TGF-β type I receptor inhibitor suppresses polarization of tumor-associated macrophages to the M2 phenotype and exerts antitumor effects on colorectal cancer

doi:10.21203/rs.3.rs-2815318/v1

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TGF-β type I receptor inhibitor suppresses polarization of tumor-associated macrophages to the M2 phenotype and exerts antitumor effects on colorectal cancer

https://doi.org/10.21203/rs.3.rs-2815318/v1

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Background:

Macrophages can be classified into two main types based on their functions: pro-inflammatory M1-like and anti-inflammatory M2-like. Tumor-associated macrophages (TAMs) are macrophages that infiltrate tumor tissues and many TAM polarized to the M2 phenotype. M2-TAMs promote tumor progression through various mechanisms. Transforming growth factor (TGF)-β, which is abundant in tumor tissues and exacerbates advanced-stage tumors, has been implicated in the M2 polarization of macrophages. The study aimed to investigate whether TGF-β promotes the M2 polarization of TAMs and whether inhibiting TGF-β would reduce this polarization in the tumor environment of colorectal cancer (CRC).

Methods:

In this study, macrophages were prepared from THP-1 cells by treating them with phorbol 12-myristate 13-acetate, and were used to analyze the polarization of human macrophages by TGF-β and TGF-β type I receptor inhibitor SB431542. The degree of polarization was assessed using both RT-qPCR and flow cytometry. To evaluate the effect of SB431542 in vivo, a CRC model was generated by implanting CT26 cells into balb/c mice. Tumor volume and weight were measured, and the percentage of M2-TAM was analyzed using flow cytometry.

Results:

The TGF-β type I receptor inhibitor SB431542 suppressed the M2 polarization of THP-1–derived macrophages in CRC conditioned medium. In syngeneic mouse models of CRC SB431542 reduced tumor volume and weight with the decrease of the proportion of M2-TAMs. The antitumor effect of SB431542 was abolished under conditions of TAM depletion.

Conclusions:

The study suggests that TGF-β promotes the M2 polarization of TAMs in the tumor environment of solid tumors, including CRC. Therefore, the anti-tumor effect of SB431542 on CRC is attributed to the reduction of M2-TAMs. The results of this study are expected to contribute significantly to the development of therapies for CRC that regulate TAM polarity.

colorectal cancer

TGF-β

TGF-β type I receptor inhibitor

tumor-associated macrophage

M2 polarization

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and the second leading cause of cancer death worldwide [1]. Although progress has been made in understanding the pathogenesis of CRC and various therapies have been developed, including cytotoxic and molecular-targeted agents, many of these therapies have yet to show satisfactory efficacy [2]. Furthermore, immune checkpoint therapy, which has been widely used in recent years, is reportedly less effective against CRC [2]. Thus, more-effective approaches to therapeutic intervention for patients with CRC are needed.

Transforming growth factor (TGF)-β contributes to the development and maintenance of homeostasis via diverse physiological functions, including mediating immunity, inhibiting cell proliferation and apoptosis, and promoting cell motility, differentiation, and angiogenesis [3]. In the early stages of cancer, TGF-β inhibits tumor growth through inhibitory effects on cell proliferation [4]. However, advanced cancers become resistant to the inhibitory effect of TGF-β on cell proliferation. TGF-β enhances the motility and epithelial-mesenchymal transition (EMT) of cancer cells in advanced cancers, resulting in increased metastatic potential and further malignant transformation [4]. In addition, TGF-β creates a immunosuppressive tumor environment favorable for cancer survival by suppressing the activity of antitumor dendritic cells, cytotoxic T cells, and natural killer cells and by promoting angiogenesis [5].

