Bidirectional regulatory role of IDO1 in hepatocellular carcinoma：Promotion of Occurrence but Suppression of Progression

doi:10.21203/rs.3.rs-5300369/v1

Aims To investigate the clinical significance of indoleamine 2,3 dioxygenase 1 (IDO1) in the development of hepatocellular carcinoma (HCC).

Methods In vitro, CCK-8, Key Fluor 488 Click-iT Edu ,wound healing, and Transwell assays were performed to explore the effect of IDO1 on the viability, proliferation, migration and invasion ability of HCC cell line SK-HEP1. In vivo, a subcutaneous xenograft tumor model of nude mice was established by subcutaneously inoculating SK-HEP1 and treated with IDO1 inhibitor epacadostat (EPA) to observe the effect of IDO1 on tumor growth and immune cells infiltration. Multiplex immunohistochemical staining was applied to detect the expression level of IDO1 and the infiltration of DCs in the HCC tissue microarray, including total 96 human HCC samples and 82 samples of matched adjacent normal tissues.

Results The in vitro cellular and in vivo animal experiments in this study showed that inhibition of IDO1 expression helped to improve the malignant biological behavior of HCC and enhance the response of immune cells to tumor cells. Results of clinical tissue microarrays showed that compared with poorly differentiated HCC cancer tissues, the expression level of IDO1 in highly differentiated HCC cancer tissues was significantly increased.Meanwhile, compared with corresponding paracancer tissues, the infiltration of mature DCs were significantly reduced in highly differentiated HCC cancer tissues with high expression of IDO1, whereas in poorly differentiated HCC cancer tissues with low expression of IDO1, the infiltration of mature DCs were significantly increased. And IDO1 was highly expressed in HCC cancer tissues with pathological grade I-II, high AFP levels (≥ 200ug/L), HBV-positivie, cirrhosis, distant metastasis and recurrence.

Conclusions IDO1 was abnormally expressed in HCC and was related to the degree of differentiation of HCC and the infiltration of immune cells and it might have bidirectional regulatory role in HCC: Promotion of Occurrence but Suppression of Progression.

Hepatocellular carcinoma

Indoleamine 2

3 dioxygenase 1

Dendritic cells

T cells

Differentiation

Primary liver cancer is a common and serious liver malignant tumor. Globally, its morbidity and mortality rank fifth and fourth respectively^[1]. It is one of the more common and serious diseases in the digestive system. According to the data reported by the National Cancer Center of China, in 2022, the number of new cases of primary liver cancer was 367,700, ranking the fourth among the number of new cases of various cancers and the fifth in the morbidity rate^{[2, 3]}. Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and mixed hepatocellular carcinoma-cholangiocarcinoma (HCC-CC), which are quite different in terms of their pathogenesis, biological behaviours, pathohistology, therapeutic methods, and prognosis, etc. HCC accounts for 75%-85% of the incidence rate of primary liver cancer, and ICC accounts for 10%-15%^{[4, 5]}. Risk factors for the development of HCC include various factors such as viral infection, excessive alcohol consumption, aflatoxin, and metabolic-associated fatty liver disease^{[6, 7]}. Although in recent years, people have made rapid progress in the diagnosis and treatment of liver cancer, it has not been able to effectively improve the prognosis of HCC. For early-stage HCC and ICC limited to the liver, treatment is mainly carried out through surgical resection and ablation. However, the recurrence rate within 5 years after treatment can be as high as 40%-70%^[8]. However, approximately 70% of patients are already in an advanced stage of the disease or have complications when diagnosed and cannot undergo radical surgery^{[9, 10]}. Therefore, exploring the mechanism of occurrence and development of HCC and finding more appropriate molecular markers is still the key research direction at present.

