Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel

2.2. Cell Culture

Human pancreatic cancer cell lines BxPC3, Capan1, Capan2, CFPAC1, MiaPaCa2, Panc1 and primary human pancreatic cells (HPNE) were procured from ATCC. Cell lines BxPC3, Capan2 and CFPAC1 were cultured in RPMI-1640 with L-Glutamine (Lonza, Basel, Switzerland). MiaPaCa2 were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 2.5% Horse serum. Panc1 were cultured in DMEM with 10% FBS. Primary pancreatic cell line hTERT HPNE were cultured in ATCC recommended media. Capan1 were maintained in Iscove’s modified dulbecco’s medium (IMDM) + 20% FBS.

2.3. Lentiviral Stable Cell Line Generation

For stable knockdown generation, human specific shOrai3 and shNT were cloned in the lentiviral pGIPZ vector (Dharmacon, Lafayette, CO, USA) were used. As reported earlier, the lentiviral constructs pCMV-VSVG, pCMV- dR8.2 and pGIPZ-shNT/shOrai3 were co-transfected in a flask containing 95% confluent HEK293FT cells [34]. Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) was used as a transfection reagent to transfect HEK293FT cells. Viral particles containing medium were collected at 48 and 72 h after transfection and was concentrated using Amicon filters through centrifugation. These concentrated viral particles were used to transduce cells seeded at 50% confluency and knockdown was confirmed by performing Western blot analysis.

2.4. MTT Assay

Panc1 and HPNE lentiviral stables carrying either shNT or shOrai3 were seeded in a 96 well plate. After adherence, MTT reagent was added to cells at 4 different time points (24, 48, 72 and 96 h) and incubated at 37 °C for 3 h. After incubation for 3 h, media was aspirated and 100 µL of DMSO was added to dissolve purple colored formazan crystals. The absorbance was measured at 570 nm with a microplate reader.

2.5. Cell Cycle Analysis and Apoptosis Assay

Cell cycle analysis by quantitation of DNA is a standard application of flow cytometry. Panc1 lentiviral stables were seeded at a density of 80,000 cells/well in a 24 well plate. On the next day, the cells were incubated in serum free media for 12 h. The media was replaced by DMEM after 12 h. Cells were trypsinized and washed with PBS. For cells fixation, 70% ethanol was added for overnight and next day cells are washed with PBS carefully. Cells were incubated with RNase A at 37 °C for 40 min. Propidium iodide (PI) was added after RNase A treatment and FACS was performed. For the apoptotic assay, cells were stained with annexin V and PI and incubated for 30–60 min.

2.6. Calcium Imaging

Calcium imaging was performed as reported earlier [34,35]. Briefly, cells were cultured on confocal dishes for performing Ca²⁺ imaging. Cells are incubated at 37 °C for 30 min in a culture medium containing 4 μM fura-2 AM. After incubation, cells were washed 3 times and bathed in HEPES-buffered saline solution (140 mM NaCl, 1.13 mM MgCl₂, 4.7 mM KCl, 2 mM CaCl₂, 10 mM d-glucose, and 10 mM HEPES; pH 7.4) for ≥5 min before Ca²⁺ measurements were made. A digital fluorescence imaging system (Nikon Eclipse Ti2 microscope coupled with CoolLED pE-340 Fura light source and a high speed PCO camera) was used, and fluorescence images of several cells were recorded and analyzed. Fura-2AM was excited alternately at 340 and 380 nm, and the emission signal was captured at 510 nm. Figures showing Ca²⁺ traces are an average from several cells (the number of cells is denoted as “n” on each trace) attached on a single imaging dish. Each experiment was performed at least 3–4 times and the final data are plotted in the form of bar graphs. In the case of Panc1 stables, 9–11 runs on 3 independent days were performed. The data shown in a particular trace originates from multiple cells on a single imaging dish. The exact number of cells and traces for each condition are specified in the respective figure.

