Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-β1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.


Introduction
Tumor-associated macrophages (TAMs), mainly derived from monocytes, abundantly infiltrate solid tumors. They promote tumorigenesis, cancer metastasis, and the acquisition of anti-cancer drug resistance [1]. The localization of TAMs in the tumor microenvironment (TME) correlates with poor clinical outcomes in solid tumors [1]. Most TAMs have M 2 -like characteristics in the TME (up to 50% of the tumor mass in solid tumors) and secrete proinflammatory and anti-inflammatory cytokines/chemokines (i.e., IL-8/CXCL8 and IL-10) and angiogenic growth factors (i.e., transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF)) during tumor progression [2]. IL-10 promotes tumor immune surveillance escape and IL-8 is a key mediator of angiogenesis, tumorigenicity, and metastasis. Macrophages differentiated from human monocytic leukemia THP-1 cells are used as human in vitro models to study M 1 -and M 2 -polarized macrophages [3]. The manipulation of TAM functions is a useful clinical therapeutic approach to prevent tumor metastasis [4].
The intermediate-conductance Ca 2+ -activated K + channel, K Ca 3.1, decreases intracellular K + concentrations ([K + ] i ) by facilitating K + efflux, and the resulting membrane hyperpolarization increases the driving force for Ca 2+ influx, leading to a sustained increase in intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in immune cells such as macrophages. K Ca 3.1-mediated [Ca 2+ ] i increases are essential for proliferation, differentiation, apoptosis, and gene expression in immune cells and represent attractive therapeutic targets for autoimmune and inflammatory diseases [5,6]. K Ca 3.1 indirectly regulates the production of pro-and anti-inflammatory cytokines/chemokines and macrophage polarization [7,8].
Hypoxic TME-induced death in tumor cells induces an isotonic elevation in extracellular K + concentrations ([K + ] e ) of up to approximately 50 mM, and elevated [K + ] e within tumor interstitial fluid contributes to immunosuppression by inhibiting the functions of cytotoxic CD8 + T cells [9]. K + -driven cancer immunotherapy with a focus on K + homeostasis is a potential therapeutic strategy for cancer [9,10]; however, the mechanisms underlying intracellular K + -driven cytokine/chemokine regulation in tumor-infiltrating immunosuppressive cells such as TAMs have not yet been elucidated.
IL-8 plays a critical role in (1) the recruitment and accumulation of TAMs and the other immunosuppressive cells, such as regulatory T (T reg ) cells, into the TME [2,11] and (2) neovascularization/tumor angiogenesis, accelerating tumor proliferation and metastasis in the TME [12]. Increased numbers of IL-8-expressing TAMs at high levels have been detected in higher clinical stage tumors and were associated with an increased risk of a poor prognosis in cancer patients [13,14]. Bidirectional communication between M 2 macrophages and tumor cells pathologically promotes M 2 macrophages to secrete IL-8 into the TME. Like IL-8, the immunosuppressive cytokine IL-10 is abundantly expressed in TAMs, promoting cancer cell proliferation and metastasis [15]. Previous studies demonstrated that the transcription of IL-8 and IL-10 was critically regulated by the ERK (extracellular signalregulated kinase) and JNK (c-Jun N-terminal kinase) signaling pathways in epithelial cells, monocytes, and fibroblasts [16][17][18]. Furthermore, several transcription factors (i.e., CREB, AP-1, and/or NF-κB) that bind to the promoters of the IL-8 and IL-10 genes have been identified [19,20].
We examined the effects of the selective K Ca 3.1 activator, SKA-121, on the expression and secretion of IL-8 and IL-10 in THP-1-derived M 2 macrophages. We also investigated the effects of SKA-121 on high K + -induced increases in IL-10/IL-8 expression and secretion in THP-1-derived M 2 macrophages under in vitro experimental conditions mimicking the TME of solid tumors. The obtained results provide emerging mechanistic insights into the therapeutic potential of K Ca 3.1 activators in cancer immunotherapy.