The human indoleamine 2,3-dioxygenase-1(IDO1) gene is located on chromosome 8, has 10 exons, and is about 15 kb in length; the human IDO1 protein is composed of 403 amino acids with a relative molecular mass of about 45 000, and consists mainly of two α-helices and their encapsulated ferrous haemoglobin moieties^[11].Physiologically, IDO1 can be expressed in tissues such as placenta, lung, small intestine, liver, kidney and brain. And it has been reported that IDO1 can be abnormally highly expressed in a variety of tumor tissues, including gastric cancer^[12], esophageal cancer^[13], pancreatic cancer^[14], breast cancer^[15], and lung cancer,etc.^[16]IDO1 is the first rate-limiting enzyme in the pathway that catabolizes tryptophan (Trp) to kynurenine(Kyn).The depletion of Trp will lead to a reduction in humoral immune function, and the accumulation of Kyn induce tolerogenic immune response, which is conducive to the immune escape of tumor cells, so IDO1 has an important role in anti-tumor immune response. Studies have shown that tumor cells, dendritic cells (DCs), tumor-associated fibroblasts, myeloid-derived suppressor cells, and tumor-associated macrophages in the tumor microenvironment can highly express IDO1 and decompose tryptophan through IDO1, inducing the formation of an immunosuppressive microenvironment, which is related to the poor prognosis of various tumors^[17–20].As an important immune organ, the liver has a complex immune microenvironment composed of many kinds of immune cells and intercellular communication-related molecules, which are involved in the immunoregulation of the liver, in which the cells have their own roles but are interrelated with each other. The immune microenvironment not only plays an important role in maintaining liver homeostasis, but also plays an irreplaceable role in the occurrence and development of liver diseases^[21]. It has been reported that IDO1 can be overexpressed in a variety of liver diseases, including alcoholic liver injury, immune liver injury, liver fibrosis, hepatic ischemia-reperfusion injury etc., and is involved in the regulation of the antigen presentation function of intrahepatic DCs and macrophages, the differentiation of T cells, and the secretion of inflammatory factors, which affects the development of liver diseases^[22–26].In fact, IDO1 has a dual regulatory effect on the immune function. Physiological levels of IDO1 are related to the innate immune mechanism that protects the body from pathogenic microbial infections, but the specific mechanism still needs in-depth study. However, overexpressed IDO1 will promote the metabolism of Trp along the Kyn pathway and inhibit the activation of T cells, thereby inhibiting immune function, and this action is one of the important mechanisms for inducing immune escape of tumor cells. In addition, IDO1 also plays a dual regulatory role in different immune cells. For example, IDO1 can inhibit the antigen-presenting ability of DCs and macrophages, which in turn inhibits T-cell activation; whereas in NK cells, IDO1 can significantly increase the killing effect of NK cells on tumor cells and promote the anti-tumor immune response^[27–30].DCs are specialized antigen-presenting cells, which plays an important role in activating naïve T cells and initiating adaptive immune responses, and are an important bridge between innate and adaptive immunity. When the self-regulatory mechanism is impaired and the inflammatory homeostasis is disrupted under the action of external pathogenic factors, uncontrolled inflammation can drive the malignant transformation of the organism to form tumors. However, we still lack an in-depth understanding of the pathological mechanisms and nodal molecules of hepatic inflammatory malignant transformation, and the specific functions and regulatory mechanisms of DCs in it need to be further clarified. Therefore, studying the relationship between IDO1 and DCs will be of great significance and is expected to provide a new strategy for the diagnosis and treatment of HCC.

Here, We conducted in vitro and in vivo experiments to explore the effects of IDO1 on the malignant biological behavior and immune cell infiltration of HCC. Multiplex immunohistochemical staining was used to detect the expression of IDO1 and the infiltration of DCs in HCC patients, as well as analyze the correlation between IDO1 and clinicopathological features of HCC patients

Cell culture and treatment

Human-derived hepatocellular carcinoma cell line (SK-HEP1) were purchased from Wuhan Servicebio Technology Co., Ltd (Hubei, China),and have been authenticated by STR profiling.

Cell lines were cultured in SK-HEP1 cell-specific culture medium (Servicebio, China) in humidiffed cell incubator at 37 ◦C with 5% CO₂. IDO1 inhibitor epacadostat (EPA) was purchased from MedChemExpress LLC (Shanghai, China). According to the reagent instructions, EPA solutions with concentrations of 2.5μM, 5 μM, 10 μM, and 20 μM were prepared using the recommended solvent.Cells were planted in 6-well plates or 96-well plates, and were randomly divided into control group (0μM EPA) and EPA treatment groups with EPA at final concentrations of 2.5, 5,10 and 20 μM in the light of the test data proposed in reference^[31-33].According to different experimental purposes, cells were collected for fixation or extraction of RNA and proteins for subsequent assays after incubation for an appropriate period of time.

Tumor models and treatments

All animal experiments were conducted in accordance with the protocols approved by the Laboratory Animal Ethics Committee of Shenzhen Zhongxun Precision Medicine Research Institute and compliance with the relevant ethical regulations for animal research (Approval number:ZXJZ202306100001). Male BALB/c nude mice, 4 - 6 weeks old, were purchased from

Guangdong Pharmalab Biotechnology Co., Ltd (Guangdong, China) and kept under specific pathogen-free conditions. For in vivo xenograft model established and the IDO1 inhibitor treatment was performed as reported in the literature^{[34, 35]}. SK-HEP1 (4 x10⁶cells/100 μL) in logarithmic growth phase was injected into the right dorsal side of nude mice.Tumor size was measured every 3 days until the end of the experiment, and the tumor volume was calculated according to the formula V = (a × b²)/2, where a is the largest superficial diameter and b is the smallest diameter.When the tumor volume reached approximately 90 mm³, mice were randomly distributed to control and experimental group (five mice in each group), and treatment was initiated. The experimental and control group received intraperitoneal injection of EPA (100 mg/kg) and DMSO, respectively,every 3 days for a total of 3 injections. At the end of the experiment, mice were sacriffced, the blood and fresh tumor tissues were collected and processed for further detection.

