Additional file 1: of Tumor regression mediated by oncogene withdrawal or erlotinib stimulates infiltration of inflammatory immune cells in EGFR mutant lung tumors

Figure S1. MRI images, histology and representative flow cytometry plots of normal or tumor-bearing lungs before and after erlotinib. (A) Coronal images of CCSPrtTA; TetO-EGFRL858R mouse lungs before (left panel) and after (right panel) treatment with erlotinib. (B) Hematoxylin and eosin (H&E) stain of lungs from control (normal) and tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks. Bar: 50 μm. Absolute number of (C) CD4 and (D) CD8 T cells normalized to weight of lungs of control (normal) and tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks. Representative FACS plot showing percentage of (E) FoxP3+ and FoxP3- CD4+ T cells (F) PD1+ FoxP3+ T cells. (G) Immunofluorescence (IF) stain of lung epithelial cells (green), CD3 T cells (red) and FoxP3 Tregs (Cyan). Nuclei were counterstained with Dapi (blue) (H) Quantification of FoxP3+ CD3 T cells in lung tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks stained by IF. Data are shown as mean ± SD and * is P < 0.05 in a student’s t-test. NS, non-significant. Figure S2. Representative flow cytometry plots of cytokine producing T cells and gene expression profile of T cells isolated from tumor-bearing lungs before and after erlotinib. Representative FACS plots showing the percentage of (A) TNF-α+, IFN-γ+, and IL-2+ CD4 T cells. (B) Quantification of GzmB+ CD4 and CD8 T cells after PMA/ionomycin stimulation and intracellular cytokine staining of cells in the lungs of tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks. (C) Representative FACS plot showing percentage of GzmB+ splenic CD8 T cells after PMA/ionomycin stimulation and intracellular cytokine staining of cells. (D) CD8 and (E) CD4 T cells isolated from CCSP-rtTA; TetO-EGFRL858R tumor bearing mice untreated or treated with erlotinib. Heatmap was generated using normalized expression values. NS, non-significant. Figure S3. MRI images, histology and representative flow cytometry plots of erlotinib sensitive and resistant tumors. (A) Coronal images of: CCSP-rtTA; TetO-EGFRL858R mouse lungs before (left panel) and after (right panel) cessation of doxycyline and CCSP-rtTA; TetO-EGFRL858R + T790M mouse lungs before (left panel) and after (right panel) treatment with 2 erlotinib. (B) Hematoxylin and eosin (H&E) stain of lungs from tumor bearing: CCSP-rtTA; TetO-EGFRL858R untreated or taken off doxycycline diet for 2 weeks and CCSP-rtTA; TetOEGFRL858R+ T70M mice in the absence (−) and presence (+) of erlotinib for 2 weeks. Bar: 50 μm. Absolute number of (C) CD4 and (D) CD8 T cells normalized to weight of lungs of tumor bearing CCSP-rtTA; TetO-EGFRL858R or CCSP-rtTA; TetO-EGFRL858R + T790M mice in the absence (−) and presence (+) of erlotinib for 2 weeks or taken off doxycycline diet. Data are obtained from three independent experiments, (n = 4–6 mice per group) * is P < 0.05 in a student’s t-test. Quantification of (E) CD4 and CD8 T cells and (F) FoxP3 positive CD4 T cells in the lungs of control (normal) mice in the absence (−) and presence (+) of erlotinib for 2 weeks. Data are shown as the mean ± SD. NS, non significant. Figure S4. Quantification of circulating and proliferating T cells. (A) Absolute number and (B) Fold change in number of circulating lung CD4 and CD8 T cells of tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks. (C) Ki-67+ CD4 and CD8 T cells of tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks or mice taken off doxycycline for 2 weeks. Data are shown as the mean ± SD and * is P < 0.05 in a student’s t-test. NS, non-significant. Figure S5. Experimental outline of CFSE labeling and analysis. (A) Flow chart for isolation, labeling and treatment of T cells from lungs and spleens of tumor bearing CCSP-rtTA; TetO-EGFRL858R mice. FACS plot showing (B) as a control for the technique, unlabeled vs CFSE labeled splenocytes (Day 0) and (C) Untreated CD4 and CD8 T cells from lungs and spleens at Day 0 as well as CD4 and CD8 T cells from lungs and spleens treated with 10 μm erlotinib or DMSO after 5 days (120 h). Figure S6. Quantification of proliferating alveolar macrophages and myeloid cells in healthy lungs before and after erlotinib treatment. (A) Ki-67 positive AMs, (B) Cxcl2 expression in AMs, (C) Cd274 mRNA expression in Epcam+ tumor cells from lungs of control (normal) and tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib for 2 weeks. (D) Quantification of myeloid cell populations in the lungs of control (normal) mice in the absence (−) and presence (+) of erlotinib for 2 weeks. Data are shown as the mean ± SD and * is P < 0.05 in a student’s t-test. NS, non-significant. Figure S7. Tumor volume measurements and survival analysis. Quantification of (A) CD8+ T cells, (B) Ki-67+ CD8+ T cells and (C) Eomes+ CD8+ T cells in the lungs of tumor bearing CCSP-rtTA; TetO-EGFRL858R mice in the absence (−) and presence (+) of erlotinib, CD40 agonist or erlotinib plus the CD40 agonist for 2 weeks, (n = 4–6 mice per group). (D) Tumor volume quantified at 1 and 2 weeks after treatment measured by MRI normalized to pretreatment tumor volume. Change in tumor volume (E) pre-treatment and 1–4 weeks after treatment with erlotinib alone or erlotinib and immunotherapy combination and (F) pretreatment, 1–4 weeks after treatment with erlotinib alone or erlotinib and immunotherapy combination and 1–3 weeks after stopping erlotinib (relapse), (n = 5–10 mice per group). Data are shown as the mean ± SD and * is P < 0.05 in a student’s t-test. NS, non-significant. (XLSX 66 kb)