D-MT Prompts the Anti-Tumor Effect of Oxaliplatin by Inhibiting IDO Expression in A Mouse Model of Colon Cancer

Colon cancer is one of the most common malignant tumors in the digestive system. Although oxaliplatin, a chemotherapy drug, has been clinically used to treat colon cancer, its therapeutic effect is unsatisfactory. It has been proved that indoleamine dioxygenase 2,3 (IDO), an immune checkpoint, is a result of tolerance to chemotherapy. Herein, an IDO inhibitor, D-MT (indoximod, 1-Methyl-D-tryptophan), was combined with oxaliplatin to treat colon cancer in mice. T cell infiltration in tumor tissues, the ratios of immune cells in the spleens, and the tumor growth and survival of the mice were detected and recorded. The results showed that the combination of oxaliplatin and D-MT significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. More importantly, the combination treatment increased the ratios of CD4+ T, CD8+ T and NK cells from the spleen in tumor-bearing mice, and prompted T cell infiltration in tumor tissues. This study provided a new therapeutic strategy for colon cancer treatment in the clinic, especially for patients with oxaliplatin resistance.


Introduction
Colon cancer is one of the most common malignant tumors of the gastrointestinal tract, with its incidence rising. Worldwide, colon cancer ranks the third in the incidence of male malignancies and fourth in mortality, and ranks the second in the incidence of female malignant tumors and third in mortality 1,2 . In 2018, about 1.8 million colon cancer cases were diagnosed, with 881,000 patients dying of the disease. In the last decade, the incidence and mortality rates of colon cancer have been increasing in certain countries, such as China 1 . Colon cancer is a major burden for patients worldwide. At present, the main treatment for colon cancer is surgery, with chemotherapy often performed following surgery for patients with metastasis. The response rate of chemotherapy could reach up to 50%, but drug resistance has been reported in nearly all patients with colon cancer 3 . Therefore, it is important to find a more effective treatment.
Oxaliplatin is a new anticancer platinum-based drug commonly used in the treatment of metastatic colorectal and liver cancer. Due to the heterogeneity of a tumor, a single drug often does not achieve satisfactory results. As reported, oxaliplatin treatment would lead to a reduction in macrophages and raised the expression of HMGB1 related to immunosuppression in the colon 4 . Therefore, studies have been trying to combine oxaliplatin with other therapies to treat tumors, improving the therapeutic effect of oxaliplatin [5][6][7] . Studies have shown that Huaier can effectively improve the anti-tumor effect of oxaliplatin by downregulating the expression of YAP protein which located in cell nucleus and related with cell proliferation, apoptosis and migration 8 . With the development of immunology, tumor immunotherapy has received more and more attention 9 . It has been found that anti-tumor immunity in colon cancer patients is inhibited to varying degrees, and thus enhancing the anti-tumor immune response may become an effective means of treating colon cancer. Consequently, chemical and immune combination treatment has become a new approach to cancer treatment 10 .
Studies have confirmed that the tumor microenvironment plays an important role in the tumor development stage 11,12  IDO is a rate-limiting enzyme of the immunosuppressive tryptophan (Trp)/kynurenine (Kyn) metabolic pathway, including IDO1 and IDO2, which are highly expressed by tumor tissues, thereby, inhibiting T cell anti-tumor immunity. IDO has been found to be highly expressed in several types of human cancer 16,17 , such as melanoma [18][19][20] , and colon 21,22 , brain 23 and ovarian cancer 24 . In tumors such as acute myeloid leukemia [25][26][27] , the high expression of IDO inhibits anti-tumor immunity; therefore, IDO can serve as a new target for the treatment of tumors 28,29 . A variety of IDO inhibitors have been approved for clinical trials by the US FDA. D-MT has been shown to be effective in inhibiting the expression of IDO 30 . A previous study found that the inhibition of the IDO expression in melanoma-bearing mice enhances the anti-tumor effect of pimozide 31 , but it remains unclear whether IDO inhibition can promote the anti-tumor effect of oxaliplatin by increasing the anti-tumor immune response in tumor-bearing mice.
The present study explored the therapeutic effect of oxaliplatin combined with D-MT, an IDO inhibitor, on colon cancer, as well as the underlying mechanism. This study will provide a new method and experimental and theoretical basis for the clinical treatment of colon tumor.