Key Fluor 488 Click-iT Edu assay

Effects of IDO1 inhibitor EPA on the proliferation of SK-HEP1 was detected using the Key Fluor 488 Click-iT Edu kit (RiboBio, China) according to the product instructions.

CCK-8 assay

CCK-8 kit (Bioswamp,China) was used to detect the effect of EPA on the viability of SK-HEP1 cells.After co-culturing with medium containing different concentrations of EPA for 24 hours, 10 μL of CCK-8 solution was added to the cells, and the cells were incubated for 4 h at 37 ℃. The absorbance of each group of cells was detected at 450 nm using a multifunctional microplate detector (TECAN Spark, Switzerland). The cell viability was calculated in each group according to the instructions.

Wound healing assay

Total 7×10^⁵SK-HEP1 cells were seeded in a 6-well plate. When the cells reached 85% density, a 10 μL sterile pipette tip was used to draw a straight line vertically on the cell layer. The cells were washed twice with PBS to remove the scratched cell debris. Then fresh complete medium containing different concentrations of EPA was added for culture. The 6-well plates were observed under a microscope and photographed at 0, 24 h and 48 h, respectively.

Transwell assay

SK-HEP-1 cell suspension was prepared by using FBS-free medium and the cell density was adjusted to 1 x10⁶cells/mL. 200 μL of cell suspension was taken from each group and placed on the upper surface of Transwell, 600 uL of complete medium containing 10% FBS was put into the lower surface of each group. After being cultured in a constant temperature incubator for 24 hours, the medium was aspirated from the lower surface of the chamber, and the cells were washed three times with PBS, and the lower surface of each group was fixed with 4% paraformaldehyde for 30 min, 0.1% crystal violet (Beyotime Biotechnology,China ) staining for 30 min. The cells on the upper chamber surface were wiped off. Under the microscope, six fields of view were randomly selected from each group to observe and count the cells.

RNA extraction, cDNA synthesis, and qPCR

Total RNA was extracted from the cells according to the instructions of the RNA Extraction Kit (Fastagen, China), and then cDNA was synthesized by reverse transcription of total RNA in accordance with the instructions of the HiScript III RT SuperMix for qPCR (Vazyme, China ). qPCR was performed with RealStar Green Fast Mixture (GenStar, China) in Applied Biosystems QuantStudio Dx system. GAPDH was used as endogenous control. Sequence-specific primers for the genes detected are listed as following:

GAPDH (human)-F, 5’-AGAAGGCTGGGGCTCATTTG-3’,

GAPDH (human)-R, 5’-GCAGGAGGCATTGCTGATGAT-3’;

E-cadherin (human)-F, 5’-ATTTTTCCCTCGACACCCGAT-3’,

F-E-cadherin (human)-R, 5’-TCCCAGGCGTAGACCAAGA-3’;

Vimentin (human)-F, 5’-AGTCCACTGAGTACCGGAGAC-3’,

Vimentin (human)-R, 5’-AGTCCACTGAGTACCGGAGAC-3’;

IDO1 (human)-F, 5’- GCCTGATCTCATAGAGTCTGGC-3’,

IDO1 (human)-R, 5’-TGCATCCCAGAACTAGACGTGC -3’;

PCNA (human)-F, 5’- AGAGGAGGAAGCTGTTACCATAGAG-3’,

PCNA (human)-R, 5’-CATACTGAGTGTCACCGTTGAAGAG -3’.

Western blot analysis

Total cellular protein was extracted and quantified according to the instructions of Total Protein Extraction Lysate(Beyotime Biotechnology, China) and BCA protein assay kit (EpiZyme, China) . Then total proteins were separated by 10% SDS-PAGE (EpiZyme, China) and then transferred onto a PVDF membrane (Millipore, USA). After membrane transfer, the membrane was blocked for 10 min with NcmBlot blocking buffer (NCM Biotech, China) . After washing with TBST, incubate the primary antibody overnight at 4 °C, including IDO1 (rabbit, CST), E-cadherin (rabbit, CST), Vimentin (rabbit, CST), GAPDH (rabbit, proteintech) and α-tubulin (rabbit, proteintech) , followed by incubation with HRP-conjugated secondary antibodies (rabbit, proteintech). After imaging by chemiluminescence, Quantity One was used to analyze the gray value of the image and calculate the relative expression level of protein.

