IFN-α facilitates the effect of sorafenib via shifting the M2-like polarization of TAM in hepatocellular carcinoma

Abstract

Tumor-associated macrophages (TAMs) and how they are activated play critical roles in tumor progression and metastasis, and in hepatocellular carcinoma (HCC), they are associated with sorafenib resistance. Reprogramming of TAMs into M1-like macrophages has been proposed as an approach to stimulate tumor regression. Here we studied the collective effects of interferon-alpha (IFN-α) and sorafenib on HCC. We found that IFN-α delayed tumor growth and inhibited pulmonary metastasis in an orthotopic HCC implantation model. Via in vitro studies, we found that IFN-α treatment could reprogram M2-like RAW264.7 and THP-1 macrophage cells toward M1-like cells. In addition, we also found that IFN-α combined with a low dose of sorafenib has a synergistic inhibitory effect on HCC tumor growth and pulmonary metastasis without obvious toxicity in an in vivo mice model. Moreover, IFN-α increased sorafenib’s therapeutic efficacy by shifting TAM polarization to an M1-like phenotype, increasing and activating intratumoral CD8⁺ T cells in HCCs. In conclusion, a combination of IFN-α and sorafenib have synergistic inhibitory effects on HCC growth and metastasis resulting from a shift in TAM polarization rather than their depletion. Our study supports the future clinical use of a combination of IFN-α and sorafenib for the treatment of advanced HCC.

Introduction

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies globally, and many HCC patients have reached advanced stages at the time of their diagnosis and are, therefore, not suitable for curative treatments [1,2]. Currently, sorafenib has been established as a first-line therapy for advanced HCC. Sorafenib is a multi-kinase inhibitor that affects a number of protein kinases, including VEGFR, PDGFR, and RAF, and it can promote cell apoptosis, reduce angiogenesis and inhibit tumor proliferation [3]. However, it has only a limited effect on overall survival (OS) and exhibits a poor response rate [4]. Therefore, new strategies are urgently needed to improve the effectiveness of targeted HCC therapies.

Immunotherapy is now hailed as a major advance in cancer treatment. In some cancer patients, immune checkpoint blockade, CAR-T therapy, and cytokine therapy treatment, have been shown to have robust benefits [5-9]. Unfortunately, only a small fraction of HCC patients benefit from these therapeutic approaches, and the low response to immunotherapy is partly due to the formation of immunosuppressive tumor microenvironments and T-cell exhaustion [10]. Infiltration by multiple types of immune cells, including TAMs, promotes the development of immunosuppressive microenvironments that can support the initiation and progression of HCC [11].

Macrophages are extremely versatile, multifunctional cells. Currently, two macrophage subtypes have been identified, i.e., classically activated macrophages (M1) and alternatively activated macrophages (M2) [11]. Inhibition of inflammatory cytokine secretion by HCC cells can “educate” recruited peripheral blood monocytes such that they differentiate into macrophages exhibiting an M2-like phenotype. Recently, accumulating evidence has revealed that TAMs can modulate tumor immune microenvironments and promote HCC progression; for example, the polarization of TAMs into the M1 phenotype can limit tumor growth and extend survival in tumor-bearing mice [12,13], and TAM depletion can enhance sorafenib’s antitumor effect in HCC models [14].

IFN-α has been widely used to treat malignancies, including HCC [15-17]. Multiple clinical trials have revealed that adjuvant IFN-α therapy can increase survival in HCC patients following curative resection [18]. The limited clinical application of IFN-α is mainly due to the inability to identify patients who will respond effectively [18,19]. The anti-tumor effect of IFN-α is due to its indirect inhibitory effects on tumor cells, including its immunomodulatory effects [20]. The immune-modulatory effects of IFN-α include the regulation of macrophage polarization and immune cell infiltration [21]. Recently, IFN-β was demonstrated to dramatically enhance the antitumor effect of anti-EGFR Ab [22].

Here we found that IFN-α has a synergistic effect with sorafenib on inhibiting HCC growth and metastasis. Further investigation revealed that this synergistic effect might result from TAM polarization toward the M1-like phenotype and attenuation of the immune suppressive microenvironment. These findings support the potential clinical use of sorafenib in combination with IFN-α for HCC treatment.