In Vitro Differentiation of Human Monocytic THP-1 Cells into M2-like Macrophages
We initially confirmed the increased transcriptional expression of M 2 macrophage markers in THP-1-derived M 2 -like macrophages. As reported by Javasingam et al. (2020) [21], the expression levels of CD163 and Arg1, which are widely used as markers for human M 2 macrophages, were increased by the IL-4/IL-13-induced differentiation of M 0 macrophages into the M 2 phenotype ( Figure 1A,B). The expression levels of the anti-inflammatory cytokine, IL-10, the pro-inflammatory chemokine, IL-8, and the angiogenic growth factor, VEGF-A, were higher in THP-1-derived M 2 macrophages ('M 2 ') ( Figure 1C,D,E) than in native, undifferentiated THP-1 ('native') and PMA-differentiated M 0 macrophages ('M 0 ') (n = 4 for each, p < 0.01). The C-C motif chemokine ligands, CCL17, CCL18, and CCL22, which are secreted from activated TAMs, are associated with the enlargement of tumor size and the promotion of tumor metastasis, and the recruitment of T reg cells [22]. Among these CCLs, CCL22 transcripts were highly expressed in THP-1-derived M 2 macrophages ( Figure 1F); however, the expression levels of the other CCLs were less than 0.001 in arbitrary units normalized by β-actin (ACTB) expression. TGF-β1 transcripts were highly expressed in both THP-derived 'M 0 ' and 'M 2 ' macrophages ( Figure 1G).

Functional Expression of K Ca 3.1 in THP-1-Derived M 2 Macrophages
To perform functional analyses of K Ca 3.1 in THP-1-derived M 2 macrophages, we measured selective K Ca 3.1 activator (1 µM SKA-121)-induced hyperpolarizing responses and increases in [Ca 2+ ] i by simultaneously monitoring DiBAC 4 (3) and Fura 2 signals, and representative time-course plots of the three different cells are shown in Figure 2A,B. In the presence of SKA-121, the selective K Ca 3.1 blocker, TRAM-34 (1 µM), almost completely suppressed them (Figure 2A,B). SKA-121-induced hyperpolarization responses positively correlated with increase in [Ca 2+ ] i (correlation coefficient, R = 0.81, n = 14). The application of TRAM-34 alone did not elicit any significant changes in the membrane potential in THP-1-derived M 2 macrophages, compared with the vehicle control ( Figure S1A). Additionally, SKA-121-induced [Ca 2+ ] i increases almost disappeared with the removal of extracellular Ca 2+ ( Figure S1B). Of the several candidates of Ca 2+ -permeable channels in macrophages [5,[23][24][25], the expression levels of the Orai1, TRPM2, and TRPM7 transcripts were high ( Figure S1C).