Multiplex immunohistochemistry analysis

We performed multiple immunohistochemical staining according to the instructions for the Opal 7-color Manual IHC Kit (PerkinElmer ,USA).The tissue microarray (HLivH180Su31) employed in this study, including total 96 human hepatocellular carcinoma samples and 82 samples of matched adjacent normal tissues, was purchased from Shanghai Outdo Biotech Co. Ltd (Shanghai, China). Tissue microarrays were routinely deparaffinized and antigenically repaired. After blocking, primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used for staining sequentially, and stained with one of the seven Opal reagents, followed by microwave intervention to remove the primary and secondary antibodies before another round of staining. Anti-IDO1(rabbit, CST), anti-CD11C (rabbit, Proteintech), anti-CD80 (rabbit, Abcam), anti-CD40 (rabbit, Abcam), anti-MHCII (rabbit, HUABIO) and anti-keratin (rabbit, Abcarta) antibody were used as primary antibodies. The dyes Opal520, Opal650, Opal480, Opal690 , Opal570, Opal620 and DAPI were used for staining. Samples were visualized and analyzed using the Tissue Gnostics (TissueFAXS SpectraS, Peking, China).

Histopathology, immunofluorescence and immunohistochemical staining

For histopathological staining, tumor tissues were fixed with 4% paraformaldehyde for 24 h , routinely dehydrated in gradient, embedded in parafﬁn sections, cut into 4 μm-thick sections. HE staining were conducted according to the reagent instructions (Servicebio, China).

For immunohistochemical staining, we perform routine baking, dewaxing to water for paraffin sections of tumor tissues, antigen repair, blocking endogenous peroxidase. Then the slices were incubated with PCNA antibody(rabbit, proteintech) at 4°C overnight in a wet box.The subsequent steps were carried out according to the instructions of the immunochromatography kit (Servicebio, China).The slides were scaned under the Digital Pathology Slice Scanner (KFBIO, China).

For immunofluorescence staining, before staining, performed routine dewaxing and rehydration, microwave antigen heat repair, and blocking. Next, primary antibodies were added and samples were incubated overnight at 4 °C. After washing with PBS three times, added the corresponding secondary antibodies, incubated at room temperature in the dark for 50 min, followed by staining the nuclei with DAPI (Solarbio Life Science, China) then observed under the fluorescence microscope (Nikon Eclipse Ti-SR, Japan).The following primary antibodies were used: anti-IDO1(rabbit, HUABIO), anti-CD11C (rabbit, Servicebio), anti-CD80 (mouse, Proteintech), anti-CD40 (rabbit, HUABIO), anti-MHCII(rabbit, HUABIO) , anti-CD4 (rabbit, Servicebio) and anti-CD8 (mouse, Servicebio).

Statistical analyses

All data are presented as means ± SEM. Statistical analyses were performed through SPSS22 (IBM Corp, USA) and GraphPad Prism 9 (GraphPad Software, USA). Data were compared using independent-samples t-test for two groups or one-way ANOVA for multi-group variable comparisons. Statistically significant differences were considered at ^*p < 0.05,^**P < 0.01 and ^***P < 0.001.

Inhibition of IDO1 expression could reduce the viability, proliferation, invasion and migration of SK-HEP1 cells in vitro.

To investigate the potential role of IDO1 in the development of HCC, CCK-8, Edu, transwell and wound healing assays were performed to assess IDO1 inhibitor EPA on the viability, proliferation, invasion and migration of human-derived HCC cell line (SK-HEP1) in vitro. As shown in Fig. 2A, 20 μM, 5μM, 2.5 μM EPA could decrease IDOl expression level in SK-HEP1 cells significantly. Treatment with 20 μM and 10μM EPA reduced proliferation level of SK-HEP1 cells dramatically(Fig. 1A-B). Consistently, CCK-8 assays also revealed that 20 μM, 10μM and 5μM EPA could significantly reduce the viability of SK-HEP1 cells(Fig. 1C). Furthermore, through transwell and wound healing assays, we found that, the invasion and migration ability of SK-HEP1 cells in the 20 μM and 10μM EPA treatment group were significantly reduced compared with that in the control group (Fig. 1D, E).These results suggest that the improved malignant biological behavior of SK-HEP1 cells observed were likely to be attributed to the inhibition of IDO1.

Suppression the expression of IDO1 could attenuate the EMT and malignant proliferation of SK-HEP1 in vitro.

