Fig 2: Lesinurad, an SLC22 inhibitor suppressed IFN signaling and EMT in Mes cells and in combination with gemcitabine restrained tumor growth, and metastasis in mouse models of PDAC(A) Transport of [glycine-2-3H]-glutathione measured in the presence of SLC22 inhibitors (20 µM each) in Mes cells. Error bars, mean ± SD. n = 3 biological replicates for each condition was examined and three independent experiments were performed.(B) Structure of lesinurad.(C) Representative phase contrast images of the Mes cells treated with vehicle or lesinurad (20 µM) for 48 h. Scale bar, 50 µm n = 5 random fields from each condition was photographed and analyzed and data represent three independent experiments.(D) Western blots showing the protein expression of players in the IFN-STAT3-ROR1 signaling axis in Mes clones treated with indicated concentrations of lesinurad for 48 h ß-Actin was used as a loading control.(E) Western blots illustrating the protein expression of epithelial and mesenchymal markers in Mes clones treated with indicated concentrations of lesinurad for 48 h ß-Actin was used as a loading control. Data presented in (D and E) represent three independent experiments.(F) Design of an orthotopic mouse pancreatic tumor xenograft study conducted to evaluate the anti-tumor and antimetastatic abilities of gemcitabine and lesinurad alone and the combination of both drugs at the indicated doses detailed in the STAR Method section.(G) Quantification of weights of the primary tumors dissected from above groups of animals. Error bars, mean ± SEM. n = 5 mice per group; each point represents one animal.(H) Quantification of metastatic foci in the viscera and cavities from the above groups of animals. Tumor associated metastatic foci and lesions >1 mm3 were scored and included in the analysis. Error bars, mean ± SEM. n = 5 mice per group; each point represents total number of metastatic nodules quantified from one animal.(I) Design showing KPC mice study conducted to assess the efficacy of gemcitabine alone and in combination with lesinurad on survival, tumor growth, and metastasis.(J) Changes in individual tumor volumes at 6 (T1) and 12 weeks of age (T2) as determined by micro-CT imaging analyses. Animals that received gemcitabine alone (n = 5 tumors) versus the combination of gemcitabine + lesinurad (n = 7 tumors) are compared. Each line represents one tumor and the average change in the collective tumor volume is presented in lines with open circles. n = 3 KPC mice per group were monitored by micro-CT imaging for tumor burden.(K) Kaplan–Meier survival analysis showing the elapsed survival time of groups of KPC mice treated with saline (n = 12 mice), gemcitabine (n = 14 mice), or gemcitabine + lesinurad (n = 14 mice) at the indicated doses (detailed in STAR Method section) twice a week.(L) The incidence of metastasis in KPC mice enrolled in the saline (n = 12 mice), gemcitabine monotherapy (n = 14 mice), and gemcitabine + lesinurad groups (n = 14 mice), as determined by post-survival necropsy examination.(M) Model of the proposed mechanism of action of SLC22A10 and SLC22A15 in promoting EMT and pancreatic cancer progression.Data in (A) were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Data in (G and H) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Data in (J) were analyzed by two-way ANOVA with Sidak’s multiple comparisons test. For the data presented in (K), the differences in median overall survival times of KPC mice in different groups were analyzed using log rank (Mantel–Cox) test. Data presented in (L) were analyzed by Fisher’s exact test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Fig 3: Identification of SLC22A10 and SLC22A15 as promoters of EMT and metastasis in pancreatic cancer(A) Representative phase contrast images of the stable clones transduced with vector and overexpressing the SLCs showing their morphological features and cell scattering. Scale bars, 50 µm n = 5 random fields were photographed and analyzed from each condition.(B) Relative mRNA levels of epithelial markers Cytokeratin 8 and Cytokeratin 18 in SLC22-OE clones determined by RNA sequencing of these clones and subsequent DE analysis of the genes.