1 Background
Colorectal cancer (CRC) is one of the most lethal human malignancies in the world [
1]. In recent years, immunotherapy has rapidly developed and benefits some cancer patients by affecting immune checkpoints, including the cytotoxic T lymphocyte associated protein 4 (CTLA-4) or the programmed cell death 1 (PD-1) signaling pathway [
2,
3], which is a promising approach to activate antitumor immunity and can improve T cell function and reduce tumor burden by strengthening cytotoxic CD8
+ T cell (CTL) infiltration to slow tumor progression and prolong patient survival. However, the majority of CRC patients with mismatch-repair-proficiency and microsatellite instability-low (pMMR-MSI-L) tumors are unresponsive to treatment [
4,
5]. Thus, the mechanism of immunotherapy resistance must be elucidated, and novel markers to predict immunotherapy resistance need to be identified.
Tumor-associated macrophages (TAMs) are critical drivers of the immunosuppressive microenvironment and are recruited to tumors by colony stimulating factor (CSF), transforming growth factor (TGF-β1) and chemokines, such as CCL2 (MCP-1) [
6], and these cells mostly exhibit an alternatively activated macrophage (M2)-like phenotype [
7]. M2-TAMs can directly stimulate tumor cell proliferation and invasion and suppress T cell recruitment and activation through the production of cytokines, such as interleukin (IL)-10 or epidermal growth factor (EGF)[
8]. Hence, modifying the properties and function of TAMs in tumors can enhance antitumor immune responses. In addition, resistance to immunotherapies can be mediated by abundant infiltration of tumor-associated myeloid cells, including macrophages [
9]. However, accumulating evidence suggests that programmed death ligand 1 (PD-L1)
+ TAMs in tumors could better predict a patient’s response to immunotherapy than PD-L1
− TAMs in non-small-cell lung cancer patients [
10,
11]. These data suggested that PD-L1
+ TAMs were related to anti-PD-1/PD-L1 therapy resistance in the tumor microenvironment (TME). A better understanding of the mechanism by which PD-L1 regulates immune checkpoints in TAMs is necessary [
12,
13].
MicroRNAs (miRNAs) widely participate in macrophage polarization in the TME, and their aberrant expression could affect the balance of macrophage polarization [
14]. Evidence from our laboratory has demonstrated that microRNA-146b (miR-146b) inhibits tumor progression in a colitis-associated cancer (CAC) mouse model by targeting TAMs with miR-146b mimic-containing nanoparticles [
13], but the mechanism remains unclear. In the current study, we demonstrated that
N6-methyladenosine (m
6A) is a key regulator that modifies the transcription of pri-miR-146b to mature miR-146b in macrophages. MiR-146b deficiency increased the number of M2-TAMs accompanied by the upregulation of PD-L1 expression through PI3K/AKT pathway activation, which inhibited T cell infiltration in the TME and resulted in immune suppression and tumor development. Importantly, the combination of miR-146b depletion and a PD-1/PD-L1 checkpoint inhibitor was shown to enhance the antitumor activity of effector immune cells. In summary, our findings revealed the mechanism by which miR-146b functions in colorectal cancer to promote PD-1 blockade, thereby providing potential new biomarkers and a therapeutic avenue for tumors refractory to ICI treatment.
2 Materials and methods
2.1 Mouse husbandry
WT C57BL/6 mice (6–8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), and miR-146b deficient (miR-146b−/−) mice on a C57BL/6 background were housed in the Guangzhou Medical University Animal Experiment Center (Guangzhou, China). All animal protocols were approved by the Animal Ethics Committee of Guangzhou Medical University. The animals were maintained under specific pathogen-free conditions in the Guangzhou Medical University Animal Experiment Center (Guangzhou, China).
2.2 Tumor studies
Six- to eight-week-old female or male WT and
miR-146b−/− mice were implanted with 5 × 10
5 MC38 cells in 100 μl of phosphate-buffered saline (PBS) by subcutaneous injection in the right flank, and tumor growth was monitored for up to 21 days. Tumor volumes were determined by measuring the length (
l) and width (
w) and were calculated as follows: tumor volume = (
l2 x
w)/2. All in vivo monoclonal antibodies (mAbs) were from BioXCell (New Hampshire, USA). Animals were euthanized before the maximum IACUC allowable tumor burden of 2 cm
3/mouse was exceeded [
12]. Tumor dimensions were measured 2 times per week beginning on day 7.
