ABSTRACT
Within tissues, cells secrete protein signals that are subsequently interpreted by neighboring cells via intracellular signaling networks to coordinate a cellular response. However, the oncogenic process of mutation and selection can rewire these signaling networks to confer a fitness advantage to malignant cells. For instance, the melanoma cell model (B16F0) creates a cytokine sink for Interleukin-12 (IL-12) to deprive neighboring cells of this important extracellular signal for sustaining anti-tumor immunity. Alternatively, oncogenesis may also rewire intracellular signaling networks. To test this concept, we asked whether IL-12 provides an intrinsic advantage to B16F0 melanoma cells. Functionally, stimulation with IL-12 promoted the survival of B16F0 cells that were challenged with a cytotoxic agent but had no rescue effect on normal Melan-A melanocytes. We also explored a mechanistic basis for the functional results by surveying the phosphorylation intracellular proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt. In contrast to T cells that exhibited a canonical response to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells phosphorylated both STAT4 and Akt. Collectively, the data suggests that B16F0 cells have shifted the intracellular response to IL-12 from engaging immune surveillance to favoring cell survival. In short, identifying how signaling networks are rewired can guide therapeutic strategies to restore effective immunosurveillance.
INTRODUCTION
Responses of the different cell types within tissues, such as T cells and Langerhans cells within the epidermal layer of skin, are coordinated by the extracellular release of protein signals. One such extracellular signal is the cytokine Interleukin-12 (IL-12) 1. IL-12 is produced by antigen presenting cells, like Langerhans and Dendritic cells, to boost natural killer (NK) and cytotoxic T lymphocyte (CTL) activity and to polarize T helper cells towards a type 1 phenotype2. Activating NK, CTLs and type 1 T helper cells play important roles in controlling viral infections and defending against malignancy. Towards this aim, local delivery of IL-12 to the tumor microenvironment has been shown to promote tumor regression in both mouse models and human melanoma 3–9.
Inside the cell, these extracellular signals are processed through a series of protein-protein interactions that transmit this information from the cell membrane to the nucleus to orchestrate a cellular response. In the case of IL-12, these extracellular signals are transduced by the canonical Janus-Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) signaling pathway10. The IL-12 receptor, composed of a β1 and β2 subunit, lacks intrinsic enzymatic activity and forms a complex with two JAKs, JAK2 and TYK2. Autophosphorylation of the receptor complex results in binding and activation of STAT4. Phosphorylated STAT4 dimerizes and then translocates to the nuclear to function as a transcription factor 11. As a transcription factor, STAT4 promotes the expression and release of Interferon-gamma (IFN-γ)12, which upregulates MHC class I antigen presentation13. An increase in antigen presentation enables CTLs to target and kill virally infected and malignant cells present within the tissue14.
While most of our understanding of how information is transmitted between cells is based on normal biology, cancer cells arise through a process of mutation and selection15, 16. This implies that during oncogenesis cancer cells develop ways to block these extracellular signals or rewire intracellular signaling pathways to gain a fitness advantage17, 18. Following from this premise, the mouse melanoma cell line B16F0 overexpresses one component of the IL-12 receptor, IL12RB2, to form a cytokine sink for IL-12, which can deprive neighboring immune cells of this important antitumor cytokine19. As rewiring of intracellular cell signaling networks is another way that tumor cells can gain advantage, the objective of this study was to assess whether IL-12 provides an intrinsic advantage to B16F0 tumor cells. We hypothesized that overexpression of IL12RB2 in melanoma cells, such as the mouse model B16F0, has an intrinsic benefit for tumor cell survival. To test our hypothesis, we investigated the copy number of IL-12 receptor subunits, IL12RB1 and IL12RB2, on the surface of a mouse model of “normal” melanocytes and two melanoma models, B16F0 and Cloudman S91, and compared that to the expression of the receptor on the surface of an IL12-dependent mouse T helper cell model, 2D6. By observed the cytotoxic activity of small molecule inhibitor drug imatinib on melanoma cells, we found that IL-12 enhanced cell viability of B16F0 cells but not normal melanocytes in the presence of this cytotoxic agent. Finally, we explored a mechanistic basis for these functional assay results by surveying the activation intracellular proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt.