Macrophages that infiltrate tumor tissue are known as tumor-associated macrophages (TAMs), and these cells comprise the tumor immunosuppressive environment along with regulatory T cells, myeloid-derived suppressor cells, and tumor-associated fibroblasts [6]. Macrophages can be broadly classified as either M1-like or M2-like based on their activation state: M1-like macrophages mediate defense activities involved in the enhancement of inflammatory responses, whereas M2-like macrophages mediate the termination of inflammatory responses by releasing anti-inflammatory cytokines, and they also mediate tissue repair via scavenger receptors (e.g., CD206) [7]. TAMs are similarly classified as M1 or M2. In the early stages of tumor development, M1-like TAMs are induced as a result of the defense response, but as the tumor grows, the proportion of M2-like TAMs (M2-TAMs) increases [8]. M2-TAMs promote tumor growth by supplying nutrients and oxygen to cancer cells, and they also induce EMT by producing extracellular matrix metalloproteinase (MMP), which reduces the adhesiveness of cancer cells, thereby promoting invasion and metastasis [9]. M2-TAMs also release vascular endothelial growth factor (VEGF), which induces angiogenesis and promotes tumor growth [10], and they also contribute to the formation of an immunosuppressive environment by releasing TGF-β [11]. Thus, M2-TAMs have been implicated as playing roles in tumor growth, invasion, metastasis, and the formation of an immunosuppressive tumor environment. For this reason, research into cancer therapies targeting M2-TAMs has been intensive in recent years [12–14].

Cytokines such as interleukin (IL)-4, IL-13, and TGF-β are thought to play a role in the differentiation of M2-like macrophages in situations in which inflammation is suppressed [15–18]. We hypothesized that TGF-β, which is abundant in tumor tissues, promotes M2 polarization of TAMs and that the inhibition of TGF-β would diminish this polarization in the tumor environment of solid tumors, including CRC. Therefore, we examined the effect of SB431542, a TGF-β type I receptor inhibitor, on M2 polarization of TAMs using THP-1–derived macrophages and syngeneic mouse models of CRC. We expect that the results of this study will contribute to the development of new therapeutic strategies for CRC that involve inhibition of the M2 polarization of TAMs.

Preparation of conditioned medium (CM)

HCT116 human CRC cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured for 72 h in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin at 5.0 × 10⁴ cells/ml in a 10-cm dish. The used medium was then collected and filtered through a 0.45-µm pore size filter (Sartorius, Goettingen, Germany) to obtain CRC CM.

Differentiation of THP-1 cells into macrophages

THP-1 cells derived from a patient with acute monocytic leukemia as a human monocytic cell line were purchased from the ATCC. THP-1 cells were cultured in RPMI-1640 medium (containing l-glutamine and phenol red, Fujifilm Wako Pure Chemicals, Osaka, Japan) containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For cell stimulation, THP-1 cells were seeded in 6-well plates (Corning, Corning, NY, USA) at 5.0 × 10⁵ cells/ml per well and stimulated with 20 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The cells were then cultured in PMA-free RPMI-1640 medium for 1 h, followed by continued culture or addition of 20 ng/ml of TGF-β (Pepro Tech, Cranbury, NY, USA), CRC CM, or CRC CM containing the TGF-β inhibitor SB431542 (Cayman Chemical, Ann Arbor, MI, USA) at 20 µM and continued culture for 48 h.

Quantitative RT-PCR

Total RNA was extracted using RNAiso Plus (Takara Bio, Otsu, Japan) from differentiated macrophages. Reverse transcription was performed using 5× PrimeScript™ RT Master Mix (Takara Bio). A total of 2 µl of 5× PrimeScript RT Master Mix was mixed with RNase-free dH₂O to make a 10-µl solution containing 500 ng of total RNA and incubated at 37°C for 15 min (reaction conditions of 37°C for 15 min [reverse transcription reaction]) and 85°C for 5 s (thermal inactivation of reverse transcriptase). Quantitative RT-PCR was performed by mixing 2.5 µl of cDNA, 6.25 µl of Go Taq qPCR Master Mix (Promega, Madison, WI, USA), 0.5 µl of forward primer (10 µM), 0.5 µl of reverse primer (10 µM), and 2.75 µl of RNase-free dH₂O. The total volume was made up to 12.5 µl, and the reaction was performed at 95°C for 30 s, followed by amplification for 40 cycles consisting of 95°C for 5 s and 60°C for 30 s using a StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA). After the reaction was completed, amplification and melting curves were prepared, and relative quantification was performed using the ΔΔCt method to confirm differences in gene expression. Relative target gene transcript levels were normalized to the transcript level of an endogenous housekeeping gene (β-actin). The sequences of primers used for gene expression analysis are shown in Table S1.

