Interplay between thyroid cancer cells and macrophages: effects on IL-32 mediated cell death and thyroid cancer cell migration

The co-culture in vitro experiments were performed using TPC-1 cells (papillary, RET/ PTC rearrangement) [22]. TPC-1 cells were grown in RPMI-1640 culture medium, Dutch modification (Life Technologies, Carlsbad, CA, USA) supplemented with gentamycin 50 μg/ml, pyruvate 1 mM, GlutamMAX 2 mM and 10% Fetal Calf Serum (FCS, Gibco, Life Technologies). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-plaque (GE Healthcare, Diegem Belgium) from buffy coats obtained from Sanquin Bloodbank, Nijmegen, The Netherlands. Ethical approval was provided by CMO Arnhem-Nijmegen (CMO 2010–104) and all experiments were performed in accordance with the principles expressed in the Declaration of Helsinki. For transcriptome analysis, an additional step of purification using CD14-labelled magnetic beads was performed after PBMC isolation in order to obtain a highly purified monocyte population.

A trans-well system with ThinCert™ cell culture inserts in a 24-well plate (Greiner Bio-One GmbH, Austria) was used for co-culture of TC cells and human monocytes. Cell counts were performed using a Coulter particle counter (Beckman Coulter Inc. Pasadena, CA, USA). A total of 1.0 × 10⁵ TPC-1 cells in 250 μl culture medium was added to the upper compartments of the trans-well system. Upper compartments solely with medium were used as negative controls. Culture medium was added to the lower compartments and the cells were incubated for 24 h at 37 °C, 5% CO₂. Next, the cells were washed with phosphate buffered saline (PBS, Braun Melsungen, Germany) after which fresh co-culture medium, containing a physiological concentration of glucose, was added: RPMI-1640 without glucose (Life Technologies, Carlsbad, California, USA) supplemented with glucose 5 mM, pyruvate 1 mM, gentamicin 50 μg/ml and HEPES 10 mM (Life Technologies, Carlsbad, California, USA).

Monocytes were isolated as described above and resuspended in the co-culture medium. A total of 5.0 × 10⁵ monocytes in 500 μl was added to the lower compartments of the trans-well system. After adhesion for 1 h in the 24-well plates, the non-adherent cells were discarded, and the adherent monocytes were incubated further with TPC-1 cells or medium alone for 24 h at 37 °C in co-culture medium. After 24 h incubation, the adherent monocytes, now called naïve monocytes or TC-induced macrophages, respectively, were stimulated for 24 h with RPMI-1640 or 10 ng/ml LPS (E. coli strain O55:B5, Sigma Chemical Co, St. Louis, MO, USA) as substitute for endogenous TLR4 ligand signalling, while leaving the trans-wells containing medium or TPC-1 cells in place. At the end of the incubation period, TPC-1 cells were lysed in 200 μl Trizol reagent (Invitrogen, Carlsbad, CA, USA) and stored at −80 °C for transcriptome analysis. Supernatants where collected and stored at −80 °C for subsequent conditioned medium experiments. A schematic representation of the experimental set-up is provided in Supplementary Fig. S1.

RNA isolation was performed according to the Trizol manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). 50 ng RNA was converted into cDNA libraries according to the TruSeq RNA library preparation kit v2. After cluster generation on a cBot (Illumina), a 75 bp single read (SR) rapid run was performed on an Illumina HiSeq 1500 system.

After base calling and de-multiplexing using CASAVA version 1.8, the 75 bp single-end reads were aligned to the human reference transcriptome hg38 from UCSC by kallisto v0.44.0 using default parameters. Data were imported into DESeq2 (v.1.10.1) using the TXimport (v1.2.0) package. DESeq2 was used for the calculation of normalized counts for each transcript using default parameters. After DESeq2 normalization, all normalized transcripts with a maximum over all group means lower than 10 were excluded resulting in 17,099 present genes. Unwanted or hidden sources of variation, such as batch and preparation date, were removed using the sva package [23]. The normalized rlog transformed expression values were adjusted according to the two surrogate variables identified by sva using the function removeBatchEffect from the limma package [24]. Differentially expressed (DE) genes were defined by a |fold change| > 1.5 and an adjusted p value (FDR) < 0.05. Log₂ fold changes were plotted against –log₁₀ adjusted p values in a volcano plot. Top 10 up- and down-regulated genes were marked in the volcano plot. Sashimi plots were generated by visualizing the aligned reads using the Integrative Genome Viewer (IGV) [25], and the function sashimi plot. Principal component analysis was performed on all present genes. Detailed data have been deposited in the GEO database with accession number GSE120391.