Materials and methods

Cell lines and animal models

Mouse Hepa1-6 HCC cells, human Huh7 HCC cells, and murine monocyte/macrophage RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Human THP-1 monocytes were cultured in 1640 with 10% FBS. All cell lines were provided by the Shanghai Institute of Cell Biology (Chinese Academy of Sciences, Shanghai, China).

Six-week-old male C57BL/6 mice were purchased from the SLAC Laboratory Animal Company (Shanghai, China). All animal experiments were approved by the Ethics Committee of Fudan University. The subcutaneous tumor model is established via subcutaneous implantation of 1×10⁶ Hepa1-6 cells into the flanks of mice. Mice bearing tumors of approximately 50 mm³ in volume were randomly partitioned into 4 groups (n = 6 mice/group): (a) oral use of vehicle and intratumoral injection of PBS (vehicle group); (b) oral use of sorafenib (30 mg/kg/day) (sorafenib group); (c) intratumoral injection of IFN-α (1.5×10⁷ U/kg/day) (IFN-α group); (d) oral use of sorafenib (30 mg/kg/day) in combination with intratumoral injection of IFN-α (1.5×10⁷ U/kg/day) (sorafenib and IFN-α combination group).

To generate the orthotopic model, subcutaneous tumors were harvested and dissected into pieces of approximately 1 mm³, which were then orthotopically implanted into the liver parcel of other mice. 14 days after orthotopic implantation, the mice were randomly partitioned into 4 groups (n = 5 for each group) that received the same treatments used in the subcutaneous HCC model.

Flow cytometry analysis (FACS)

Single cell suspensions were prepared from fresh mouse tumors by using the mouse tumor dissociation kit following the manufacturer’s instructions (Miltenyi Biotec). After red blood cell lysis, the cells were centrifuged for 5 minutes at 500×g before being suspended in FACS buffer. To avoid non-specific binding, cells were incubated with anti-mouse CD16/32 antibody for 15 minutes. The cells were then incubated with primary antibody at room temperature (RT) for 30 minutes in the dark, washed twice in phosphate-buffered saline (PBS), suspended in 300 μl of PBS, and then analyzed with a FACS Aria II instrument (BD Biosciences, San Jose, CA). The following antibodies from Biolegend (San Diego, California, USA) were used for cell staining to detect tumor-infiltrated immune cells: FITC-CD11b, PE-CD206, APC-CD4, PE-cy7-CD8, and APC-F4/80.

Quantitative real-time polymerase chain reaction (qPCR)

Trizol (Invitrogen, California, USA) was used for extraction of total RNA from cells and frozen samples. The RNA was then reverse transcribed into cDNA. Real-time PCR was performed with SYBR Green PCR Master Mix (DBI Bioscience, Denmark) in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). To quantify the mRNA levels, the measurements were normalized to GAPDH. The primer sequences used in this study are provided in Supplementary Table 1.

Enzyme-linked immune-sorbent assay (ELISA)

The culture supernatant of the cells to be examined was collected and centrifuged to remove interfering particles. ELISA kits were used to detect the IL-1b, IL-10, TGF-β1, IFN-γ and TNF-α levels following the manufacturer’s instructions (R&D Systems, Minneapolis, Minnesota, USA).

M2-like macrophage polarization of RAW264.7 and THP-1

20 ng/ml IL-13 (Invitrogen Inc., Carlsbad, CA, USA) was used to stimulate RAW264.7 and THP-1 macrophages to polarize into the M2 phenotype, and the levels of the murine M2 macrophage marker CD206, the human M2 macrophages marker CD163, and the human M1 macrophage marker HLA-DR were detected via flow cytometry. Next, cytokines were detected via ELISA and the expression levels of macrophage polarization-related genes were measured via qPCR.

Cell counting Kit-8 (CCK-8)

5×10 cells/well were seeded in 96-well plates. After culturing to a specific time point, the numbers of viable cells were determined via the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technology, USA). The optical density (OD) of the medium at a wavelength of 450 nm was measured with a spectrophotometer.

Colony formation assay

1000 cells per well were inoculated in 6-well plates. Next, approximately 2 ml of culture medium was added to each well, and even distribution of the cells was confirmed. The medium was changed once a week, being careful not to disturb the cells. After approximately 4 weeks, the medium was discarded, and the cells were washed thrice in PBS, fixed with 4% paraformaldehyde for 15 minutes, and then washed thrice in PBS. The cells were then stained with crystal violet (0.5%) for 10 minutes at RT, followed by rinsing with distilled water. After drying at RT, the cell clones were counted.

