Abstract
Background/Aim: In a previous study, we have demonstrated heightened Pyra–Metho–Carnil (PMC) efficacy in nude mice with intact innate immunity that lack T and B cells. This has prompted hypothesizing that PMC may target macrophages that promote cancer growth. In this study, we conducted co-culture experiments with macrophages derived from THP-1 human monocyte cell lines and spheroids representing normal and cancer microenvironments. We then performed RNA sequencing and flow cytometry analysis to elucidate the mechanisms by which PMC affects macrophage differentiation and maturation. Materials and Methods: THP-1 cells were differentiated by phorbol 12-myristate 13-acetate (PMA) and matured by PMA and lipopolysaccharide (LPS) either with or without PMC. Co-cultures were performed using stimulated THP-1 cells and HKe3-wild-type KRAS or HKe3-mutant (mt) KRAS spheroids. We then performed RNA-seq analysis of THP-1 cells stimulated by PMA (either with or without PMC) and flow cytometry analysis of mice peripheral blood obtained after PMC administration. Results: THP-1 cells matured by PMA and LPS specifically increased the area of HKe3-mtKRAS cancer spheroids and the addition of PMC to THP-1 cells was found to inhibit cancer spheroid growth. RNA-seq data suggested that PMC treatment of THP-1 cells stimulated with PMA suppressed cell motility regulatory functions via down-regulation of the NF
B pathway. Furthermore, flow cytometry results showed that PMC treatment suppressed monocyte maturation in B6 mice. Conclusion: The high level of in vivo tumor suppression caused by PMC may be due to inhibition of the differentiation and maturation of tumor-associated macrophages via the NF
B signaling pathway.
In a previous study, we established HKe3 cells from human colorectal cancer HCT116 cells by disrupting mutant (mt) KRAS (G13D), resulting in the presence of wild type KRAS alone (1). Using both HKe3 cells and HCT116 cells in 3D culture, we have elucidated the signaling pathway mediated by mtKRAS in cancer microenvironment (2, 3), however the genetic background of HCT116 and HKe3 cells is not completely same. To solve this problem, we established HKe3-wild type (wt) KRAS (normal model) and HKe3-mtKRAS cells (cancer model) re-expressing wtKRAS and mtKRAS, respectively (1, 4), to facilitate an informative comparison of mtKRAS-mediated signaling in two lines that are isogenic, except for a single KRAS mutation (5). Using this pair of cells, we conducted a screening of natural compounds to selectively and effectively eliminate cancer spheroids (6, 7). As a result, we identified Pyra–Metho–Carnil (PMC) as a compound capable of specifically inhibiting the growth of HKe3-mtKRAS spheroids (8). Notably, PMC exhibited growth-restricting properties against cancer spheroids with various genetic mutations (8), suggesting that it is effective across a variety of different tissue types. Moreover, we also found that PMC suppresses aerobic glycolysis in HKe3-mtKRAS spheroids via down-regulating the hypoxia-inducible factor-1α (HIF-1α) pathway (9), however the precise mechanism by which PMC exerts its effects remains unknown. The anti-tumor effects of PMC were more pronounced compared to other anti-cancer drugs in nude mice with intact innate immunity that lacked functional T and B cells (8). Therefore, we hypothesized that PMC may target mechanisms of cancer evasion that result from macrophage-mediated innate immunity that promotes cancer growth. Therefore, this study aimed to elucidate the mechanism by which PMC regulates macrophage differentiation, maturation, and reprogramming in various microenvironments. To do so, we used THP-1 cells derived from human monocyte-type leukocytes as a model for macrophage differentiation and maturation. THP-1 cells can differentiate and mature into macrophages upon treatment with phorbol 12-myristate 13-acetate (PMA) and they can be further activated by lipopolysaccharide (LPS) stimulation (10). Finally, they can then be reprogrammed by exocrine factors found in the microenvironment (11). Here, we tested which exocrine factors support macrophage reprogramming by co-culturing THP-1 cells with HKe3-wtKRAS spheroids (i.e., a “normal microenvironment” model) or with HKe3-mtKRAS spheroids (i.e., a “cancer microenvironment” model) (4). In addition, we performed RNA sequencing (RNA-seq) analysis and flow cytometry on mice peripheral blood samples to more accurately elucidate the mechanism of action by which PMC affects macrophage differentiation and maturation in normal microenvironments.
