Bio-Plex IL-6 assay

The concentration of IL-6 in cell supernatants and PC patient sera was determined using the Bio-Plex human IL-6 Assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocol. Briefly, 50 μl of the supernatant/cytokine standard was added to the plate coated with beads coupled to IL-6 antibody. After a series of washes, a biotinylated detection antibody, specific for a different IL-6 epitope was added to the reaction resulting in formation of antibodies assimilated around the target protein. Streptavidin–phycoerythrin (streptavidin–PE) was then added to bind to the biotinylated detection antibody on the bead surface. The data from the reaction were analyzed by using Bio-Plex suspension array system (or Luminex 100 system) from Bio-Rad Laboratories.

Real-time polymerase chain reaction

MSLN, IL-6, IL-6R and sIL-6R (alternatively spliced form), ADAM10, ADAM17 and GAPDH messenger RNA (mRNAs) were analyzed by real-time reverse transcription–polymerase chain reaction using the SYBR Green supermix kit (Bio-Rad Laboratories) as described previously (2). The relative mRNA levels were normalized to GAPDH mRNA and presented as unit values of 2[C_t(β-actin)−C_t(gene of interest)], where C_t is threshold cycle. Primer sequences for the genes are as follows: h-IL-6 sense: 5′-TCCAGAACAGATTTGAGAGTAGTG-3′; h-IL-6 antisense: 5′-GCATTTGTGGTTGGGTCAGG-3′; h-sIL-6R-sense: 5′-GCGACAAGCCTCCCAGGTTC-3′; h-sIL-6R-antisense: 5′-GTGCCACCCAGCCAGCTATC-3′; hADAM17-sense: 5′-GGTGGTAGCAGATCATCGCTT-3′; hADAM17-antisense: 5′GTGGAGACTTGAGAATGCGAA-3′; hADAM10-sense: 5′-ATGGGAGGTCAGTATGGGAATC-3′ and hADAM10-antisense: 5′-TTTGGCACGCTGGTGTTTTTG-3′.

MSLN/IL-6 shRNA/small interfering RNA transfections

For MSLN shRNA transfection experiments, a plasmid encoding MSLN shRNA (TR311377; Origene, Rockville, MD) and a control plasmid encoding GPF shRNA (TR30003) were used. MIA-MSLN/MIA-V cells were transfected with the above or an MSLN-specific small interfering RNA (siRNA) oligonucleotide or a pool of four human IL-6-specific siRNA oligonucleotides (IL-6 siRNA Pool #1; Thermo Scientific, IL) using Lipofectamine 2000 (Invitrogen, CA). Negative controls included cells treated with transfection reagent (mock) and control scramble siRNA (for MSLN siRNA experiments) or a pool (Non-Targeting siRNA Pool #1; Thermo Scientific) of scrambled siRNAs (for IL-6 siRNA experiments). Cells/supernatants were collected 24/48 h after transfection for mRNA/protein extraction for real-time polymerase chain reaction/Bio-Plex detection and functional studies done as indicated.

Immunoblot analysis

Cells were lysed in lysis buffer (Cell Signaling Technology Laboratories) with phosphatase and protease inhibitors. Total proteins (60 μg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad Laboratories). Different proteins were detected using specific primary antibodies, appropriate horseradish peroxidase-conjugated secondary antibodies and the ECL detection system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Nuclear/cytoplasmic extracts were prepared using N-PER reagents (Pierce Biotechnology, Rockford, IL) and used for studying nuclear translocation of p65. For detecting sIL-6R in the supernatant, protein from serum-free supernatants were acetone-precipitated overnight at −20°C, solublized in protein loading dye and subjected to immunoblot analysis.

Treatment with inhibitors

MIA-MSLN/MIA-V cells were treated with 0, 12.5 or 25 μM of the IKK inhibitor wedelolactone (Calbiochem, La Jolla, CA). Cells were collected after 24 h and whole cell and cytoplasmic/nuclear extracts were prepared according to procedures already described (21) and used for western blot. Supernatants were collected for Bio-Plex detection of secreted IL-6. After a 36 h serum starvation, TAPI-1, the TACE inhibitor was used at 10–20 μM final concentration for 36 h (for sIL-6R in supernatant) and 4 days [to check proliferation by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT)].