2.7. Western Blotting

Total protein was extracted with NP40 lysis buffer along with protease inhibitor cocktail. Total protein concentration was quantified by BCA assay. Protein extracts were separated by 8–12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, and then incubated overnight at 4 °C in TBST with primary antibody, including rabbit anti-Orai3 (1:500, Abcam), rabbit anti-cyclin D1 (1:1000, Cell Signaling) and rabbit anti-Cdk4 (1:1000, Santa Cruz). Following incubation with horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (1:5000, NA934V, Sigma, St. Louis, MO, USA) for 2 h, the membranes were detected using the enhanced chemiluminescence (ECL) kit. β-tubulin (1:5000, Abcam) was used as a loading control. Densitometric analysis was done using ImageJ software and data are graphically represented as mean ± SEM.

2.8. Wound Healing Assay

Panc1 lentiviral stable cells were seeded in a 24 well plate at a density of 2 × 10⁵ cells per well. After 48 h of incubation, cells reached 100% confluency and a wound was created at the center of the well by gently scrapping the cells with a sterile P200 tip. After removing the incubation media and washing with PBS, new media were added to each well. Each well was observed under the bright field microscope for migration at different time points. The migration rate of individual conditions (shNT and shOrai3) was determined by measuring the distance covered from the initial timepoint to selected timepoints. Different widths were normalized to the 0 h timepoint. Graph was plotted for percentage wound healing vs. time (h) using GraphPad Prism software. Data are presented as mean ± SEM.

2.9. Migration Assay

Cell migration was assessed using 24-well corning costar inserts with 8-μm pores. Cells (5 × 10⁴) were added in the upper chamber in DMEM media (without FBS) and incubated at 37 °C and migration was assessed at 24 h. Non-migrating cells were removed from the upper chamber with the help of cotton buds. Cells adhered at the bottom of the transwell were fixed with formaldehyde, permeabilized with 100% methanol and stained with crystal violet and bright field images of 5 different fields were quantified using ImageJ. Experiments were performed in triplicate in three independent biological samples and data are reported as mean ± SEM.

2.10. In Vivo Experiments

Animal protocols were approved by the Institutional Animal Ethics Committee (IAEC), Regional Center for Biotechnology (Protocol #RCB/IAEC/2020/076). Four-to-six weeks old female NOD SCID mice were acclimated at the Small Animal Facility (SAF) for 2 weeks before any intervention. 1 × 10⁶ Panc1 stables either shNT or shOrai3 in a volume of 100 µL with 100 µL of Matrigel were injected subcutaneously into the left flank of 5 mice/condition. Tumor volume was assessed weekly using Vernier calipers. Tumor volume was calculated using the formula V = W² × L/2, where V is tumor volume, W is tumor width, and L is tumor length [24]. Animals were euthanized 12 weeks post injection and pictures of isolated tumors were taken. Tumor weights were measured using a digital scale.

For metastasis studies, we performed whole body fluorescence imaging using the SPECTRUM In Vivo Imaging System (IVIS) (PerkinElmer, Santa Clara, CA, USA) by setting the excitation filter at 500 nm and the emission filter at 540 nm. Animals were anesthetized using 1.5% isoflurane in the anesthesia chamber and were transferred to IVIS for imaging. Whole body and thoracoabdominal region fluorescence imaging was performed for evaluating the tumor development and metastasis, respectively.

2.11. Tumor Lysates Preparation

Tumors were harvested from two groups (shNT and shOrai3) NOD SCID mice and were snap frozen using liquid nitrogen. Subsequently, they were thawed on ice. A portion of the tumor was cut with the help of a sterile scalpel and weighed before adding an appropriate amount of protein lysis buffer (NP40 + protease inhibitor cocktail). Tumor samples suspended in lysis buffer were homogenized using hand-held tissue homogenizer on ice and further centrifuged at 13,000 rpm at 4 °C to obtain protein lysate. The supernatant obtained after centrifugation was used to estimate the total protein concentration by performing BCA assay and protein lysates were used for performing Western blotting.