Materials and Methods
Compound. PMC (IUPAC Name: 1-{3-[(3,5-dimethylpyrazol-1-yl)methyl]-4-methoxyphenyl}-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole) was synthesized by Namiki Shoji Co., Ltd. (Tokyo, Japan) (8).
Cell culture. Hke3-wtKRAS and HKe3-mtKRAS cultures were established and maintained as per a previously described method (4, 12).
3D floating cell culturing and THP-1 co-culturing. Cells were seeded in 96-well round bottom plates with ultralow attachment surfaces (Product No. 7007; Corning Inc., Corning, NY, USA) for 3D floating (3DF) culture. Cancer cells were cultured for seven days in a CO2 incubator as per a previously described method (4, 8, 12). THP-1 cells were collected and resuspended in RPMI1640 + 10% FBS + 1% penicillin/streptomycin as per a previously described method (13). PMA (10 ng/ml) and/or LPS (100 ng/ml) were then added to the THP-1 cell culture medium and the cells incubated at 37°C for six hours. Subsequently, DMSO or PMC (10 μM) was added to the culture medium, and cells were further incubated at 37°C for an additional 66 h. After this incubation period, THP-1 cells were stained with Calcein-AM (10 μM), and co-culture of spheroids and THP-1 cells was initiated on day 0. Cell images were then taken using a BIOREVO BZ9000 microscope (Keyence, Osaka, Japan), and spheroid area was measured using a BZ Analyzer (Keyence) as per a previously described method (12, 14). The number of mature macrophages was then measured by counting THP-1 cells using the hybrid cell counting function of a BZ Analyzer. Specifically, cells with a high fluorescence intensity (i.e., >600~1,000, with the exact threshold adjusted depending on levels of background fluorescence) and with large size (>100 μm2) were counted on days 3, 7, and 9.
RNA-seq. RNA-seq libraries were prepared using NEBNext rRNA Depletion Kits (Human/Mouse/Rat) (E6310, NEB, Ipswich, MA, USA) and NEBNext Ultra Directional RNA Library Prep Kits for Illumina (E7420, NEB), with all procedures performed as described previously (9).
RNA-seq analysis. To identify genes that were up-regulated in THP-1 cells stimulated by PMA, we compared the expression levels of genes between THP-1 cells treated with PMA and THP-1 cells treated with a dimethyl sulfoxide (DMSO) negative control. Genes with expression levels 1.5 times higher than the control (p<0.05) were identified as significantly up-regulated. In addition, we compared PMA-treated THP-1 cells with PMA- and PMC-treated THP-1 cells to identify genes that were significantly suppressed by PMC (i.e., those with expression levels 0.75 times lower than the control at p<0.05). Overall, all RNA-seq analyses were conducted as previously described (9).
Gene ontology enrichment analysis. Gene ontology enrichment analysis for 151 genes was performed using Metascape.
Flow cytometry. Flow cytometry for peripheral blood samples obtained from B6 mice was conducted under the following conditions. First, we obtained four 5 ml samples from control groups or the PMC-treated group. Next, blood samples were collected and placed in a 10-fold volume of 1×red blood cell lysis buffer (Thermo Fisher Scientific, Waltham MA, USA) at room temperature. Flow cytometry was performed as per a previously described method (12) and the gating strategy used was also performed as per a previously described protocol (15).
Results
Differentiation and maturation of THP-1 cells in a co-culture system with or without PMC. We utilized a 3D co-culture system to assess the impact of PMC on the differentiation and maturation of THP-1 cells from monocytes to macrophages as well as how this process differs in the presence of normal and cancer spheroids. The time course of the co-culture experiment is shown in Figure 1A. Compared to co-cultures with THP-1 cells alone, those treated with PMA and LPS showed an increase in the number of maturated THP-1 cells in both the HKe3-wtKRAS and HKe3-mtKRAS groups (Figure 1B-D). Moreover, the number of THP-1 cells differentiated by PMA were significantly decreased by PMC; this reduction was approximately 37% in the HKe3-wtKRAS group and 60% in the HKe3-mtKRAS group (Figure 1D). Similarly, the number of THP-1 cells matured by PMA and LPS were decreased by PMC by approximately 25% in the HKe3-wtKRAS group and 38% in the HKe3-mtKRAS group (Figure 1D). Taken together, these results suggest that PMC suppresses macrophage differentiation and maturation in our co-culture system. Notably, maturated THP-1 cells treated with both PMA and LPS increased the area of cancer spheroids relative to those treated with THP-1 cells (p=0.018) or with differentiated THP-1 cells exposed to PMA by day 7 (p=0.033) (Figure 1B, C, and E). These results suggest that cancer spheroids altered the phenotypes of matured macrophages to those that provide support for the further growth of these spheroids. Moreover, the addition of THP-1 cells treated by PMC during maturation before co-culturing resulted in reduced spheroid area by day 3 (p=0.001) and day 7 (p=0.048; Figure 1E). Overall, these results suggest that the addition of PMC suppresses macrophage maturation, thereby decreasing the ability of reprogramed THP-1 cells to support tumor growth.