To examine whether K Ca 3.1 expression levels are modified during the differentiation of M 0 into M 2 macrophages, the expression levels of K Ca 3.1 in THP-1-derived M 0 and M 2 macrophages were measured using real-time PCR and Western blot assays. Those K Ca 3.1 transcripts and proteins were almost the same between both groups (n = 4 for each, p > 0.05) ( Figure 2C-E). Correspondingly, no significant difference in SKA-121-induced hyperpolarizing responses was found between both groups (p > 0.05) ( Figure 2F). In addition, we measured 1 µM SKA-121-activated K Ca 3.1 currents in THP-1-derived M 2 macrophages by whole-cell patch-clamp recordings [26]. Cells were held at −80 mV and 200 msec ramps from −120 to +40 mV were delivered every 10 sec with a rate of 0.8 mV/ms. Figure 3A represents plots of current density versus voltage after the addition of vehicle (black), 1 µM SKA-121 (red), and 1 µM SKA-121 plus 1 µM TRAM-34 (blue). As shown in Figure 3B, SKA-121-sensitive K Ca 3.1current components showed typical voltage independence and reversal potential of −71.9 ± 5.8 mV (n = 9). Outward currents at +40 mV were significantly increased by the addition of 1 µM SKA-121 (n = 9, p < 0.05 vs. vehicle control), and SKA-121-activated current components were reduced by the application of 1 µM TRAM-34 (n = 9, p < 0.01 vs. SKA-121-treated group) ( Figure 3C). To examine whether KCa3.1 expression levels are modified during the differenti of M0 into M2 macrophages, the expression levels of KCa3.1 in THP-1-derived M0 an macrophages were measured using real-time PCR and Western blot assays. Those K transcripts and proteins were almost the same between both groups (n = 4 for each 0.05) ( Figure 2C-E). Correspondingly, no significant difference in SKA-121-induced perpolarizing responses was found between both groups (p > 0.05) ( Figure 2F). In a

Downregulation of IL-10 and IL-8 by the Treatment with SKA-121 in THP-1-Derived M2 Macrophages
We previously reported that the activation of KCa3.1 with SKA-31 suppressed IL-10 expression and production in human T-cell lymphoma HuT-78 cells [27]. In this study, we examined the effects of treatment with SKA-121 (1 μM) for 24 h on the expression of IL-10, IL-8, VEGF-A, and TGF-β1 in THP-1-derived M2 macrophages ( Figure 4). The expression levels of IL-10 and IL-8 transcripts were significantly suppressed by the SKA-121 treatment (n = 4 for each, p < 0.01), and simultaneous treatment with the KCa3.1 blocker, TRAM-34 (10 μM), reversed this downregulation ( Figure 4A,B). TRAM-34 alone did not elicit significant changes in the transcription of IL-10 and IL-8 ( Figure S1D,E). On the other hand, the expression of VEGF-A and TGF-β1 was not affected by the SKA-121 treatment (n = 4 for each, p > 0.05) ( Figure 4C,D). Consistent with the results shown in Figure 4A,B, the secretion of IL-10 and IL-8 was decreased by the SKA-121 treatment (n = 4 for each, p < 0.01) ( Figure 4E,F). These suggest that the KCa3.1 activator suppressed the immunosuppressive and pro-tumorigenic functions of TAMs by repressing the transcription of IL-10 and IL-8.

Downregulation of IL-10 and IL-8 by the Treatment with SKA-121 in THP-1-Derived M 2 Macrophages
We previously reported that the activation of K Ca 3.1 with SKA-31 suppressed IL-10 expression and production in human T-cell lymphoma HuT-78 cells [27]. In this study, we examined the effects of treatment with SKA-121 (1 µM) for 24 h on the expression of IL-10, IL-8, VEGF-A, and TGF-β1 in THP-1-derived M 2 macrophages ( Figure 4). The expression levels of IL-10 and IL-8 transcripts were significantly suppressed by the SKA-121 treatment (n = 4 for each, p < 0.01), and simultaneous treatment with the K Ca 3.1 blocker, TRAM-34 (10 µM), reversed this downregulation ( Figure 4A,B). TRAM-34 alone did not elicit significant changes in the transcription of IL-10 and IL-8 ( Figure S1D,E). On the other hand, the expression of VEGF-A and TGF-β1 was not affected by the SKA-121 treatment (n = 4 for each, p > 0.05) ( Figure 4C,D). Consistent with the results shown in Figure 4A,B, the secretion of IL-10 and IL-8 was decreased by the SKA-121 treatment (n = 4 for each, p < 0.01) ( Figure 4E,F). These suggest that the K Ca 3.1 activator suppressed the immunosuppressive and pro-tumorigenic functions of TAMs by repressing the transcription of IL-10 and IL-8.

