1-Methyl-D-tryptophan Reduces Tumor CD133+ cells, Wnt/β-catenin and NF-κβp65 while Enhances Lymphocytes NF-κβ2, STAT3, and STAT4 Pathways in Murine Pancreatic Adenocarcinoma

1-Methyl-D-tryptophan (1-MT) is extensively utilized in preclinical trials to deplete indoleamine 2,3-dioxigenase (IDO) activity and kynurenine pathway. Since IDO related signaling pathways aren’t well understood, some clinical reports affirmed IDO inhibiting therapeutic significance. Therefore, we did use direct tumor autologous antigens vaccination and 1-MT without chemotherapy to explore biological mechanisms and immunomodulations of 1-MT that motivate antitumor responses. However, DCs antigen-uptake capability, anti-tumor efficiency, intra-tumor and intracellular cytokines were assessed. Besides, CD133+ cells viability and tumor biomarkers were investigated. Splenocytes responses and their signaling pathways such TLRs 2 to 9, NF-κβ1-2, Wnt/β-catenin and TGF-β were dissected. Results evinced that a regimen of 1-MT and TAAs significantly reduced CSC CD133 + viability inside tumor microenvironment, besides increasing tumor cells necrosis and apoptosis. Expression of TGF-β, IDO, RANTES, and PDL-1 was also significantly reduced. Interestingly, 1-MT enhanced lymphocytes TLR2, TLR7, TLR8, and TLR9 pathways. It motivated lymphocytes’ NF-κβ2, STAT3, and STAT4 pathways, while reduced tumors’ NF-κβp65 and Wnt/β-catenin signaling pathways. We found that periphery and intra-tumor Treg cells were significantly decreased. In conclusion, depletion of indoleamine 2,3-dioxigenase activity evidenced IDO relation with tumor stem cells proliferation pathways. Furthermore, 1-MT supports immunotherapeutic vaccines susceptibility and tumor specific targeting by reducing tumorgensis signaling pathways.

projects consider IDO work motifs, which could explain clear working itinerary of IDO related pathways to help identification of critical therapeutic target. Because, a lot of human aggressive cancers such as pancreatic, colorectal, lung, ovarian, prostatic, cervical, gastric, head, etc. demonstrated high expression of this enzyme or tryptophan metabolic derivatives 13,14 during tumor development stage.
Furthermore, a study of IDO-tryptophan catabolism and immune mechanisms in the tumor microenvironment is cloudy and needs more understanding 2 , subsequent studies represented that IDO has multiple immunological functions. It promotes different signaling pathways to suppress T-cell responses, and enhancing Treg cells 15,16 . Collecting all these details together, we find that IDO knockout or inhibition without chemotherapy applications could represent IDO biological signaling pathways in tumor cells and lymphocytes. Nevertheless, IDO relation with immune cells is also complicated and needs deep understanding. It mainly produced from Mesenchymal stromal cells, endothelial cells, fibroblasts, and myeloid-derived antigen-presenting cells 17,18 . Recent reports conducted that mature and immature DCs rather than plasmacytoid DCs could overexpressed IDO in some metastatic cancers 19,20 . Thus signaling pathways involved IDO during tumor related immunomodulations are versatile and poorly understood. Moon Yong et al. 21 revealed that IDO provokes tryptophan shortage, which leads to mechanistic target of rapamycin inhibition (mTORC1) and general control non-depressible 2 (GCN2) activation, that causes anergy of Th1. Meanwhile, bioactive kynurenine pathway stimulates Aryl hydrocarbon receptor (AHR), resulting in promotion of Treg differentiation 21 . Moreover, It was thought that a mechanism of tryptophan degradation happened by base-catalyzed abstraction 22 , while recently was concluded that this mechanism involves formation of a transient ferryl 23 . Whereas, Li, Fang xuan et al. 24 summarized that IDO1 could induces most of immune cells participations. Particularly APCs and cancer related cells through NF-κβ canonical and non-canonical, Jak/STAT pathways, PKC and TGF-β signaling pathways 25 .
Therefore, we blocked IDO activity by using 1-Methyl-D-tryptophan, and used a biological vaccine (tumor autologous antigens) to explore clear evidences regarding virtual work mechanisms and pathways ascribed to IDO activity, without chemotherapy that could confuse results interpretation, because of its harmful side effects 26 .
We considered pancreatic adenocarcinoma model in the mice, because recently it's one of the most aggressive cancers in the world, which showed high rates of IDO overexpression associated tumor aggressiveness 27,28 .