2.12. qRT-PCR Analysis

For mRNA extraction Panc1 cells stably expressing either shNT or shOrai3 were processed with Qiagen RNeasy kit (Catalog #74106). mRNA was then converted to cDNA using high-capacity cDNA reverse transcription kit from ThermoFisher (Waltham, MA, USA) (Catalog #4368814). Real-time PCR reactions were performed using SYBR green in Quant Studio 6 Flex from Applied Biosystems. The data were analyzed with Quant Studio real-time PCR software version 1.3. The expression of Orai1, Orai2 and Orai3 in shNT and shOrai3 cells were normalized to that of the housekeeping gene GAPDH. The sequences of primers used in the study are listed in the Table 2.

3.2. Orai3 Is Expressed in “Normal” as Well as Cancerous Pancreatic Cell Lines and Forms a Functional Ca²⁺ Influx Channel

First of all, we examined the Orai3 protein expression in a battery of PC cell lines (BxPC3, Capan1, Capan2, CFPAC1, MiaPaCa2 and Panc1) and compared it with the Orai3 levels in immortalized “normal human pancreatic epithelial cells-HPNE”. All these cell lines were procured from “American Type Culture Collection” (ATCC). We performed Western blot analysis on the protein lysates of “normal” pancreatic epithelial cells and 6 PC cell lines for evaluating Orai3 expression (Figure 1D). Similar to publicly available datasets, we observed a differential expression of Orai3 in these cell lines. In comparison to HPNE cells, it was found that: (1) Orai3 levels were higher in Capan1, CFPAC1, and Panc1; (2) Capan2 and MiaPaCa2 showed lower Orai3 expression; and (3) while in BxPC3, Orai3 levels were comparable to that in HPNE cells (Figure 1D,E). This expression analysis corroborates the unbiased largescale publicly available datasets from different sources, i.e., GEPIA, TCGA and The Human Protein Atlas. Taken together, it suggests that Orai3 is overexpressed in a certain proportion of PC tumors and it is not universally upregulated in all PC samples.

We next evaluated the functional significance of Orai3 expression in “normal” pancreatic epithelial cells and PC cell lines by performing live cell Ca²⁺ imaging with ratio-metric Fura2-AM dye. We employed standard thapsigargin (Tg) activated SOCE protocol [34,35] wherein ER Ca²⁺ stores were depleted by Tg in absence of extracellular Ca²⁺ and subsequent addition of Ca²⁺ in the bath solution resulted in SOCE. Further, we utilized the pharmacological tool 2-Aminoethoxydiphenyl borate (2APB) to differentiate between functional Orai1 v/s Orai3 channels. It is well reported that 2APB in the same concentration range of 30–50 µM blocks Orai1 and activates Orai3 channels [36,37,38,39]. In the past, several groups used 2APB (50 µM) to demonstrate Orai3 activation as a functional SOCE channel in breast and lung cancers [20,23,24,40,41]. Therefore, in order to examine the functionality of Orai3 as a SOCE channel in pancreatic cells, we used 2APB (50 µM) in our live cell Ca²⁺ imaging experiments. Interestingly, our Ca²⁺ imaging studies in the “normal” HPNE and PC cell lines show that the SOCE is potentiated by 2APB in these cells suggesting that Orai3 is most likely the functional SOCE channel in these cells (Figure 2A–F). We performed these experiments multiple times with >100 cells and further analyzed the Ca²⁺ imaging data in detail. The Ca²⁺ imaging data was analyzed as per the standards of the field, which are followed by several leading groups across the globe. Briefly, the basal cytosolic levels were calculated at the start of the experiment and plotted as ratio of fluorescence emission intensity acquired upon excitation of Fura-2 at 340 nm and 380 nm. The ER Ca²⁺ release was measured by calculating the rise in the ratio of fluorescence emission intensity upon addition of Tg. We then waited until the cytosolic Ca²⁺ levels reached to the baseline and subsequently added Ca²⁺ in the bath solution. SOCE was quantitated by determining the increase in the ratio of fluorescence emission intensity upon the addition of Ca²⁺ in the bath solution. Upon the plateauing of SOCE, we added 2APB (50 µM) for examining the consequence of 2ABP addition on the SOCE. Finally, 2APB potentiation was measured by analyzing the rise in the ratio of fluorescence emission intensity upon 2APB addition. Our analysis shows that: (1) basal cytosolic Ca²⁺ levels were comparable across these cells (Figure 2G); (2) ER Ca²⁺ release (the indirect measure of ER Ca²⁺ stores) was higher in PC cells, except MiaPaCa2, as compared to HPNE cells (Figure 2H); (3) similarly, in comparison to HPNE cells, SOCE was greater in PC cells except MiaPaCa2 (Figure 2I); (4) finally, SOCE was potentiated by 2APB in all these cell lines wherein highest potentiation was observed in case of Panc1 and lowest in case of MiaPaCa2 (Figure 2J). It is important to note that the Panc1 cells express higher Orai3 expression in comparison to HPNE whereas MiaPaCa2 cells express lower Orai3 levels. Since Orai3 is overexpressed and most likely forms a functional channel in Panc1 cells, we performed further validation studies in Panc1 cells.