RESULTS
Inspired by our previous observation of an increase in IL12RB2 expression in the B16F0 cell line, we first asked whether the subunits of the IL-12 receptor are similarly upregulated in other melanoma cell lines that do not depend on IL-12 for their growth and proliferation. We compared two mouse melanoma cell lines, B16F0 and Cloudman S91, with Melan-A cells, a model of “normal” melanocytes. The 2D6 T cells, a mouse type 1 T helper cell line, were used as a positive control for IL-12 receptor expression19. IL12RB1 and IL12RB2 abundance was assayed by flow cytometry and calibrated to copy number using antibody calibration beads (Figure 1).
In 2D6 cells, IL12RB1 and IL12RB2 were present in almost a 2:1 copy ratio, with 43,000 copies of IL12RB1 and 19,500 copies of IL12RB2 and consistent with previous findings19. Interestingly, all three melanoma-related cell lines stained brightly for IL12RB2. The copy number of IL12RB2 was 1.5 times higher in the melanoma cell line versus the “normal” melanocyte line, where the Cloudman S91 and B16F0 cell lines expressed over 330,000 copies of IL12RB2 and Melan-A cells had 214,000 copies. In contrast, IL12RB1 abundance was barely detectable in all three cell lines, as staining was similar to the unstained controls. Only the B16F0 cell line was quantified at 1000 copies, while the Melan-A and Cloudman cell lines were below the detection level of 200 copies per cell. These data suggest that expression of components of the IL-12 receptor, IL12RB1 and IL12RB2, are differentially regulated in mouse cell models of melanoma compared with T cells. This differential regulation is manifested as a high copy number of IL12RB2 and an absence of IL12RB1, such that copies of the heterodimeric IL-12 receptor are limited.
While creating a cytokine sink by overexpressing IL12RB2 provides an indirect survival advantage to malignant melanocytes, we also wondered whether overexpression of IL12RB2 provides a direct survival advantage to malignant melanocytes. To test for an intrinsic survival advantage, we used an apoptosis and necrosis assay to assess cell viability during a chemotherapy challenge with imatinib in the B16F0 melanoma (Figure 2) in combination with an IL-12 rescue. We used imatinib, as it is currently being investigated as a possible chemotherapy for metastatic melanoma20, 21.
To determine whether IL-12 could “rescue” the melanoma cells from the cytotoxic effects of chemotherapy, we incubated the cells with three different concentration of IL-12 (40, 100, or 200 ng/mL) for 18 hours, which correspond to concentrations of IL-12 in the serum of various mouse models at approximately 3-134 ng/mL22. We then treated with three different concentrations of imatinib (5, 10, or 20 µM) for 24 hours, with 0.1% DMSO in DPBS serving as a vehicle control. Cell viability was assayed by flow cytometry, whereby cells were identified based on forward and side scatter characteristics. Gating on “cells”, the presence of Annexin-V staining on the cell surface and no PI staining indicates a cell undergoing apoptosis, while cells that are positive for PI and negative for Annexin-V indicates a cell that underwent necrotic cell death22.