(C) Western blotting analyses of whole-cell lysates from SLC22-OE clones showing the protein expression of epithelial and mesenchymal markers. ß-Actin was used as an endogenous loading control. Data represent three independent experiments.(D) Representative crystal violet-stained images of migrated and invaded SLC22-OE clones. Scale bars, 50 µm n = 5 random fields from each condition were photographed and analyzed.(E and F) Quantification of the number of migrated and invaded SLC22-OE clones depicted in fold-change values relative to the control clone. Error bars, mean ± SD.(G) Representative necropsy images of mice harboring tumors from vector-transduced and SLC22A10- and SLC22A15-OE clones showing the primary tumors (red arrows) and secondary tumors (green arrows) (middle panel). Images of dissected primary tumors (upper panel) and metastatic foci in the liver and spleen (lower panel) are shown separately. Arrows point at tumors, metastatic foci, or related lesions. n = 5 mice per group.(H) Quantification of the weights of primary tumors dissected from groups of animals harboring SLC clones at the end of the experiment. Error bars, mean ± SEM. n = 5 mice per group; each point represents one animal.(I) Quantification of the average number of metastatic foci in the vital organs and the abdominal cavities of animals in each group in the study. Error bars, mean ± SEM. n = 5 mice per group; each point represents one animal.(J) Representative phase contrast images of scramble-, shSLC22A10-, and shSLC22A15-transduced clones. Scale bars, 50 µm n = 5 random fields photographed and analyzed from each condition.(K) Western blotting analyses of whole cell lysates showing the protein expression of epithelial and mesenchymal markers in scramble-, shSLC22A10-, and shSLC22A15-transduced clones. ß-Actin was used as an endogenous loading control for the Western blots. Data represent three independent experiments.(L) Quantification of the weights of primary tumors dissected from mice harboring tumors from scramble-, shSLC22A10-, and shSLC22A15-transduced clones. Error bars, mean ± SEM. n = 5 mice per group; each point represents one animal.(M) Quantification of the average number of metastatic foci in the abdominal viscera of animals in each group in the study. Error bars, mean ± SEM. n = 5 mice per group; each point represents total number of metastatic nodules from one animal. Data presented in (E, F, H, I, L, and M) were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig 4: SLC22A10 and SLC22A15 transport glutathione that stimulates IFN-STAT3-ROR1 signaling and EMT(A) Heat maps depicting metabolites that are significantly altered in SLC22A10-OE (left panel) and SLC22A15-OE (right panel) clones compared with the vector-transduced clone, as determined by untargeted metabolomics utilizing LC-MS/MS in ESI + mode. Metabolites in bold are related and significantly increased in both SLC22A10- and SLC22A15-OE clones compared with the vector-transduced clone. n = 5 biological replicates and n = 3 technical replicates of each sample (pLV-Control, pLV-SLC22A10, and pLV-SLC22A15) were analyzed.(B–D) Levels of total cellular glutathione (GSH + GSSG), reduced glutathione (GSH), and oxidized glutathione (GSSG) in primary tumor tissues dissected from mice described in Figure 2 G. Error bars, mean ± SD. Tissues from n = 3 tumors for each condition was processed and assayed.(E) Transport of radiolabeled [glycine-2-3H]-glutathione was determined in PANC-1 cells transduced with control, SLC22A10, and SLC22A15 lentiviruses. Error bars, mean ± SD.(F) Transport of [glycine-2-3H]-glutathione in Mes cells transduced with scramble, shSLC22A10, and shSLC22A15 lentiviruses. Error bars, mean ± SD.(G) Transport of [glycine-2-3H]-glutathione in PANC-1 cells transduced with control, SLC22A10, and SLC22A15 lentiviruses or co-transduced with SLC22A10 and SLC22A15 lentiviruses. Cells were pretreated with BSO (100 µM) for 24 h before the transport study. GSSG (20 mM) was added to the transport buffer for the duration of transport period. Error bars, mean ± SD.(H) Transport of [glycine-2-3H]-glutathione in the presence of Na+-containing buffer at pH 7.4 or N-methyl-D-glucamine chloride (NMDG) buffer or HEPES buffered saline (HBS) or Na+-containing buffer at pH 5.5 in PANC-1 cells transduced with control, SLC22A10, and SLC22A15 lentiviruses. Error bars, mean ± SD.