TL efficiently activates DCs maturation and T cell during 24 hours. The analysis of DCs tumor anti-
gens (TL) uptake potential showed that DCs significantly pulsed in response to tumor lysate in comparison to PBS. It presented high maturation responses; CD40 + CD80 + cells that were gated from CD11c gate represented 74.7% under the impact of TL in comparison to PBS 21.9% (Fig. 1A). Meanwhile, MHCII, CD40 and CD80 genes expression showed significant increasing in comparison to PBS (Fig. 1B); these findings suggested that DCs effectively recognized tumor antigens and efficiently presented to T cell. On the other hand, the determination of optimal time point to activate T cell kinetic and polarization toward tumor antigens showed that accumulation of intracellular INF-γ+ in CD3 + CD4 + cells was increased during 9 hours to reveal 3.15%, but a significant level of INF-γ was detected after 24 hours 6.35% as represented in (Fig. 1C); whereas the accumulation of INF-γ in CD3 + CD8 + cells was also showed significant raising after 24 hours 7.65% as seen in (Fig. 1D). Later, we confirmed these results by analysis INF-γ + gene expression in splenocytes by qRT-PCR, which showed a significant expression of INF-γ + after 24 hours of incubation (Fig. 1E).

1-MT and Tumor lysate inhibit tumorgensis and elicit tumor inflammatory necrosis. Subsequently,
in vivo tumorgensis progression dynamics were significantly inhibited under 1-MT+TL, and 1-MT+DCs-TL regimens effect. We noted that tumor growth of 1-MT, 1-MT+TL, and 1-MT+DCs-TL groups was significantly reduced in comparison to PBS group (2.16 mm 3 /1.30 g, 1.68 mm 3 /1.12 g, 2.14 mm 3 /1.27 g, and 2.5 mm 3 /2.51 g) respectively ( Fig. 2A,C,D). Moreover, the survival rate of 1-Mthyl-D-tryptophan treated groups were significantly improved (88days/38.64%, 78days/30.77%) respectively in comparison to PBS (54days) as showed in (Fig. 2B). Furthermore, the analysis of tumor histology showed that 1-MT and 1-MT+TL significantly elicited inflammations and tumor necrosis inside tumor tissue more than 1-MT+DCs-TL ( Fig. 2E(a,b,c)). These results indicated that 1-MT effectively enhanced the performance of TL vaccine to elicit lymphocytes responses against tumor. Also, pathologist report described destruction of tumor stromal fibers in groups treated by 1-MT which illustrated an interesting impact of 1-MT on tumor fibrosis.

1-MT and 1-MT+TL effectively reduces tumor fibrosis. Tumor fibrosis was critically assessed by using
Picro-Sirius red staining technique (PSR) to determine tumor collagen fibers area under 1-MT effect; stained sections showed that 1-MT, 1-MT+TL and 1-MT+DCs-TL groups significantly reduced collagen fibers distribution around tumor tissue in comparison to PBS, it measured 1.77%, 0.99%, 2.69%, and 12.38% respectively ( Fig. 2F(a,b,c)). So, collagen fibers deposition were decreased in tumors treated 1-MT. Collagen folding and deposition of PBS group 12.38% (( Fig. 2F(g)) was found higher than 1-MT treated groups that interestingly indicate a high impact of 1-MT on tumor fibrosis.