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Figure 2

Characterization of SOCE in a battery of Pancreatic cancer cell lines. (A–F) Representative Ca²⁺ imaging traces of HPNE, BxPC3, MiaPaCa2, Capan1, Panc1 and CFPAC1 cells where “n” denotes the number of cells in that particular trace. (G) Quantitation of basal Ca²⁺ levels across different cell lines. (H) The amplitude of ER Ca²⁺ release was calculated from a number of experiments and data are presented in dot plot graphs. (I) The extent of SOCE was calculated from several experiments and data are presented in dot plot graphs. (J) 2-APB induced potentiation of Orai3 mediated SOCE was calculated from several experiments and data are presented in dot plot graphs. The total number of cells imaged in (G–J) are reported as “n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded.

For corroborating the role of Orai3 in SOCE, we generated lentiviral-based stable Panc1 cell lines expressing either non-targeting control shRNA (shNT) or shRNA specifically targeting Orai3 (shOrai3). We used the shRNA vectors co-expressing GFP for fluorescence-based identification of transduced cells and for performing in vivo bio-fluorescence studies (Figure 3A,B). First of all, we confirmed Orai3 silencing in shOrai3 stable Panc1 cell line by performing Western blot analysis (Figure 3C). We observed a significant knockdown of Orai3 in shOrai3 stable cells in comparison to shNT cells (Figure 3D). Next, we examined the specificity of shOrai3 by analyzing the mRNA and protein expression of Orai3 homologs, i.e., Orai1 and Orai2 in shOrai3 Panc1 stable cells. As presented in Supplementary Figure S1A, in Panc1 shOrai3 stables Orai3 mRNA levels were decreased by over 50% in comparison to that in shNT Panc1 cells. Whereas Orai1 and Orai2 mRNA levels remain comparable between shNT and shOrai3 Panc1 cells (Figure S1A). We also corroborated shOrai3 specificity at protein levels by performing Western blot analysis for Orai1 and Orai2 in Panc1 stable cells. As expected, we observed similar levels of Orai1 and Orai2 in shNT versus shOrai3 Panc1 stable cells (Figure S1B–E). Taken together, these experiments clearly demonstrate that shOrai3 is specific to Orai3. After confirming specificity, we used shNT and shOrai3 Panc1 stable cells for performing live cell Ca²⁺ imaging experiments. We performed Ca²⁺ imaging experiments in around 250 cells/condition. As presented in Figure 3E, shNT Panc1 stable cells showed SOCE that was potentiated upon 2APB application whereas shOrai3 Panc1 stable cells displayed lower SOCE as well as reduced 2APB mediated potentiation (Figure 3F). The detailed analysis of Ca²⁺ imaging traces demonstrate that Orai3 knockdown in Panc1 cells significantly decrease both SOCE and 2APB induced SOCE potentiation (Figure 3G,H). Interestingly, 2APB (50 µM) can activate Orai3 independent of Ca²⁺ store depletion. Therefore, we performed additional Ca²⁺ imaging assays in shNT and shOrai3 Panc1 stable cells wherein we directly stimulated cells with 2APB (50 µM) without Tg pre-exposure. As shown in Supplementary Figure S2A,B, 2APB activated Ca²⁺ influx in shNT Panc1 stables and this influx was significantly decreased in shOrai3 Panc1 cells. We analyzed 2APB stimulated store independent Ca²⁺ entry in around 150 cells/condition. The quantitation of this data is presented in Supplementary Figure S2C. Collectively, our live cell Ca²⁺ imaging experiments clearly show that Orai3 is a functional SOCE channel in Panc1 cells.