At baseline, the viability of B16F0 cells was greater than 90%. Upon treatment with imatinib, the viability was decreased in a dose-dependent manner, as expected. At 24 hours, very few of the cells were observed during the early stages of apoptosis (Annexin-V+, PI-) and the majority of non-viable cells either underwent necrotic cell death (Annexin-V-, PI+) or were in the late stages of apoptotic cell death (Annexin-V+, PI+). The addition of IL-12 reduced the cytotoxic efficacy of imatinib such that the maximum shift in viability of cells pre-treated with IL-12 before imatinib challenge was about 15%. To quantify the effects of imatinib and IL-12 on the viability of the B16F0 cells, we analyzed the data using a mathematical model and inferred the model parameters using a Markov Chain Monte Carlo (MCMC) approach. In theory, the model is identifiable as there are more data points (16) than model parameters (3). However given the non-linear nature of the model, the MCMC approach was used to assess whether the model parameters could be practically identified from the available data in addition to estimating parameter values. The MCMC results suggested that the effective concentration of imatinib (EC50) and the rescue effect of IL-12 could be practically identified from the data as they exhibited symmetric bounded distributions. The posterior distributions provided a median value of the EC50 of imatinib equal to 40 nM for imatinib and of α equal to 1 (Figure 2d). Moreover, the posterior distributions indicated that the rescue effect of IL-12 from the cytotoxic actions of imatinib was significant (p = 0.0095 that α<0). Quantitatively, a saturating concentration of IL-12 would increase the EC50 for imatinib by 100% (maximum likelihood value of α=1), thereby shifting the imatinib dose-response curve to the right. We repeated this experiment using a plate-based viability assay (ATPlite) using both B16F0 and Melan-A cells (Figure 2e). While the variability was higher than the flow cytometry-based assay, IL-12 did not provide a rescue effect when Melan-A cells, an immortalized normal melanocyte cell line, were exposed to imatinib (i.e., maximum likelihood value of α = 0). While cell viability was assayed after only 24 hours, the observed difference observed with B16F0 cells implies that, over a 1 week period, tumor load would increase by about 10-fold in the IL-12 pre-treated cells versus chemotherapy alone.
To confirm the role of IL12RB2 in the “rescue” effect of IL-12 to cytotoxic stress with imatinib, we knocked down IL12RB2 in the B16F0 cell line using a CRISPR-Cas9 construct (Figure 3a). Generally, knockdown clones grew at similar rates, showed minor differences in color, and displayed similar phenotypes to the wild-type cell line. Knockdown of IL12RB2 was confirmed by flow cytometry. To compare the functional response, wild type (wt) and IL12RB2 knockdown (KD) B16F0 cells were treated with increasing concentrations of imatinib for 12 hours and pre-stimulation with 100 ng/mL IL-12 overnight was included as a second independent variable. Cell viability was assessed using an ATPlite assay. Consistent with data shown in Figure 2, imatinib dose-dependently reduced viability in the wild-type B16F0 cell line that was prevented with pre-treatment with IL-12 (Figure 3b). The rescue effect of IL-12 was most pronounced at the higher concentrations of imatinib. In comparison, imatinib also dose-dependently reduced viability of the IL12RB2 knockdown cells but IL-12 provided no benefit (Figure 3c). Visually, we also noticed that IL-12 had no effect on preventing cell detachment or inhibiting growth in IL12RB2 KD cells. Collectively, the results suggested that IL-12 enhances the survival of B16F0 cells in the presence of cytotoxic stressors, like imatinib, and its effect is dependent on IL12RB2 expression.
Given the severe imbalance of IL-12 receptor expression in B16F0 cells, it was unclear what signals were transduced in B16F0 cells in response to IL-12 stimulation. Moreover, activating STAT4, which plays a role in Interferon-γ gene expression and not survival, is thought to be the canonical signaling response to IL-12. To investigate, we assayed the phosphorylation of STAT4, a canonical signal transducer, and Akt, a non-canonical signal transducer involved in promoting cell survival and therapeutic resistance23, in response to IL-12 stimulation in both live B16F0 and 2D6 cells by flow cytometry (Figure 4). The 2D6 T helper cell line was used as a model for canonical IL-12 signal transduction. Following 12-24 hours of preconditioning in media free of IL-12 (2D6 only) and serum (2D6 and B16), we stimulated B16F0 and 2D6 cells with increasing concentrations of IL-12 (0, 40, 100, and 200 ng/mL; data shown for the 100 ng/mL treatment). Pre-conditioning ensured that activation of the IL-12 signaling pathway was due to IL-12, and not due to factors in the serum or basal IL-12. PBS was used as a negative vehicle control for both cell lines.