Migration and invasion assay

The ability of the HCC cells to migrate and invade was assessed in transwell chambers with 8-µm pore size polycarbonate membranes between the upper and lower chambers (BD Pharmingen). The bottom chamber contained DMEM with 10% FBS, which acts as a chemoattractant. 5×10⁴ cells (for migration, without prepared matrigel) and 10×10⁴ cells (for invasion, with prepared matrigel) were suspended in serum-free medium and seeded into the upper chambers. The assay plates were incubated at 37°C in a humidified incubator maintained with 5% CO₂.

Statistical analyses

SPSS 16.0 for Windows (IBM, Armonk, NY) was used for the statistical analyses. Between-group differences were analyzed via an unpaired two-tailed Student’s t test or one-way ANOVA. The chi-square test or Fisher’s exact test were used to analyze categorical data. Survival curves were calculated via the Kaplan-Meier method and compared via the log-rank test. P values less than 0.05 indicated statistically significant differences.

Results

IFN-α enhances the anti-tumor effect of sorafenib on HCC

We established subcutaneous implantation models in C57BL/6 mice with Hepa1-6 cells, which we then treated with IFN-α, sorafenib, or a combination of both. We found that sorafenib alone could inhibit HCC tumor growth, but that a much more robust inhibition was obtained when sorafenib was used in combination with IFN-α (Figure 1A-D and Supplementary Figure 1A).

Figure 1.

IFN-α enhances the anti-tumor effects of sorafenib. (A) Schematic description of IFN-α and sorafenib treatment in the subcutaneous tumor model. (B-D) Representative micrographs of the subcutaneous liver cancer model after treatment with vehicle, IFN-α, sorafenib, or the combination. Tumor growth was measured as (C) the tumor volume at different time points. (D) Tumor weight at the time of sacrifice. (E) Schematic description of IFN-α and sorafenib treatment in the orthotopic liver cancer model. (F, G) Representative micrographs of orthotopic liver tumors after treatment with vehicle, IFN-α, sorafenib, or the combination. (G) Tumor volume at the time of sacrifice. (H) Survival of mice bearing orthotopic Hepa1-6 tumors following IFN-α and sorafenib treatment. Significance was determined via the log rank test. (I) Left, representative images of Hematoxylin and Eosin (HE)-stained lung sections in the treatment groups. Scale bar, 200 μm. Right, the numbers of lung metastatic foci for each group. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001. IFN-α, interferon-alpha; P.0., per os; S.C., subcutaneous injection.

To further evaluate the effects of these treatments on HCC growth and metastasis, we established orthotopic implantation models based on the above Hepa1-6 model (Figure 1E). Consistently, we observed that administration of sorafenib alone could inhibit tumor growth and lung metastasis (Figure 1E-G), but that a combination induced more robust decreases in tumor size and lung metastasis (Figure 1I) and increased the survival time (P < 0.01; Figure 1H) in the tumor-bearing mice compared with the effects of sorafenib or IFN-α alone. Moreover, we did not observe any appreciable differences in treatment toxicity (as indicated by body weight) among the different treatment groups (Supplementary Figure 1B). These results suggested that IFN-α dramatically enhanced sorafenib’s antitumor effect on HCC and that it had synergetic effects with sorafenib on HCC growth and metastasis. The combined treatment was more effective in combating HCC.

IFN-α inhibits macrophage activation and M2-like polarization in tumor microenvironments

To explore the underlying mechanism, we performed flow cytometry analysis (FACS) using F4/80, CD11b and CD206, and immunohistochemical (IHC) staining to detect intratumoral macrophages in orthotopic implantation models. We found that IFN-α treatment reduced the number of infiltrated M2-like macrophages (F4/80⁺CD206⁺ cells and F4/80⁺CD11b⁺ cells) (Figure 2A, 2B) and increased the number of M1-like macrophages (iNOS) in Hepa1-6 tumors (Figure 2C). These results indicated that IFN-α treatment induced a shift in TAM polarization from an M2- to an M1-like phenotype in HCC.

Figure 2.

