Extended Data Fig. 10: circGigyf2 elicits anti-tumour immunity against mouse breast cancer via encoding cryptic antigenic peptides.

(a) Heatmap of Z-score normalized log₂(count+1) expression of the selected differentially expressed circRNAs between mouse normal breast epithelial cell line EpH4-Ev and breast cancer cell line 4T1 (n = 3). (b) Flowcharts indicating key steps involved in TSA discovery for details. Numbers in the charts indicate the number of circRNAs upregulated in 4T1 cells. (c) The expression of circGigyf2, normalized to Actb expression, in EpH4-Ev and 4T1 cells, as evaluated by RT-qPCR. **P = 0.0079. (d) Relative quantitation of circGigyf2 and linGigyf2 levels by RT-qPCR is shown. **P = 0.0022, ***P = 0.0003. (e) Relative abundance by RT-qPCR of circGigyf2 in different cell fractions of 4T1 cells. (f) FISH with junction-specific probes indicates the cellular localization of circGigyf2 in EpH4-Ev and 4T1 cells. Scale bars, 5 μm. (g-k) 4T1 cells were transfected with siGFP, circGigyf2 siRNA-1 and siRNA-2 (sicirc-1 and sicirc-2, respectively). Relative quantitation of circGigyf2 (g) and linGigyf2 levels (h) by RT-qPCR is shown. ***P = 0.0003. (i) CCK-8 assays were used to detect 4T1 cell viability. Abs, Absorbance. (j) Percentages of Annexin V⁺ cells are shown, evaluated by flow cytometry. (k) Migrated cell counts per field are shown, determined by Transwell migration assays. (l, m) BALB/c mice were immunized with liposome-encapsulated in vitro circularized circGigyf2 or linGigyf2 RNA, respectively. (l) The splenic T cells from immunized mice were rechallenged with BMDCs transfected with linGigyf2 and circGigyf2 in vitro, respectively. Quantitation of the spot count per 5 × 10⁵T cells determined by IFNγ ELISpot. (m) The cytotoxic effect on 4T1 cells induced by splenic T cells was assessed by LDH assay. (n) The putative IRES activity in circGigyf2 was determined by relative luciferase activity of Luc/ Rluc. (o) Immunoblotting for Flag expression in HEK293T cells with indicated transfection. (p) MS identified circGigyf2-encoded unique peptide sequences in HEK293T cells transfected with circGigyf2. (q) Immunoblotting for circGigyf2-104 in EpH4-Ev and 4T1 cells. (r) MS analysis identified circGigyf2(95-103) as the MHC-I binding peptide from 4T1 cells pulled down by H-2-K^d. (s) MHC peptide binding predictions for circGigyf2 peptides to H-2-D^d, H-2-K^d or H-2-L^d using IEDB algorithm (Rank, Score¹) and SYFPEITHI (Score²). The unique amino acid sequence encoded by circGigyf2 are shown in red. (t-w) BALB/c mice were immunized with peptides encoded by circGigyf2 and linGigyf2 in combination with adjuvant, poly(I:C). (t, u) The splenic T cells were re-challenged with BMDCs pulsed with peptides encoded by circGigyf2 and linGigyf2 in vitro. (t) Quantitation of the spot count per 5 × 10⁵ T cells is shown, determined by IFNγ ELISpot. (u) Percentages of T cells stained for IFNγ, IL-2 and TNF are shown, evaluated by flow cytometry. (v) Percentages of PI⁺ 4T1 cells induced by the splenic T cells were determined by flow cytometry. (w) Cytotoxic effect on 4T1 cells was assessed by LDH assay. Representative image of n = 3 independent experiments (f, o-r). For gel source data of Extended Data Fig. 10o, q, and r, see Supplementary Fig. 8. Results are mean ± s.d. of n = 5 (c), n = 3 (d, e, g-n, t-w) independent experiments producing similar results. ****P < 0.0001. P values, compared with Eph4-Ev cells (c), cells with indicated treatment (d), untreated 4T1 cells (-) (g-k, m, w), T cells from mice injected with PBS (l, t-v), HEK293T cells transfected with empty vector (vec) (n), were determined by two-tailed Wilcoxon rank-sum tests (c), two-tailed one-way ANOVA with Tukey’s multiple-comparisons test (d) or Dunnett’s multiple-comparisons test (g-n, t-w).

Source Data