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Figure 3

Orai3 encodes a functional SOCE channel in Panc1 cells. (A,B) Lentiviral transduced Panc1 cells stably expressing either GFP tagged shNT control or GFP tagged shRNA targeting Orai3 (fluorescence and merged images of bright field plus fluorescence are presented). (C) Representative Western blot showing knockdown of Orai3 in shOrai3 Panc1 stables in comparison to shNT control Panc1 stable cells. (D) Densitometric analysis of Orai3 silencing in 3 independent shNT and shOrai3 Panc1 stable clones. (E) Representative Ca²⁺ imaging trace of shNT Panc1 stables where “n = 40” denotes the number of cells in that particular trace. (F) Representative Ca²⁺ imaging trace of shOrai3 Panc1 stables where “n = 42” denotes the number of cells imaged in that particular trace. (G) The extent of SOCE was calculated from 281 shNT and 247 shOrai3 cells, which were imaged from several autonomous experiments/traces (11 traces of shNT and 9 traces of shOrai3 originating from 3 independent clones of shNT and shOrai3 Panc1 stables) and data from autonomous traces are presented in dot plot graphs. (G’) Data for extent of SOCE from all individual cells from 3 independent clones are presented as clone N°1 yellow rounds, clone N°2 blue squares, clone N°3 green diamond. (H) 2-APB induced potentiation of Orai3 mediated SOCE was calculated from around 250 cells/condition. These cells were imaged during several autonomous experiments/traces (10 traces of shNT and 9 traces of shOrai3 originating from 3 independent clones of shNT and shOrai3 Panc1 stables) and data are presented in dot plot graphs. (H’) Data for 2-APB induced SOCE potentiation from all individual cells from 3 independent clones are presented as clone N°1 yellow rounds, clone N°2 blue squares, clone N°3 green diamond. The total number of cells imaged are reported in (G,H) as “n  =  x, y” where “x” denotes total number of cells imaged and “y” denotes number of traces recorded. Data presented are mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”; p-value < 0.01 is presented as “**”and p-value < 0.0001 is presented as “****”.

3.3. Orai3 Regulates PC Cell Viability, Cell Cycle Progression and Apoptosis

For understanding the role of Orai3 in PC progression, we started by evaluating Orai3 contribution to PC cell viability. We performed a colorimetry-based MTT assay for examining the significance of Orai3 in Panc1 viability. As presented in Figure 4A, the cell viability was significantly decreased in shOrai3 Panc1 stables in comparison to shNT stables. We next sought to determine if Orai3′s role in regulating cell viability is specific to PC cells or is it conserved in “normal” pancreatic epithelium. Therefore, we generated lentiviral-based stable silencing of Orai3 in HPNE cells. We used similar strategy as discussed above for Panc1 cells and generated shNT control stable HPNE cells and shOrai3 stable HPNE cells (Figure 4B,C). We performed Western blot analysis to confirm the knockdown of Orai3 in HPNE cells (Figure 4D) and observed that there was a significant reduction in Orai3 expression in shOrai3 HPNE stable cells in comparison to shNT control cells (Figure 4E). We then performed MTT assays on these HPNE stable cells and found that Orai3 knockdown only marginally (non-significantly) affected the viability of “normal” pancreatic cells (Figure 4F). This suggests that Orai3 specifically contributes to the cellular viability of PC cells. We thus performed further functional assays with Panc1 shNT and shOrai3 stable cells.