To further explore the influence of IDO1 on the malignant biological behaviors of SK-HEP1 cells , we detected the change level of epithelial-mesenchymal transition (EMT) markers in SK-HEP1 cells after inhibiting the expression of IDO1. Results of western blot showed that reducing IDO1 expression caused an increase in the expression of the epithelial marker E-cadherin and a decrease in mesenchymal marker Vimentin as compared with control cells (Fig. 2A, B). We also determined that the RNA level of E-cadherin in EPA treatment SK-HEP1 cells was remarkably increased and Vimentin was decreased as compared with control cells (Fig. 2C). Additionally, proliferating cell nuclear antigen (PCNA) plays a crucial role in the initiation of cell proliferation and is an excellent indicator of the proliferative state of cells. Higher levels of PCNA expression in tumor cells indicate greater proliferative activity and higher degrees of malignancy^[36]. As expected, both PCNA protein and RNA expression level were down-regulated in EPA treatment SK-HEP1 cells as compared with control cells(Fig. 2B, C). In summary, these results indicate that down-regulation of IDO1 expression could alleviate the degree of EMT and malignant proliferation of SK-HEP1 cells.

Down-regulation of IDO1 expression could delay the subcutaneous tumor formation of SK-HEP1 cells in vivo.

Given that inhibition of IDO1 expression could attenuate the malignant biological behavior of SK-HEP1 in vitro, we further established a xenograft tumor model by inoculating SK-HEP1 cells subcutaneously on the right back of nude mice to explore the effect of IDO1 on HCC cell growth in vivo. The experimental procedure is shown in Fig. 3A. When the subcutaneous tumors of nude mice reached a visible volume of 90 mm³, they were randomly divided into control and experimental groups. The experimental and control groups received intraperitoneal injection of EPA (100 mg/kg) and DMSO, respectively, every 3 days for a total of three injections. As reflected in Fig.3D, compared with the control group, the protein expression level of IDO1 was reduced in the tumor tissues of the experimental group significantly intervened with EPA. According to the tumor growth curves in Fig.3B and Fig.3C, both the volume and weight of the tumors in the experimental group of tumor-bearing mice were significantly reduced compared with control group. HE staining of tumor tissues in the two groups revealed that in the control group, the tumor cells were in good growth status, abundant and tightly arranged between cells, with unequal cell sizes and deeply stained nuclei, and there were obvious necrotic foci, which was in line with the morphologic characteristics of tumor cells. In the experimental group, the number of tumor cells were significantly reduced, the length and diameter of the tumor was significantly shorter than that of the control group, and the arrangement between cells was loose (Fig.3E).We have previously found that both PCNA protein and RNA expression level was decreased in EPA treatment SK-HEP1 cells in vitro. The protein expression level of PCNA in tumors were further analyzed by immunohistochemical staining. We detected a signiﬁcant decrease on the expression intensity of PCNA in the tumor tissues from the experimental group compared to those from controls (Fig.3F).Taken together, these data indicated that, inhibition of IDO1 expression could significantly reduce the growth rate of SK-HEP1 cells in vivo and delay SK-HEP1 xenograft tumor growth.

Inhibition of IDO1 expression could enhance immune cells response to tumor cells in vivo.

It is known that the tumor immune microenvironment is closely related to the occurrence and development of tumors. Our previous research have shown that IDO1 can be used as a key biological target for liver fibrosis, the early pathological process of HCC.By mediating the Trp metabolic pathway, IDO1 could inhibit the maturation of the immune phenotype of DCs, affects the immune microenvironment in the liver, and participates in regulating the process of liver fibrosis^[25]. Therefore, we reasoned that IDO1 inhibition may be able to enhance immune cells response to tumor cells. In this study, immunofluorescence staining was further performed to compare the infiltration of DCs and T cells in the tumor tissue microenvironment between the two groups. We then verified that a increased proportion of inﬁltrated CD11C⁺, MHCII⁺,CD80⁺,CD40⁺DCs cells, and CD4⁺, CD8⁺T cells were observed in tumor tissues after administrating EPA (Fig. 4A, B), indicated that IDO1 was involved in the regulation of DCs and T cells response to tumor cells.

Expression level of IDO1 in HCC tissues correlated with the degree of differentiation.

To evaluate the possible implication of IDO1 in the occurrence and development of HCC, We further applied immunofluorescence staining to detect the expression level of IDO1 in the HCC tissue microarray, including total 96 human HCC samples and 82 samples of matched adjacent normal tissues.Of note, we failed to detect any difference in the fluorescence intensity of IDO1 in HCC tissues and paracarcinoma tissues (Fig. 5A), which was inconsistent with our experimental results in vitro and in vivo.But interestingly, we divided the patients into highly, moderately, and poorly differentiated group according to the degree of differentiation in pathological diagnosis, and then compared the differences in the fluorescence intensity of IDO1 among these groups, we found that the fluorescence intensity of IDO1 increased with the degree of differentiation of HCC (Fig. 5B). In poorly differentiated HCC, the fluorescence intensity of IDO1 in cancer tissues was lower than that in adjacent tissues (Fig. 5C,F). In moderately differentiated HCC, there was no significant difference in the fluorescence intensity of IDO1 between liver cancer and adjacent tissues (Fig. 5D,G) and in highly differentiated HCC, the fluorescence intensity of IDO1 was significantly increased compared with that in paracancer tissues (Fig. 5E, H).These observations suggest IDO1 might has a bidirectional effect in the occurrence and development of HCC: it may play a pro-cancer role in the process of oncogenesis and an inhibitory role in the process of malignant progression, and the relevant mechanisms need to be further clarified.