In vitro flow cytometry analysis

Differentiated macrophages were detached from wells using TrypLe Express (Gibco, Gaithersburg, MD, USA). The number of cells was then determined, after which Human BD Fc block (BD Biosciences, San Jose, CA, USA) was added, and the samples were placed on ice for 10 min. Aliquots of 1 × 10⁶ cells per sample were prepared, and the samples were then incubated with antibodies (FITC human anti-CD206: BioLegend, San Diego, CA) or isotype control (FITC mouse IgG1, κ Isotype Control: BioLegend) and placed on ice for 30 min. Finally, the cells were washed with phosphate-buffered saline (PBS) and suspended in Stain buffer (BD Biosciences), followed by addition of 7-aminoactinomycin D (7-AAD) (BD Biosciences) and analysis using a FACS Verse system (BD Biosciences).

Preparation of syngeneic mouse models of CRC

All animal experimental protocols were approved by the Animal Care and Use Committee of Nagoya City University. All experiments were performed in accordance with relevant guidelines and regulations of the institutional and U.S. National Institutes of Health guide for the care and use of laboratory animals. All methods were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. Female mice (BALB/cCrSlc) aged 4–6 weeks were purchased from Japan SLC (Shizuoka, Japan). Mice were allowed to acclimatize for 1 week in the animal facility before any interventions were initiated. CT26 mouse CRC cells were cultured in DMEM low-glucose (containing l-glutamine and phenol red, Wako) containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. A total of 2 × 10⁶ CT26 cells were implanted subcutaneously into the right flank of female BALB/c mice (5–11 weeks old) as previously reported [12]. After all experiments, animals were sacrificed by cervical dislocation in deep CO₂ anesthesia, primary tumors were surgically removed and weighted (g).

Analysis of therapeutic effect in syngeneic CRC model mice

To examine the therapeutic effect of the TGF-β type I receptor inhibitor SB431542, treatment was initiated in the DMSO group and the SB431542 group when the tumor volume reached approximately 100 mm³. SB431542 was administered intraperitoneally at a concentration of 10 mg/kg [19]. The DMSO group received intraperitoneal administration of 20 µl of DMSO dissolved in 180 µl of PBS. The SB431542 group received intraperitoneal administration of 20 µl of DMSO containing SB431542 dissolved in 180 µl of PBS. Drug administration and tumor volume measurements were performed every 2 days for 2 weeks. To examine the therapeutic effect of SB431542 with clodronate liposome administration, clodronate liposome small (Hygieia Bioscience, Woodland, CA, USA) was administered intraperitoneally once every 4 days at a concentration of 9.75 mg/kg to induce macrophage depletion. Tumor volume was calculated using the following formula: long diameter × short diameter × short diameter/2. Tumor weight was measured 2 weeks after the start of treatment.

In vivo flow cytometry analysis

Tumors were removed 10 days after the start of drug administration, treated with collagenase (Wako), and dispersed using a gentleMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The tumor cells were counted, Mouse BD Fc block (BD Biosciences) was added, and the samples were placed on ice for 10 min. Aliquots of 1 × 10⁶ cells per sample were prepared, and antibodies (APC rat anti-CD11b [BD Biosciences], Brilliant Violet 421 anti-mouse CD206, FITC anti-mouse F4/80 [BioLegend]) or isotype controls (APC rat IgG2b, κ Isotype Control [BD Biosciences], Brilliant Violet 421 rat IgG2a, κ Isotype Control, FITC rat IgG2a, κ Isotype Control [BioLegend]) were added and placed on ice for 30 min. The samples were then washed with PBS and suspended in Stain buffer (BD Biosciences), followed by addition of 7-AAD (BD Biosciences) and analysis using a FACS Verse system (BD Biosciences).

Statistical analyses

All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R. Specifically, EZR is a modified version of R Commander designed to add functions frequently used in biostatistics [20]. Two-tailed non-paired Student’s t-tests were used for comparisons between two groups. One-way analysis of variance followed by Tukey’s multiple comparison test was used for multiple comparisons. Data are presented as the mean ± standard error (SE). A p value of < 0.05 was considered statistically significant.