IFN-α inhibits tumor growth by altering tumor-associated macrophage polarization. (A, B) Representative flow cytometry plots (A) and quantification (B) showing the proportion of M2-like macrophages (F4/80⁺CD206⁺ and F4/80⁺CD11b⁺) in Hepa1-6 orthotopic tumor samples from C57BL/6 mice. (C) Representative immunohistochemistry images (left panels) and quantified data (right panels) of F4/80, CD206, Arg1, and iNOS in liver cancer tissues. Scale bar: 100 µm. Error bars indicate mean ± SEM. *P < 0.05 and **P < 0.01.

To explore the effect of IFN-α on macrophage polarization, we used IL-13 to induce M2-like polarization and then treated the polarized cells with IFN-α. As shown in Figure 3A, we observed a significant upregulation of CD206 in RAW264.7 murine macrophages treated with 20 ng/ml of IL-13 for 72 h that was robustly reduced after IFN-α treatment (10⁴ U/mL). Via qPCR, we found that the mRNA levels of M2 markers, i.e., Arg1 and IL-10, were significantly downregulated, while those M1 markers, i.e., iNOS, IL-1b, and TNFα, were upregulated after IFN-α treatment (Figure 3B). ELISAs demonstrated a reduction in the IL-10 protein level (an M2 marker) in the supernatant of IFN-α-treated macrophages, while the IL-1b level (an M1 marker) was clearly elevated (Figure 3C). In addition to its effect in murine macrophages, IFN-α treatment also significantly inhibited the M2-like shift of phorbol-12-myristate-13-acetate (PMA)-treated human THP-1 cells induced by IL-13 (Figure 3D-F). These results provided further evidence supporting the notion that IFN-α treatment can effectively inhibit M2-like macrophage polarization, thus inducing a shift of TAMs from an M2- to an M1-like phenotype in the tumor microenvironment.

Figure 3.

IFN-α inhibits M2-like polarization of TAMs induced by IL-13 in vitro. A. RAW264.7 cells were treated with IL-13 (20 ng/ml), IFN-α (10⁴ U/mL), or the combination for 72 h. The expression level of the M2 marker CD206 was analyzed via FACS. B. qPCR analysis of the expression levels of M2 markers (Arg-1, IL-10) and M1 markers (iNOS, IL-1b and TNF-α). C. The supernatant IL-10 and IL-1b levels measured via ELISA. D. Human THP-1 cells were treated with IL-13 (20 ng/ml), IFN-α (10⁴ U/mL), or the combination for 72 h. The expression level of the M2 marker CD163 and the M1 marker HLA-DR were analyzed via FACS. E. qPCR analysis of the expression levels of M2 markers (IL-6, IL-10, CCL22, MRC1, and MSR1). F. Supernatant IL-10 and TGF-β1 levels measured via ELISA. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

IFN-α neutralizes the promoting effects of M2-like macrophages on HCC cell proliferation and invasion

M2-like macrophages have been previously shown to promote the progression and metastasis of HCC; therefore, we further explored the effects of IFN-α on the functional roles of macrophages in HCC cell behaviors. Murine (RAW264.7) and human (PMA-treated THP-1) macrophages were treated for 72 h with IL-13, IFN-α, or a combination. The culture medium (CM) was then replaced with fresh medium without serum, and the supernatant was collected 24 h later. The CM from IL-13-treated macrophages significantly enhanced cell viability (Figure 4A and 4D), as well as the colony formation (Figure 4B and 4E), migration, and invasion (Figure 4C and 4F) abilities of HCC cells (Hepa1-6 and Huh7). By contrast, the CM from the IL-13 and IFN-α combination group did not have any significant promoting effects on HCC cells. These results indicated that IFN-α could neutralize the promoting effect of M2-like macrophages on HCC cell proliferation and invasion.

Figure 4.