For the in vivo depletion study, mice were pretreated by an intraperitoneal injection (i.p.) with anti-CD8 mAbs (200 μg/mouse), anti-NK1.1 mAbs (200 μg/mouse), clodronate (CLO, 1 mg/mouse) or control liposomes (Ctrl, 1 mg/mouse) (Clodronateliposomes.com, Amsterdam, The Netherlands) for 1 week (twice/week) and then subcutaneously injected with MC38 cells, after which the mice were treated with mAbs or CLO for an additional three weeks.
For miR-146b agomir administration, 6- to 8-week-old miR-146b−/− mice were implanted with 5 × 105 MC38 cells, and after 1 week, the mice were intraperitoneally administered 10 mg/kg miR-146b agomir twice per week, while the control group was administered the same dose of NC for 2 weeks.
In vivo macrophage adoptive transfer experiments were performed. Primary bone marrow-derived macrophages (BMDMs) were polarized to the M2 phenotype as described in the Supplemental Material. M2 cells were mixed 1:1 with MC38 tumor cells, and a total of 1 × 106 cells were injected subcutaneously. Primary BMDMs were polarized to the M2 phenotype, and macrophage-conditioned medium (CM) was harvested, centrifuged at 800 × g for 10 min and filtered through a 0.22-μm syringe filter (Millipore, Billerica, MA, USA). Then, CM mixed with 5 × 105 MC38 tumor cells was injected subcutaneously, and inoculated mice were further treated by intradermal injection with conditioned medium at 3 and 6 days post inoculation.
For inhibitor treatment, miR-146b−/− BMDMs were pretreated with inhibitors of AKT (GSK2141795, 30 μM), phosphoinositide 3-kinase (PI3K) (LY294002, 20 μM) and p110β (TGX221, 10 μM) for 30 min, polarized to the M2 phenotype, and then mixed with MC38 tumor cells. In some experiments, morpholino (MO) was dissolved in PBS, and WT BMDMs were preincubated with MO–nc (5 μM) and MO-methyltransferase-like 3 (Mettl3) (5 μM) for 12 h, polarized to the M2 phenotype, and then mixed with MC38 tumor cells. All adoptive transfer experiments were carried out in new WT mice.
For anti-PD-1 mAb treatment, 5 × 10
5 MC38 cells were subcutaneously injected into the flanks of the mice. Tumors were allowed to grow for seven days, and then the mice were treated i.p. with 200 μg of anti-PD-1 mAb (Clone J110) or rat IgG2a isotype control every 3 days for 2 weeks. In the colitis-associated cancer model, mice were intraperitoneally injected with 10 mg/kg azoxymethane (AOM, Sigma-Aldrich Corp, St Louis, MO, USA). One week later, the mice were administered 2% dextran sodium sulphate (DSS, MW 40000–50,000) (MP Biomedicals, Solon, Ohio, USA) in distilled water for 5 days, followed by 14 days of normal drinking water [
13]. This cycle was repeated three times. After 5 weeks, the mice were administered 200 μg of anti-PD-1 mAbs or Rat IgG2a isotype control every 3 days for the indicated time. The mice were sacrificed after the third cycle ended.
4 Discussion
In this study, we demonstrate that the METTL3/miR-146b axis reprograms macrophages to exert protumor effects. METTL3 knockdown or miR-146b ablation alone significantly increases the TAM population, reduces T cell infiltration, and promotes tumor progression. However, METTL3 or miR-146b deficiency increases PD-L1 expression in TAMs, and the therapeutic effect of anti-PD1 mAb is markedly improved and leads to the regression of even well-established CRC tumors. Mechanistically, we demonstrated that miR-146b deficiency promoted the polarization of TAMs by inducing oncogenic PI3K-AKT signaling; specifically, miR-146b expression is decreased in TAMs, which significantly increases p110β expression and promotes PI3K/AKT signaling activation. Moreover, we found that the m6A modification was the main factor associated with miR-146b expression and determined that knockdown of the m6A “writer” protein METTL3 and the “reader” protein HNRNPA2B1 significantly decreased miR-146b expression and promoted M2 macrophage polarization. These data suggest that the METTL3/miR-146b axis may represent an effective therapeutic target to reprogram TAMs, improve the immunosuppressive microenvironment of CRC tumors, and increase the efficacy of immunotherapy.