1-MT significantly activates caspase-3 and apoptosis.
The impact of 1-MT on tumor cell apoptosis was investigated by testing an immune reactivity of cleaved caspase-3 pathways of 1-MT treated and untreated groups. We found that 1-MT extremely increased caspase3 reactivity in treated tumors ( Fig. 2G(a,b,c)), while other groups showed no apoptotic reactions ( Fig. 2G(d,e,f,g)). This indicates that 1-MT has a potential role to enhance tumor cell destruction by activating apoptotic signaling pathways.  . This is proved that DCs successfully uptake tumor antigens and proceeded presenting potency. Besides that, determination of specific time point to activate Th1 and CTL cells responses by tumor lysate was performed by Flowcytometer interval 3 hours; it showed a highest INF-γ + accumulation at 24 hour, as showed in (C), CD4 + INF-γ + gated from CD3+ significantly raised after 9, 12, and 18 hours, but the highest response showed at 24 hours of incubation. While CD8 + INF-γ + activation significantly rose at 18 hours and the highest response was noted at 24 hours of incubation (D), these results were confirmed by RT-qPCR to determine a relative INF-γ mRNA expression after activation of splenocytes by (TL = 20 ug/ml). Results showed that high response started at 18 hours to reach a highest at 24 hours (E). The statistical significant differences were represented from at least three independent experiments, ***P < 0.001.  and 12.9%) respectively (Fig. 3A,B). Whereas a screening of CD133 + cells in tumor microenvironment showed that 1-MT, 1-MT+TL, and 1-MT+DCs-TL were significantly reduced a population of CD133 + cells in the tumor microenvironment in comparison to PBS (1.0%, 0.5%, 0.7%, and 4.6%) respectively ( Fig. 3C,D). These results were ensured by determination of relative CD133 mRNA gene expression among total tumor cells RNA. We found that expression of mRNA was significantly reduced by 1-MT in comparison to PBS (Fig. 3E).

1-MT and tumor lysate inhibits tumor regulatory pathways. Total protein contents of tumor tissues
were obtained to evaluate signaling pathways expression; total extracted proteins were loaded by protein loading buffer (PLF) (Blossom, China) for 10 min at 100 °C. Then 5 ug/ml of each protein samples was tested by western blot with Page Ruler Protein marker (Thermo scientific, USA) to test TGF-β, PDL-1, NF-κβ2, IDO, CD133 and β-catenin in comparison to GAPDH positive control. Results showed that 1-MT, tumor lysate, and DCs-pulsed tumor lysate were significantly reduced expression of TGF-β, PDL-1, NF-κβ2, IDO, and β-catenin in comparison to PBS as showed in (Fig. 5A,B). These outputs accumulated more evidences of 1-MT related mechanisms in tumor cells, which confirmed results of genes expression profile screening of intracellular signaling pathways. It interpreted why 1-MT could modulate various humoral and cellular responses.

Immunofluorescence imaging evidences blocking of NF-κβp65 signaling pathway by 1-MT.
Laser imaging of intracellular NF-κβp65 expression of Pan02 tumor cell line which was exposured to 1-MT or non exposured to 1-MT showed that IDO depletion effectively inhibited NF-κβp65 signaling pathway (Fig. 6), that ensured a role of IDO in NF-κβ pathway activation in response to regulatory polarization, the inhibiting of this pathway by 1-MT leads to breakdown tumor tolerance which facilitate modulation of immune responses inside tumor microenvironment.

1-MT and TL or DCs-TL promotes splenocytes inflammatory responses.
The analysis of CD4 + and CD8 + T cells' ex vivo kinetics of all immunized groups according to the determined time point (24 hs) showed that 1-MT itself significantly enhanced high accumulation of INF-γ in CD4 + cells rather than its efficacy with TL stimulation and also DCs-TL in comparison to PBS group as shown in (Fig. 7A), As well as, CTL (CD8+) cells were also showed significant increasing of INF-γ in response to TL and DCs-TL stimulation in comparison to PBS (Fig. 7B). On the other hand, genes expression of regulatory and inflammatory mediators illustrated that 1-MT elicited increasing of IL-12, 1L-6, and IL-4 expression (Fig. 7C,D,E), while it declined TGF-β, and IL-10 expression (Fig. 7F,G). These results suggested that 1-MT enhanced Th1 CD4+, and CTL CD8+ activation, to initiate proinflammatory immune mediators, while it reduced Treg cells expanding which ascribed to decline of regulatory mediators.  Further tumor genetic screening showed that NF-κβ2, and β-Catenin pathways were interestingly reduced (H,J) by1-MT, 1-MT+TL and 1-MT+DC-TL regimens, also TRAF1, and IKKβ mediators were significantly inhibited (I,L), while IKKα was significantly induced (K). These findings clearly evidenced that 1-MT effectively enhanced inflammatory immunomodulations inside tumor microenvironment in response to TL/DCs-TL vaccines, it also reduced tumorgensis enhancers that lead to prevent tumor homing Treg cells as showed in the Fig. 2, which ensured a breakdown of tumor immunotolerance, and recognition of tumor cells by immune surveillance. The statistical significant differences were represented from at least three independent experiments, ***P < 0.001 and **(P < 0.001).

