Extended Data Fig. 3. Characterization of in vitro tumor-immune cell co-culture system.

a, MHC-I (H-2K^d/H-2D^d) presentation in parental Py8119 cells were analyzed by flow cytometry. Isotype IgG served as negative control. b, Stable expression of Ovalbumin (OVA) in the resulted Py8119-OVA cells were confirmed with western blot. c, Surface presentation of OVA (H-2K^b-SIINFEKL) in parental Py8119 and Py8119-OVA cells with or without OT-1 splenocytes co-culture were analyzed with flow cytometry. d, Splenocytes isolated from OT-I mice were treated with PBS or 2 μg/ml of Ovalbumin peptide (OVAp257) for 2 hr. The cells were washed with PBS and plated in fresh media for another 24 hr followed by flow cytometry analysis. % of CD137⁺ or IFN-γ⁺ cells in live populations are shown. e, Splenocytes on the CD8 population were gated and the mean fluorescence intensity (MFI) of CD137 and IFN-γ were measured. n=3 independent experiments. f, The indicated cells with endogenous Mtdh knockdown and vector (Vec) or wild type MTDH rescue were confirmed with western blot. g, The tumorigenesis ability of indicate cell lines were evaluated. Indicated cells were inoculated into the mammary fat pad of C57BL/6 female mice. Ten weeks after injection, lung metastatic nodules were counted. shCtrl, Py8119-shCtrl; KD, Py8119-shMTDH; shCtrl-OVA, Py8119-OVA-shCtrl; KD-OVA, Py8119-OVA-shMTDH; KD-OVA-Vec, Py8119-OVA-shMTDH rescued with vector; KD-OVA-MTDH, Py8119-OVA-shMTDH rescued with wild type MTDH. n=5 mice per group. h, Py8119-OVA cells with/without endogenous Mtdh knockdown or with/without wild type MTDH rescued were employed for mammary fat pad injections. The injected OT-I female mice were treated with/without anti-CD8 neutralization antibody or IgG. Six weeks after treatment, lung metastasis was determined. n=9 mice per group. i, Splenocytes co-cultured with indicated Py8119-OVA tumor cells (same as in Fig. 2b) for 24 hr were harvested for flow cytometry analysis. The expression of Granzyme B in CD8⁺ T cells were examined. MFI, Mean Fluorescence Intensity. n=3 independent experiments. Data represent mean ± SEM. Significance determined by two tailed Student’s t-test (e,g), or one-way ANOVA analysis with Sidak’s’s test for multiple comparisons (h,i).Numerical source data for e, g, h, i, and uncropped blots for b, f are provided.