Cell proliferation measurement by cell viability assay (MTT)

Cell proliferation was determined by measuring cell viability using MTT. Briefly, 2000 cells were plated and serum starved for 24 h. Serum free or 0.2% serum media ± treatments were added to the plates and the cells were continuously cultured for 2, 3 or 6 days and viability measured by MTT. Optical density (OD) was measured at 590 nm using a 96-well multi-scanner (EL-800 universal microplate reader; BioTek, Winooski, VT). Proliferating capacity of a cell was measured by dividing its viability at a certain time point by its viability at 0 day (day after plating cells). For antibody blocking experiments, anti-sIL-6R Abs or isotype control Abs were added to the starvation media at a final concentration of 5 or 25 μg/ml. Further treatment with serum free or 0.2% serum media plus other treatments were added the next day while maintaining antibody concentrations. After 3 days, viabilities were measured as indicated above. To study effect of IL-6 siRNA on MIA-MSLN proliferation, MIA-MSLN/MIA-V cells were transfected using IL-6-specific siRNA pool or negative control non-targeting siRNA pool in six-well plates. The next day cells were trypsinized, and 4000 cells were plated in replicate in 96-well plates and serum starved for 24 h. Fresh medium with 0.2% serum was added to the plates and continuously cultured for another 48 h, and the viability was measured by MTT as above. For IL-6 treatment experiment, IL-6 was used at 0.1, 1, 10 or 100 ng/ml. For the combined IL-6 and sIL-6R treatment, IL-6 and sIL-6R were used at concentrations of 100 ng/ml and 2 μg/ml, respectively.

Cell cycle analysis

Cells were harvested and processed using the CycleTEST™ PLUS DNA Reagent Kit from Beckton Dickinson (San Jose, CA) according to the manufacturer’s instructions. Flow cytometry [fluorescence-activated cell sorting (FACS)] analysis was carried out to examine the cell cycle distribution by a FACSCalibur (Becton Dickinson). Data were further analyzed using the software FLOWJOW Ver. 6.1.1 (Tree Star, San Carlos, CA).

Serum IL-6 levels in PC patients positively correlate with the level of MSLN expression in the cancer tissue

To further test the MSLN-IL-6 correlation, we examined serum IL-6 levels in six PC patients using the Bio-Plex assay. The tumor/normal tissue ratio of MSLN expression showed a high correlation with the serum IL-6 level (r = 0.982, P < 0.001, Figure 1C). The mean IL-6 level in these patients was 7.2 ± 4 pg/ml, which was higher than the previously reported (mean IL-6 level 3.4 pg/ml) in PC patients (22).

Forced overexpression of MSLN in PC cell lines leads to enhanced IL-6 production

Our previous study showed that MSLN overexpressing stable MIA PaCa-2 cells (MIA-MSLN) had increased migration and proliferation under serum-reduced conditions (2,3). To decipher the mechanism of these enhanced functions, we studied the MIA-MSLN cell line in detail for expression of various growth factors, cytokines and their receptors (data not shown). We found that the MIA-MSLN cell line secreted ~18 times higher quantity of IL-6 compared with untransfected (MIA), vector control, (MIA-V) and GFP-expressing control cells (MIA-GFP) (Figure 2A, P < 0.01). Similarly, IL-6 mRNA was also significantly higher in MIA-MSLN cells than in MIA-V cells (P < 0.01) (Figure 2B). When we blocked MSLN expression (Figure 2C, P < 0.05) in MIA-MSLN cells using specific shRNA/siRNA against MSLN, the IL-6 expression level was drastically reduced at the mRNA level (Figure 2B, P < 0.01) and protein level (Figure 2D, P < 0.05). These data strongly suggest that MSLN expression leads to increased IL-6 production in PC cells. We also tested a second MSLN overexpressing stable cells line Panc-1, for IL-6 production. Figure 2E shows that the Panc-1-MSLN cells expressed significantly higher (P < 0.01) MSLN than the control cells. Just like the MIA-MSLN cells, the Panc-1-MSLN cells also expressed significantly higher quantities of IL-6 mRNA compared with untransfected (Panc-1), vector control cells (Panc-1-V), and GFP-expressing control cells (Panc-1-GFP) (Figure 2F, P < 0.01).

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Fig. 2.

