Clinical information was retrospectively obtained from 74 untreated patients diagnosed with CLL according to the World Health Organization (WHO) classification [19], and 13 healthy volunteers at the Brest University Hospital. Disease assessment included Binet stage determination, progression free survival (PFS), treatment free survival (TFS), CD38 expression, lymphocyte counts, lymphocyte doubling time (LDT), cytogenetic risk-status, and IgHV mutational status, which were performed as previously described [20]. Consent was obtained from all individuals and the protocol approved by the Ethical Board at the Brest University Hospital (clinicaltrials: NCT03294980; cohort OFICE; CRB Biobank collection 2008–2014), in accordance with the Declaration of Helsinki.

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation (Eurobio, Courtaboeuf, France) and B cells were further enriched using the Pan B-cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Cell purity was assessed by fluorescence-activated cell sorting (FACS) analysis and was over 95% for B cells (CD19+).

All monoclonal antibodies (mAb) were from Beckman-Coulter (Brea, CA, USA) unless specified: FITC-conjugated anti-IgM (clone: SA-D4), cyanin (Cy)5.5-conjugated anti-CD38 (LS198.4.3), electron coupled dye (ECD)-labelled anti-CD5 (BL1a), allophycocyanin (APC)-conjugated anti-IgD (1A6–2, BD Biosciences, Franklin-Lakes, NJ), APC-Alexa Fluor 700 (AF700)-conjugated anti-CD19 (J4–119), Pacific blue (PB)-conjugated anti-CD21 (1A4CD27) mAbs with saturating concentrations were incubated for 10 min at RT with 10 μl of PBS-washed blood. Versalyse solution (Beckman- Coulter) was added for 10 min in order to lyse red blood cells. The determination of the mean fluorescence intensity (MFI) of all markers required a minimum of 5000 events. The results were standardized to those obtained with isotype controls. Data were analysed using Kaluza 1.5 software (Beckman-Coulter).

For intracellular staining, preliminary fixation and permeabilization were performed with the cytofix/cytoperm kit (BD Biosciences) followed by a 30 min incubation at 4 °C with Phyco-Erythrin (PE)-conjugated anti-STIM1 (Gok, BD Biosciences), PC7-conjugated anti-CD19 and APC-AF700-conjugated anti-CD5 mAbs. The same protocol was applied for plasma membrane STIM1_PM with the omission of the permeabilization step. In the transfection experiments a polyclonal rabbit anti-human Orai1 Ab (Sigma-Aldrich, Saint-Louis, MO) or anti-TRPC1 Ab (Alomone, Jerusalem, Israel) were used after the permeabilization step, and mAb staining was assessed with a FITC-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, Ely, UK). To assess PLCγ2 phosphorylation, 5 × 10⁵ cells were fixed and permeabilized with cold 80% methanol solution during 30 min. After 2 washes in PBS-BSA 0.05%, APC-conjugated anti-pPLCγ2 mAb (clone K86–689.37, BD Biosciences) was added.

For each patient, B-CLL cells were incubated 48 h in 24-well plates at 37 °C with either (i) 10 μg/ml murine IgG2a (Biolegend, San-Diego, CA) (ii) 10 μg/ml anti-STIM1 (Gok), (iii) 10 μg/ml RTX (Roche, Paris, France), or (iv) 2x10μg/ml anti-STIM1 + RTX. All cultures were set at 5 × 10⁵ B cells/mL in RPMI-1640 (Sigma-Aldrich) supplemented with 2 mM L-glutamine, antibiotics, and 20% of decomplemented normal human serum AB (Invitrogen, Carlsbad, CA). B cells were recovered, washed and stained for 15 min with FITC-conjugated annexin-V (AV) / propidium iodide (PI) and AF700-conjugated anti-CD19 antibody according to the Beckman-Coulter apoptosis kit protocol. The decrease in the percentage of live cells (CD19⁺ AV-PI-) was recorded.