Reversal Effects of Treatment with SKA-121 on High K + Level-Induced Increases in IL-10 and IL-8 in THP-1-Derived M2 Macrophages
In a hypoxic TME, K + released from necrotic cancer cells accumulates in the extracel lular compartments [9]. An in vivo K + imaging analysis by Tan et al. (2020) showed tha K + levels were elevated in the TME, with an average concentration of approximately 2 mM [28]. The concentration of K + was 5 mM in the RPMI 1640 medium used in this study Media with high K + (final concentration: 25 mM) were prepared by KCl supplementation Real-time PCR and ELISA assays showed the increased transcriptional expression (n = for each, p < 0.01) ( Figure 5A,B) and secretion (n = 4 for each, p < 0.01) ( Figure 5C,D) of IL 10 and IL-8 in THP-1-derived M2 macrophages exposed to high [K + ]e (25 mM). Supple mentation with 20 mM NaCl instead of 20 mM KCl did not affect their expression (n = for each, p > 0.05); however, 10 mM K2HPO4 showed a similar effect to 20 mM KCl sup plementation (n = 4 for each, p < 0.01) ( Figure S2A,B).
We then investigated the effects of a 24 h treatment with SKA-121 on high [K + ]e-in duced increases in IL-8 and IL-10 in THP-1-derived M2 macrophages. High [K + ]e-induced increases in the expression ( Figure 5A,B) and secretion ( Figure 5C,D) of IL-8 and IL-1 were not affected by simultaneous treatment with 1 μM SKA-121 (n = 4 for each, p > 0.05) however, was significantly reduced by simultaneous treatment with 10 μM SKA-121 (n  In a hypoxic TME, K + released from necrotic cancer cells accumulates in the extracellular compartments [9]. An in vivo K + imaging analysis by Tan et al. (2020) showed that K + levels were elevated in the TME, with an average concentration of approximately 29 mM [28]. The concentration of K + was 5 mM in the RPMI 1640 medium used in this study. Media with high K + (final concentration: 25 mM) were prepared by KCl supplementation. Real-time PCR and ELISA assays showed the increased transcriptional expression (n = 4 for each, p < 0.01) ( Figure 5A,B) and secretion (n = 4 for each, p < 0.01) ( Figure 5C,D) of IL-10 and IL-8 in THP-1-derived M 2 macrophages exposed to high [K + ] e (25 mM). Supplementation with 20 mM NaCl instead of 20 mM KCl did not affect their expression (n = 4 for each, p > 0.05); however, 10 mM K 2 HPO 4 showed a similar effect to 20 mM KCl supplementation (n = 4 for each, p < 0.01) ( Figure S2A,B).
showing P-ERK1/2, total ERK1/2 (ERK1/2) (A,B), P-JNK, and total JNK (JNK) (C,D) in 1 μM SKA-121-treated (A,C) and high [K + ]e-exposed (B, D) THP-1-derived M2 macrophages. Specific band signals were observed at 42 (P-ERK2), 42 (ERK2), 43/50 (P-JNK), and 43/50 (JNK) kDa. E-H: Summarized results of the relative protein expression of P-ERK2/ERK2 (E,F) and P-JNK/JNK (G,H) in 1 μM SKA-121-treated (E,G) and high [K + ]e-exposed (F,H) THP-1-derived M2 macrophages. After compensation with the optical density of the ACTB signal (43 kDa), the expression level in the vehicle control or 5 mM K + is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control and 5 mM K + .  