MSLN overexpression leads to increased IL-6 production in PC cells. (A). MIA, MIA-V, MIA-GFP and MIA-MSLN cells were seeded at 1 × 10⁶ cells per well in six-well plates and cultured until 60% confluence. Thereafter, the medium was replaced and the supernatants were harvested at 48 h of further incubation and the levels of IL-6 were determined by using the Luminex-based IL-6 assay kit. Y-axis represents IL-6 concentration in pg/ml. (B) Silencing MSLN using MSLN-specific shRNA plasmid. MIA-V and MIA-MSLN cells were transfected with MSLN-specific shRNA plasmids or GFP shRNA control plasmids. The cells collected 48 h posttransfection were used to detect MSLN mRNA by real-time polymerase chain reaction. The MSLN-expressing levels were detected by using real-time polymerase chain reaction. Y-axis represents MSLN mRNA level normalized to the GAPDH mRNA level. Relative mRNA level is presented as 2[C_t(GAPDH)−C_t(IL-6)]. The bars denote SD of duplicate data. Experiment was performed several times using separate MIA-MSLN pools and different siRNA/shRNAs against MSLN with similar results. (C) Silencing MSLN decreases IL-6 mRNA production in MIA-MSLN cells. MIA-V and MIA-MSLN cells were transfected with MSLN-specific shRNA plasmids or GFP shRNA control plasmids. The cells collected 48 h posttransfection were used to detect IL-6 mRNA by real-time polymerase chain reaction. Y-axis represents IL-6 mRNA level relative to GAPDH. The bars denote SD of duplicate data. Experiment was performed several times using separate MIA-MSLN pools and different siRNA/shRNAs against MSLN with similar results. (D) Silencing MSLN decreases IL-6 secretion in MIA-MSLN cells. MIA-V and MIA-MSLN cells were transfected with MSLN-specific siRNA or scrambled siRNA. The cells collected 48 h posttransfection from 24-well plates in 2 ml of medium were used to detect IL-6 by Luminex-based IL-6 assay kit. Values on Y-axis show the amount of IL-6 in pg/ml, bars denoting SD of duplicate data. (E) Stable overexpression of MSLN in human PC cell Panc1. The GAPDH-normalized MSLN expression levels in the stably MSLN expressing (Panc1-MSLN) and control MIA cells generated by retroviral gene transfer and subsequent puromycin selection are shown. (F) Panc1, Panc1-V, Panc1-GFP and Panc1-MSLN cells were seeded at 1 × 10⁶ cells per well in six-well plates and cultured until 80% confluence, cells collected were used to detect IL-6 mRNA by real-time polymerase chain reaction. Y-axis represents IL-6 mRNA level relative to GAPDH. The bars denote SD of duplicate data. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. * and # denote comparison to different base lines and hence are denoted differently.

MSLN-induced NF-κB activation is responsible for the high IL-6 secretion in MIA-MSLN cells

Various reports indicated that aberrantly activated NF-κB leads to induction of IL-6 in many cancers (23), including PC. We examined NF-κB activation in the MIA-MSLN cells and control MIA-V cells and found that nuclear translocation of NF-κB was indeed substantially increased in MIA-MSLN cells (Figure 3A). We could block this increased p65 nuclear translocation with a specific IKK blocker, wedelolactone, with a corresponding decrease in the phosphorylated as well as total IκB-α in the cytoplasm of treated MIA-MSLN cells (Figure 3A). We then measured IL-6 in the supernatants from the MIA-V and MIA-MSLN cells treated with wedelolactone using Bio-Plex. We found that blocking NF-κB activation decreased the secretion of IL-6 significantly from 251.4 pg/ml in the untreated MIA-MSLN cells to 117 pg/ml (a 53% reduction) and 75 pg/ml (a 70% reduction) (Figure 3B, P < 0.01) in cells treated with 12.5 and 25 μM of wedelolactone, respectively. These data show that MIA-MSLN cells have aberrantly activated NF-κB, which leads to an increased production of the inflammatory cytokine IL-6.

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Fig. 3.

Blocking NF-κB activation in MIA-MSLN cells leads to decrease in IL-6 expression. (A) NF-κB activation in MIA-MSLN cells was blocked by IKK inhibitor wedelolactone. For the nuclear extract, 20 μg of nuclear total proteins from control MIA-V and MIA-MSLN cells treated with or without IKK inhibitor wedelolactone at the indicated concentrations for 24 h was subjected to immunoblot analysis with antibodies against p65 and lamin A. For the cytosolic proteins, 30 μg of cytoplasmic protein from the same cells were subjected to immunoblot for p-IκBα, IκBα and β-actin. Data from a representative blot is shown. (B) Blocking NF-κB activation by wedelolactone reduced IL-6 production in MIA-MSLN cells. At 48 h posttreatment with wedelolactone in MIA-V and MIA-MSLN cells at the indicated concentrations, the supernatants were collected and tested for IL-6 using the Luminex-based IL-6 assay kit. Values on Y-axis show the amount of IL-6 in pg/ml, bars denoting SD of duplicate data. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test.

