Fig 2: Representative images of immunoreactivity for LAT1, 4F2hc, and Ki67 in human ATC and AG tissues. (a) immunoreactivity 0 for LAT1 in ATC tissues; (b) immunoreactivity 1 for LAT1 in ATC tissues; (c) immunoreactivity 2 for LAT1 in ATC tissues; (d) immunoreactivity 3 for LAT1 in ATC tissues. (e) immunoreactivity 0 for 4F2hc in AG tissues; (f) immunoreactivity 1 for 4F2hc in AG tissues; (g) immunoreactivity 2 for 4F2hc in ATC tissues; (h) immunoreactivity 3 for 4F2hc in ATC tissues. (i) immunoreactivity 0 for Ki67 in AG tissues; (j) immunoreactivity 1 for Ki67 in ATC tissues; (k) immunoreactivity 2 for Ki67 in ATC tissues; (l) immunoreactivity 3 for Ki67 in ATC tissues. (m) Cause-Specific Survival (CSS) rates of ATC patients according to LAT1 expression.

Fig 3: IL1RAP forms a complex with CD98 and xCT of the system Xc- cystine transporter and promotes cystine uptake. A, Volcano plots of proteins identified in the IL1RAP interactome in A673. B, Representative immunoblots of the indicated co-IP samples in A673 cells. The experiments were repeated twice with similar results. C, Representative IF staining of IL1RAP and CD98 in A673 cells. D, PLA of IL1RAP and CD98 in A673 cells (n = 5; >100 cells were analyzed for each condition). E, PLA of IL1RAP–CD98 or IL1RAP–xCT in TC32-derived Ewing sarcoma (EwS) xenografts in the murine renal subcapsular implantation model. Scale bar, 30 µm; red arrows indicate positive PLA signals in Ewing sarcoma tumor cells. F, Representative immunoblots of the indicated co-IP samples in A673 cells expressing wild-type (WT) or mutant CD98 (C109S or C330S). Rabbit (Rb) IgG and goat (Gt) IgG were used as isotype controls for the co-IP experiments. The experiments were repeated twice with similar results. G, Representative immunoblots of the indicated co-IP samples in HEK293 cells transfected with WT or mutant IL1RAP (C362S) and WT HA-tagged CD98. H and I, Representative images showing the IF staining of IL1RAP and xCT (H) and PLA assay of IL1RAP and xCT (I) in A673 cells. Scale bars, 10 µm. For I, n = 5–7; =50 cells were analyzed for each condition. J, The impact of IL1RAP knockdown (by sh#39 or #40) on cystine uptake was measured by cystine consumption assay in media containing regular (200 µmol/L) or low (10 µmol/L) levels of cystine. K and L, Intracellular cysteine and GSH pools were measured in the indicated cell lines (K) or Ewing sarcoma xenografts derived from TC32 (L) with or without IL1RAP knockdown (by sh#39 or #40). M, Rescue of WT or mutant IL1RAP (C362S) in Ewing sarcoma cells with IL1RAP depletion was determined by immunoblot analysis. N, Cystine uptake and glutamate secretion were determined in the indicated Ewing sarcoma cells cultured in media containing 10 µmol/L cystine. Data presented are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001;P values were determined by two-tailed unpaired Student t test.

Fig 4: Anti-tumor effect of JPH203 in 8505C-inoculated athymic BALB/c nude mice. An equal number of 8505C cells (1 × 106 cells) were injected into the flanks of each mouse before treatment. When tumors started to develop (average tumor size reached 100 mm3), JPH203 or vehicle was administered intraperitoneally for 18 day (12.5 mg/kg/d). JPH203 treatment decreased (A) tumor growth ratio, (B) tumor size. ‡: #4 mouse was just died before euthanasia. Therefore, this mouse was excluded from following analysis. (C) Representative images of H&E-stained tumor sections (panels a and b). There was no difference between vehicle treated mice (panel a) and JPH203 treated mice (panel b). Immunohistochemical analysis showed the LAT1 and 4F2hc express expression at cell surface, and their expressions had little effect on JPH203 treatment (panel c to f). However, JPH203 treatment decreased the Ki67 immunoreactivity (panel g and h). (D) The counting data showed the number of Ki67 immunoreactive positive cells were decreased in the group of JPH203 treated mice (***p < 0.001). (E) Western blot for LAT1, 4F2hc, cyclin D1, phosphorylated S6, total S6 and α-tubulin was carried out to access mTOR signal and cell cycle regulators. (F) The band intensities of these proteins detected in (E) were quantified and compared. JPH203 treatment suppresses the expression of cyclin D1 (*p < 0.05) and the phosphorylation of S6 (*p < 0.05).