Both 2D6 and B16F0 cells phosphorylated STAT4 in response to IL-12 stimulation (p<0.0001 for 2D6 and p<0.05 for the B16), with the response more pronounced in 2D6 cells, as expected. Basal STAT4 phosphorylation was minimal in both cell lines for the vehicle only-treated controls. In terms of Akt phosphorylation, the B16 melanoma showed a marked activation of Akt within 3 hours at all concentrations of IL-12 tested, with a significant increase (p<0.05) in pAkt with 100 ng/mL IL-12 treatment. In contrast, 2D6 cells exhibited no significant shift in phosphorylation of Akt above baseline in response to IL-12 treatment. Pre-conditioning cells in the absence of serum was critical to observe differences in Akt phosphorylation.
To determine whether Akt activation in the B16 melanoma line was specific, we repeated the experiment and used a Western Blot assay to determine basal levels of Akt and test several inhibitors against the IL-12-induced effect on Akt phosphorylation (Figure 5). To serve as a negative control and to determine whether the PI3K pathway may be cross-activated in response to IL-12 stimulation, we used a PI3K inhibitor LY294002. In addition, we used a selective pAkt inhibitor, MK-2206, to investigate whether the observed effect was solely through possible direct activation of Akt by the IL-12 receptor. Two different phosphorylation sites on Akt were assessed, threonine 308 (Thr308) and serine 472/473 (Ser472/473). We also measured total Akt, and β-actin was used as a loading control. We found no change in phosphorylation of Thr308 in response to IL-12 stimulation, whereas baseline phosphorylation was turned off by both LY294002 and MK-2206. However, IL-12 stimulation produced a marked increase in phosphorylation of the Ser472/473 site, and again, phosphorylation was blocked by both the PI3K and Akt-selective inhibitors. Collectively, the results suggest that IL-12 provides a survival advantage to B16F0 cells and that IL-12 induced a different cellular response in B16F0 cells compared to 2D6 T cells, whereby phosphorylation of Akt, a non-canonical signal transducer that is associated with enhanced cell survival is favored over STAT4, which is the canonical signal transducer.
To extend our focus beyond mouse cell models, we also wondered whether similar patterns of gene expression are observed in human melanoma. Towards this aim, we determined whether IL12RB1 and IL12RB2 gene levels are significantly different in human melanoma and are comparable to our protein level findings in mouse models of melanoma (Table 1). From data retrieved from Oncomine, we found that IL12RB2 is significantly upregulated by approximately 5 fold in human melanoma, compared to normal tissue and at a higher level than IL12RB1, which was also elevated by approximately 2.26 fold, compared to normal. Benign nevus samples did not produce significant differences in IL12RB1 or IL12RB2 gene levels. While normal tissue samples were not included in the Cancer Genome Atlas (TCGA) study, there was no significant difference in the levels of IL12RB2 between the primary or metastatic sites. However, IL12RB1 was significantly upregulated by more than 1.5 times in metastatic versus primary tumors. These results state that significant gene level changes in the IL-12 receptor subunits occur within development of human melanoma and are exacerbated in metastatic sites versus primary tumors. Because the TCGA and Oncomine data sets reflect homogenized tissue samples that may contain a mixture of tumor and immune cells, we also examined data sets from the Cancer Cell Line Encyclopedia (CCLE), which reflects mRNA expression in cellular models of human cancer. In the reported melanoma cell lines, expression of IL12RB2 was significantly greater than IL12RB1, such that the ratio of IL12RB2 to IL12RB1 gene expression was 1.06 (p<0.028, ratio<1) (Table 1).