Increased IL-6 in MIA-MSLN cells is responsible for their enhanced survival and proliferation under serum-reduced conditions in an autocrine manner

A number of studies have indicated a growth-promoting property of IL-6 in various cancer cells (5,24–27). IL-6 has been reported as both an autocrine and paracrine factor supporting growth factor independence and/or lower growth factor requirement for cancer cells. We found that MIA-MSLN cells proliferated more than control MIA-V cells, even in serum-free medium. MTT assay showed that MIA-MSLN cells proliferated almost 1.8 times faster than the MIA-V cells after day 4 (P < 0.01) and almost 2.6 times faster at day 6 (P < 0.01, Figure 4A). Clearly, MIA-MSLN cells had a lesser requirement for growth factors for their growth. We observed that MIA-MSLN cells, which secreted high IL-6, produced even higher quantities of IL-6 while on starvation. After 24 h of starvation, the MIA-MSLN cells produced ~590 pg/ml of IL-6 and 48 h of serum deprivation increased the concentration of IL-6 in the supernatant to 3029 pg/ml (Figure 4B). However, MIA-V cells produced only negligible amounts of the cytokine IL-6 at all time points. This indicated that IL-6 could be responsible for the growth factor-independent survival/growth of these cells. We then used specific siRNA against IL-6 to block IL-6 expression and observe its effect on cell proliferation. Figure 4C clearly shows that IL-6 siRNA significantly reduced proliferation of MIA-MSLN cells (P < 0.01) to a rate similar to that of MIA-V cells after 2 days in 0.2% fetal bovine serum (FBS) containing media. We then examined whether IL-6 affects cell proliferation or survival. As shown in Figure 4D, 24 h IL-6 siRNA treatment significantly (P < 0.05) reduced S phase cells from 33 (mock transfected) and 29% (scrambled siRNA control) to 21%. Simultaneously, we saw a leap in G₁ cells (68%) compared with only 51% in mock-transfected cells. Next, we examined apoptosis induction by IL-6-blocking at day 5. Figure 4E clearly shows under serum-free conditions, MIA-V cells underwent apoptosis as indicated by decreased intact (uncleaved) caspase3, whereas the MIA-MSLN cells were relatively viable. siRNA against IL-6 on the other hand clearly decreased survival of MIA-MSLN cells with increased cleavage of caspase3. We then checked a number of pro- and anti-apoptotic proteins regulated by IL-6/stat3 pathway and found reasonable reduction in levels of the anti-apoptotic mcl-1 and bcl-2 as also the c-myc protein (Figure 4F), indicating a major role of these in MIA-MSLN cell survival.

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Fig. 4.

IL-6 is an autocrine growth and survival factor for the MIA-MSLN cells. (A) Overexpression of MSLN promotes PC cell proliferation in serum-free conditions. MIA-MSLN and control MIA-V cells were seeded in 96-well plates (2 × 10³ cells per well), serum starved (0% FBS) for 24 h before changing to fresh serum-free medium and cultured for 6 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. Proliferation after initial starvation followed by low serum supplementation was performed several times, serum-free survival was also performed multiple times, and data shown is a representative one. (B) Serum starvation increases IL-6 production by MIA-MSLN but not by MIA-V cells. MIA-V or MIA-MSLN cells (4 × 10⁶) were plated in T75 flasks and cultured till 90% confluent. Growth medium was then replaced by serum-free medium and supernatants were collected after 24 and 48 h and assayed for IL-6 using the Luminex-based IL-6 assay kit. Values on Y-axis show the amount of IL-6 in pg/ml, bars denoting SD of duplicate data. (C) IL-6 siRNA reduced MIA-MSLN cell proliferation. MIA-V and MIA-MSLN cells were transfected in six-well plates using a pool of four IL-6-specific siRNAs or a negative control pool of scrambled siRNAs. At 24 h after the transfection, cells were trypsinized, counted and seeded in 96-well plates (2 × 10³ cells per well), serum starved (0% FBS) for 24 h before changing to fresh medium with 0.2% FBS and cultured for 2 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. Experiment was performed several times using various serum conditions with similar results shown is a representative data. (D) IL-6 siRNA reduced cell cycle distribution of MIA-MSLN cells. MIA-MSLN cells treated with IL-6-specific siRNA as previously were allowed to grow in serum-free media for 24 h after initial growth media replenishing for 12 h posttransfection and collected for Propidium Iodide (PI) staining. The numbers of cells in each phase are plotted along the Y-axis. (E) IL-6 siRNA transfected and control MIA-MSLN cells were continued in culture for 96 h and subjected to Caspase3 activation detection by western blot. (F) In order to see which IL-6/stat3 regulated genes were affected by IL-6 siRNA treatment, control and siIL-6 treated MIA-MSLN cells were subject to western blot detection of various IL-6/stat3 regulated genes.

