin and PI staining was realized using a flow cytometer (Navios, Beckman Counter) to evaluate cell apoptosis and necrosis.

Example 10: Association of an Anti-STIM1 Antibody Targeting the C Terminal Fraction of STIM1 with Gemcitabine Potentiates the Effect of Gemcitabine on Pancreatic Cell Survival

[0131] A low dose (5 μg/ml) of the anti-STIM1 antibody targeting the C terminal fraction of STIM1 (clone CDN3H4, Santa Cruz) or a low dose of Gemcitabine (15 nM) slightly but significantly reduce the survival and proliferation of pancreatic cancer cells (see FIG. 17), without inducing cell cytotoxicity. Association of this anti-STIM1 antibody targeting the C terminal fraction of STIM1 with the same low dose of Gemcitabine induces a significant and important potentiation of the effects of a treatment with Gemcitabine alone.

[0132] For cell proliferation, cytotoxicity, and cell survival measurements, 10.sup.6 cells were seeded in 96 wells plates in 100 μL of DMEM medium. Cells were incubated for 48H hours at 37° C. with 5 μg/ml of the anti-STIM1 antibody targeting the C terminal of STIM1 (CDN3H4, Santa Cruz) alone, with 15 nM Gemcitabine alone or with both 5 ug/ml of the anti-STIM1 clone CDN3H4 and 15 nM Gemcitabine. Relevant controls (isotype or/and DMSO treatments) were also realized. Cell proliferation was evaluated using the Cell Titer kit proliferation assay (Promega). Cytotoxicity was evaluated using the Cell Tox green cytotoxicity assay (Promega) and cell survival measured using the CCK8 kit from Sigma. All these kits were used following manufacturer recommendations and using a plate reader

Example 11: Method of Screening Anti-C Terminal Fragment of mSTIM1

[0133] Screening of the molecules modulating the STIM1 fraction localized to the plasma membrane (mSTIM1) with its C terminus out is carried out on cells expressing sufficient amount of mSTIM1 with its C terminus out such as Panc-1 pancreatic cancer cells. Two types of Panc-1 cells are used: Panc-1 cells stably transfected with STIM1 or Panc-1 with endogenous amount of STIM1. These cells display a measurable constitutive calcium entry. Screening consists in a first approach of measuring the effects of the molecules targeting the fraction of STIM1 localized to the plasma membrane of the cells on the constitutive entry of extracellular calcium. The effects of these molecules on calcium entry dependent on the release of reserves SOCE (Store Operated Calcium Entry) are also evaluated in order to determine the effect of the molecules on the influx SOCE of the molecules acting on constitutive calcium entry. The amplitude of these two calcium flows is measured by monitoring the variations in intracellular calcium concentration using a fluorescent probe (Fura-2 QBT, Molecular Devices).

[0134] For constitutive Ca.sup.2+ entry (CCE) measurements, Panc cells were loaded with 2 μM of the Fura-2 QBT fluorescent dye for 60 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 1.8 mM CaCl.sub.2). Cells were then washed and displayed on coverslips for single cell fluorescence imaging. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl.sub.2) for 100 s and again with the original 5 mM CaCl.sub.2-containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.

[0135] For SOCE and Constitutive Ca2+ entry measurements, 10.sup.6 cells were seeded in 96 wells. Cells are loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™, Molecular Probes) fluorochrome according to the manufacturer's protocol. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca.sup.2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. Changes in fluorescence are monitored by using the FlexStation 3™ (Molecular Devices, Berkshire, UK). Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340 nm (bound to Ca.sup.2+) and 380 nm (unbound to Ca.sup.2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca.sup.2+ concentrations. The SOCE was elicited by releasing the Ca.sup.2+ stores from the endoplasmic reticulum with thapsigargin (2 μM) solution under Ca.sup.2+—free conditions to determine the magnitude of intracellular Ca.sup.2+ release (Hepes-buffered solution). Next, cells were returned to a Ca.sup.2+-containing Hepes-buffered solution to measure SOCE. The magnitude and speed of SOCE were estimated. Constitutive entry is measured in the absence of any stimulation by following the quench of Fura 2 fluorescence recorded at 360 nm wavelength excitation when manganese (Mn.sup.2+) is added to the extracellular medium. The rate of fluorescence quenching represents a good approximation of Ca.sup.2+ entry.

[0136] The cells are put in contact with the test compound at the moment of loading the cells with the fluorescent probe and throughout measurement of the variations in intracellular calcium concentration.

[0137] The effects of screened compounds on STIM1 location at the plasma membrane is realized for lead candidates. Screening is also done by following the effects of promising molecules on cell migration and cell survival.

REFERENCE LIST

[0138] 1. Mignen O, Thompson J L, Shuttleworth T J. STIM1 regulates Ca2+ entry via arachidonate-regulated Ca2+-selective (ARC) channels without store depletion or translocation to the plasma membrane. J Physiol. (2007) 579:703-15. [0139] 2. Remington Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985.