DISCUSSION
Given the importance of IL-12 in promoting type 1 anti-tumor immune response, we had previously observed that, by overexpression one component of the IL-12 receptor IL12RB2, B16 melanoma cells could create a “cytokine sink” and deprive tumor-infiltrating lymphocytes of this local signal to sustain an anti-tumor response19. Here, our results indicate that IL-12 can act directly on B16 melanoma cells to phosphorylate STAT4 (Figure 4a and 4c), although less pronounced as observed in T helper cells (Figure 4b and 4d). In contrast to T helper cells, IL-12 provided an intrinsic survival advantage to B16 melanoma cells by engaging the non-canonical signal transducer Akt, that is B16 cells re-wired the canonical immune cell response to IL-12 to enhance cell survival. The survival benefit of IL-12 appears to be mediated through IL12RB2, as knock down of IL12RB2 abrogated the effect. As the IL-12 rescue was not observed with Melan-A cells, this observation indicates that melanoma cells may rewire signal transduction to create a non-canonical signaling pathway to promote survival in response to cytokines within the tumor environment.
As summarized in Figure 6, we propose a mechanism by which rewiring of the canonical IL-12 pathway could occur that is initiated by IL-12 mediated homodimerization of IL12RB2. IL-12 has been shown to promote a low but significant level of homodimerization of IL12RB2 subunits24. Overexpression of IL12RB2 may make this association more likely to occur in the melanoma cell line. Following IL12RB2 homodimerization, the receptor associated Janus kinase, JAK2, interacts to promote cross phosphorylation and activation38. Activated JAK2 can then recruit and activate PI3K via the p85a subunit25, 26. Activated PI3K can then activate Thr308-primed Akt via phosphorylating the Ser 472/473 loci. Our findings support the direct role of PI3K in the observed phosphorylation of Akt to affect cell survival, as inhibitors directed against PI3K pathway like LY294002 ameliorated this effect.
Rewiring of signaling pathways during oncogenesis is emerging as an important concept in cancer biology27. For instance, a recent systems biology study across all known human kinases showed there are six types of network-attacking mutations that perturb signaling networks to promote cancer, including mutations that dysregulate phosphorylation sites to cause constitutive activation or deactivation, rewire networks through upstream or downstream interactions, and dysregulate network dynamics by activated or deactivating nodes of response28. As for precedence of non-canonical signaling within the JAK-STAT pathway, STAT3 has been reported as a non-canonical activator of the NF-κB pathway to promote myeloid-derived suppressor cells in the tumor microenvironment29. Beside oncogenic fusion proteins, increased abundance of intracellular signaling proteins through either copy number amplifications or epigenetic changes can also create non-canonical protein interactions that could redirect the flow of intracellular information30. Knowing the basis for cancer cell rewiring can be used for therapeutic advantage. For instance, the non-canonical use of signaling cross-talk downstream of the EGF receptor sensitized resistant triple negative breast cancers to DNA-damaging chemotherapies by inhibition of EGFR31. Similar approaches could be applied to circumvent rewiring of the IL-12 pathway.
While additional studies may help clarify the mechanistic details and generalize the finding, several prior studies lend support that IL-12 can elicit a direct response in tumor cells and that the IL-12 receptor can activate non-canonical signaling elements. In vitro, IL-12 had a direct effect to upregulate MHC complexes on human melanoma cell lines 32. A similar response was not observed in mouse tumor cell lines although the concentrations of IL-12 used were rather low (0.3 − 1 ng/ml)33. Su et al. also reported that the expression of IL12RB1 was absent in B16 cells compared to other mouse tumor cell lines. However, results for IL12RB2 are inconclusive as IL12RB2 amplicons were unable to be observed in any of the tumor cell lines. IL-12 did activate NF-κB in two cell lines (MC-38 and Colon-26-NL-17); yet, this could be an artifact of the platform used for producing recombinant IL-12, such as endotoxin derived from E coli34. Using the B16 model in IL12RB2 KO mice, Airoldi et al. show that IL-12 directly acts on B16 tumor cells such that, after 12 days, tumor growth is reduced by a factor of 2.5 35. This difference in tumor growth was attributed to a release of anti-angiogenic factors by B16 cells following IL-12 stimulation; yet, a specific mechanism for this observation remains unclear.