Differentiation and maturation of THP-1 cells in a co-culture system with or without Pyra–metho–carnil (PMC). A) Time course of 3D co-cultures. B and C) Images of HKe3-wtKRAS (B) or HKe3-mtKRAS (C) spheroids on days 3 and 7 with THP-1 cells fluorescently labeled with Calcein-AM (Green). D) The numbers of differentiated and matured macrophages cultured with THP-1 cells. Results are shown for macrophages treated with PMA alone or with PMA and LPS with or without PMC on days 3 and 7. *p<0.05; **p<0.01 (Student’s t-test). he areas of HKe3-wtKRAS or HKe3-mtKRAS spheroids co-cultured with THP-1 cells, either treated with PMA alone or with PMA and LPS with or without PMC as quantified on days 3 and 9. *p<0.05; **p<0.01 (Student’s t-test).
Analyses of genes up-regulated in differentiated THP-1 cells with PMA that were repressed following PMC treatment. Next, to examine the effects of PMC on THP-1 differentiation, we performed RNA-seq using RNA extracted from differentiated THP-1 cells treated with either PMA or DMSO and with or without PMC (Figure 2A). Analysis of RNA-seq data identified 151 genes that may be associated with the PMC-induced suppression of THP-1 differentiation (Table I). Thus, to identify the biological processes affected by PMC administration on THP-1 cells, we performed ontological analysis of 151 genes identified from the RNA-seq data. Further ontological analysis revealed that biological processes such as cell migration and cytokine activity were significantly enriched among those genes that were down-regulated in PMC-treated THP-1 cells relative to the control (Figure 2B). Further in silico analysis of transcription factor targets revealed an abundance of pathways controlled by the NF-kB subunit (RELA: p65) and NF
B1 (p50) (Figure 2C). Collectively, these data suggest that PMC treatment of THP-1 cells decreases the transcriptional levels of genes associated with cell migration and cytokine activity, including those involved in the NF
B pathway as mediated by two main subunits, p65 and p50 (16).
RNA-seq analysis of THP-1 cells during differentiation with or without PMC. A) Methods used to select genes. B) Genes specifically down-regulated by PMC in THP-1 cells. Bar graph of enriched terms across input gene lists. Colors reflect p-values. C) Summary of enrichment analysis for transcription factor targets of genes found to be specifically down-regulated by PMC in THP-1 cells. Enrichment analysis was conducted using text mining of the Transcriptional Regulatory Relationships Unraveled by Sentence-based Text (TRRUST), a database designed for the analysis of gene transcriptional regulatory relationships. Enriched terms are depicted as bar graphs and are color-coded according to p-value.
Genes up-regulated in THP-1 cells differentiated with PMA that were repressed following PMC treatment. Signal ratios calculated from the mean of four independent RNA-seq experiments are shown. Reported ratios include: PMA-treated THP-1 cells/DMSO-treated THP-1 cells, and PMA and PMC-treated THP-1 cells/PMA-treated THP-1 cells. Genes related to cell migration are highlighted in bold, and genes associated with cytokine activity are shown in italics.
Effects of PMC on macrophage differentiation and maturation in vivo. Next, we investigated the effect of PMC on in vivo macrophage differentiation and maturation by examining the distribution of blood cells in the peripheral blood of B6 mice. We sampled four mice from an untreated (control) group and a group intraperitoneally administered with PMC (10 mg/kg) every day for a week. In the control group, the number of large monocytes was higher than that of small monocytes, while in the PMC administration group, the number of large monocytes was lower than that of small monocytes (Figure 3A and B). Specifically, the ratio of large to small monocytes in the PMC group was less than half of that in the control group (Figure 3C), thereby suggesting that PMC inhibits macrophage maturation in vivo.