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Figure 4

Orai3 regulates Panc1 but not HPNE cell viability. (A) MTT assay-based cell viability analysis (24–96 h time points) in Panc1 shNT and Panc1 shOrai3 stable cells (n = 5). (B,C) Lentiviral transduced HPNE cells stably expressing either GFP tagged shNT control or GFP tagged shRNA targeting Orai3 (fluorescence and merged images of bright field plus fluorescence are shown). (D) Representative Western blot showing knockdown of Orai3 in shOrai3 HPNE stables in comparison to shNT control HPNE stable cells. (E) Densitometric analysis of Orai3 silencing in 4 independent shNT and shOrai3 HPNE stable clones. (F) MTT assay-based cell viability analysis (24–96 h time points) in HPNE shNT and HPNE shOrai3 stable cells (n = 4). Data presented as mean  ±  S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”; p-value < 0.01 is presented as “**”and p-value < 0.001 is presented as “***”.

The decrease in cell viability observed upon Orai3 knockdown in Panc1 cells could be either due to decrease in cell cycle progression or increase in basal cell apoptosis. Thus, we performed both cycle cell analysis and cell apoptosis estimation in shNT and shOrai3 Panc1 cells. We carried out standard propidium iodide-based FACS measurements of the cell-cycle progression in Panc1 shNT and shOrai3 cells (Figure 5A,B). Interestingly, we observed that Orai3 silencing led to accumulation of Panc1 cells in the G₁ phase of cell cycle and there were substantially less cells in G₂ phase in comparison to shNT stable cells (Figure 5C). In shNT Panc1 stables, cells were able to transit to the S and G₂ phases of the cell cycle while in the case of shOrai3 stables, the majority of cells (>50%) were stalled in the G₁ phase suggesting that Orai3 silencing induces cell-cycle arrest in the G₁ phase (Figure 5C). To further corroborate this observation, we examined the levels of key G₁ phase transition regulators, i.e., cyclin D1 and cyclin dependent kinase 4 (Cdk4) in shOrai3 stables. We performed Western blot analysis in shNT and shOrai3 stables for evaluating cyclin D1 and Cdk4 expression. As expected, the expression of both cyclin D1 (Figure 5D,E) and Cdk4 (Figure 5F,G) was significantly decreased in shOrai3 stables in comparison to shNT control cells suggesting that Orai3 plays a critical role in the G₁ phase transition of PC cells.

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Figure 5

Orai3 contributes to Panc1 cell-cycle progression. (A,B) Representative data showing FACS-based cell-cycle analysis of Panc1 shNT and Panc1 shOrai3 stable cells. Cell-cycle analysis was performed with three independent biological experiments using propidium iodide. (C) Quantitative analysis of % cells in different phases of cell cycle in the case of Panc1 shNT and Panc1 shOrai3 stable cells (n = 4). (D) Western blot analysis for cyclin D1 expression in shNT and shOrai3 Panc1 stable cell lines. (E) Densitometric analysis of cyclin D1 levels in shNT and shOrai3 Panc1 stable cells. (F) Western blot analysis for Cdk4 expression in shNT and shOrai3 Panc1 stable cell lines. (G) Densitometric analysis of Cdk4 levels in shNT and shOrai3 Panc1 stable cells. Data presented as mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”.

Next, we examined the role of Orai3 in modulating PC cell apoptosis by performing standard annexin V-based FACS assays. Since our stable cells were constitutively expressing GFP, we used TRITC conjugated annexin V to ensure that there was no spectral overlap between live and apoptotic cells in our experiments. This strategy helped in ruling out the possibility of any false positive apoptotic signals during FACS analysis. We used unstained cells and cells heated at 95 °C as negative and positive controls, respectively, in the apoptosis assays. As presented in Figure 6A,B, we observed 0% and 95.6% early apoptotic cells in the negative and positive controls, respectively. It suggests that our assay was working efficiently, and we were able to capture correct biological signals using TRITC conjugated annexin V. We then evaluated basal cellular apoptosis in shNT (Figure 6C) and shOrai3 Panc1 stables (Figure 6D) and found that there was a two-fold increase in the number of apoptotic cells in shOrai3 condition as compared to shNT cells (Figure 6E). Taken together, this data suggests that Orai3 plays a vital role in PC cell survival and therefore, Orai3 silencing leads to significant increase in the cellular apoptosis of Panc1 cells.