Many innate and adaptive immune cells are recruited to the TME, and macrophages are the most abundant immune cells [
27]. Macrophages are the first line of defense against pathogenic insults, suggesting that these cells can be tumoricidal after being activated; however, cancer-associated macrophages are usually polarized to a protumoral M2 phenotype at the time of tumor initiation, and these cells are named TAMs [
28]. Therefore, the polarization of TAMs can indirectly stimulate CTL activation and synergize with checkpoint inhibitors, suggesting improved cancer outcomes [
8]. MiRNAs have also been identified as important determinants of the protumoral activity of TAMs. In our study, we observed that miR-146b participated in the initiation and progression of CAC, which significantly reduced tumor numbers and tumor areas [
13]. Accordingly, Arg-1 expressing macrophages were sharply reduced [
13].
miR-146b−/− mice had significantly increased M2-TAM numbers, reduced T cell recruitment, and promoted immune suppression and tumor progression. Our in vitro study further confirmed that the miR-146b mimic could inhibit M2 macrophage polarization under M2 conditions.p110γ is the most highly expressed PI3K isoform in myeloid cells, and PI3K/AKT is involved in the macrophage phenotype transitions in the TME [
8,
12]. We found that miR-146b ablation promoted PI3K/AKT signaling pathway activation, exacerbated the upregulation of the PI3K/AKT signaling pathway-associated target C/EBPβ in M2 macrophages and modulated Arg-1 expression by binding to the response element of Arg-1. These results suggest that miR-146b repressed the polarization of TAMs by targeting the PI3K/AKT signaling pathway. In our study, miR-146b deficient BMDMs promoted p110β expression but not p110γ expression. Intriguingly, both p110β and p110γ contribute to AKT activation [
15]. Taken together, these results suggest that miR-146b can regulate the polarization of TAMs through PI3K/AKT signaling and that these activities may be highly dependent on p110β expression.
TAMs can suppress T cell recruitment and activation, thereby exacerbating immunosuppression [
8]. We found that miR-146b deficiency reduced the T cells recruited to tumors. In addition, PD-L1 expression can be mediated by PI3K/AKT signaling [
18].These effects indicate that miR-146b may affect T cell-based immunotherapy. David L. Rimm et al. recently showed that macrophages expressing PD-L1 can enhance anti-PD-1 mAb therapy [
10]. We assessed the percentage of PD-L1 in TAMs and showed that PD-L1 expression in TAMs was significantly increased in macrophage bearing tumors. Furthermore, we found that high expression of PD-L1 in
miR-146b−/− macrophages indicated a better immunotherapeutic effect. Given that these outcomes occurred in
miR-146b−/− mice, we hypothesize that miR-146b deficiency-mediated polarization of TAMs promotes tumor progression, whereas increased PD-L1 expression potentially drives a response to anti-PD1 immunotherapy.
RNA m
6A modification is a common epigenetic regulation that is involved in a variety of cellular processes [
29,
30]. The decrease in the m
6A “reader” METTL14 in TAMs determines T cell phenotypes in tumors [
26]. In our study, m
6A modification was decreased in M2 macrophages by the m
6A methyltransferase METTL3, and we found that m
6A methylation mediated the maturation process of pri-miR-146b. Knockdown of both METTL3 and the m
6A “reader” HNRNPA2B1 impaired the maturation process of pri-miR-146b and led to a decrease in mature miR-146b expression. Furthermore, knockdown of both METTL3 and HNRNPA2B1 induced M2 macrophage polarization. When miR-146b mimics were transfected into METTL3-knockdown cells, the miR-146b mimic rescued the M2 macrophage polarization induced by METTL3. An in vivo study further confirmed that knockdown of METTL3 could decrease miR-146b expression, leading to an increase in TAMs and ultimately promoting tumor progression. More interestingly, we showed that METTL3 knockdown increased PD-L1 expression via miR-146b in M2 macrophages, thereby activating the p110β/PI3K/AKT pathway and inducing an immunosuppressive status. More interestingly, METTL3 knockdown led to sensitization of macrophages to anti-PD-1 therapy. These results suggested that METTL3 promotes the maturation of pri-miR-146b in CRC TAMs in an m
6A-dependent manner. Targeting the enzymatic activity of METTL3 may be an anti-CRC strategy. For example, STM2457, the first bioavailable inhibitor of METTL3, may be useful, though future research is needed. In addition, we only quantified the m
6A level in M2 macrophages using a colorimetric method, which was not quite accurate. Further might quantify m
6A levels by mass spectrometry, and these results need to be confirmed in TAMs from human CRC tissues.
In conclusion, we have identified the role of the m6A-miR-146b/p110β/PI3K/AKT axis in the polarization of TAMs in CRC tissue, which might be associated with CRC development and progression. Our results also suggest that m6A modification may play an important role in CRC and other types of cancer linked with miR-146b decrease.
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