Several transcription factors (i.e., CREB, AP-1, and/or NF-κB) that bind to the promoters of the IL-8 and IL-10 genes have been identified to date; however, the ERK1/2-CREB pathway is a candidate that is commonly associated with the transcription of IL-8 and IL-10 [19,20]. We, therefore, examined the effects of the potent and selective CREB inhibitor, 666-15 (1 μM), on the expression levels of IL-10 and IL-8 transcripts in THP-1derived M2 macrophages (Figure 9). The expression and secretion of IL-10 and IL-8 were significantly decreased by the 24 h treatment with 666-15 ( Figure 9A-D). The ratio of P-CREB to total CREB in THP-1-derived M2 macrophages was decreased by the treatment with 1 μM SKA-121 (n = 4, p < 0.01) ( Figure 9E,G), but was increased by the exposure to high [K + ]e (25 mM) (n = 4, p < 0.01) ( Figure 9F,H). Therefore, ERK-CREB and JNK-c-Jun cascades may be involved in KCa3.1-regulated IL-10 and IL-8 in TAMs.  SKA-121-treated (E) and high [K + ] e -exposed (F) THP-1-derived M 2 macrophages. Specific band signals for P-CREB and CREB were observed at approximately 40 kDa. G, H: Summarized results of the relative protein expression of P-CREB/CREB in 1 µM SKA-121-treated (G) and high [K + ] eexposed (H) THP-1-derived M 2 macrophages. After compensation with the optical density of the ACTB signal, the expression level in the vehicle control or 5 mM K + is expressed as 1.0 (n = 4 for each). **: p < 0.01 vs. the vehicle control or 5 mM K + . Tumor-conditioned media are associated with the generation and infiltration of TAMs [33]. We examined the effects of exposure to human prostate cancer PC-3 spheroidcultured media for 24 h on the expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 in THP-1-derived M 2 macrophages. Media were prepared by the 3D spheroid formation of PC-3 cells for 7 days using ultra-low attachment surface coating cultureware. As shown in Figure 10A,B (left columns), IL-10 and IL-8 expression levels in THP-1-derived M 2 macrophages were increased by approximately 3-fold following the exposure to PC-3 media. Simultaneous treatment with 1 or 10 µM SKA-121 resulted in significant decreases in the expression levels of both transcripts (n = 4 for each, p < 0.01) ( Figure 10A,B) without changes in those of VEGF-A or TGF-β1 (n = 4 for each, p > 0.05) ( Figure 10C,D). Furthermore, elevated IL-10 and IL-8 secretion levels by approximately 1.5-fold following the exposure to PC-3 media were significantly decreased by simultaneous treatment with SKA-121 (n = 4, p < 0.01) ( Figure 10E,F). The 24 h exposure to PC-3 media did not alter the viability of THP-1-derived M 2 macrophages (n = 5 for each, p > 0.05) ( Figure S2E). Tumor-conditioned media are associated with the generation and infiltration of TAMs [33]. We examined the effects of exposure to human prostate cancer PC-3 spheroidcultured media for 24 h on the expression levels of IL-10, IL-8, VEGF-A, and TGF-β1 in THP-1-derived M2 macrophages. Media were prepared by the 3D spheroid formation of PC-3 cells for 7 days using ultra-low attachment surface coating cultureware. As shown in Figure 10A,B (left columns), IL-10 and IL-8 expression levels in THP-1-derived M2 macrophages were increased by approximately 3-fold following the exposure to PC-3 media. Simultaneous treatment with 1 or 10 μM SKA-121 resulted in significant decreases in the expression levels of both transcripts (n = 4 for each, p < 0.01) ( Figure 10A,B) without changes in those of VEGF-A or TGF-β1 (n = 4 for each, p > 0.05) ( Figure 10C,D). Furthermore, elevated IL-10 and IL-8 secretion levels by approximately 1.5-fold following the exposure to PC-3 media were significantly decreased by simultaneous treatment with SKA-121 (n = 4, p < 0.01) ( Figure 10E,F). The 24 h exposure to PC-3 media did not alter the viability of THP-1-derived M2 macrophages (n = 5 for each, p > 0.05) ( Figure S2E).