(I) Transport of [glycine-2-3H]-glutathione in PANC-1 cells transduced with control, SLC22A10, and SLC22A15 lentiviruses in the presence of 100 µM each of top differentially altered metabolites (identified from the metabolomic analysis of pLV-Control, pLV-SLC22A10, and pLV-SLC22A15 clones). For the data presented in (E-I), three biological replicates for each condition were tested and n = 3 independent experiments were performed.(J) Representative phase contrast images of PANC-1 cells treated with indicated concentrations of GSH for 48 h. Scale bar, 50 µm. Three random fields from each condition were photographed and analyzed. n = 3 independent experiments performed.(K) Western blots depicting the protein expression of epithelial and mesenchymal markers in PANC-1 cells treated with indicated concentrations of GSH for 6 h ß-Actin were used as a loading control. Western blots shown represent three independent experiments.(L) Western blots illustrating the protein expression of the markers of IFN-STAT3-ROR1 signaling axis in PANC-1 cells treated with increasing concentrations of GSH for 6 h ß-Actin were used as a loading control. Western blots shown represent three independent experiments.(M) Western blots assessing the protein expression of markers of the IFN-STAT3-ROR1 signaling axis in vector-transduced and SLC22A10- and SLC22A15-OE clones treated with the indicated concentrations of BSO and GSH. Cells were treated with BSO or vehicle for 24 h and followed by GSH or vehicle for 6 h ß-Actin was used as a loading control. Western blots represent at least three independent experiments.Data presented in (B–G) were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test and data presented in (H and I) were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig 5: Increased expression of SLC22A10 and SLC22A15 in human pancreatic cancers(A) Representative images of IHC staining in human normal pancreatic tissues showing the expression of SLC22A10 (green) in ductal epithelial cells (I) and pancreatic islet cells (II). Insets display the enlarged view of boxed regions. Nuclei stained with DAPI are blue. Scale bars, 50 µm n = 3 samples per condition.(B) Representative images of IHC staining in human normal pancreatic tissues showing the expression of SLC22A15 (cyan) in ductal epithelial cells (III) and pancreatic stellate cells surrounding the acini (IV). Insets display the enlarged view of boxed regions. Nuclei stained with DAPI are blue. Scale bars, 50 µm n = 3 samples per condition.(C) Representative images of IHC staining in human pancreatic cancer tissues showing expression of SLC22A10 or SLC22A15 (green), Pan Cytokeratin (PanCK) (red), and their colocalization (yellow). Nuclei stained with DAPI are blue. Scale bars, 50 µm.(D) Representative images of multiplexed IHC staining in human pancreatic cancer tissue microarray (TMA) illustrating the predominant expression of SLC22A10 in tumor (Pan-CK positive; yellow) and SLC22A15 in stromal (Pan-CK negative; green) regions, respectively. Nuclei stained with DAPI are blue. Scale bars, 50 µm.(E) H-scores of SLC22A10 and SLC22A15 in human normal pancreatic tissues and human pancreatic cancer TMA as determined by quantification of the IHC images using inForm software. The intensity and frequency of expression of these two SLCs in human pancreatic tumor tissues in comparison with human normal pancreatic tissues are presented. For the data presented in C-F, n = 86–91 patient-derived pancreatic cancer tissue samples were analyzed.(F) Histoscores (H-scores) of SLC22A10 and SLC22A15 expression intensity in human pancreatic tumor and stromal compartments from quantitative IHC multiplexing of human pancreatic cancer TMA. Data presented in (E and F) were analyzed by two-sided aStudent’s t-test and bU rank sum test with level of significance 0.05.(G) Western blots depicting the expression of SLC22A10 and SLC22A15 in normal human pancreatic ductal epithelial (HPDE), and a panel of six human PDAC cell lines (HPAF-II, Capan-1, L3.6PL, PANC-1, MiaPaca-2, and AsPC-1). ß-Actin was used as a loading control. Data represent three independent experiments.