TGF-β type I receptor inhibitor suppresses CRC CM–induced polarization of THP-1–derived macrophages to M2-TAMs

After THP-1 cells were induced to differentiate into macrophages using PMA, the effect of TGF-β on M2 polarization was evaluated by quantitative RT-PCR (Fig. 1A). The expression of genes encoding the M2 markers CD206, arginase-1 (ARG1), TGF-β, and IL-10 was significantly upregulated in the TGF-β group (Fig. 1B). Furthermore, the expression of CD206 on the cell surface was significantly upregulated in the TGF-β group (Fig. 1C and S1A). THP-1 cells were induced to differentiate into macrophages using PMA and then cultured in CRC CM alone or CRC CM plus the TGF-β type I receptor inhibitor SB431542, followed by analysis of M2 polarization using quantitative RT-PCR (Fig. 1D). Expression of genes encoding the representative M2 markers CD206, ARG1, TGF-β, and IL-10 was significantly upregulated in the CRC CM group, whereas expression of the CD206, ARG1, and IL-10 genes (but not the TGF-β gene) was significantly downregulated in the CRC CM plus SB431542 group to the same levels as in the control group (Fig. 1E). Furthermore, the expression level of CD206 on the cell surface was significantly elevated in the CRC CM group but significantly decreased to the same level as the control group in the CRC CM plus SB431542 group (Fig. 1F and S1B).

TGF-β type I receptor inhibitor suppresses the growth of CRC tumor tissue in syngeneic model mice

When the tumor volume reached approximately 100 mm³, tumor model mice were divided into DMSO and SB431542 groups and treated accordingly every other day (Fig. 2A). The tumor volume in the SB431542 group was significantly lower than that in the DMSO group (Fig. 2B and C). In addition, the tumor weight in the SB431542 group was significantly lower than that in the DMSO group (Fig. 2D).

Changes in populations of M2-TAMs in CRC tumor tissue of syngeneic model mice induced by treatment with the TGF-β type I receptor inhibitor were evaluated using flow cytometry analysis of CD206⁺ and CD11b⁺ cells. The SB431542 group exhibited a significantly lower proportion of CD206⁺CD11b⁺ cells among all live cells compared with the DMSO group (Fig. 3A and B). Furthermore, the proportion of CD206⁺CD11b⁺ cells among all CD11b⁺ cells was lower than that in the DMSO group (Fig. 3A and C).

Clodronate liposome–mediated macrophage depletion diminishes the antitumor effect of the TGF-β type I receptor inhibitor

When the tumor volume reached approximately 100 mm³, model mice were treated with DMSO or SB431542 every other day, and all mice were treated with clodronate liposomes every 4 days to deplete macrophages in the tumor tissue (Fig. 4A). Flow cytometric analysis confirmed that administration of clodronate liposomes reduced the number of CD11b⁺ F4/80⁺ macrophages in tumor tissue (Fig. S2). No significant changes in tumor volume or weight were observed in the SB431542 group compared with the DMSO group under macrophage-depleted conditions (Fig. 4B–D).

In this study, we showed that the TGF-β type I receptor inhibitor SB431542 reduces the number of M2-TAMs within tumors and exerts antitumor effects. IL-4, IL-13, and TGF-β reportedly induce M2 polarization of macrophages [12–14]. In this study, in vitro experiments using THP-1–derived macrophages showed that TGF-β increased the expression of genes encoding M2 markers such as CD206, ARG1, TGF-β, and IL-10 and increased the expression of CD206 on the cell surface, suggesting that TGF-β alone promotes M2 polarization of macrophages. These results are consistent with previous reports [18]. TGF-β is abundant in the tumor environment of various types of solid tumors, including CRC. We hypothesized that TGF-β, rather than IL-4 and IL-13, promotes the polarization of TAMs toward the M2 phenotype in the intratumor environment. CRC CM increased the expression of genes encoding the M2 markers CD206, ARG1, TGF-β, and IL-10 in THP-1–derived macrophages, suggesting that CRC CM contains factors that promote the M2 polarization of TAMs. The upregulated expression of M2 markers other than TGF-β was completely abolished by SB431542. These results suggest that TGF-β is the primary factor involved in M2 polarization in CRC CM and that this polarization can be suppressed by the administration of SB431542.

In a study using a mouse model of bladder cancer, the potent TGF-β type I receptor inhibitor SB431542 reduced tumor volume [21]. SB431542 also reportedly inhibited metastasis to the lungs in a mouse model of breast cancer, although it did not reduce tumor volume [22]. Furthermore, our study also showed a reduction in tumor volume in a mouse model of CRC, suggesting that the TGF-β type I receptor inhibitor exerts antitumor effects against a variety of cancers. Importantly, administration of SB431542 to CRC tumor model mice significantly reduced the proportion of M2-TAMs in tumor tissue. In vitro and in vivo experimental data indicated that TGF-β significantly contributes to the M2 polarization of TAMs in the tumor environment and that inhibition of TGF-β signaling suppresses M2 polarization of TAMs in tumor tissues.