Discussion
The intermediate-conductance Ca 2+ -activated K + channel, K Ca 3.1, is a key regulator of the polarization and tumor infiltration of TAMs. In the TME, tumor-infiltrating, M 2differentiated TAMs promote escape from cancer immune surveillance, thereby enhancing invasive and metastatic potentials and angiogenesis in cancer [1,2]. The various stresses preventing antitumor immune responses in the TME, such as hypoxia, high lactate levels, and high [K + ] e levels, affect TAM functions [34]. Accumulated K + in the TME overcomes antitumor immune surveillance by inhibiting cytotoxic lymphocyte functions [9,10]. K + channels as an 'ionic checkpoint' are the potential targets for cancer immunotherapy because activators of these channels, specifically expressing immune cells, may overcome the evasion of immune surveillance. In this study, we demonstrated that selective K Ca 3.1 activators can potentially treat increased TAM-mediated cancer metastatic potential and angiogenesis in the TME using THP-1-derived M 2 macrophages. The main results of this study are as follows: (1) pro-tumorigenic IL-8 and IL-10 expression and secretion in THP-1-derived M 2 macrophages were significantly decreased by treatment with a K Ca 3.1 activator (Figure 4), (2) high [K + ] e level-induced increases in IL-8 and IL-10 levels were reversed by treatment with a K Ca 3.1 activator ( Figure 5), (3) the transcription of IL-10 and IL-8 was promoted through the ERK-CREB and/or JNK-c-Jun cascades with increases in their phosphorylation levels ( Figures 6-9), and (4) prostate cancer cell-cultured mediuminduced increases in IL-10 and IL-8 levels were reversed by treatment with a K Ca 3.1 activator ( Figure 10). Alternatively, the treatment with a K Ca 3.1 activator did not affect the expression levels of VEGF-A or TGF-β1, which promote the immunosuppressive activity of T reg cells and their recruitment to the TME ( Figure 4C,D). We previously reported K Ca 3.1 activatorinduced reductions in the expression and secretion of IL-10 in IL-10-producing T-cell lymphoma, HuT-78 cells [27]. In contrast to native T reg cells naturally present in the immune system, the NF-κB signaling pathway is continuously active in HuT-78 cells, and IL-10 transcription is regulated through the TGF-β-mediated Smad2/3 signaling pathway [27]. However, as shown in Figure 6A,D, the TGF-β signaling pathway did not contribute to the transcription of IL-10 or IL-8 in THP-1-derived M 2 macrophage. No changes in the expression levels of IL-10 and IL-8 were found by the treatment with 10 ng/mL TGF-β1 for 24 h [0.0342 ± 0.0002 and 0.0336 ± 0.0009 (in arbitrary units) in vehicle-and TGF-β-treated groups, respectively, n = 4 for each, p > 0.05].
TAMs play a pivotal role in impairing the functions of antitumor, cytotoxic CD8 + T cells, and natural killer cells by releasing the anti-inflammatory cytokine IL-10 [1,4]. Multiple distinct signaling pathways were found to be responsible for the transcriptional regulation of IL-10 in immune and non-immune cells [35,36]. The IL-10 promoter was activated by CREB through the ERK1/2 signaling pathway in native THP-1 cells [37]. Correspondingly, this study showed the transcriptional repression of IL-10 in THP-1-derived M 2 macrophages by a single treatment with an ERK1/2 inhibitor or a CREB inhibitor (Figures 6A and 9A) and the reduced phosphorylation levels of ERK1/2 and CREB in THP-1-derived M 2 macrophages treated with a K Ca 3.1 activator ( Figures 7A,E and 9E,G). We recently reported that the inhibition of K Ca 3.1 upregulated the expression of IL-10 through the JNK/c-Jun cascade in T reg cells [38]. In this study, the JNK/c-Jun cascade was involved in the K Ca 3.1 activation-induced down-regulation of IL-10 ( Figures 7C,G and 8A,C). Collectively, the present results represent the first time that K Ca 3.1 activators are a possible target for overcoming IL-10-mediated escape from tumor immune surveillance in the TME, through their inhibition of the IL-10 in TAMs via the ERK-CREB and JNK-c-Jun cascades.