While this may seem at odds with our observations, this in vivo study doesn’t rule out the possibility that IL-12 may have a pleiotropic effect on tumor cells such that it may both promote survival in the presence of cytotoxic stressors and inhibit angiogenesis. Of note, cellular stress, such as hypoxia, is an important trigger for angiogenesis36. In terms of signal transducers, Goreilik et al. observed the activation of both Akt and STATs upon treating human ovarian cancer cell lines with 40 ng/ml of IL-1237. Alternatively another cytokine receptor type could associate with IL12RB2, which contains binding sites for JAK2 and STAT4 and primarily interacts with the IL12p35 subunit of IL-12. For instance, IL-35 signals through a receptor comprised of the IL12RB2 and the gp130 subunits, but does not bind nor transduce signals in response to IL-12, which binds cytokines IL-6, IL-11, and several other cytokines to activate STATs 24.
In summary, IL-12 provides an important function in coordinating an anti-tumor immune response. In melanoma, overexpression of one component of the IL-12 receptor, IL12RB2, by malignant melanocytes may not only serve to deprive immune cells of this important cytokine, but malignant melanocytes may also use IL-12 as a means of activating cell survival pathways. Supported by prior studies, we present evidence showing that IL-12 increased the dose of imatinib chemotherapy needed to induce cell death in B16 melanoma cells and that IL-12 activated a non-canonical cell survival protein, Akt, in a melanoma cell line, but not in the 2D6 T helper cell line. While subsequent studies will help clarify the mechanistic details to improve the translational impact, these studies highlight the importance of non-canonical pathway activation in cancer.
METHODS
Cell Culture
B16F0 mouse melanoma (ATCC, Manassas, VA), Cloudman S91 mouse melanoma (ATCC), Melan A mouse melanocytes (V. Hearing, National Cancer Institute, Bethesda, MD) and 2D6 cells (M. Grusby, Harvard University, Cambridge, MA) were cultured at 37.5°C with 5% CO2. Complete medium for B16F0 cells consisted of Dulbecco’s Modified Eagle Medium (DMEM, Corning, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS-HI, Hyclone, Logan UT, USA), 1% penicillin-streptomycin (Pen-Strep, Hyclone), and L-Glutamine (2 mM final concentration, Medtech Inc., Herndon, VA, USA). Cloudman S91 complete culture medium was DMEM with 10% FBS-HI and 1% Pen-Strep. Melan A culture medium was DMEM with 10% FBS, 20 mM HCl, 10mM HEPES, 1% Pen-Strep, and fresh 12-O-tetradecanoyl phorbol-13-acetate (TPA; 200 nM) and phenylthiourea (PTU; 200µM). Cells were detached using either Trypsin-Versene or magnesium and calcium-free Dulbecco’s Phosphate-Buffered Saline (DPBS, Corning). 2D6 cells were maintained in RPMI 1640 supplemented with 10% FBS-HI, 1% Pen-Strep, 2 mM L-Glutamine, 50 mM HEPES, 49 mM b-mercaptoethanol (Sigma Chemical, St Louis, MO), 100 mM sodium pyruvate (Fisher Scientific, Pittsburgh, PA, USA), 1% MEM non-essential amino-acid solution (100x Fisher Scientific) and 6.7 ng/ml recombinant mouse IL12p70 (IL-12, eBioscience, San Diego, CA, USA).