Discussion
It's well-known, that intracellular indoleamine-2,3-dioxygenase IDO by the depletion of tryptophan exerts an immunosuppressive pathways to induce tumor immune escaping 21 . Clinical trials (NCT02077881, NCT00567931) showed that combining IDO inhibitors such as 1-MT with other autologous antigens, chemotherapy, or itself could restore immune immunosurveillance performance 9,29 . To date many cancer regimens were tested, all of them showed limited clinical efficacy, because recruiting T cells against tumor isn't enough, it becomes functionally inactive when exposure to tumor microenvironment, which was ascribed to overexpression of IDO and regulatory mediators 2,5,30 . Meanwhile, signaling pathways that being orchestrated by IDO mechanisms in tumor microenvironment or myeloid cells are yet poorly understood 21 . Here, we investigated an efficient anti-tumor responses of TL/DCs-TL vaccination, which enhanced by 1-MT performance and its related signaling pathways to understand prognostic IDO work mechanisms related tumor growth, proliferation, and immunosuppressive pathways. We found that IDO mechanisms are complex, there are many signaling pathways mediated by this factor. Blockage of this enzyme enhanced significant humoral and cellular pathways responses to tumor lysate vaccine and/or DCs pulsed tumor lysate, besides significant improvements showed in clinical trials with chemotherapy used 1-MT checkpoint inhibitor 21,31,32 , which clearly evidenced that depletion of IDO activity instantly leads to increase therapeutic susceptibility. Interestingly, we didn't found documented information about the role of IDO inhibitor on CD133 + cancer stem cells (CSC), while our results showed that 1-MT with/without tumor lysate vaccine significantly reduced CD133 + cells viability in treated tumor tissues, which suggests that IDO motif role is orchestrated by CSC in the tumor microenvironment. Also, PDL-1 expression was significantly reduced under the effect of 1-MT in comparison to PBS group. This consequently led to inhibit tumorgensis activities, which indicated that cancer stem cells mainly involve IDO pathways to develop tumorigenesis. As the analysis of these results showed that 1-MT is the main responsible of immune inflammatory responses to TL/DCs-TL antigens elicitation, that showed no significant responses without 1-MT presence. The statistical significant differences were represented from at least three independent experiments, ***P < 0.001 and **(P < 0.001).
in vivo tumor apoptosis and tumor fibrosis under 1-MT effect showed that 1-MT significantly activates cleaved caspase3 and increased apoptotic potential, while it extremely reduced tumor fibrosis. These findings confirmed a role of IDO in tumor survival and progression, because knocking out IDO activity enhances tumor destruction and promotes tumor apoptosis that illustrates an impact of 1-MT on tumorgensis pathways. This expresses a  A,B). ELISA analysis of periphery cytokines showed that circulating TGF-β was significantly reduced in the mice immunized by 1-MT, 1-MT+TL, and 1-MT+DCs-TL (D), while analysis of IL-12 showed significant rising in comparison to PBS (E). This is proved that tumor immunosuppressive pathways were completely blocked, while immunosurveillance and inflammatory immunomodulations were efficiently activated. Statistical significant differences were represented from at least three independent experiments, ***P < 0.001 and **(P < 0.001).
SCieNTiFiC RepoRts | (2018) 8:9869 | DOI:10.1038/s41598-018-28238-8 significance of IDO blockage during tumor therapy. Furthermore, our results accumulated sequential evidences from splenocytes, DLN, and periphery that confirmed a significant promoting role of 1-MT for inflammatory responses and invasion of xenografted tumor models in groups vaccinated tumor lysate, and DCs pulsed TL in case of 1-MT administration, which were on the same line of several studies that investigated likely similar regimens [33][34][35][36] . In this study we found that 1-MT significantly reduces tumor cells IKKα, NF-κβ2, and Wnt/β-catenin pathways, besides modulation of its regulatory cytokines in the tumor microenvironment, which significantly inhibited tumor growth and tumor immune escaping potency, that was on the same line of several previous studies 37,38 , but our findings showed that 1-MT significantly enhances IKKα, NF-κβ2, STAT3, and STAT4 pathways of lymphocytes in response to TL/DCs-TL vaccination, which indicated that 1-MT activates intracellular signal transducer, in response to vaccination to induce tumor specific targeting, because activated DCs efficiently trigger presentation of tumor associated antigens to elicit specific tumor targeting. Because we found that TLR2, 7, 8, and 9 were significantly activated in the splenocytes of TL/DCs immunized mice during 1-MT addressing, which elucidated non-enzymatic activity of IDO engage tumorigenesis proceeds. These evidences apparently revealed that 1-MT completely forbids tumor tolerance pathways, which interprets the reduction of regulatory mediators and Treg cells in the tumor microenvironment and periphery analysis. These analyses represented a virtual antitumoral kinetics during vaccination period, which provided some biological evidences that could optimize solid tumor treatment strategies in the near future