For CE measurements, B cells were loaded with 2 μM Fura-2/AM dye (Molecular Probes, Leiden, Netherlands) and 2 μM Pluronic acid (Gibco, Waltham, MA) for 30 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 (Buffer A) supplemented with 5 mM CaCl₂. Cells were washed and left to attach in the same buffer on 12 mm Cell-Tak (Corning, NY) precoated coverslides for 20 min, allowing the de-esterification of the dye. Fura-2 was excited alternatively at 340 and 380 nm (Polychrome V, TILL photonics), and fluorescence emission was recorded at 510 nm using a fluorescence microscope (IX71, Olympus) equipped with a dichroic mirror (415DCLP) and a 14-bit CCD camera (ExiBlue, Qimaging). After the stabilization of basal fluorescence, the extracellular medium was replaced with Buffer A supplemented with 0.5 mM CaCl₂ for 100 s and again with the original 5 mM CaCl₂-containing Buffer A after curve stabilization. Excitation/emission ratio (F_340nm/F_380nm) was calculated for each time point and each cell with the Metafluor 6.3 Software (Universal Imaging, West Chester, USA). The amplitude of CE was calculated after normalization to the basal ratio (ΔF/F₀), as the difference between average values of basal ratio measured in 5 mM external Ca²⁺ and the average ratio value in 0.5 mM Ca²⁺.

For anti-IgM and Thapsigargin (TG)-induced calcium entry, B cells were loaded in Buffer A containing 1.8 mM CaCl₂ and 2 μM Fura-2/AM (Fura-2 QBT Kit, Molecular Devices) for 1 h at 37 °C in Cell-Tak precoated 96-well plates, and fluorescence acquisition (excitation 340 and 380 nm; emission 510 nm) was performed on the Flexstation 3 microplate reader with SoftMax Pro 5.4.5 software (Molecular Devices, San Josa, CA). For anti-IgM induced Ca²⁺ response, the extracellular medium was replaced with Buffer A supplemented with 10 mM CaCl₂, before reading, and 10 μM of polyclonal goat anti-human IgM (Jackson Immunoresearch) were injected after 150 s. TG-induced ER Ca²⁺ release, extracellular medium was replaced with Buffer A supplemented with 100 μM EGTA just before starting the reading protocol. A stimulation with 2 μM of TG was performed after 100 s of recording, and 1.8 mM CaCl₂ was added after 700 s in order to quantify SOCE entry. Ca²⁺ entry were quantified after value normalization (ΔF/F₀) with the exception of basal Ca²⁺ concentrations estimated as the average of initial F_340nm/F_380nm values.

In selected experiments, 1 μM Ibrutinib (SelleckChem, Munich, Germany), 5 μM LY294002 (Sigma Aldrich), 2.5 μM Synta66 (Sigma Aldrich), DMSO, 10 μg/ml anti-STIM1 (clone GOK) or 10 μg/ml IgG2a mAb were added to the loading buffer.

The small interfering RNAs (siRNA) targeting Orai1 (GCAACGUGCACAAUCUCAAtt, Sigma), STIM1 (AGGUGGAGGUGCAAUAUUAtt, Dharmacon, Lafayette, CO), TRPC1 (s7311), and the control siRNA (4390843) were purchased from Ambion unless specified and transfected in B-CLL cells from 3 CE+ CLL patients by nucleofection (Lonza, Basel, Switzerland) as previously described [17, 21]. After a 48 h-incubation, the knock-down was controlled by flow cytometry and intracellular Ca²⁺ analysis was performed.

Protein extraction was performed by incubating 10⁷ B cells for 30 min on ice in 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 protease inhibitor cocktail (Roche). Protein extracts were sonicated and centrifuged for 12 min at 16,000 g. The protein concentration of the cell lysates was determined using the micro-bicinchoninic acid assay, and 50 μg were run on SDS-PAGE (10% polyacrylamide gels) in denaturing conditions, and transferred onto PVDF membrane sheets (Bio-Rad, Hercules, CA). After unbound sites were blocked, blots were probed overnight with 5% fat milk in PBS, 0.1% tween 20, and either mouse monoclonal anti-STIM1 (GOK; 1:1000), rabbit polyclonal anti-Orai1 (1:1000; Sigma Aldrich), mouse monoclonal anti-TRPC1 (E6; 1:1000; Santa Cruz, Dallas, TX) or mouse monoclonal anti-GAPDH antibody (6C5; 1:10,000; Abcam, Cambridge, UK). Washed blots were incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-mouse or rabbit IgG Ab (1:10,000; Abcam). Image acquisition was performed on a Chemi-Smart 5100 system (Vilber-Lourmat) with the Chemi-Capt 5000 software and analysed on Image J. All results were normalized upon GAPDH quantification.