Apoptosis and Necrosis Assay
Cells were plated at a density of 1×105 cells/well in a 12 well tissue culture plate (BD Falcon, BD Biosciences, San Jose, CA). Cells were allowed to adhere to the plate for 12 hours prior to the beginning of the experiment, then pre-treated with complete culture media with a final concentration of 40, 100, or 200 ng/mL IL-12. After 18 hours of pretreatment, cells were treated with 0, 5, 10, or 20 µM Imatinib (Cayman Chemical, Ann Arbor Michigan). A 0.1% DMSO vehicle control in complete media was used as a negative control, while cells heated to 50°C for 5-10 minutes served as a positive control cell death. After 24 hours of incubation with imatinib or other treatment, cell viability was assessed using either a flow cytometry-based assay or an ATPlite assay (PerkinElmer, Shelton, CT), which was performed according to the manufacturer’s instructions. For the flow cytometry-based assay, cell media and floating cells were pooled with cells detached with DPBS without magnesium or calcium, centrifuged in 15 mL conical tubes at approximately 250xg, supernatant removed, and cells transferred to a 96 well round bottom plate (BD Falcon) for staining. Cells were then washed with DPBS with magnesium and calcium. After centrifuging at approximately 250xg again and removing the supernatant, cells were then stained with an apoptosis and necrosis staining kit (ABD Serotech, Bio-Rad, Raleigh, NC) according to manufacturer’s instructions. Samples were then analyzed shortly thereafter by flow cytometry on a BD LSRFortessa. Data was analyzed in the BD FACSDiva 3.0 software and in Microsoft Excel. For the ATPlite assay, pre-treated cells were lysed with 50μL/well of mammalian cell lysis solution, followed by 5 min. shaking. The substrate solution was added (50μL/well), shaked for another 5 min., and incubated 10 min. at room temperature protected from light. Finally, the luminescence was measured using a Modulus Microplate Multimode Reader (Turner BioSystems). B16F0 cells cultured in base media alone and exposed to 10% DMSO were included as controls. Statistical significance in the difference in viability upon IL-12 treatment for a given imatinib concentration was assessed using a two-tailed homoscedastic t-Test, where a p-value less than 0.05 was considered significant.
Flow Cytometry
Cells were grown at a density of 1×105 cells/well in a 12-well tissue culture plate if adherent (B16F0, Melan-A, Cloudman S91) or in a 96 well round bottom plate if non-adherent (2D6) (BD Falcon). At each time point, adherent cells were detached from the plate with Trypsin-EDTA or DPBS without calcium/magnesium. For IL12RB1 and IL12RB2 copy number, cells were stained for 30 minutes with Yellow or Violet Live/Dead Fixable Stain (BD Biosciences), fixed with Lyse/Fix Buffer (BD Biosciences), then surface stained for PE-IL12RB1 (BD Biosciences) and AlexaFluor 488-IL12RB2 (R&D Systems, Minneapolis, MN) antibodies separately. Counting beads were also stained with IL12RB1 or IL12RB2 antibody within the same experiment and used to assess receptor copy number. Fluorescent spillover was assessed using single stain controls for IL12RB1 and IL12RB2 antibodies and Live/Dead stains. To assay intracellular activity of STAT4 and Akt, B16F0 and 2D6 cells were starved of FBS and IL-12p70 for 12-24 hours after plating and then treated for 1hr or 3hr with 100 ng/mL IL-12p70 or vehicle. Positive control cells were treated with 10% FBS and 40 ng/mL IL-12p70. Cells were then stained using Violet Fixable Live/Dead (Invitrogen) to assess cell viability, and then fixed (BD Biosciences Lyse/Fix), permeabilized with Perm Buffer III (BD Biosciences), blocked with Mouse IgG (1:100, concentration, Jackson Labs, West Grove, PA), and intracellular stained with AF647-pAKT and AF488-pSTAT4 (BD Biosciences). Cells were stored at 4°C in PBSaz before analyzing via flow cytometry. Cells were analyzed using the BD LSRFortessa flow cytometry analyzer, FCSExpress, and BD FACSDiva 3.0 software. Following acquiring 20,000 events on average, forward and side scatter were used to gate cell populations. Live-Dead staining was used as a gate for live cells.