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Figure 6

Orai3 regulates basal apoptosis of Panc1 cells. (A–D) Representative FACS-based analysis of apoptosis using propidium iodide and TRITC conjugated annexin V for unstained, i.e., negative control Panc1 cells (A); positive control Panc1 cells (B); shNT Panc1 stable cells (C) and shOrai3 Panc1 stable cells (D). (E) Quantitative analysis of % early apoptotic cells in shNT and shOrai3 Panc1 stable cell lines from 4 independent experiments. Data presented as mean ± S.E.M. Paired t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and is presented as “*”.

3.4. Orai3 Controls PC Cell Migration In Vitro

Since most of the PC associated deaths are due to PC metastasis to the secondary sites, we started evaluating the relevance of Orai3 in PC cell migration. We first utilized scratch wound assays for examining the role of Orai3 in Panc1 cell migration. We allowed shNT and shOrai3 stable Panc1 cells to form a monolayer in 24-well plates and then created a wound in the monolayer using 200 µL pipette tip. We observed the wound closure, which gave an estimation of cellular migration at regular intervals until the 24-h timepoint. It is important to mention that the doubling time of Panc1 cells is >50 h (documented at the ATCC website as well). Therefore, in this assay our observations were specific to Panc1 cell migration. As shown in Figure 7A,B, shNT Panc1 stable cells were able to migrate and close the wound more efficiently than shOrai3 Panc1 stables. We performed this assay in triplicates with four independent biological samples and observed that shNT Panc1 stables were able to close around 80% of the wound in 24 h whereas shOrai3 cells closed just over 40% of the wound in the same time (Figure 7C). This suggests that Orai3 could be playing an important role in driving PC-cell migration. To further substantiate this observation, we performed trans-well Boyden chamber-based migration studies with shNT and shOrai3 stable Panc1 cells. We seeded either shNT or shOrai3 cells in the top chamber (with 8 µm pores) containing culture media without serum and used complete media including serum in the bottom chamber. Therefore, in these assays, serum which is rich in growth factors served as a chemoattractant. We allowed cells to migrate towards the lower chamber for 24 h and fixed the migrated cells using formaldehyde. Eventually, migrated cells were stained with crystal violet and quantitation of number of migrated cells was performed using ImageJ software. As presented in Figure 7D,E, the cell migration (crystal violet staining) is substantially less in the case of shOrai3 stables in comparison to shNT stables. Further, the quantitation of data from three independent biological samples suggests that Orai3 silencing results in around 35% decrease in the Panc1 cells migration in trans-well assays (Figure 7F). Taken together, both wound healing and trans-well migration assays highlight a crucial role for Orai3 in regulating PC-cell migration.

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Figure 7

Orai3 controls migration of Panc1 cells. Scratch wound healing (A–C) and transwell migration assays (D–F) were performed for evaluating the role of Orai3 in regulating Panc1 migration. (A) Representative wound healing images at 0, 6, 12 and 24 h time points in the case of shNT Panc1 stable cells. (B) Representative wound healing images at 0, 6, 12 and 24 h time point in case of shOrai3 Panc1 stable cells. (C) Quantitative analysis of %wound healing over the period of 24 h in shNT and shOrai3 Panc1 stable cells from 4 independent experiments. (D) Representative micrographs of crystal violet stained transwell migrated shNT Panc1 stable cell line. (E) Representative micrographs of crystal violet stained transwell migrated shOrai3 Panc1 stable cells. (F) Quantitative analysis of number of migrated cells in the transwell migration assay from three independent experiments. Data presented as mean ± S.E.M. Unpaired Student’s t-test was performed for statistical analysis. p-value < 0.05 was considered as significant and p-value < 0.01 is denoted as “**”.

Authors thank Manoj Garg from Amity University, Noida; CV Srikanth from RCB and Guruprasad Medigeshi from THSTI for help with reagents/antibodies. S.A. and N.S. acknowledge their Junior Research Fellowship from DBT, India; S.S. acknowledges his Junior Research Fellowship from CSIR, India and J.T. acknowledges her Senior Research Fellowship from CSIR, India.