To test whether induction of IL-32 mRNA expression in TPC-1 was caused by paracrine factors from the co-culture medium, fresh TPC-1 cells were stimulated with supernatants from TPC-1-monocyte co-culture experiments for 24 h. Since TNFα is an important inducer of IL-32 [1, 4] and TNFα is abundantly present in the co-culture medium [21], TPC-1 cells were also stimulated with TNFα (100 ng/ml R&D Systems, Minneapolis, MN, USA) for 24 h. Briefly, 500.000 cells/well were seeded in a 24-well plate (Corning, New York, USA) and left to adhere for 18 H. Medium was replaced with fresh medium mixed with supernatants from co-culture experiments in a 1:1 ratio or fresh medium with TNFα (100 ng/ml). To test whether IL-32 could also be induced in other TC cell lines, two additional cell lines were included, i.e., BC-PAP (papillary TC, BRAF V600E mutation) and FTC-133 (follicular TC, PTEN deficient) [22]. To confirm the role of TNFα, specific TNFα inhibitors, etanercept (decoy receptor, 10 μg/ml, Enbrel©, Pfizer, New York, USA) and adalimumab (monoclonal antibody, 10 μg/m, Humira©, Abbott GmbH & Co. Wiesbaden, Germany) were added to pre-selected experiments.

pcDNA3-based vectors for the expression of IL-32β and IL-32γ_mutant were generated by standard PCR and restriction based cloning methods. The expression plasmids were constructed with a Kozak sequence (5′-GCCGCCACC-3′) immediately upstream of the ATG start codon followed by full-length cDNAs of human IL-32β, IL-32γ and eGFP or an empty DNA (control). An IL-32γ_mutant was created that cannot be spliced into other IL-32 isoforms like regular IL-32γ, to investigate the effect of IL-32γ without the effect of over-expression of other IL-32 splice variants. The IL-32γ_mutant was created by mutation of a donor splice site from GU to AU as described previously [2].

TPC-1 cells were transfected with pCDNA3 plasmids expressing IL-32β, IL-32γ_mutant or empty DNA, together with pCDNA3 plasmids expressing eGFP in a 10:1 ratio. Briefly, cells were seeded at a density of approximately 40.000 cells per cm² in a 10 mm dish (VWR, Radnor, PA, USA). Medium was refreshed after 24 h and cells were transfected using Fugene (Promega Corporation, Madison, WI, USA) according to the manufacturers’ protocol. After 24 h, cells were collected using trypsin/EDTA (Life Technologies), counted using a Bürker-Türk counter chamber (Sigma Aldrich Chemie, Zwijndrecht, The Netherlands) and Tryphan blue staining (Sigma), and only live cells were reseeded for further experiments.

To quantify the transfection efficiency, 200.000 cells were reseeded after transfection. After 40 h and 64 h, the cells were trypsinised, centrifugated and fixed through resuspension in Fix and Perm buffer (Life technologies) for 45 min. After washing in Perm buffer, the cells were stained with an anti-Ki-67 monoclonal antibody (SolA15, invitrogen, Carlsbad, CA, USA) as a marker for proliferation activity, and incubated for 30 min in the dark at 4 °C. Next, the cells were washed and resuspended in PBS containing 1% Bovine Serum Albumin (BSA, Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands) for analysis by flow cytometry. A Cytoflex flow cytometer (3 lasers, Beckman Coulter Inc.) was used to determine the percentage of transfected cells (i.e., GFP expressing cells) by setting the excitation at 488 nm, the emission at 525 nm (FITC channel) and recording 100.000 events per sample. The percentage of Ki-67 positive cells was determined by setting the excitation at 488 nm and the emission at 610 nm (ECD-A channel). The percentage of positive cells was obtained using a fluorescence intensity histogram, which represents the number of events at a given fluorescence intensity.

To assess cytotoxicity, we performed a Cytotox96 assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

To assess IL-32 protein expression, cells were seeded at a density of 300.000 cells/well in a 24-well plate (Corning). At two different time points, 40 and 64 h after transfection, cells were lysed in lysis buffer containing phosphatase and protease inhibitors (Roche, Basel, Switzerland) after which proteins were loaded on a pre-casted 4–15% gel (Biorad, CA, USA) for polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Biorad) after which incubation overnight at room temperature with a goat-anti-IL-32 (AF3040) antibody (R&D systems, Minneapolis, Minnesota, USA) was used to detect IL-32 protein. Detection of actin with a rabbit anti-actin antibody (Sigma) was used to verify protein concentrations. Polyclonal secondary antibodies (Dako, Begium) and a Clarity Western ECL Blotting Substrate (Biorad) were used to visualize protein expression.