Continuous data are described as mean ± standard error of the mean (SEM). Following normality and equality of variance tests, nominal values were compared to controls using the student’s t test or alternatively by using a nonparametric test (Mann-Whitney rank sum test). Differences among groups were analyzed by one-way ANOVA in a non-parametric test and the Dunn’s test was used for post-hoc comparisons. For categorical data the Fisher’s exact test was used, and for correlation analysis the Pearson’s coefficient r was calculated. The profile likelihood method using a Cox regression model of PFS was used in univariate analysis to determine the optimal threshold and stratify patients into two groups as previously described [22]. PFS, TFS and LDT analyses were next performed using Kaplan–Meier curves and prognosis differences between groups were assessed with a log-rank test. Receiver operating curves (ROC) were generated to determine the area under the curve (AUC) and the optimal cut-off values were chosen by using the upper left corner value (100% specificity). P values under 0.05 were considered significant. Statistical analyses and the correlation matrix were performed using GraphPad Prism 7.0a (La Jolla, CA).

As deregulation in Ca²⁺ signaling is an important hallmark of B-CLL cells, and suspected to vary during CLL disease progression [2], Ca²⁺ entry in the absence of BCR engagement, designated as CE, was evaluated in resting B-CLL cells. To this end 30 untreated CLL patients were selected and, as reported in Fig. 1a, CE was significantly enhanced in a subset of B-CLL cells when compared to B cells from 8 healthy controls (ΔF/F₀: 0.10 ± 0.01 in B-CLL cells versus 0.06 ± 0.01 in controls, P = 0.03). CLL patients were further dichotomized into CE+ (high levels) versus CE- (normal/low levels) using the profile likelihood method in a Cox regression model of PFS for optimal cut-off identification (cut-off = 0.083).Fig1Fig. 1

An elevated level of constitutive calcium entry (CE) is relevant for chronic lymphocytic leukemia (CLL) clinical outcome. a- Representative kinetic plots of CE in representative healthy control B cells (n = 8, Ctrl), CE- B-CLL (n = 13) and CE+ B-CLL (n = 17) samples. A Cox regression model of progression free survival was used to dichotomize CLL patients in CE- and CE+ (dash line in the islet). Kaplan-Meier plots showing: b- time to progression free survival; c- time to treatment free survival; and d- lymphocyte doubling time between CE+ and CE- CLL groups. P values are indicated when significant

One step further, to test BCR pathway dependence in CE+ B-CLL cells, the BCR capacity to mobilize Ca²⁺ was tested within B-CLL cells from 16 CE+ CLL patients, 13 CE- CLL patients, and 13 healthy controls (Fig. 2a and Additional file 2: Figure S2). As previously described [2, 3], Ca²⁺ mobilization in response to BCR engagement was reduced in B-CLL cells when compared to controls (P = 0.002 for both CE subgroups), however no difference was observed when comparing the two CE subgroups within CLL patients. Interestingly, by conducting a bivariate analysis of PFS on both CE and IgM Ca²⁺ mobilization, we further observed that CLL patients with disease progression were restricted to CE+/IgM+ (n = 11) and CE+/IgM- (n = 5) CLL patients but not to CE−/IgM+ (n = 4) and CE−/IgM- (n = 9) CLL patients (P = 0.006, Fig. 2b). To dissect heterogeneity between the 4 subgroups of patients (Additional file 2: Table S1), we next examined whether these differences resulted from differential expression of the membrane surface (s) IgM, sIgD, and co-receptors (CD19, CD21, CD38, and CD5). No differences were observed between the 4 subgroups for these markers that participate or modulate the proximal BCR signaling. As well, no differences were reported when considering CE+ and CE- CLL patients. Accordingly, we concluded that there is independence of CE from proximal BCR signaling and BCR co-activators.Fig2Fig. 2

Constitutive calcium entry (CE) is independent from anti-IgM capacity to induce Ca²⁺ signaling in chronic lymphocytic leukemia (CLL). a- Normalized ratio of peak to baseline Ca²⁺ flux in response to anti-IgM stimulation of healthy control (n = 13, Ctrl), CE- CLL (n = 13) and CE+ CLL (n = 16) samples. b- Bivariate analysis by Kaplan-Meier curves of Ca²⁺ signaling in the context of CLL subsets according to the CE (+/−) and anti-IgM capacity to induce Ca²⁺ signaling (IgM +/−). A Cox regression model of progression free survival (PFS) was used to dichotomize CLL patients in CE- and CE+, on one hand, and IgM- and IgM+, on the other hand. c/d- the effects of Ibrutinib (BTK inhibitor, 2.5 μM) and LY294002 (PI3K inhibitor, 5 μM) on CE in CE+/IgM+ B-CLL samples (n = 3). e/f- the effects of Ibrutinib (BTK inhibitor) and LY294002 (PI3K inhibitor) on anti-IgM capacity to induce Ca²⁺ signaling in CE+/IgM+ B-CLL samples (n = 3). P values are indicated when significant