Combination treatment with D-MT + oxaliplatin significantly inhibits IDO and p-Stat3 expression in CT-26 cells
To detect the effectiveness of D-MT + oxaliplatin on the expression levels of IDO and p-Stat3 in colon cancer cells, the dosage of D-MT or oxaliplatin was 2.5 μg/ml (the data not shown). As shown in Fig. 1 indicating that combination treatment has the synergic action.

Combination treatment with D-MT + oxaliplatin significantly increases T cell infiltration in tumor tissues
It has been shown that the activation of IDO in the tumor microenvironment can impair the survival and function of T cells 32 . it was therefore detected herein that the T cells infiltrated tumor tissues by IF. The data showed that single-agent treatment with oxaliplatin or D-MT increased CD4 + and CD8 + T cell infiltration in tumor tissues ( Fig.   5a-d). Of note, as compared with other groups, combination treatment with D-MT + oxaliplatin could significantly increase CD4 + and CD8 + T cell infiltration, indicating that combination treatment could significantly prompts the survival of T cells in tumor-bearing mice.

Combination treatment with D-MT + oxaliplatin significantly increases the ratio of immune cells in spleens from tumor-bearing mice
In order to determine whether combination treatment improved the whole anti-tumor effect of tumor-bearing mice, the ratio of immune cells in the spleen, the largest peripheral immune organ in the body, was detected. The results of flow cytometry showed that the ratio of CD8 + T and NK cells were increased in the spleens of mice treated with D-MT ( Fig. 6b-f). Although treatment with oxaliplatin did not significantly raised the ratio of immune cells (Fig. 6 a-f), but the ratio of CD4 + T, CD8 + T and NK cells were significantly raised in mice treated with D-MT and oxaliplatin, as compared with the D-MT or oxaliplatin group (Fig. 6 a-f).

Combination treatment with D-MT + oxaliplatin significantly increases the concentration of TNF-α or IFN-γ in the sera of tumor-bearing mice
Finally, the concentration of TNF-α or IFN-γ was detected to play an anti-tumor role.
The ELISA results showed that, as compared with the PBS group, the concentration of TNF-α or IFN-γ in the sera of tumor-bearing mice in the D-MT, oxaliplatin and combination groups was raised. Of note, the concentration of TNF-α or IFN-γ in the combination group was higher than that in other groups (Fig. 7).

