Extended Data Fig. 3: Generation and immunogenicity of Eml4-Alk^PGPGRVAKI immortalized cell lines.

(a) Representative H&E image of lung tumors generated in syngeneic BALB/c mice injected intravenously with mEml4-Alk. 2x magnification (left panel); 40x magnification (right panel) (n = 3 independent mice). Scale bar left = 1.5 mm; Scale bar right= 100 µm. (b) Schematic illustration of mouse and human ALK short peptide 7 sequences differences and CRISPR/Cas9-edited DNA bases (Red). NetMHC4.0 showing peptide affinity (predicted IC50 values in nM) for H-2D^d and NetMHCpan4.1 showing the rank of the elution ligand likelihood (%Rank_EL) for H-2D^d. (c) Sanger sequencing chromatogram showing the cDNA sequence of mEml4-Alk (upper panel), Eml4-Alk^PGPGRVAKI-1 (middle panel), and Eml4-Alk^PGPGRVAKI-2 (lower panel) of PGPGRVAKI peptide. (d,e) Dose response curves of Eml4-Alk^PGPGRVAKI-1 (d) and Eml4-Alk^PGPGRVAKI-2 (e) cell lines to different ALK inhibitors (crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib). Two independent experiments were performed for each ALK inhibitor in each cell line. (f) Representative immunoblot for the indicated proteins in Eml4-Alk^PGPGRVAKI-1 cells treated with lorlatinib at the indicated concentrations for 6 h (N = 2 independent experiments with similar results). (g) Representative H&E of lung tumors generated in syngeneic BALB/c mice injected intravenously with Eml4-Alk^PGPGRVAKI-1. 2x magnification (left panel); 40x magnification (right panel) (n = 3 independent mice). Scale bar left = 1.5 mm; Scale bar right= 100 µm. (h) IFN-γ-ELISPOT analysis of freshly isolated splenocytes from tumor-bearing BALB/c mice 15 days after been injected either subcutaneously (flank) or intravenously (lung) with mEml4-Alk (left panel), Eml4-Alk^PGPGRVAKI-1 (middle panel), and Eml4-Alk^PGPGRVAKI-2 (right panel) cells. Data is shown as average number of spot forming units (SFU ± SEM). Unpaired two-tailed Student’s t test, not significant, P = 0.2038; **P < 0.005; ***P < 0.001. The N shown on the figure corresponds to the number of mice per group. (i,j) Dextramer staining of PGPGRVAKI-specific CD8⁺ T cells isolated from splenocytes (i) and PGPGRVAKI-specific tumor infiltrating T lymphocytes (TILs) isolated from lung tumors (j) at day 15 post subcutaneous (flank) and intravenous (lung) injection. Mice were injected with mElm4-Alk (left panel), with Eml4-Alk^PGPGRVAKI-1 (middle panel), and with Eml4-Alk^PGPGRVAKI-2 (right panel) cells (%±SEM). Unpaired two-tailed Student’s t test. The N shown on the figure corresponds to the number of mice per group. (k) Tumor growth of mice injected subcutis in the flank with mEml4-Alk¹ cells and treated as indicated. Data are shown as average tumor volume (mm³ ± SEM). (l) Kaplan-Meier curves showing overall survival of mice injected intravenously with Eml4-Alk^PGPGRVAKI-1 and treated as indicated. Log-rank test), not significant, P = 0.6040).

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