Since CE could be attributable to an antigen-independent autonomous BCR pathway [15], this Ca²⁺ entry was recorded in the presence of two BCR signalosome inhibitors Ibrutinib, a covalent inhibitor of BTK, and LY294002, a selective inhibitor of PI3Kδ. As shown in Fig. 2c/d, CE+ B-CLL cells from 3 patients were selected and CE was unaffected by the addition of the BCR signaling inhibitors. In parallel and as a positive control, the Ibrutinib and LY294002 capacity to inhibit Ca²⁺ response following BCR activation was demonstrated (Fig. 2e/f). Such a concept was further reinforced by the analysis of basal pPLCγ2, an indicator of BCR signalosome activation, in resting B-CLL cells showing that pPLCγ2 levels were similar between the CE+ and CE- CLL subgroups (Additional file 2: Table S1).

Next, 29 B-CLL cells (10 CE−/IgM-, 4 CE+/IgM+, 4 CE+/IgM- and 11 CE+/IgM+) were selected and a correlation matrix was performed for all in order to compare CE with (i) the basal intracellular Ca²⁺ level estimated by the initial F340/380 ratio, (ii) the anti-IgM Ca²⁺ response; (iii) the ER Ca²⁺ release by thapsigargin (TG), an inhibitor of the ER Ca²⁺ ATPase pumps, that artificially and maximally deplete Ca²⁺ stores in the absence of extracellular Ca²⁺; and (iv) the TG SOCE response observed after Ca²⁺ reffiling.

Results from the correlation matrix were effective to highlight two groups of Ca²⁺ responses in B-CLL cells (Additional file 2: Figure S3A/B). First an association based on the correlation observed between CE and the basal Ca²⁺ level (r = 0.591; P = 0.001), but not CE with anti-IgM and Tg SOCE response (data not shown). Second, a proximal BCR-InsP3R signaling pathway as the anti-IgM Ca²⁺ response was correlated with both TG ER Ca²⁺ release and TG SOCE (P = 3 × 10^− 4 and 1 × 10^− 7, respectively) but not with basal Ca²⁺ levels and CE.

Based on our previous work showing a role for Orai1, TRPC1 channels and STIM1 [17, 23] in CE, and to better characterize the autonomous Ca²⁺ channel influx in CE+ B-CLL cells, three strategies were developed using (1) the Orai1 channel blocker, Synta66 (S66); (2) specific siRNA for Orai1, TRPC1 and STIM1 to modulate CE amplitude; and (3) a quantitative analysis of Orai1, TRPC1 and STIM1 expression by Western-blot.

First, specific blockade of Orai1 channels with S66 at 2.5 μM significantly reduced CE (P = 0.03), the anti-IgM Ca²⁺ response (P = 0.01), and TG SOCE (P = 0.05) but not TG ER Ca²⁺ release in CE+/IgM+ B-CLL cells compared to control conditions (Fig. 3a/b and data not shown). Second, and another way in which to further test our hypothesis, was to reduce the expression of Orai1, TRPC1 and/or STIM1 by transfecting specific siRNA into B-CLL cells (1 CE+/IgM- and 2 CE+/IgM+). In contrast to the negative siRNA control, a reduction was seen at the protein level when using specific siRNAs for STIM1, Orai1, and TRPC1 (FACS representations are depicted Fig. 3c). As a result, CE was reduced in the presence of siRNA to Orai1, TRPC1 and STIM1 (P < 0.05 for all) (Fig. 3d). These results suggest that Orai1 together with TRPC1 both contribute to CE regulated by STIM1.Fig3Fig. 3

Constitutive calcium entry (CE) is related to STIM1, Orai1 and TRPC1. a/b- The effects of the Orai1 channel inhibitor Syntha(S)66 on CE (a) and on anti-IgM Ca²⁺ response (b) in CE+/IgM+ CLL samples (n = 3). c- siRNA control analysis by FACS. The mean fluorescence intensities (MFI) of representative CE+ transfected B-CLL cells are shown in the upper left corner of each plot for the siRNA control, and in the lower left corner for the specific siRNAs. d- Average time course of CE in siRNA transfected CE+ B-CLL cells (n = 3). P values are indicated when significant

