IDO-1 inhibitor INCB24360 elicits distant metastasis of basal extruded cancer cells in pancreatic ductal adenocarcinoma

Neoplastic cells of non-immunogenic pancreatic ductal adenocarcinoma (PDAC) express indoleamine 2,3-dioxygenase 1 (IDO-1), an immunosuppressive enzyme. The metabolites of IDO-1 in cancers provide one-carbon units that annihilate effector T cells, and recruit immunosuppressive cells. In this study we investigated how IDO-1 affected the neoplastic cell behaviors in PDACs. Using multiple markers co-labeling method in 45-µm-thick tissue sections, we showed that IDO-1 expression was uniquely increased in the neoplastic cells extruded from ducts’ apical or basal domain, but decreased in lymph metastatic cells. IDO-1+ extruding neoplastic cells displayed increased vimentin expression and decreased cytokeratin expression in PDACs, characteristics of epithelial-mesenchymal transition (EMT). However, IDO-1 expression was uncorrelated with immunosuppressive infiltrates and clinicopathological characteristics of grim outcome. We replicated basal extrusion with EMT in murine KPIC PDAC organoids by long-term IFN-γ induction; application of IDO-1 inhibitor INCB24360 or 1-MT partially reversed basal extrusion coupled EMT. Ido-1 deletion in KPIC cells deprived its tumorigenicity in immunocompetent mice, decreased cellular proliferation and macropinocytic ability, and increased immunogenicity. KPIC organoids with IFN-γ-induced basal extrusion did not accelerate distant metastasis, whereas inhibition IFN-γ-induced IDO-1 with INB24360 but not 1-MT in KPIC organoids elicited liver metastasis of subcutaneous KPIC organoid tumors, suggesting that lower IDO-1 activity accelerated distant metastasis, whereas IDO-1 was indispensable for tumorigenicity of PDAC cells and supports the survival of extruding cells.


Figure legends
b. IDO-1 and E-cadherin antibodies immunostaining in lymph nodules or the capsule of nodules in two patients (white arrows, the neoplastic cells on the capsule; pink arrows, IDO-1 + and E-cadherincells with multiple processes).
c. IDO-1 and E-cadherin antibodies immunostaining in liver metastatic PDAC lesions revealed that metastatic neoplastic cells expressed lower level IDO-1 than its pancreatic lesions. Stuendet t-test, **, p<0.001. III, a lymph nodule without metastatic cells, IV, precursor lesion; V, noninvasive ducts; VI, invasive PDAC ducts with the disrupted basal surface; yellow arrows, immune cells; white arrows, metastatic cells in I and II, ducts in V). Sample size, n=10. The 3D rendered tomography showed the cellular shape (inner panels, the upper panels).       groups by Flow Cytometry. In IFN-γ group, KPIC cells have been exposed to 20 ng/mL IFN-γ for 24 h; In IFN-γ+INCB24360, the KPIC cells have been exposed to 20 ng/mL IFN-γ for 12 h and exposed to 20 µM INCB24360 for another 12h h without withdrawing IFN-γ. One-way ANOVA. Data, mean ± SD b. Comparing the count of effete cells in IFN-γ+INCB24360 group with other groups. One-way ANOVA. Data, mean ± SD. **, p<0.01.

Isolation of KPIC cells from organoids
After scratching the KPIC organoids from the culturing well, the organoids were moved to a dish with DMEM/F12 on ice, blew with 1ml Pipette Tips three times, precipitated naturally for 1 minute， discarded supernatant， and then used the precipitant for LC-MS analysis.

Protein Digestion and Mass Spectrometry
Two milligrams of protein were mixed by buffer containing 8 m urea, 400 mm NH4HCO3 to 1.2 ml, and diluted with 5 mm dithiothreitol for 30 min at 50°C and alkylated by addition of 10 mm iodoacetamide. After 30 min of incubation in the light-proof room at RT, the first digestion was done by adding Lys-C at an enzyme/protein ratio of 1:100 and incubated for 4 h at 37°C. Subsequently, the digest was diluted by water to a final urea concentration of 1 m, and a second digestion with trypsin at an enzyme/protein ratio of 1:50 was done at 37°C overnight. Finally, the digestion was stopped with 5% formic acid. The digested protein was desalted by 200-ml Sep-Pak C18 cartridges (Waters Corp.), dried in vacuo, and stored at −20°C.
As described previously (34), trapping was performed at 5 μl/min solvent C (0.1 m acetic acid in water) for 10 min, and elution was achieved with a gradient of 10-35% (v/v) of solvent D (0.1 m acetic acid in 80:20 acetonitrile/water) in 140 min with a total analysis time of 180 min. When performing the elution, the flow rate was passively split from 0.60 ml/min to 100 nl/min. Nanospray was achieved using a distally coated fused silica emitter (360-μm outer diameter, 20-μm inner diameter, 10-μm tip inner diameter; constructed in-house) biased to 1.7 kV. Survey full-scan MS spectra were acquired from m/z 350 to m/z 1500 in the Orbitrap with a resolution of 60,000 at m/z 400 after accumulation to a target value of 500,000 in the linear ion trap with a target value of 30,000. The 10 most intense peaks were fragmented in the linear ion trap using collision-induced dissociation.

Data Analysis
All MS/MS spectra were centroided and merged to a single peak list file using

LC-ESI-MS/MS analysis
LC-ESI-MS/MS analysis was performed using a nanoflow EASY-nLC 1000 system coupled to an LTQ-Orbitrap Elite mass spectrometer. A two-column setting was adopted for all analyses. Samples were firstly loaded onto an Acclaim PepMap100 C18 Nano Trap Column (5 μm, 100 Å, 100 μm id x 2 cm, (Thermo Fisher Scientific, Sunnyvale, CA, USA)) and then analyzed on an Acclaim PepMap RSLC C18 column (2 μm, 100 Å, 75 μm id x 25 cm. The mobile phases consisted of Solution A (0.1% formic acid) and Solution B (0.1% formic acid in ACN). The derivatized peptides were eluted using the following gradients: 2-40% B in 40 min, 40-90% B in 5 min, 90% B for 5 min, 90-0% B in 1 min, and 0% B for 9 min at a flow rate of 200 nL/min. Data-dependent analysis was employed in MS analysis: the 15 most abundant ions in each MS scan were automatically selected and fragmented in HCD mode to achieve the high mass accuracy in MS/MS spectra. The resolution was set to 60 000 for MS and 15 000 for MS/MS, and the AGC target was set as 106 for MS and 5×104 for MS/MS. For MS/MS analysis, the isolation window was set as 2.0 Da, normalized collision energy as 35.0, activation time as 0.1 ms, and the starting mass as 100.0 Da. For each analysis, 0.5 μg of E. coli sample was injected.

Data analysis
Proteome Discoverer (Version 1.4, Thermo Fisher Scientific) using an in-house MASCOT server (Version 2.3, Matrix Science, London, UK) applied for analyzing raw data.