Exogenous IL-6 can stimulate MIA-MSLN cell proliferation with upregulated soluble IL-6 receptor but not surface IL-6 receptor

Because IL-6 seemed to act in an autocrine fashion to support MIA-MSLN cell survival/proliferation, we examined whether exogenous IL-6 could induce MIA-MSLN growth. Treatment with increasing concentrations (0, 0.1, 1, 10 or 100 ng/ml) of recombinant human IL-6 (rIL-6) did not have any effect on the growth of control cells (Figure 5A). However, rIL-6 significantly increased proliferation of MIA-MSLN cells at 10 and 100 ng/ml (Figure 5A, P < 0.01).

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Fig. 5.

Auto/paracrine IL-6 trans-signaling is enhanced in MIA-MSLN cells through upregulated soluble IL-6 receptors. (A) Exogenous rIL-6 induces proliferation in MIA-MSLN cells. MIA-MSLN and control MIA-V cells were seeded in 96-well plates (2 × 10³ cells per well), serum starved (0% FBS) for 24 h and treated with indicated concentrations of IL-6 in 0.2% FBS containing medium for 3 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells and is representative of at least two similar experiments. (B) IL-6 receptor gp80 and gp130 expression patterns in MIA-V and MIA-MSLN cells. IL-6 receptor gp80 and gp130 mRNA expression levels were determined by using real-time polymerase chain reaction in both MIA-V and MIA-MSLN cells. Y-axis shows GAPDH-normalized mRNA levels for gp80 and gp130 in MIA-V and MIA-MSLN cells. Relative mRNA level is presented as 2[C_t(GAPDH)−C_t(IL-6)]. The bars denote SD of duplicate data. (C) Cell surface IL-6R (gp80) and soluble IL-6 receptor (sIL-6R) expression patterns in MIA-V and MIA-MSLN cells. (a) Cell surface gp80 expression was analyzed by FACS using anti-gp80 antibody. The results are depicted as histograms, the percentage denotes the positive cell population. sIL-6R expression in MIA-V and MIA-MSLN supernatants (b) was analyzed by western blot with anti-IL-6R antibody. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test. (D) Effect of ADAM inhibitor TAPI-1 on the generation of sIL-6R in MIA-MSLN cells. Supernatant collected after 48 h treatment with the indicated concentrations of TAPI-1 or dimethyl sulfoxide were concentrated and used to detect the 50 kD sIL-6R band by western. (E) Treatment with TAPI-1 concurrently decreased the cell proliferation of MIA-MSLN cells as indicated. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test.

To study why IL-6 failed to induce growth in control cells, we examined expression of the two receptors involved in IL-6 signaling, the specific IL-6R gp80 and the signal-transducing partner, gp130. The mRNA level of gp80 (IL-6R) was low in both cells (Figure 5B). FACS analysis using anti-gp80 antibody that could detect membrane-bound IL-6 receptor (mIL-6R), revealed both cells had low mIL-6R expression, although it was marginally elevated in MIA-MSLN cells (Figure 5C, a). Western blotting using a IL-6R (alpha chain)-specific antibody revealed elevated levels of a lower molecular weight band at ~50 kD, consistent with a sIL-6R, in supernatants from serum starved MIA-MSLN cells but not MIA-V cells, (Figure 5C, b). This suggested the interesting possibility that IL-6/sIL-6R trans-signaling was involved.

sIL-6Rs could be generated by two mechanisms: by alternatively spliced mRNA for p80 or by intramembrane enzymatic cleavage by metalloproteinases ADAM10 (TACE) and ADAM17 (28). To ascertain the mechanism of sIL-6R generation in MIA-MSLN cells, we used primers that specifically amplify the alternatively spliced mRNA and tried to detect it by real-time polymerase chain reaction. There were very low levels of this form in both cells (data not shown). Thus, the increase in sIL-6R in MIA-MSLN cells was not due to different levels of alternatively spliced IL-6R transcript. We then checked expression levels of the ADAM10 and ADAM17. mRNA expression of these two proteases was similarly high in both cells (data not shown). Treatment with a TACE (ADAM17) inhibitor TAPI-1 could clearly reduce sIL-6R in MIA-MSLN cells (Figure 5D) with a concomitant decrease in cell proliferation of these cells (P < 0.01, Figure 5E). The data indicates that MIA-MSLN cells have increased protease mediated cleavage of membrane IL-6R which might be responsible for an enhanced autocrine trans-signaling in combination with the autocrine IL-6 produced by these cells, helping them survive under growth factor devoid situations.