Since glycosylation is required for STIM1 localization at the plasma membrane [24, 25], and given that the pool of STIM1 located in the plasma membrane (STIM1_PM) regulates store-independent Ca²⁺ influx [26], this raises the possibility that STIM1_PM controls CE and contributes to its enhancement in CE+ B-CLL cells. To address this issue (Fig. 5a), B-CLL cells from 28 patients (11 CE+ and 17 CE-) were tested by FACS for STIM1 expression using STIM1 mAb following permeabilization of the cells (total-STIM1 expression determination) or not (STIM1_PM quantification). In agreement with WB results, FACS analysis revealed that both total-STIM1 and STIM1_PM were increased in CE+ B-CLL cells (P = 0.01 and < 10^− 4, respectively), and their levels correlated with CE amplitude (P = 0.01 both, Fig. 5b). A ROC analysis was performed in order to establish the cut-off for positivity (Fig. 5b left). We next sought to determine STIM1_PM involvement in CE regulation, and this was tested by exploring the capacity of the anti-STIM1 mAb (GOK, 10 μg/mL) to inhibit CE. In contrast to the IgG2a isotype control mAb that had no effect on CE (Fig. 5c), the anti-STIM1 mAb inhibits CE (P = 0.03), while no effects were reported on the anti-IgM Ca²⁺ response, TG ER Ca²⁺ release and TG SOCE responses (Fig. 5d and Additional file 2: Figure S4B). This is in agreement with the observed correlation between STIM1_PM levels and basal Ca²⁺ but not with TG ER Ca²⁺ release and IgM/TG SOCE results (Additional file 2: Figure S4A). Altogether this reinforces our hypothesis that CE and basal Ca²⁺ are regulated by STIM1_PM and supported by Orai1 and TRPC1 channels in a unique and alternative influx pathway distinct from SOCE and downstream the BCR-InsP3R pathway.Fig5Fig. 5

The pool of STIM1 in plasma membrane controls constitutive calcium entry (CE). a- Cytoplasmic (after permeabilization, total STIM1[t]) and plasma membrane staining of STIM1 (STIM1_PM) on B-CLL cells from CE+ (n = 7) and CE- (n = 12) CLL patients. The isotype control is shown (black line). The mean fluorescence intensities (MFI) of representative CE- B-CLL cells are shown in the upper left corner of each plot in blue, and in the lower left corner in blue for CE+ B-CLL cells. b- Correlation between CE and tSTIM1_T (upper panel) or STIM1_PM (lower panel). c- Receiver operating curves (ROC) were generated to determine the area under the curve (AUC) and the optimal cut-off value to discriminate STIM1 high from STIM1 low patients. d- The effects of the anti-STIM1 mAb clone GOK on CE in CLL samples (n = 6). e- No effect of the anti-STIM1 mAb on anti-IgM Ca²⁺ response in CLL samples (n = 10). The r² coefficient and P values are indicated when significant

As CE determination is difficult to manage in routine practice, we further compared the patient’s characteristics according to their plasma membrane STIM1 status in 74 untreated CLL that included those tested for Ca²⁺ signaling. As depicted in the Kaplan-Meyer curves (Fig. 6a), the CLL STIM1_PM high subgroup had shorter PFS and TFS (P = 0.0007 and P = 0.02, respectively). Characteristics of STIM1_PM high and low patients are presented in Table 1 (right part) showing that lymphocytosis (p = 0.05), but not the other parameters tested, was increased in the CLL STIM1_PM high subgroup.Fig6Fig. 6

In the whole CLL cohort (n = 74), an elevated level of STIM1 at plasma membrane (STIM1PM) is relevant for CLL clinical outcome and influence in vitro cell survival. a Kaplan-Meier plots showing progression free survival and treatment free survival for STIM1PM dichotomize into high and low levels. b Increase in the density of STIM1PM improves the efficacy of rituximab (RTX) in the STIM1_PM high CLL subgroup (n = 9) when used in combination with the anti-STIM1 mAb (both 10 μg/mL, 48 h), effect which was not observed in the STIM1_PM low CLL subgroup (n = 8). P values are indicated when significant