Extended Data Figure 6 |. Autophagy inhibition modulates anti-tumour immunity in both orthotopic tumours and liver metastasis.

a, Immunoblots showing autophagy inhibition in mSt-Atg4B^C74A expressing cells. Mouse PDAC cells carrying doxycycline (Dox)-inducible mStrawberry (mSt) or mSt-Atg4B^C74A (4B) were treated with Dox (1 μg/mL) for the indicated days. mSt or mSt-Atg4B^C74A was detected by anti-RFP antibody. A representative of two independent experiments is shown. b-j, (Related to Fig. 3e–h) Mouse PDAC cells shown in (a) were orthotopically transplanted into syngeneic (C57Bl/6) mice. HY15549 cells (mSt, n = 8; 4B, n = 7) and HY19636 cells (n = 8 per group) were injected. b, Study design. c, Images of tumours at end point. d-g, HY19636 tumour weight (d), cell surface MHC-I levels (e) and PD-L1 levels (f) on PDAC cells and (g) tumour-infiltrating CD8⁺ T cells measured by flow cytometry. h,i, Representative H&E staining (h) and immunofluorescent staining (i) of HY15549 tumours (mSt, n = 8; 4B, n = 7). Scale bars, 100 μm. j, Quantification of tumour-infiltrating immune cells by flow cytometry (HY15549, n = 8 and 7; HY19636, n = 8 per group). Gating strategies are shown in Extended Data Fig. 7k and Supplementary Table 2. Treg, T regulatory cells; MDSC, myeloid-derived suppressor cells; TAM, tumour-associated macrophages. k-o, Autophagy-inhibition by Atg7 knockdown elicits similar anti-tumour T cell responses. k, Immunoblots for Atg7, LC3, and β-actin in PDAC cells (HY15549) expressing shRNAs against GFP or Atg7. A representative of at least two independent experiments is shown. l-o, Mouse PDAC cells shown in (k) were orthotopically transplanted into syngeneic mice (n = 7 per group). Images of tumours harvested on day 22 (l). Tumour weight (m). Tumour-infiltrating immune cells as measured by flow cytometry (n). Correlation between CD8⁺ T cell frequency among CD45⁺ cells and tumour weight (o). p-t, (Related to Fig. 3i–l) Autophagy inhibition modulates anti-tumour immunity in metastatic tumours in the liver. Mouse PDAC cells (HY15549) carrying Dox-inducible mSt or 4B were injected into the spleen of syngeneic (C57Bl/6) mice that were pre-fed with Dox-containing diet (n = 4 per group). PDAC cells were pre-treated with Dox (1 μg/mL) for 7 days before injection. Study design (p). Images of the liver (q). Representative images of H&E staining (r) and immunofluorescent staining (s) (n = 4 per group). Scale bars, 200 μm (r) and 100 μm (s). Quantification of immune cells in the liver metastasis as measured by flow cytometry (t). Data are mean ± s.e.m. n indicates individual mice. Statistical differences were determined by unpaired two-tailed t-tests (d-g,j,m,n,t), and Pearson correlation analysis (o). For gel source data of a,k, see Supplementary Fig. 1.