CRISPR/Cas9 Constructs, Transfection and Cloning Validation
Two vectors were prepared by GenScript (Piscataway, NJ) with puromycin resistance genes and the flanking guide RNA (gRNA) sequences TCCGCATACGTTCACGTTCT and AGAATTTCCAAGATCGTTGA and transfected into wild-type B16F0 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). Transfection was confirmed via microscopy visually using a green fluorescent protein control. Derivatives of the B16F0 cell line were obtained from single clones isolated using puromycin selection and single cell plating. Approximately 10-25% of clones were estimated at having some level of IL12RB2 knockout, which was confirmed by flow cytometry with PE-IL12RB12 (R&D Systems).
Western Blot
B16 and 2D6 cells were grown in complete media at a density of 5×105 cells/well in 6 well plates 24 hours before the beginning of the experiment. At 12 hours prior, cells were starved of FBS and IL-12. One hour before start of experiment, cells were treated with control solution, PI3K inhibitor LY294002 (50 µM, Cell Signaling Technology, Danvers, MA), or with Akt inhibitor MK-2206 (2.4 ug/mL or 5 µMol/L, Cayman Chemical, Ann Arbor, MI). Cells were then treated with 100 ng/mL IL-12 at t=0 or stimulated with FBS as a positive control. After 30 minutes or 3 hours, cells were collected for cell lysates for western blot analysis for total Akt (1:1000, Cell Signaling Technology), pAkt (1:1000, Cell Signaling Technology), and β-actin (1:1000, Sigma Aldrich, St. Louis, MO). The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM NaVO4, 10 mM NaF, and protease inhibitors), and the protein concentration in the lysates was determined by a spectrophotometer. Equal amounts of the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and the immunoblotting was performed with use of the primary antibodies listed above and secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Proteins were visualized by chemiluminescence.
Data Analysis and Statistics
The impact of IL-12 on decreasing the cytotoxic efficacy of imatinib was inferred from the data using a mathematical model: where CIm is the concentration of imatinib, CIL12 is the concentration of IL-12, EC50 is the effective concentration required to elicit a 50% drop in viability, Kd is the equilibrium binding constant of IL-12 and set equal to 40 ng/mL, and α is a parameter that quantifies the impact of IL-12 in shifting the EC50 of imatinib. As described previously19, a Markov Chain Monte Carlo approach based on a Metropolis-Hasting algorithm was used to obtain converged Markov Chains that represent samples from the distributions in the model parameters that were consistent with the data, that is posterior distributions in the model parameters. The posterior distribution in parameter α was used to infer whether the effect was greater than zero and significant. A p-value (P(α < 0 |M, Y)) of less 0.05 was considered as significant, that is the data support that IL-12 provides a rescue effect to imatinib. The significance associated with differences in STAT4 and Akt phosphorylation was assessed using a two-tailed homoscedastic t-Test in GraphPad Prism 7, where a difference with a p-value of less than 0.05 was considered significant and not explained by random noise.
Author contributions
C.B. and D.K. wrote the manuscript. O.M., W.D., P.W., and Y.R. contributed to the methods and assisted with preparation of figures. All authors reviewed the manuscript.
Competing financial interests
Christina N. Byrne-Hoffman, Wentao Deng, Owen McGrath, Peng Wang, Yon Rojanasakul, and David J. Klinke declare that they have no conflict of interest or competing financial interests.
Acknowledgements
The authors would like to acknowledge Cassidy Bland and Adam Palmer for their assistance in manuscript preparation and Vanessa Cuppett for technical assistance. Sources of funding that contributed to this work include NIH/NCI R01CA193473, NSF 1644932, WVU Flow Cytometry Core funding: MBRCC CoBRE grant GM103488/RR032138, ARIA S10 grant RR020866, Fortessa S10 grant OD016165, and WV InBRE grant GM103434.