Relationship of IDO1 expression with clinicopathologic and prognostic features of HCC.

To define the potential functional role of IDO1 to the outcome of HCC, we further studied the association of IDO1 expression with clinicopathologic and prognostic features in HCC. As displayed in Fig.6A, there was no remarkable association between IDO1 expression and age, gender, tumor size, HBcAb. Similarly, IDO1 expression was lower in HCC cancer tissues with pathologic grade III-IV compared to those with pathologic grade I-II.Moreover, HCC tissues from patients with high AFP levels (≥200ug/L), HBV-positive, cirrhosis, metastasis, and relapse presented higher expression levels of IDO1 compared with those from patients with low AFP levels (< 200ug/L), HBV-negative and the absence of cirrhosis, metastasis, and relapse.

Infiltration of DCs in HCC cancer tissues with different degrees of differentiation.

Based on the above experimental results, we next evaluated the infiltration of DCs based on the degree of differentiation in HCC. As shown in Fig.6B, in the HCC cancer tissues from highly differentiated HCC patients, the percentage of CD11C⁺MHCII⁺, CD11C⁺CD80⁺ and CD11C⁺CD40⁺DCs were significantly lower than those of the corresponding paracancer tissues. In the HCC cancer tissues from patients with moderately differentiated, there was no significant difference between CD11C⁺MHCII⁺ and CD11C⁺CD40⁺DCs, except that the percentage of CD11C⁺CD80⁺DCs was significantly lower than that of the corresponding paracancer tissues. Proportions of CD11C⁺MHCII⁺ and CD11C⁺CD40⁺DCs were significantly higher than those of the corresponding paracancer tissues, whereas there was no significant difference in the proportions of CD11C⁺CD80⁺DCs, in HCC cancer tissues from patients with poorly differentiated HCC. Together, these results suggest that IDOl may be responsible for establishing the immune tolerance mechanism of HCC and affect the prognosis of patients with HCC.

Analysis of risk factors affecting the prognosis of patients with HCC.

In order to understand the correlation between various clinicopathological parameters, the expression level of IDO1 and the overall survival rate of HCC patients, we conducted univariate and multivariate Cox proportional hazards model analyses on these factors. Univariate Cox regression proportional risk model analysis revealed that CD11C⁺MHCII⁺DCs , CD11C⁺CD80⁺ DCs , CD11C⁺CD40⁺ DCs and HBcAb were protective factors affecting the prognosis of HCC patients. Age and distant metastasis were risk factors for poor prognosis in patients with HCC. Multifactorial Cox regression proportional risk model analysis confirmed that age and distant metastasis were independent risk factors for poor prognosis in HCC patients (Table 1).

HCC is an invasive primary liver tumor, most commonly associated with chronic liver inflammation or cirrhosis. HCC has an insidious onset, lacks effective early diagnostic methods, as well as being easy to metastasize and recurrence, so surgical resection or transplantation is still the most effective treatment for HCC patients.However, HCC patients are often in an advanced stage or have distant metastasis at the time of diagnosis and have lost the opportunity for surgery. Even after surgical resection, the 5-year recurrence rate is as high as 50%-70%, and about 50% of HCC patients relapse within 2 years and lack effective therapeutic drugs. Therefore, at present, there is still a lack of effective treatment for HCC, and the overall 5-year survival rate of HCC patients is still less than 20%^{[37, 38]}.This is also mainly due to the fact that the molecular pathogenesis of HCC has not been fully clarified, and there is a lack of sensitive early diagnostic and surveillance methods. Therefore, it is crucial to elucidate the molecular mechanisms of HCC development and to explore reliable biomarkers.

Previous studies have reported that IDO1 is aberrantly expressed in various types of tumors, including colorectal cancer, gastric cancer, lung cancer and so on, and it often indicates a poor prognostic factor^[39–41]. Our previous studies have shown that IDO1 is highly expressed in the pre-pathological processes of HCC, including liver injury, liver fibrosis and cirrhosis, and it plays a role in exacerbating the pathological process during the development of these diseases^{[25, 26, 42]}.