IL-6/sIL-6R trans-signaling may be responsible for the enhanced cell proliferation in MIA-MSLN cells

To clearly establish the functional significance of upregulated sIL-6R in MIA-MSLN cells, we used a sIL-6R-specific neutralizing antibody to block sIL-6R and examined both endogenous and exogenous IL-6-induced proliferation. Blocking the sIL-6R decreased MIA-MSLN cell viability by almost 25% (P < 0.05), whereas the isotype antibody had no effect (Figure 6A). Similarly, blocking sIL-6R also blocked the IL-6-induced proliferation of MIA-MSLN cells (Figure 6B).

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Fig. 6.

Blocking the IL-6/sIL-6R trans-signaling axis affects the growth/survival of MSLN expressing PC cell proliferation. (A) sIL-6R antibody blocking can reduce the endogenous IL-6-induced proliferation of MIA-MSLN cells. MIA-MSLN cells were seeded in 96-well plates (2 × 10³ cells per well), serum starved (0% FBS) for 24 h with or without anti-sIL-6R or isotype control antibody. The cells were then treated with 0.2% FBS containing medium for 3 days. Viability was measured with MTT. Relative viability was measured by dividing viability after a treatment by viability of same cells without treatment (or dimethyl sulfoxide treated) and is plotted along Y-axis. Data plotted show mean of triplicate wells and is representative of at least two similar experiments. (B). Soluble IL-6R antibody blocking can reduce the exogenous IL-6-induced proliferation of MIA-MSLN cells. MIA-MSLN cells were serum-starved (0% FBS) for 24 h with or without anti-sIL-6R or isotype control antibody and then treated with a cocktail of rIL-6/rsIL-6R in 0.2% FBS containing medium for 3 days. Viability was measured with MTT. Relative viability was measured by dividing viability of treated cells (treated with rIL-6/rsIL-6R cocktail with or without anti-sIL-6R antibody blocking) by that of untreated cells and is plotted along Y-axis. Data plotted show mean of triplicate wells. (C) IL6/sIL-6R trans-signaling axis is operative in MIA cell proliferation. Both MIA-V and MIA-MSLN cells were serum starved (0% FBS) for 24 h with or without anti-sIL-6R or isotype control antibody and then treated with a cocktail of rIL-6/rsIL-6R in 0.2% FBS containing media for 3 days. Viability was measured with MTT. Relative increase in viability was measured by dividing viability of treated cells (treated with rIL-6/rsIL-6R cocktail with or without anti-sIL-6R antibody blocking) by that of untreated cells and is plotted along Y-axis. Data plotted show mean of triplicate wells. *, # denote P < 0.05, and **, ## denote P < 0.01, compared with controls, t-test.

Since IL-6 alone could not induce proliferation of low-MLSN-expressing MIA-V cells, we asked if this was due to lack of sIL-6R in these cells. So, we treated MIA-V/MIA-MSLN cells with a cocktail of exogenous rIL-6 and recombinant sIL-6R (rsIL-6R). The rIL-6/rsIL-6R mixture indeed induced a significant increase in proliferation of the MIA-V cells (~1.5 times in 3 days, P < 0.01, Figure 6C). These data demonstrate that IL-6/sIL-6R trans-signaling was involved in the MIA-V cell proliferation/survival as well. Expectedly, rIL-6/rsIL-6R induced increased proliferation in MIA-MSLN cells to a greater extent (~1.9 times in 3 days, P < 0.01, Figure 6C). Most importantly, blocking sIL-6R with sIL-6R-specific neutralizing antibody completely abolished this growth-promoting effect (P < 0.01, Figure 6C) but a matched isotype control antibody could not block the effect. Altogether, the data clearly established IL-6 trans-signaling to be a major factor affecting survival/proliferation of MSLN-expressing PC cells.

Conflict of Interest Statement: None declared.