Similar to IL-10, IL-8 secreted from TAMs plays a critical role in promoting tumor immunosuppression [39]. IL-8 has also been shown to play a role in the establishment of cancer stemness, chemoresistance, and tumor neovascularization in the TME [39,40]. In this study, the K Ca 3.1 activator suppressed the production of IL-8 by repressing its transcription in THP-1-derived M 2 macrophages ( Figure 4B,F). Importantly, the K Ca 3.1 activator also suppressed high [K + ] e exposure-induced IL-8 overexpression ( Figure 5B,D). These results suggest that the down-regulation of IL-8 by the K Ca 3.1 activator in TAMs not only prevented cancer migration and metastasis but also reduced microvessel density in cancer. However, further studies will be needed to prove this hypothesis in the future. Additionally, IL-8 transcription was distinctly repressed by ERK and JNK inhibitors in THP-1-derived M 2 macrophages ( Figure 6D). Correspondingly, IL-8 transcription was promoted by the ERK and JNK signaling pathways with their phosphorylation in several epithelial cells and synovial fibroblasts [18,41]. As shown in Figures 7-9, the treatment with SKA-121 reduced the phosphorylation levels of ERK1/2, JNK, c-Jun, and CREB in THP-1-derived M 2 macrophages, while the exposure to high [K + ] e exerted the opposite effects. These results suggest that the mechanisms underlying the K Ca 3.1 activator-induced down-regulation and high [K + ] e -induced upregulation of IL-8 and IL-10 are mediated via the same signaling pathways.
Angiogenic inhibition therapy is a promising strategy for cancer. TAMs secrete angiogenic factors such as VEGF-A and TGF-β1; however, the K Ca 3.1 activator did not alter their expression in this study ( Figure 4C,D). IL-8 cooperates with VEGF-A to promote tumor neovascularization [39]. Therefore, K Ca 3.1 activators may be a novel strategy to terminate/initiate tumor neovascularization by reducing IL-8 secreted from tumorinfiltrating TAMs. Intertumoral IL-8 leads to the recruitment and accumulation of TAMs in the TME [39,40,42]. Bilaterally, IL-8 secreted from TAMs contributes to the epithelialmesenchymal transition (EMT) process with cancer stem cell properties such as chemoresistance in the TME via a paracrine pathway [42,43]. In this study, IL-8 and IL-10 mRNA expression and secretion levels in THP-1-derived M 2 macrophages increased in the culture media of prostate cancer PC-3 cells ( Figure 10A,B,E,F). Bidirectional crosstalk between TAMs and cancer cells promotes TAMs to secrete IL-8 and IL-10 into the TME, resulting in the tumorigenesis, metastasis, and angiogenesis of solid cancers. A recent study indicated that exosomal microRNA from cancer cells promoted the M 2 polarization of TAMs [44]. miR-21 mimicked increases in IL-10 mRNA levels and a miR-21 inhibitor prevented this alternation [44]. Indeed, several studies have shown that miR-21 is highly expressed in PC-3 cells and is upregulated by the spheroid formation and EMT of PC-3 cells [45,46]. Therefore, the PC-3-medium-induced upregulation of IL-10 and IL-8 in TAMs may be attributed to microRNA(s) in cancer cells, promoting EMT in the TME. Feng et al. (2018) reported that the activation of Nrf2 upregulated the expression of M 2 markers (CD163 and Arg1) [47]. Therefore, the upregulation of IL-10 and IL-8 following the M 2 differentiation of THP-1 cells may be due to the activation of Nrf2. The expression levels of Nrf2 were high in THP-1-derived M 2 macrophages ( Figure S3A); correspondingly, the transcriptional and secretory levels of IL-10 and IL-8 were significantly decreased by the 24 h treatment with ML385 (n = 4 for each, p < 0.01) ( Figure S3B-E). These results suggest that Nrf2 is involved in the upregulation of IL-10 and IL-8 but not in the SKA-121-induced down-regulation of them in THP-1-derived M 2 macrophages ( Figure S3F). CCL22, which is secreted from TAMs, is responsible for the recruitment of T reg cells to tumors, and the prevention of cytotoxic CD8 + T cell activation. No significant changes were observed in the expression levels of CCL22 transcripts following the 24 h treatment with SKA-121 ( Figure S3G).