Therefore, we suspected that IDO1 might also be involved in the development of HCC and carried out relevant cellular, animal and clinical experiments for verification. First, we explored the effects of IDO1 on the cell viability, proliferation, migration invasion ability and EMT of HCC cell line SK-HEP1 in vitro. Our results showed that the IDO1 inhibitor EPA significantly suppressed the cell viability, proliferation, migration, invasion ability and EMT of SK-HEP1, suggesting that inhibition of IDO1 expression could improve the malignant biological behavior of SK-HEP1. Then, SK-HEP1 was further selected in this study to establish a subcutaneous xenograft tumor model in nude mice to explore the effect of IDO1 on the tumorigenic ability of HCC cells in vivo. PCNA is a cell cycle regulatory protein related to cell proliferation level that exists in eukaryotes and plays an important role in cell events such as DNA replication, damage repair, and chromatin remodeling. As reported in the literature^{[43, 44]}, PCNA is abnormally highly expressed in various cancer tissues such as laryngeal cancer, esophageal cancer, gastric cancer, colorectal cancer, and bladder cancer. Moreover, the expression level is closely related to the degree of malignant proliferation, invasive metastasis, and prognosis of tumors. Moreover, the higher the expression level of PCNA, the higher the histological type of HCC and the more serious the condition. PCNA can be used as a biomarker for predicting poor prognosis in HCC patients^[45–47]. In this study, immunohistochemical staining of PCNA was performed on tumor tissues of nude mice. Results showed that PCNA was highly expressed in tumors. After inhibiting the expression level of IDO1, the expression level of PCNA in tumor tissues could be reduced suggesting that down-regulating the expression of IDO1 can inhibit the proliferation of HCC cells in vivo and delay the growth rate of subcutaneous tumors, which is consistent with the results of in vitro experiments.

As early as 1863, Rudolf Virchow, a German pathologist, had already put forward the theory that tumors may originate from chronic inflammation. Now, many scholars have pointed out that there exists a malignant feedback loop between inflammation and tumor, in which "inflammatory microenvironment → malignant transformation (tumor) → tumor cells secrete inflammatory factors autocrinally and recruit inflammatory cells → create inflammatory microenvironment" ^[48–50]. As a typical inflammation-associated tumor, the immune microenvironment is particularly critical in the pathogenesis of HCC and is closely related to the poor prognosis of HCC patients

Therefore, exploring the hepatic immune microenvironment as a whole and searching for key molecules regulating the hepatic immune microenvironment will not only enrich the theoretical basis of immunity and tumor pathogenesis, but also provide important target sites for the prediction, diagnosis and drug development of HCC. Studies have shown that IDO1 was an enzyme with both catalytic function and immunomodulatory effect. It can intervene in Trp-Kyn metabolism in the microenvironment and regulate the function of DCs and T cells, which play an important role in regulating liver immunity and inflammatory responses^{[22, 51]}. Elevated IDO1 expression levels result in Trp deficiency and enrichment of the toxic metabolite Kyn, which contribute to malignant tumors evading host immune killing, as well as inhibiting the production and activation of DCs, T cells, NK cells and regulatory T cells, and promoting tumor angiogenesis. In recent years, major advances have been made in the immunological mechanisms related to tumor pathogenesis. Multiple immunotherapies, especially those that can improve and restore the function of T cells, have begun to be applied in clinical treatment^{[52, 53]}. In this study, we further analyzed the changes of DCs and T cell subsets in SK-HEP1 HCC-bearing mice after down-regulation of IDO1 expression. Results showed that the markers of DCs, CD11C and surface co-stimulatory molecules, MHCII, CD80, CD40, as well as CD4⁺ T cells and CD8⁺ T cells, were in low numbers in cancer tissues, whereas down-regulation of the expression of IDO1 could increase the infiltration of these immune cells in cancer tissues, suggesting that inhibition of the expression of IDO1 may be able to enhance the response of immune cells to tumor cells.