For mRNA expression analyses, TPC-1 cells (200.000/well) were lysed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and stored at −80 °C. RNA isolation was performed according to the manufacturer’s instructions and transcribed into cDNA by reverse transcription using an iScript cDNA Synthesis Kit (Bio-Rad). A Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) was used for RT-qPCR in a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). Expression data were normalized to the housekeeping genes human β₂M or GAPDH. The primers used were purchased from Biolegio (Nijmegen, The Netherlands) and the sequences used for RT-qPCR are listed in Supplementary Table S1. In the different stimulation experiments the mRNA expression levels of IL-32 isoforms α, β and γ were assessed. After transfection, the mRNA expression levels of migratory factors associated with IL-32 expression were assessed, including IL-8, MMP2, 3 and 9, the EMT-marker E-cadherin and VEGF [13‐16]. The mRNA expression level of IL-32α was also assessed, as this is an IL-32 splicing product and expression of IL‐32β and IL‐32γ could, therefore, also result in an increased IL‐32α mRNA level.

In vitro gap closure assays were conducted using silicone cell culture inserts (Ibidi GmbH, Martinsried Germany) attached to culture plates. TPC-1 cells were seeded into the inserts (28.000 cells/insert chamber) and left to adhere overnight to form a confluent monolayer. Next, the inserts were removed with tweezers, and the cells were rinsed with PBS once to remove detached cells. Normal RPMI-1640 culture medium containing either 0% or 10% FCS was added after which gap closure was assessed at different time points using conventional light microscopy. Gap closure was quantified by ImageJ, using the MRI wound healing tool (National Institute of Health, Bethesda, MD, USA).

All analyses were performed in Graphpad prism 5 (CA, USA). Differences in mRNA expression levels, GFP and Ki-67 positivity, cytotoxicity and differences in gap closure, were analysed using Kraskal Wallis test with Dunnets multiple comparison test, or Mann Whitney test to compare different conditions to control condition, or paired Wilcoxon signed rank test to compare effects within conditions. * p < 0.05, ** p < 0.01. Data are shown as means ± SEM.

To investigate transcriptional differences of TPC-1 cells after co-culture with inactivated (control, RPMI-1640 stimulated) or TLR-4 activated (LPS stimulated) TC-induced-macrophages on a global level, we generated genome-wide transcriptome data through quantitative RNA-sequencing in TPC-1 cells and performed bioinformatic analyses (Fig. 1a). Unbiased principle component analysis (PCA) based on 17,099 transcripts (Fig. 1b) revealed significant transcriptional changes in TPC-1 cells after co-culture with TLR-4 activated TC-induced macrophages. Differential gene expression analysis (FC ≥ 1.5) revealed 98 significantly upregulated and 30 significantly downregulated genes (Fig. 1c). Indeed, IL-32 was among the top 10 upregulated genes as visualized by Volcano Plot (Fig. 1d). Sashimi plots (Fig. 1e) show the splice junctions for TPC-1 cells co-cultured with TC-induced macrophages after re-stimulation with RPMI-1640 (control) or TLR-4 ligand (LPS), indicating that IL-32β is the most probable splice variant.

Next, we found that stimulation of TPC-1 cells with supernatants from TC-induced macrophages, naive monocytes and TPC-1 cells only, confirmed that paracrine factors from activated TC-induced macrophages induced IL-32 expression in TPC-1 cells (Fig. 1f). We also found that supernatants from TLR-4 stimulated TC-induced macrophages induced significantly higher IL-32α, IL-32β and IL-32γ mRNA expression levels in TPC-1 cells (p = 0.0156) compared to supernatants from RPMI stimulated TC-induced macrophages. Supernatants from TLR-4 stimulated TC-induced macrophages were found to induce in the highest levels of IL-32β, with a fold change of 16.77 ± 6.17 compared to 9.23 ± 5.55 in naive, TLR-4 stimulated monocytes (p = 0.0313).

We have previously shown that TC-induced macrophages produce increased levels of TNFα and IL-6 compared to naive monocytes [21]. Since TNFα is one of the main inducers of IL-32, we hypothesized that the high levels of TNFα in the co-culture supernatants might be responsible for the rise in IL-32 mRNA expression in TC. To test this hypothesis, TPC-1 cells were stimulated with TNFα (100 ng/ml), after which again increased mRNA expression levels of IL-32α (p = 0.0079) and IL-32β (p = 0.0079) were observed, whereas the IL-32γ expression level was not significantly altered (Fig. 2a). To investigate whether TNFα could also induce IL-32 in other TC-derived cell lines, BC-PAP (papillary TC, BRAF V600E mutation) and FTC-133 (follicular TC, PTEN deficient) cells were stimulated with TNFα as well. In both BC-PAP and FTC-133 cells the IL-32α and IL-32β mRNA expression levels were found to be upregulated after stimulation with TNFα, albeit to a lesser extent (Fig. 2a).