Cell Culture and Differentiation into M 2 Macrophages
The differentiation of the human monocytic leukemia cell line, THP-1, into M 0 macrophages was induced by treatment with PMA treatment (100 ng/mL) for 24 h. After removal of the medium, cells were incubated with Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with IL-4 and IL-13 (20 ng/mL each) for 72 h to induce the polarization of M 2 macrophages. Drug applications were performed 48 h after the incubation with IL-4/IL-13 [3].

Measurements of the Membrane Potential and [Ca 2+ ] i
The membrane potential was measured using the fluorescent voltage-sensitive dye, DiBAC 4 (3), as previously reported [27]. In membrane potential imaging, cells loaded with DiBAC 4 (3) were illuminated at a wavelength of 490 nm. [Ca 2+ ] i was measured using the fluorescent Ca 2+ indicator dye, Fura 2-AM, and cells loaded with Fura 2 were alternatively illuminated at wavelengths of 340 and 380 nm. Before the fluorescence measurements, cells were incubated in the extracellular solution containing 100 nM DiBAC 4 (3) and 10 µM Fura 2-AM for 20 min. The extracellular solution was composed of (in mM) the following: 137 NaCl, 5.9 KCl, 2.2 CaCl 2 , 1.2 MgCl 2 , 14 glucose, and 10 HEPES [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] (pH 7.4). A Ca 2+ -free solution was prepared by replacing 2.2 mM CaCl 2 with 1 mM EGTA [ethylene glycol bis(beta-aminoethylether)-N,N,N,N-tetraacetic acid]. Fluorescence images were recorded using the ORCA-Flash2.8 digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Data collection and analyses were performed using an HCImage system (Hamamatsu Photonics). Images were obtained every 5 sec, and fluorescent intensity values were assessed using the average for 1 min (12 images). The fluorescent intensity of Fura 2 was expressed as measured 340/380 nm fluorescence ratios after background subtraction. Drug solutions (SKA-121 alone and SKA-121 plus TRAM-34) were applied by operating the three-way stopcock.

Whole-Cell Patch-Clamp Recording
Whole-cell patch-clamp recording experiments were performed as reported previously [26]. A whole-cell patch-clamp was applied to single THP-1-derived M 2 macrophages using an Axopatch 200B patch-clamp amplifier under the control of pClamp 11 software (Molecular Devices, San Jose, CA, USA) (23 ± 1 • C). Data acquisition and analysis of to the manufacturer's protocols. Standard curves were plotted using a series of cytokine/chemokine concentrations.

Statistical Analysis
Statistical analyses were performed with the statistical software XLSTAT (version 2013.1). The unpaired/paired Student's t-test with Welch's correction or one-way ANOVA with Tukey's test was used to assess the significance of differences between two groups and among multiple groups. Results with a p-value < 0.05 were considered to be significant. Data are shown as means ± standard error.

Conclusions
In this study, elevated [K + ] e by necrotic cancer and cancer-infiltrating cells in the TME increased the expression and production of IL-10 and IL-8 in THP-1-derived M 2 macrophages. We showed the effectiveness of K Ca 3.1 activators for TME-mediated TAM dysregulation, generating the overexpression of IL-10 and IL-8. Several potent and selective K Ca 3.1 activators, which positively modulate channel-gating, have been developed [48]; however, their clinical and therapeutic importance remains unclear. The present results provide novel insights into recent advances in TAM targeting therapies as antitumor strategies and indicate that potent and selective K Ca 3.1 activators need to be considered for future therapeutic applications in cancer immunotherapy. Further studies under the TME-mimicking conditions, such as acidic pH, hypoxia, high lactate, low glucose, and so on will be needed to evaluate the clinical applications and limitations K Ca 3.1 activators in cancer immunotherapy.