IFN-α eliminates TAM-promoted HCC proliferation and migration in vitro. RAW264.7 and THP-1 cells were treated with IL-13 (20 ng/ml), IFN-α (10⁴ U/mL), or the combination for 72 h, and the culture medium was replaced with serum-free fresh medium. 24 h later, the supernatant was collected as macrophage-conditioned medium (MCM). A. Hepa1-6 cells were cultured in MCM for 96 h, and cell vitality was determined via Cell Counting Kit-8 assays. B. Hepa1-6 cells were cultured with MCM, and cell proliferation was assessed via colony-forming assays. C. The migration and invasive abilities of Hepa1-6 cells were evaluated via transwell assays 24 h post-treatment. D. Huh7 cells were cultured in MCM for 96 h, and cell vitality was determined via Cell Counting Kit-8 assays. E. Huh7 cells were cultured in CM, and cell proliferation was assessed via colony-forming assays. F. The effects of different MCMs on Huh7 migration and invasion were evaluated via transwell assays 24 h post-treatment.

To identify the effect of IL-13 or IFN-α on Huh7 cells, Huh7 cells were cultured in macrophage-conditioned medium (MCM) with/without macrophages (THP1 cells) treated with IL-13, IFN-α, or their combination (Supplementary Figure 2A-C). We found that IL-13 alone had a promoting effect on tumor proliferation and invasion, but that this promoting effect was more robust in cells co-cultured with macrophages. By contrast, we did not observe a significant pro-tumor effect of IFN-α in Huh7 cells in vitro, due to the lower expression of IFN receptors (IFN-R) in Huh7 cells (Supplementary Figure 2D). We observed a significant difference between the Huh7+MCM (IL-13) group and the Huh7+MCM (IFN-α+IL13) group, suggesting that IFN-α inhibited HCC by reprogramming the polarization of TAMs from M2 to M1 phenotypes rather than via direct effects on HCC.

IFN-α relieves TAM-mediated immunosuppression and increases intratumoral CD8⁺ T cell infiltration during sorafenib treatment

To investigate the mechanism of the synergetic anti-tumor effects of combined IFN-α/sorafenib treatment, we used FACS to examine the proportions of CD4⁺ T cells, CD8⁺ T cells, and TAMs (F4/80⁺/CD11b⁺) in orthotopic liver tumors (Figure 5A, Supplementary Figure 3A, 3B). In orthotopic Hepa1-6 tumors, we found that sorafenib alone induced a significant increase in TAM infiltration without any obvious changes in CD4⁺ T and CD8⁺ T cells, indicating a shift to an immunosuppressive status in the tumor microenvironment. IFN-α treatment significantly inhibited the M2-like polarization of TAMs and increased intratumoral infiltration by CD4⁺ T cells and CD8⁺ T cells. Moreover, treatment with a combination of sorafenib and IFN-α also resulted in a significant reduction in the proportion of TAMs and an increase in the proportion of CD8⁺ T cells, indicating that sorafenib could induce neutralization of the immunosuppressive state in the tumor microenvironment (Figure 5A). Our IHC staining also revealed a decrease in the proportion of F4/80⁺ TAMs and an increase in the proportion of CD8⁺ T cells in Hepa1-6 tumors (Figure 5B, 5C) after combined IFN-α/sorafenib treatment. Via ELISAs, we also evaluated the levels of proteins that serve as markers for activation of tumor-infiltrating CD8⁺ T cells. We found that IFN-α in combination with sorafenib significantly increased the intratumoral levels of IL-2, IFN-γ, and TNF-α compared with their levels in the controls (Figure 5D). IHC staining demonstrated a significant increase in apoptosis (via cleaved caspase-3 and TUNEL staining) and a decrease in angiogenesis (CD34) in the IFN-α/sorafenib treatment group compared with the effects in the other groups (Figure 5B, 5C and Supplementary Figure 4A, 4B).

Figure 5.

IFN-α relieves TAM-mediated immunosuppression and increases intratumoral CD8⁺ T cell infiltration during sorafenib treatment. (A) The proportions of intratumoral CD4⁺ T, CD8⁺ T cells, and TAMs (F4/80⁺/CD11b⁺) were quantified via FACS. (B, C) Representative immunohistochemistry (B) and quantification (C) of cleaved caspase-3, CD8, F4/80 and CD34 in tumors after treatment. Scale bar: 100 µm. (D) The supernatant levels of IL-2, IFN-γ and TNF-α were measured via ELISA. (E) A schematic description of the mechanisms involved in the sorafenib/IFN-α effects presented in this study. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

Our combined findings suggest that IFN-α treatment can neutralize the immunosuppressive status in tumor microenvironments induced by the M2-like polarization of TAMs after sorafenib treatment and increase the proportion of CD8⁺ T cells (Figure 5E).