Conclusion
We conclude that, our results revealed that 1-MT has a potential to breakdown tumor tolerance and significantly releases immunosurveillance potential throughout depletion of kynurenine related pathways, besides activation of functional DCs and specific T cells to recognize tumor antigens and activate inflammatory modulations efficiently. These processes involve enzymatic and non-enzymatic mechanisms that need deep analysis of metabolic products pathways as a fundamental secret to identify specific motifs could lead to design high critical therapeutic strategies. However, tumor autologous antigens vaccine with 1-MT showed a significant therapeutic potential as an immunotherapeutic vaccine towards pancreatic adenocarcinoma in the murine models, which encourage this strategy implementation in human clinical trials.

Materials and Methods
Mice details and Ethical Issue. Black females (20-25) g C57BL/6 mice were recruited from the Laboratory Animal Center of Yangzhou University. Experimental animals were housed in plastic cages under pathogen-free conditions. Experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, meanwhile it approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University.

Preparation of Tumor lysate (TL) and DCs loaded TL.
Pan02 cell line was utilized to prepare freezethawed tumor lysate, as described by W. Asavaroengchai et al. 39 . Then protein concentration of supernatant was determined by BCA protein assay kit (Beyotime, China), according to the manufacturer instructions. However, 20 ng/ml of tumor lysate was introduced to (1 × 10 6 ) bone marrow dendritic cells (DCs) for 24 hours, in comparison to 1 ng/ml LPS and PBS. Later, pulsed DCs were stained by APC-Anti-mouse anti-CD11c, FITC-Anti-mouse anti-CD40, and PE-Anti-mouse Anti-CD80 (Biolegend, USA) for 30 min at 4 C. Thereafter, DCs uptake capability was assessed by BD Accuri C6 Flowcytometer (Biosciences, USA). On the other hand, to ensure activation status of DC loaded tumor lysate, on the 6 th day DCs were co-cultured with TL, as described before for 24 hours and then total RNA was extracted by TRizol (Ambion, USA). Consequently, 500 ng/ul of RNA/sample was mixed with 4 ul of 5x All-In-one RT master mix (Abm, USA), and then final volume was completed to 20 ul. After that, RNA was reversed by reverse transcriptase to cDNA (incubate 25 °C for 10 min, then worm 42 °C for 30 min. Thereafter, reaction was stopped at 85 °C for 5 min and chilled at 4 °C) by thermo cycler (Thermofisher scientific, Finland). Later, cDNA samples were amplified by light cycler 480 II qPCR (Roche, USA) with 2x SYBR Green qPCR master mix (Bimake, China), CD11c (Itgax), MHCII (H2-Ab1), CD80, CD40 primers (Anhui, China), and cyc positive control gene which listed in the Table 1. Then gene expression was calculated according to cyc gene ct.