Using Western-blot analysis, we confirmed that TNFα induced IL-32 expression at the protein level in all three TC-derived cell lines (Fig. 2b). The highest level was found in TPC-1 cells, most likely being IL-32β as based on protein size (25.9 kDa, Supplementary Fig. S2A). We found that the use of two different inhibitors of TNFα, Humira and Enbrel, abrogated the upregulation of IL-32 in the TC cell lines, further confirming that TNFα acts as an important inducer of IL-32β mRNA and protein expression in TC (Fig. 2c, d and Supplementary Fig. S2B-D).

Since IL-32 mRNA and protein expression was highest in TPC-1 cells, we next exogenously overexpressed IL-32β and IL-32γ in TPC-1 cells. IL-32β was used because this isoform was most highly expressed in TC. Since IL-32γ is known as most potent isoform of IL-32 for several functions [3], IL-32γ was also included in these experiments. To investigate the effect of IL-32γ without the effect of overexpression of other IL-32 splice variants, an IL-32γ_mutant vector was created, which cannot be spliced into other IL-32 isoforms like regular IL-32γ. The transfection efficacy was assessed by calculating the percentage of green fluorescent protein (GFP)-positive cells at two different time points [t = 40 h (start of gap closure assay), and t = 64 h, (end of gap closure assay)]. We found that the mean percentage of GFP-positive cells at 40 h after transfection in serum-rich conditions (10% FCS) did not differ significantly, and was lowest in the control transfected cells (46.2 ± 14.1%) and highest in the IL-32γ_mutant transfected cells (74.0 ± 9.2%) (Fig. 3a). Sixty-four hours after transfection, the mean percentage of GFP-positive cells was only slightly increased (not significant) in both serum-rich (10% FCS) and serum-deprived (0% FCS) conditions. In addition, we found that transfection with either IL-32β or IL-32γ_mutant resulted in high levels of protein expression of the indicated IL-32 isoform in TPC-1 cells at both time points (Fig. 3b, t = 40 h; data for t = 64 h not shown), while control transfected cells (empty vector) showed no IL-32 protein expression. Interestingly, we detected both IL-32β and IL-32γ protein in the 32γ_mutant transfected cells.

Next, the effect of exogenous IL-32 overexpression on cytotoxicity was assessed. An increased cytotoxicity was observed at 40 h after transfection for all conditions compared to non-transfected cells (p = 0.0006) (Fig. 3c), indicating that transfection did result in increased cell death. At 64 h post transfection, we found lower percentages of cytotoxicity due to transfection, and that both IL-32β and IL-32γ overexpression induced significantly more cytotoxicity both in the presence of serum (p = 0.0043 and p = 0.0022) and in the absence of serum (p = 0.0411 and p = 0.0129). IL-32γ overexpression resulted in the highest level of cell death in the presence of serum (25.5 ± 3.3% cytotoxicity for IL-32γ transfected cells compared to 18.4 ± 0.5% in control transfected cells).

To investigate the effect of IL-32 on proliferation, we analyzed whether the expression of Ki-67, a marker for proliferation, was affected by exogenous IL‐32 overexpression. We found that > 98% of all cells were Ki‐67 positive in all conditions, and that all GFP-positive cells were also Ki-67 positive (Fig. 3d). The mean fluorescence intensity of Ki-67 staining in IL-32γ_mutant transfected cells was found to be lower compared to that in control transfected cells and IL-32β transfected cells, but the differences were not statistically significant (Fig. 3e, f). Thus, we conclude that IL-32 overexpression did not affect the percentage of Ki-67-positive cells and only slightly affected Ki-67 intensity.

Next, the effect of exogenous IL-32β and IL-32γ overexpression on TC cell migration in an in vitro gap closure assay was assessed (see example in Fig. 4a). Figure 4b shows the percentage of the gap that the cells filled in a 24 h period. As expected, we found the highest percentage of gap closure in serum-rich conditions with an abundance of growth and migratory factors. When comparing control transfected cells with IL-32β or IL-32γ transfected cells, no significant differences were found in the percentages of gap closure after 24 h, neither in serum-deprived nor in serum-rich conditions.

Finally, we assessed the mRNA expression levels of factors known to be associated with IL-32 expression and cell migration in serum rich conditions. At 64 h after transfection, we found that the mRNA expression levels of IL-32α, IL-8 and VEGF were significantly reduced in all conditions compared to those 40 h after transfection (Fig. 5). The IL-32α and IL-8 mRNA expression levels were significantly upregulated in IL-32β-transfected cells at 64 h after transfection compared to those in control transfected cells (p = 0.0003 and p = 0.0120). The IL-32α mRNA expression level was also significantly higher after 40 h in both IL-32β- and IL-32γ-transfected cells compared to control transfected cells. IL-32β and IL-32γ overexpression did not result in any significant differences in mRNA expression levels of MMP2, 3 or 9, E-cadherin or VEGF compared to control transfected cells at both time points.