Fig 2: CCL2-mediated IM recruitment is critical for LUSC metastasis. a Relative expression of CCL2 for KLN205 and sub-clones. b Quantification of luciferase signal. c Representative images obtained 10 days after cell injection of (i) KLN205-Scr ORF, (ii) KLN205-CCL2 ORF, (iii) LN4K1-Cntrl shR, (iv) LN4K1-CCL2 shR#1 and (v) LN4K1-CCL2 shR#2. Data are averages ± s.e.m. P-values were obtained with Student’s t-test, n = 10 mice/group. d Survival plots of mice following tail vein injection of KLN205 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. e Number of IMs per lung lobe, n = 12 lobes/group. f Survival plots of mice following tail vein injection of LN4K1 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. g Number of IMs per lung lobe, n = 12 lobes/group. h Schematic (left) and quantification of luciferase signal (right) of mice treated with vehicle or PF-04136309 to assess effects on established metastases. i FACS plots and (j) quantification of percent IMs in the blood and (k) right lung of LN4K1-bearing mice. Data are averages ± s.e.m. P-values were obtained with Student’s t-test, n = 5 mice/group. l, FACS analysis of percent immune infiltrates for TAMs (gated on F480), DCs (gated on SiglecF-/CD11c), CD4, CD8, Tregs (gated on TCRb + ) and NK cells (gated on SiglecF-/B220-/TCRb-). m Schematic (left) and quantification of luciferase signal (right) of mice treated at the time of cell injection to assess effects on preventing metastasis. Data are averages ± s.e.m. P-values were obtained with Student’s t-test, n = 10 mice/group. n.s. = non-significant, * P=0.05, *** P < 0.001. For panel b, * FDR < 0.05, ** FDR < 0.01

Fig 3: A fraction of tongue-associated Treg cell clones exhibit clonal expansion and reactivity to regional antigens(A and B) 6- to 8-week-old Foxp3GFP TCRßtg+ mice were placed on 4-NQO drinking water for 8 weeks, followed by 24 weeks of normal drinking water. Treg cells were subsequently isolated from tongues, single-cell sorted by fluorescence-activated cell sorting (FACS), and TCRa chains were amplified by nested PCR and subjected to Sanger sequencing (see STAR Methods). n = 5 mice.(A) Heatmap depicting all CDR3a sequences identified among all single-cell sorted Treg cells. Each row represents a unique CDR3a sequence. (Left) The number of occurrences each CDR3a sequence appears in tongue-associated Treg datasets from individual 4-NQO-treated mice is shown. Each column represents an individual mouse, with the number of cells sequenced per mouse noted in parentheses. The last column represents the cumulative data. Color denotes the total number each CDR3a sequence appears in dataset. (Right) The frequency each CDR3a sequence appears in published datasets of CD4+ Foxp3+ Treg cells or CD4+ Foxp3neg Tconv cells isolated from pooled secondary lymphoid organs (SLOs) of untreated, non-tumor-bearing Foxp3GFP TCRßtg+ mice is shown.59 Each column represents dataset from an individual mouse. Color denotes the frequency of each CDR3a sequence in dataset.(B) Select CDR3a sequences from (A), depicting those recurrent among at least three 4-NQO-treated mice. Underlined amino acid sequence refers to the 3-letter ID used to reference each TCR clone in (C) and (D).(C and D) 104 Thy1.1+ CD4+ TCR retrogenic (TCRrg) T cells were isolated from pooled SLOs of primary retrogenic mice and co-transferred with 106 RBC-lysed splenocytes from CD45.1/.1 mice intravenously into Tcrb-/- secondary recipients (see STAR Methods). 3 weeks after transfer, the fate of transferred T cells was assessed in SLOs.(C) Representative flow cytometric analysis of CD4+ T cells isolated from indicated organs of secondary recipients that received RTSrg donor T cells. The top plots depict Thy1.1 versus CD45.1 expression by CD4+ cells, whereas the middle and bottom plots depict Egr2 versus CD69 expression by donor Thy1.1+ RTSrg or CD45.1+ polyclonal CD4+ T cells, respectively. The frequency of cells within the indicated gates is denoted.(D) Summary plots of pooled data from (C), showing the frequency of Thy1.1+ CD4+ TCRrg T cells among total CD4+ T cells (top) and the frequency of Thy1.1+ CD4+ TCRrg T cells expressing Egr2 and CD69 (bottom), isolated from indicated organs of secondary recipient mice. TCR clone names depicted refer to the 3rd–5th amino acids of the CDR3a sequence, underlined in (B). Mean is indicated. Dotted line denotes y axis = 1 (top) or y axis = 0 (bottom). n = 4–7 mice (RTS TCRrg), n = 2 mice (DPY TCRrg), n = 9 mice (PHI TCRrg), and n = 3 mice (KGN TCRrg).Data are pooled from multiple independent experiments. aLN, axial lymph node; bLN, brachial lymph node; cLN, cervical lymph node; mLN, mesenteric lymph node; pLN, periaortic lymph node; Spl, spleen. One-way ANOVA with Tukey’s post-test analysis, comparing all pairs in column (D; adjusted p values from Tukey’s post-test are depicted). *p < 0.0.5, **p < 0.01, and ***p < 0.001. See also Tables S1 and S2.