Determination of T cell activation time point under TL effect. C57BL/6 black mice were immunized
by TL, and then splenocytes were harvested to separate T lymphocytes according ot density. Then separated lymphocytes were co-culture with TL for 3, 6, 9, 12, 18, and 24 hours in the presence of Brefeldin A (BFA) 1,000x (Biolegend, USA) in comparison to ConA, and PBS. Next lymphocytes were collected, washed and stained (surface and intracellular) as described by Wei Ye et al. 40 , by using Pecp-Anti-mouse anti-CD3, FITC-anti-CD4, and PE-anti-CD8 for 30 min on ice. After fixation and permeabilization intracellular staining was performed by APC-anti-INF-γ for 30 min on ice. Finally cells were assessed by BD Accuri C6 Flowcytometer. Further confirmation test of specific time points were performed by RT-qPCR. However, total RNA of lymphocytes-TL exposured was extracted by TRizol, and then RNA transcriptase was performed to get cDNA as described before, then it used to determine relative INF-γ mRNA according to forward and reverse primers (Anhui, China) listed in the Table 2.
Immunization and In Vivo Tumor Evaluation. Black C57BL/6 mice were divided 7 groups (n = 6). They were subcutaneously challenged on the right flank by 1 × 10 5 Pan02 cell line. 6 days later, 1-MT was orally administrated (2 mg/ml)/mouse interval 2days, as described by several protocols 24,31,41 to the first three group's 1-MT, 1-MT+TL+ALU, and 1-MT+DC-TL+ALU. Other groups TL+ALU, TL, ALU, and PBS were treated without 1-MT. However, mice were immunized three times subcutaneously with tumor lysate (100 ng/ml) that was mixed with adjuvant (Al(OH)3 aluminum hydroxide gel). This mixture was connected with Polyethylenimine (PEI 5 mg/ml) particles. Then it was kept at 4 °C for 30 min, thereafter, it was subcutaneously injected into mice. During the immunization programme, tumor size of all mice groups was daily measured. Thereabout, mice survival rate of another 7 groups was daily noted until the death of last mouse. However, tested groups were sacrificed and their tumor tissues, blood, spleens and draining lymph nodes were collected.

Tumor Infiltrating Lymphocytes (TIL) and HE staining. To investigate tumor necrosis and tumor
infiltrating T lymphocytes CD4 + CD25 − in comparison to CD4 + FOXP3+, tumor samples were sectioned 4 µm and introduced to Haematoxylin and Eosin (H&E) staining protocol according to Fischer et al. 42 . Thereafter, results were numbered and sent to a pathologist for consultation without any details about the treated and untreated tissues. Finally, tumor tissues were visualized by light microscope 200x. Furthermore, samples of each fresh enucleated tumor were mechanically digested to get single cell suspension. Next cells were counted and recruited to surface and intracellular staining. Briefly, 1 × 10 6 cells of tumor tissue/sample was firstly stained by Percp-anti-mouse Anti-CD3, FITC-Anti-CD4, and PE-Anti-CD25 for 30 min on ice. Then cells were fixed, permeabilized and stained by APC-Anti-FoxP3 for 30 min on ice. Finally cells were assessed by BD Accuri C6 Flowcytometer.
Picro Sirius Red Staining. To evaluate the impact of 1-MT and 1-MT+TL on tumor fibrosis, sections of enucleated tumor tissues were deparaffinized, rehydrated and processed for Sirius red staining as described by Segnani et al. 43 . Briefly, fixed slides were co-incubated with 0.1% Sirius red aqueous dye solution (Abcam, USA) for 1 hour. Then slides were washed in acidified water. Later it was dehydrated and mounted with DPX. Collagen red fibers and fibers area were measured by Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Immunohistochemistry analysis of cleaved caspase-3 pathway.
To assess an impact of 1-MT and 1-MT+TL on tumor apoptosis potency, fixed tumors were deparafinzed and rehydrated by ethanol, endogenous peroxidase activity was blocked by soaking in absolute methanol including 0.3% H 2 O 2 for 30 min. then antigen was retrieved by using 10 mmol/L Tris-HCl buffer (pH 8.0) containing 1 mmol/L EDTA. Later it was blocked with normal goat serum for 5 min. Thereafter, anti-activated caspase3 antibody which was diluted 1:20 (Cell signaling, USA) was performed for overnight. Next tissues were washed by TBS and incubated with goat anti-rabbit IgG conjugated with rhodamine for 30 min. Finally they were visualized by light microscope at 200x magnification.

