Fig 2: The topological distribution of CD4‐ and CD8‐positive cells in lymph nodes with no, small, intermediate, and large metastatic tumor burden. (A–D) One SNneg (non‐metastatic sentinel node) (patient 11) and three SNmets (metastatic sentinel nodes) (patients 37,39, and 27) were IHC stained with AE1/AE3, CD4, and CD8. Magnified area of extratumoral (orange) and intratumoral (green) regions stained with CD8 and CD4 of lymph nodes with small (B), intermediate (C), and large (D) tumor burden. Scales and magnifications are shown in images. (E, F) A scoring system, ImmunoPath (with field fraction function measuring positive and negative pixels was used to calculate the percentage positive areas) was used to objectively score IHC‐positive areas. (E) Histograms comparing tumor cell percentage and CD4/CD8 ratio measured by IHC (left axis) and mass cytometry (right axis) performed on cells from the same lymph nodes. (F) Comparison of CD4/CD8 ratio in intratumoral and extratumoral areas of the metastatic lymph node analyzed separately by ImmunoPath. (G) Immunofluorescence staining with CD8 (green) and Treg (FoxP3, red) and counterstained with DAPI on non‐metastatic sentinel node and metastatic sentinel nodes (same samples as A‐D) Scales and magnifications are shown in images.

Fig 3: Alteration of TME cell presence over time in PT and in patient-derived organoids (PDOs) at day 7 and 14 of culture. n = 9 patients. The relative expression of cellular TME components [%] was quantified in PT (blue) and at day 7 (violet) and day 14 (green) of PDO culture: (A) CD45, (B) CD7, (C) CD4, (D) CD8, (E) CD14, (F) CD68, (G) iNOS, (H) CD163, (I) CCR2, (J) CD25, (K) PD1, (L) FOXP3, (M) TIM3, (N) LAG3; Whiskers indicate standard deviation; significant differences are indicated.

Fig 4: Expression of cellular TME markers in normal PDOs (nPDOs) and enhanced PDOs (ePDOs) at day 0, 7, 14 and 21 compared to PT. n = 3 patients. The relative expression of cellular TME components [%] was quantified in PT (blue) and at day 7 (green), day 14 (orange) and day 21 (red) of PDO culture in nPDOs and ePDOs: (A) CD45, (B) CD7, (C) CD4, (D) CD8, (E) CD14, (F) CD68, (G) iNOS, (H) CD163; Whiskers indicate standard deviation; significant differences are indicated.

Fig 5: Loss of BCAT2 enhances efficacy of anti-PD-1 therapy. A) Flow diagram of treatment plan. B) Harvested tumors of different therapy regimens (n = 5 per group). C–E) Quantification of tumor volume, body weight and survival time in different therapy regimens (n = 5 per group). ns: no significance; *p< 0.05; **p < 0.01; ***p < 0.001. F) Contour plots indicate proportion of CD8+T cells; G) proportions of GZMB+CD8+T cells, IFN-? +CD8+T cells, TNF-a +CD8+T cells, and Perforin+CD8+T cells in different therapy regimens. H) Quantified scatter plots exhibit proportion of CD8+T cells; I–L) proportions of GZMB+ CD8+T cells, IFN-? + CD8+T cells, TNF-a + CD8+T cells, and Perforin+ CD8+T cells in different therapy regimens (n = 5 per group). ns: no significance; *p<0.05; **p < 0.01; ***p < 0.001. M,N) IF image and quantified histogram show densities of CD8+T cells in different therapy regimens (n = 3 per group). Scale bar: 20 µm. CD8+T cell (green) and cell nucleus (blue). ns: no significance, *p<0.05; **p < 0.01; ***p < 0.001.