Fig 4: 4-NQO-induced lesions are not significantly impacted by the endogenous T cell response or treatment with checkpoint blockade monotherapies(A and B) 6- to 8-week-old littermates of indicated genotype were exposed to 4-NQO drinking water for 20 weeks, tongues were subsequently excised, and histopathology was quantified as in Figure 2A. n = 20 mice (Tcrb+/-Tcrd+/-), n = 11 mice (Tcrb+/-Tcrd-/-), n = 18 mice (Tcrb-/-Tcrd+/-), and n = 7 mice (Tcrb-/-Tcrd-/-).(A) Tumor incidence, where each mouse was scored once based on most severe histology grade observed. Summary plot of pooled data shows the percentage of mice scored as indicated histology grade. Each bar represents the cumulative data for mice of the indicated genotype.(B) Tumor burden. Summary plots of pooled data show the percentage of tongue perimeter defined as hyperkeratosis (left plot), dysplasia (middle plot), or SCC (right plot). Each symbol represents an individual tongue from a single mouse of the indicated genotype. Median is indicated.(C–E) 6- to 8-week-old female C57BL/6 mice were exposed to 4-NQO drinking water for 20 weeks. During the last 4 weeks of 4-NQO exposure, mice were treated intraperitoneally twice weekly with 100 µg anti-PD-L1 or isotype control monoclonal antibody. Tongues were excised at endpoint, and histopathology and IHC staining was performed and quantified as in Figures 2A and 2D. n = 10 mice per group.(C) Tumor incidence, where each mouse was scored once based on most severe histology grade observed. Summary plot of pooled data shows the percentage of mice scored as indicated histology grade. Each bar represents the cumulative data for mice that received indicated antibody treatment.(D) Tumor burden. Summary plot of pooled data shows the percentage of tongue perimeter defined as the indicated histology grade. Each symbol represents an individual tongue from a single mouse that received the indicated antibody treatment. Median is indicated.(E) Summary plot of pooled data from CD3 IHC density analysis, showing the number of CD3+ cells per mm2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 6–52 lesions from 10 mice per group.Data are pooled from multiple independent experiments. Fisher’s test (A and C); one-way ANOVA, comparing all pairs in column (B); two-tailed nonparametric Mann Whitney test (D and E). NS, not significant. See also Figure S2.

Fig 5: Application of DestVI to a MCA205 tumor sample.(A) Schematics of the experimental pipeline. We performed ST (10x Visium) and single-cell RNA sequencing (scRNA-seq, single-cell MARS-seq protocol) on MCA205 tumor that contains heterogeneous immune cell populations 14 days after intracutaneous transplantation into the wild-type mouse (two sections). (B) Visualization of the ST data for two MCA205 tumor sections, after quality control (4,027 spots). Scale bar, 1000 μm. The two sections were processed on the different capture areas of the same Visium gene expression slide. (C) UMAP projection of the scRNA-seq data (8,051 cells), embedded by scVI and manually annotated. (D) Spatial autocorrelation of the CTP for every cell type, computed using Hotspot. (E) Spatial distribution of CTP for DCs, monocytes and macrophages (Mon-Mac), CD8 T cells and NK cells. (F) Immunofluorescence imaging from neighboring tumor sections, using antibodies for MHCII+ cells showing for DCs (Section-3, +20 μm from Section-2), F4/80+MHCII− cells showing for Mon-Mac (Section-3, +20 μm from Section-2), TCRb+ cells showing for CD8 T cells (Section-5, +60 μm from Section-2) and NK1.1+ cells showing for NK cells (Section-4, +30 μm from Section-2). All scale bars denote 500 μm. Red solid lines indicate the section boundary. Right side is the MCA205 tumor marginal boundary. The staining marker positive cells are segmented and annotated using QuPath and showing yellow color here with changed brightness and contrast (Supplementary Methods).