Discussion

HCC is an aggressive cancer with a very poor prognosis [2]. The outlook for the mortality rate of liver cancer patients is still not optimistic in China [23], and it is also significantly increased in the United States [24]. Even though considerable progress has been made in HCC treatment over the past decades, the current challenges in its treatment are mainly driven by the tendency of patients to have advanced stage cancer and a high incidence of metastasis at the time of diagnosis as well as by a lack of effective treatments. Angiogenesis is considered a hallmark of cancer progression, and blocking the induction of angiogenesis can inhibit tumor growth and metastasis [25]. Sorafenib, the current first line treatment for advanced-stage HCC, is an angiogenesis inhibitor [26,27]; however, therapeutic efficacy of sorafenib for HCC patients is still not satisfactory. Anti-angiogenic therapies that target VEGFR, including sorafenib, promote more invasive and metastatic behaviors in cancer cells [28,29]; therefore, because sorafenib alone is not an effective treatment for HCC patients, it is critical to identify effective combination treatments.

Many studies have revealed that IFN-α has antiviral, immunomodulatory, and anti-proliferative effects on HCC as well as on other types of cancer [30-32]. Several clinical trials have demonstrated that HCC patients derive significant benefits from IFN-α treatment in terms of both overall and disease-free survival [18,19]. IFN-α is often used as an adjunct therapy in the treatment of cancer patients, and its use in combination therapy has been extensively studied in the past decade. In particular, a recent study indicated that a combination of anti-CD20 Ab and IFN-α had a synergistic therapeutic effect in lymphoma treatment [33]. Furthermore, combined treatment with IFN-α and BRAF inhibitor enhanced the anti-proliferative and immunomodulatory effects of IFN-α on BRAF^V600E-mutant melanoma [34]. Patients with untreated metastatic renal cell carcinoma (mRCC) benefited from combined treatment with sorafenib and IFN-α [35]. Here we demonstrated that IFN-α enhances sorafenib’s anti-tumor effect in HCC treatment. In in vivo mouse models, a combination of IFN-α had synergistic inhibitory effects on tumor growth and pulmonary metastasis of HCC without obvious toxicity.

TAMs, a major component of tumor immune microenvironments, are mainly polarized into an activated M2 phenotype [11,20]. Many studies have shown that TAMs can modulate and shape HCC immune microenvironments and promote HCC growth, metastasis, and immune evasion [36-39]. The results of the current study showed that sorafenib treatment elevated TAM infiltration in HCC, resulting in an immunosuppressive HCC microenvironment. Interestingly, IFN-α could inhibit the polarization of macrophages into an M2-like phenotype, thereby neutralizing the promoting effects of M2-like macrophages on HCC cell proliferation and invasion. Moreover, IFN-α relieved TAM-mediated immunosuppression and increased intratumoral CD8⁺ T cell infiltration by shifting macrophages towards the M1 phenotype during sorafenib treatment.

Via its antiangiogenic effects, sorafenib induces a reduction of microvascular density, which can promote intratumoral hypoxia [40]. Accumulating evidence shows that sorafenib induces intratumoral hypoxia, and cellular responses mediated by hypoxia-induced factors can favor selection of resistant cells adapted to the hypoxic microenvironment [41]. Our results demonstrated that sorafenib can induce the macrophage polarization in vivo but not in vitro, and we speculate that this inconsistency is partly due to the hypoxic microenvironment induced by its antiangiogenic effects. Some evidence has shown that hypoxic conditions promote polarization of macrophages toward the M2 phenotype and modify inflammatory microenvironments by decreasing proinflammatory cytokine release [42]. Thus, we conclude that IFN-α can function as an immunomodulator that, when combined with sorafenib, results in increased therapeutic efficacy via a shift in TAM polarization and enhanced CD8⁺ T cells infiltration, thus improving CD8⁺ T cell activation in the HCC microenvironment.

In summary, we have demonstrated here that in HCC treatment, the synergistic anti-tumor effects of a combination of IFN-α and sorafenib are likely mediated by a transition from M2 to M1 macrophages induced by IFN-α. We propose that clinical use of IFN-α and sorafenib combination therapy holds promise for patients with advanced HCC.

Disclosure of conflict of interest

None.