We hypothesized that the antitumor effects of SB431542 might be related to the reduction in the proportion of M2-TAMs. Using clodronate liposomes, we administered SB431542 to tumors depleted of TAMs. As a result, neither tumor volume nor tumor weight changed significantly under TAM-depleted conditions, suggesting that the anti-tumor effect of SB431542 on CRC is partially attributed to the suppression of the polarization of TAMs to the M2 phenotype. M2-TAMs promote tumor growth, invasion, and metastasis by secreting VEGF, MMP, TGF-β, IL-6, and IL-10 [6]. The antitumor activity of SB431542 reported previously [21, 22] may be partly related to the suppressive effect of TAMs.

There are several limitations to this study that should be noted. Firstly, the study only examined the impact of the TGFβ receptor inhibitor SB431542 on macrophages, without evaluating the status of other immune cells such as T cells and NK cells. Since TGFβ has been shown to reduce the antitumor activity of these immune cells [23, 24], SB431542 could not only modify macrophage characteristics but also activate tumor immunity mediated by T cells and NK cells. Secondly, we did not explore the potential of combination therapy involving SB431542 and other drugs. Although SB431542 treatment had some inhibitory effect on tumor growth, SB431542 alone is unlikely to completely eliminate tumors. To facilitate the clinical application of SB431542, it is worth considering its use in combination with other anti-cancer agents such as immune checkpoint inhibitors, cisplatin, and etoposide. Finally, we did not assess the potential side effects of SB431542. Future research should aim to investigate the concentration of SB431542 that can be expected to have some efficacy while minimizing any adverse effects, in consideration of potential clinical applications.

The TGF-β type I receptor inhibitor SB431542 suppressed the CRC CM–induced polarization of THP-1–derived macrophages to the M2 phenotype. Furthermore, SB431542 reduced both tumor volume and weight in a mouse model of CRC and reduced the percentage of M2-TAMs. Under TAM-depleted conditions, the antitumor effect of SB431542 was abolished. These results suggest that the anti-tumor effect of SB431542 on CRC is attributed to the reduction of M2-TAMs. The results of this study could contribute significantly to the development of new cancer therapies that control TAM polarization.

Ethics approval and consent to participate

Data Availability of data and materials

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Consent for publication

Not applicable.

Competing interests

The authors of this manuscript have no conflicts of interest to report.

Funding

This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, KAKEN grant numbers 20K22715, 20K08211, 21K16403, and 21K07830.

Author contributions

Akira Sakuragi: Data curation, formal analysis, writing – original draft, and approval of the final manuscript.

Hiromasa Aoki: Data curation, formal analysis, writing – original draft, and approval of the final manuscript.

Kohki Toriuchi: Data curation, formal analysis, and approval of the final manuscript.

Riri Kondo: Data curation, formal analysis, and approval of the final manuscript.

Yasumichi Inoue: Data curation, resources, methodology, writing – original draft, and approval of the final manuscript.

Hidetoshi Hayashi: Resources, methodology, writing – original draft, and approval of the final manuscript.

Satoru Takeshita: Data curation, methodology, writing – original draft, and approval of the final manuscript.

Hiroki Kakita: Methodology, writing – original draft, funding acquisition, and approval of the final manuscript.

Yasumasa Yamada: Methodology, writing – original draft, funding acquisition, and approval of the final manuscript.

Mineyoshi Aoyama: Conceptualization, data curation, methodology, writing – original draft, funding acquisition, and approval of the final manuscript.

Acknowledgments

We acknowledge the assistance of the Research Equipment Sharing Center at Nagoya City University. All illustrations were created using Biorender.com.

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No competing interests reported.

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TGF-β type I receptor inhibitor suppresses polarization of tumor-associated macrophages to the M2 phenotype and exerts antitumor effects on colorectal cancer

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Preparation of conditioned medium (CM)

Differentiation of THP-1 cells into macrophages

Quantitative RT-PCR

In vitro flow cytometry analysis

Preparation of syngeneic mouse models of CRC

Analysis of therapeutic effect in syngeneic CRC model mice

In vivo flow cytometry analysis

Statistical analyses

Results

TGF-β type I receptor inhibitor suppresses CRC CM–induced polarization of THP-1–derived macrophages to M2-TAMs

Clodronate liposome–mediated macrophage depletion diminishes the antitumor effect of the TGF-β type I receptor inhibitor

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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