To further verify the role of IDO1 in the occurrence of HCC, we further detected the expression level of IDO1 in clinical HCC tissue samples and its relationship with clinicopathological features. Our data showed that IDO1 presented different expression patterns in HCC with different degrees of differentiation. Compared with paracancer tissues, IDO1 was highly expressed in highly differentiated HCC and lowly expressed in poorly differentiated HCC. In moderately differentiated HCC, there was no significant difference in the expression level of IDO1 between cancer tissues and paracancer tissues. And, the expression level of IDO1 was significantly higher in highly differentiated HCC cancer tissues compared with that in poorly differentiated HCC cancer tissues. Meanwhile, we found that compared to corresponding paracancer tissues, the infiltration of mature DCs, including CD11C⁺MHCII⁺ DCs, CD11C⁺CD80⁺ DCs, CD11C⁺CD40⁺ DCs, were significantly reduced in highly differentiated HCC cancer tissues with high expression level of IDO1, whereas the infiltration of mature DCs were significantly increased in the poorly differentiated HCC cancer tissues with low expression level of IDO1. We further analyzed the relationship between the expression level of IDO1 and the clinicopathological and prognostic characteristics of HCC.Results showed that IDO1 was highly expressed in HCC cancer tissues with pathological grade I-II, high AFP levels (≥ 200ug/L), HBV-positivie, cirrhosis, distant metastasis and recurrence.Most undifferentiated/poor-differentiated carcinomas are derived from the malignant progression of high-differentiated carcinomas, which is a process of tumor dedifferentiation and may be a process of cumulative mutations of malignant tumor-related genes.Based on our previous research results^{[24–26, 42]}, IDO1 was highly expressed in the early pathological processes of HCC, including liver injury, liver fibrosis, and cirrhosis suggesting that IDO1 has a complex function in the development of HCC and may have a bidirectional effect on the malignant biological behavior of tumors, which playing a role in promoting HCC occurrence and inhibiting HCC malignant progression, but the relevant mechanisms need to be further clarified.

Collectively, the in vitro cellular and in vivo animal experiments in this study showed that inhibition of IDO1 expression helped to improve the malignant biological behavior of HCC and enhance the response of immune cells to tumor cells. Results of clinical tissue microarrays suggested that IDO1 was abnormally expressed in HCC and correlated with the degree of differentiation and infiltration of immune cells in HCC, but the related potential mechanisms still need more research in the future.This study has provided a foundation for further research on IDO1 to the HCC immunity and clinical treatment. However, due to limited data information, further analysis of prospective clinical data and experimental data verification of the mechanism of action and drug therapy about IDO1 in HCC are still needed in the future to confirm its guiding significance as a guide for clinical application.

Declaration of competing interest

The authors declare no conflict of interest.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

This work was supported by the Science and Technology Planning Project of Guangdong Province (2021A1515110861) , National Natural Science Foundation of China (82204841) and

Shenzhen Medical Research Fund (A2303020).

CRediT authorship contribution statement

All authors have made substantial contributions to all of the following: Chan Mo and Xiuming Zhang, the conception and design of the study; Chan Mo and Liu Yuan, acquisition of data; Hong Min, analysis and interpretation of data; Chan Mo,Li Yunjia and Huang Danping, drafting the article; Dan Xiong, revising the article critically for important intellectual content; Chan Mo, Dan Xiong , and Xiuming Zhang, final approval of the version to be submitted.

Ethics approval statement

All animal experiments were conducted in accordance with the protocols approved by the Laboratory Animal Ethics Committee of Shenzhen Zhongxun Precision Medicine Research Institute and compliance with the relevant ethical regulations for animal research (Approval number:ZXJZ202306100001).

Consent for publication

N/A.

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Table1 Univariate Cox regression proportional risk model analysis

Variables	Univariate analysis			Multivariate analysis
Variables	HR	95%CI	P value	HR	95%CI	P value
CD11C⁺MHCII⁺DCs	0.61	0.39-0.96	0.0306	0.79	0.47-1.34	0.383
CD11C⁺CD80⁺DCs	0.60	0.36-0.98	0.0404	0.67	0.32-1.39	0.283
CD11C⁺CD40I⁺DCs	0.64	0.41-0.10	0.048	1.17	0.58-2.34	0.661
sex	0.80	0.42-1.51	0.486	/	/	/
age	2.07	1.30-3.30	0.00204	2.11	1.28-3.48	0.00358
Tumor_size	1.09	0.70-1.70	0.698	/	/	/
T	1.49	0.93-2.39	0.0966	/	/	/
N	7.41	0.96-56.95	0.0544	/	/	/
M	26.84	5.29-136.10	0.0000715	26.04	4.58-147.98	0.000235
grade	2.23	1.40-3.57	0.000818	1.43	0.82-2.48	0.209
TNM	1.49	0.93-2.39	0.0966	/	/	/
Distal_metastases	26.84	5.29-136.11	0.0000715	/	/	/
Lymph.node_metastasis	7.41	0.96-56.95	0.0544	/	/	/
Cirrhosis	1.08	0.68-1.71	0.757	/	/	/
HBsAg	1.37	0.86-2.16	0.184	/	/	/
HBcAb	0.50	0.28-0.87	0.0138	0.55	0.3-1.03	0.0606
HCVAb	0.94	0.23-3.86	0.934	/	/	/
AFP.ug.L.	1.41	0.91-2.20	0.126	/	/	/

No competing interests reported.

Bidirectional regulatory role of IDO1 in hepatocellular carcinoma：Promotion of Occurrence but Suppression of Progression

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Abstract

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Introduction

Materials and methods

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