Discussion
Colon cancer is a malignant tumor that seriously endangers human health in several countries. Despite the existence of certain drugs for the treatment of colon cancer, their therapeutic effect is generally low. Colon cancer treatment has therefore been attracting more and more attention. In the present study, it was determined that the D-MT, an IDO inhibitor, prompted an anti-tumor effect of oxaliplatin and strengthened the immune response against tumor in mice.
Oxaliplatin, a chemotherapy drug, is the standard first-line treatment for colon cancer in the clinic 33,34 . Unfortunately, clinical treatment often fails due to oxaliplatin resistance, which is associated with complex mechanisms, such as DNA adduct repair, cell death mechanisms and autophagy 35 . In addition, it was found shown that immunosuppressive mechanisms, such as the immune checkpoint or immunosuppressive factors, were also important reasons for drug resistance 36  However, despite the several factors that affect the occurrence of tumors, the therapeutic effect of oxaliplatin was limited and unsatisfactory 43 . One of the reasons is that the tumors that develope to cause immune suppression in the body, such as overexpression of the immune checkpoint 36 . IDO has been proven to catalyze the oxidative catabolism of Trp to Kyn, and regulate immune responses by impairing the survival and activity of T cells 44,45 . IDO has therefore become a therapeutic target for cancer that could either be used alone or in combination with other treatments for tumors 46,47 . However, our results showed that oxaliplatin could inhibit tumor growth and increase the apoptosis of tumor cells, but could also increase the expression of IDO, which might interfere with the therapeutic effect of oxaliplatin. We therefore detected the therapeutic effect of combination treatment with D-MT and oxaliplatin.
D-MT has been proven to effectively inhibit the IDO expression 48 and enhance the anti-tumor immune response. D-MT might play a role in disrupting tumor immune escape 30,49,50 . In fact, the present study determined that, even though oxaliplatin increased the IDO expression, the protein level of IDO could also be reduced in mice Furthermore, the present results showed that the combination treatment with D-MT + oxaliplatin not only increased T cell infiltration in tumor tissues, but also raised the ratio of T cells in the spleen. They also indicated that this strategy could strengthen the anti-tumor immune response in tumor-bearing mice. T cells were found to play an important role in tumor immunity 55 and inhibit the expression of IDO, which could reverse CD8 + T cell suppression in breast cancer cell-bearing mice 56 . Interfering with the activity of IDO could lead to strengthening the anti-tumor immune response by increasing the amount of CD8 + T lymphocyte infiltration 57 . In addition, the present data showed that combination treatment with oxaliplatin and D-MT increased the ratio of NK cells, which was also an important reason against tumor. This might be associated with the inhibition of IDO, whose activation could lead to the downregulation of the function of NK cells 58,59 .
In the present study, it was confirmed that D-MT could strengthen the anti-tumor effect of oxaliplatin, which might be associated with the inhibition of IDO.

Combination treatment with oxaliplatin and D-MT significantly inhibited tumor
growth and prolonged the survival of tumor-bearing mice. Of note, the combination strategy could prompt the anti-tumor immune response by increasing T cell infiltration in tumors. This study might provide a new therapeutic strategy for colon cancer treatment in the clinic, particularly in patients treated with oxaliplatin, in which treatment was ineffective.

Methods
All animal studies were carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org/) as detailed below.

Animal experiments
The CT-26 cells were adjusted to 1x10 7 cells/ml, and a 0.1-ml cell suspension was

Immunofluorescence (IF) detection
Indirect IF was performed as previously described 60 . Briefly, the tumor slides were incubated with the appropriate antibody (CD3, CD4 and CD8) at 4˚C overnight in a wet box. The tumor slides were restored to 25˚C at least 30 min after being incubated with the secondary antibody for 30 min. Finally, the slides were stained with DAPI and the images were captured using a fluorescence or a laser confocal microscope. The intensities of positive cells were analyzed using a scale of 0-3+: 0, no staining identified; 1+, <25% positive cells; 2+, 25-75% positive cells; 3+, >75% positive cells.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)
The cell apoptosis in tumor tissues was detected by TUNEL assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, the TUNEL detection solution was dropwise added onto the surface of tumor section.

Flow cytometry
The concentration of spleen cells was adjusted to 1x10 7 cells/ml, and each tube was added into 100 μl cell suspension. The cells were incubated with antibodies for CD3, CD4, CD8 and CD49b (BioLegend, Inc.) at 4˚C for 30 min. The ratio of cells was detected using flow cytometry (CytoFLEX; Beckman Coulter, Inc.).

Enzyme-linked immunosorbent assay (ELISA).
One week after the last treatment, the mice in each group were sacrificed and the sera collected. The concentration of tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ) was analyzed by ELISA kits (RayBiotech), according to the manufacturer's instructions.

Statistical analysis
Measurement data are expressed as the mean ± SD of three independent experiments. Data were analyzed by SPSS 19.0 (IBM Corp.). One-way ANOVA was performed to test the difference among the different groups, and the Kaplan-Meier method with a log-rank test was used to analyze survival. P<0.05 were considered to indicate a statistically significant difference.