Determination of cancer stem cells (CSC) viability in treated and non-treated tumor tissues.
Single cell suspensions were utilized to determine viability of CSC population in 1-MT treated and non-treated groups. However, 1 × 10 6 tumor cells were washed and stained by APC-Anti-mouse Anti-CD133 for 30 min on ice, and then stained cells of all groups were tested by BD Accuri C6. To confirm these results, tumor CD133 gene expression was assessed by RT-qPCR according to purchased primers (Anhui, China) listed in the Table 2 as described before.

Analysis of tumor microenvironments' signaling pathways by western blot. First, red blood cells
were disrupted, and then tumor cells were washed and fractured by using RIPA buffer (Thermo fisher, USA) with phenyl methyl sulfonyl fluoride (PMSF) a proteinase inhibitor (Sigma, USA) according to the manufacturer instructions. Total protein was assessed by BCA to determine protein concentration for every sample. Thereafter, 5 ug/ml of each protein sample was utilized to test TGF-β, IDO, PDL-1, CD133, NF-κβp65, and β-Catenin pathways expression by western blot. Briefly, samples were run on 10% gel, and then transfer on blotting membrane for 30 min by Trans-blot Turbo (Bio-Rad, Singapore). After milk blocking, membranes were incubated with primary antibodies Anti-TGF-β (Abcam, USA), Anti-IDO (Biolegend, USA), Anti-PDL-1, Anti-NF-κβp65 (Abcam, USA), Anti-CD133 (Boster, China), and Anti-β-Catenin (Affinity, USA) for overnight on shaker, and then washed three times and incubated with goat anti-rabbit secondary antibody (Abcam, USA) for 1-2 hours. Finally, results visualized by gel imaging system 5200 (Tanon, China).
Immunofluorescence imaging of tumor NF-κβp65 pathway with/without 1-MT. As  Intracellular kinetics of splenic CD4 + and CD8 + T Cells. Splenocytes and draining lymph nodes DLN of the 7 immunized groups were used to investigate intracellular kinetics by determination of T CD4 + INF-γ + , and CTL + INF-γ + cells. However, lymphocytes were stained for surface markers and intracellular for INF-γ as described above. Harvested results were presented by graph pad software. Thereafter, all CD3 + CD4 + and CD3 + CD8 + cells were collected in separate tubes by FACS Area Flowcytometer. These collected cells were fractured by TRizol to collect total RNA, which used to prepare cDNA by which expression of lymphocytes intracellular cytokines was determined under the effect of 1-MT. However, genes expression of IL-12, IL-6, IL-4, TGF-β, and IL-10 was quantitatively determined as described above with primers (Anhui, China) listed in the Table 3.
Assessment of Periphery Immunomodulations. The obtained blood samples were separated to plasma and lymphocytes. Lymphocytes were stained (surface and intracellular) to determine Treg + cells CD4 + CD25 + FOXP3 + and Th1 CD3 + CD4 + CD25 − population in treated groups in comparison to PBS. First, cells were stained by Percp-Anti-CD3, FITC-Anti-CD4, and PE-Anti-CD25 (Biolegend, USA) for 30 min on ice. Then it was fixed, permeabilized and stained by Alexa Fluor 647-anti-Foxp3 (Biolegend, USA) for 30 min on ice. Finally cells were examined by BD Accuri C6. Later, plasma samples were utilized to determine periphery TGF-β and IL-12 cytokines by ELISA kits according to manufacturer instructions (Lington Biosciences, China).   Statistical analysis. Statistical analysis was performed using GraphPad Prism 6.0 for at least three independent experiments. Data were analyzed by paired student's t-test and one-way analysis of variance (ANOVA). The significant difference level was set at p < 0.05.

Data availability.
Authors declare that all data generated or analyzed during this study are included in this published article (and its Supplementary Information files). If any interest needs more details please contact corresponding editor by private email.