Effects of Pyra–Metho–Carnil (PMC) on macrophage differentiation and maturation in vivo. A) Flow cytometry dot plots of B6 mouse peripheral blood samples. The vertical axis represents side scatter-area (SSC-A), and the horizontal axis represents forward scatter-area (FSC-A). The upper panel shows the control group, while the lower panel shows a group treated with PMC (10 mg/kg). B) Multi-color flow cytometry dot plots and the distribution of leukocyte subsets from mouse whole blood samples. “a” represents granulocytes, “b” represents small monocytes, “c” represents large monocytes, and “d” represents lymphocytes. C) Ratio of large to small monocytes in the control and PMC-treated groups, respectively. *p<0.05; **p<0.01 (Student’s t-test).
Discussion
THP-1 cell co-culture systems are valuable tools for elucidating the complex interactions between immune cells and the tumor microenvironment (TME) (17-19). In this study, we employed a 3D co-culture system with THP-1 cells and colorectal cancer-derived spheroids to mimic both normal and cancer microenvironments (Figure 1A-C). This system enabled us to observe both macrophage phenotype and cancer spheroid growth in the absence of specific growth factors or cytokines (e.g., INFγ; Figure 1D and E) (11).
Activation of THP-1 cells by PMA is widely used to induce differentiation in macrophage-like cells. As in previous studies, we observed higher numbers of differentiated THP-1 cells following stimulation with PMA (Figure 1D), thereby indicating successful differentiation (10). Notably, matured THP-1 cells treated with PMA and LPS were specifically able to support the growth of HKe3-mtKRAS spheroids (Figure 1E), suggesting that a specific factor from cancer spheroids promotes macrophage reprograming to tumor-associated macrophages (TAMs). In previous studies, mutant KRAS was found to extrinsically trigger the functional reprogramming of TAMs via stabilizing HIF-1α in a colorectal cancer (20). Subsequent RNA-seq analysis suggested that PMC treatment reduced the transcription levels of molecules linked to cell migration and cytokine activity, both of which are controlled by the NF
B pathway (Figure 2B and C). Activation of NF
B in turn induces the expression of various pro-inflammatory genes, including those encoding cytokines and chemokines, and participates in inflammasome regulation (21). In addition, NF
B plays a crucial role in regulating the survival, activation, and differentiation of innate immune cells, and inflammatory T cells (21, 22). TAMs play crucial roles in the TME and exhibit tumor-promoting or -inhibitory functions depending on how they are polarized by specific factors (23). NF
B signaling is known to induce the polarization of non-polarized macrophages toward TAM-like phenotypes, thereby contributing to tumor progression and immune suppression (24). Our results suggest that inhibition of NF
B signaling by PMC may potentially suppress differentiation of THP-1 cells and subsequently affect macrophage reprograming into TAM-like phenotypes (Figure 1E).
Further in vivo studies using mouse models showed that PMC administration altered the distribution of blood cells, especially the ratio of large to small monocytes (Figure 3A and B), suggesting that PMC suppresses macrophage maturation. This observation is consistent with the in vitro observation of the inhibitory effect of PMC on THP-1 cell differentiation and maturation (Figure 1).
Despite these promising results, several limitations should be acknowledged. First, our study focused on the effects of PMC on macrophage differentiation by quantifying the size and fluorescence intensity of colorectal cancer spheroids. Future research using macrophage surface markers should explore its effects on various cancer types and use more complex TME models. In addition, while our results suggest that there is an association between NF
B signaling and the observed effects of PMC, further studies should attempt to identify PMC target molecules in order to fully elucidate the molecular mechanisms responsible.
In conclusion, our study reveals the inhibitory effect of PMC on macrophage differentiation and explores its potential for anticancer therapies. By targeting NF
B signaling, PMC may modulate the TME and enhance anti-tumor immune responses, thereby offering a promising strategy for future use in clinical settings.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI, Grant No. 21K07161 and 22K07221) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and the support of the Fukuoka Foundation for Sound Health Cancer Research Fund. The Authors would like to thank Enago (www.enago.jp) for the English language review.
Footnotes
Authors’ Contributions
T.K., K.Y., Y.H., T.M., M.A., M.N., and T.T. performed experiments, analyzed data, and wrote the first draft of the manuscript. S.I., G.M., and H.M. participated in study design, data collection, and analysis. S.K., Y.I., F.H. and S.S. conceived the idea, designed the study, interpreted data, provided important intellectual content, and obtained final approval for manuscript submission.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received May 21, 2024.
- Revision received June 12, 2024.
- Accepted June 13, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
