Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Acute intranasal HDM and tamoxifen treatment protocol. B. Representative flow cytometry of lung intraparenchymal (anti-CD45 i.v. antibody negative) CD4 + T cells showing Foxp3 and CCR4 expression from indicated groups. C. Percent lung CCR4 + Foxp3 + Tregs among Foxp3 + Tregs from indicated groups. D. Percentage of Foxp3 + Tregs among lung CD4 + T cells and number of total lung Tregs from indicated groups. E. Number of total lung DC subsets from indicated groups. F. Number of PD-L2 + CCR7 + lung cDC2s. G. Number of lung eosinophils H. Percentage of Foxp3 - ST2 + T cells (Th2 cells) among lung CD4 + T cells and number of total lung Th2 cells . I. Representative PAS-stained lung sections. J. Mucus scores. Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were administered 10 µg i.n HDM 3 times per week for 6 weeks alongside 1 mg daily doses of tamoxifen via oral gavage on days 4-8, 18-22 and 32-36 of HDM treatment then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 42. K. Chronic intranasal HDM and tamoxifen treatment protocol. L. Representative flow cytometry of lung CD4 + T cells showing Foxp3 and CCR4 expression from indicated groups. M. Percentage of lung CCR4 + Foxp3 + Tregs. N. Number of lung Tregs. O. Number of lung DC subsets. P. Number of PD-L2 + CCR7 + lung cDC2s. Q. Number of lung eosinophils R. Number of lung Th2 cells. Data are from three independent experiments with 11-18 mice pooled (A-J) or data are from two independent experiments with 12 mice pooled (K-R). For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. One-way ANOVA analysis with Holm-Sidak’s testing for multiple comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Flow Cytometry, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A . % Ki67 + and gMFI for TIGIT, GITR, ICOS, and ST2 in lung Tregs from indicated groups. B . Percentage PD-L2 + CCR7 + cDC2s of total lung cDC2s. C . Total number of lung neutrophils. D . Percentage of lung ILC2s of lineage - cells and total number of lung ILC2s. E . Percentage and number of lung Th1 cells. F . Percentage and number of lung Th17 cells. G . Percent LN CCR4 + Foxp3 + Tregs. H . Percentage LN Tregs. I . Number of LN Tregs. J . Percentage and number of LN Th2 cells. K . Number of total LN DC subsets. L . Percentage and total number of PD-L2 + CCR7 + of LN cDC2s. M . Serum IgE levels. N-Q . Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were administered 10 µg i.n HDM 3 times per week for 6 weeks, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, 18-22 and 32-36 of HDM treatment, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 42. N . Percentage of lung Tregs of total CD4 + T cells from indicated groups. O . Percentage PD-L2 + CCR7 + cDC2s of total lung cDC2s. P . Representative PAS-stained lung sections. Q . Mucus scores. Data are from three independent experiments with 11-18 mice pooled or data are representative of one experiment with n L=L5 mice per group from three independent experiments. For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. One-way ANOVA analysis with Holm-Sidak’s testing for multiple comparisons. *, p<0.05; ****, p<0.0001, ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: CCR4 promotes the efficiency of Treg trafficking to suppress the expansion of CCR7+ cDC2-CD4+ T cell clusters during allergic inflammation. Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Representative imaging of lungs from indicated groups with staining for CD4 (Cyan), Foxp3 (Red), and FSCN1 (Yellow). B. Higher magnification of images shown in (A). C-D. Quantification of nearest distance (µm) between FSCN1 + DCs and Foxp3 + Tregs (C) and FSCN1 + DCs and CD4 + T cells (D). E-G . Mixed bone marrow chimeras were generated with Foxp3 EGFP-Cre-ERT2 Thy1.1 + Thy1.2 + control and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl (Thy1.2) bone marrow cells. Ctrl: Ccr4 cKO mixed bone marrow chimeras were treated with HDM (days 0, 7-11) and tamoxifen via oral gavage (days 4-8) then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. E. Representative flow cytometry of lung CD4 + T cells showing Foxp3 + and Thy1.1 + Thy1.2 + (Ctrl) and Thy1.2 + ( Ccr4 cKO) Tregs. F. Ratio of Ctrl: Ccr4 cKO Tregs in the lymph nodes and lungs. G. Ratio of Ctrl: Ccr4 cKO Th2 cells in the lymph nodes and lungs. H. Volcano plot showing differential gene expression analysis of bulk transcriptomes between sorted lung Tregs from Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice treated with HDM and tamoxifen. I . Asb2 relative RNA expression in iTregs cultured with or without rIL-4 for 2 days. J . Mixed bone marrow chimeras were generated with C57BL/6 control (Ctrl) and Il4r α-deficient bone marrow cells. Ctrl: Il4ra -deficient mixed bone marrow chimeras were treated with HDM (days 0, 7-11) then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. Ctrl: Il4ra KO ratio of lung T cell subsets. Images are representative of one of two independent experiments (A-B), data were pooled form two independent experiments (C-D), or data are representative of one experiment with n L=L10 mice per group from two independent experiments (E-G), or data were pooled form two independent experiments with n = 6 mice per group (H), or data were pooled from nine independent experiments (I), or data were pooled from two independent experiments with n = 10 mice (J). For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. **, p<0.001; ****, p<0.0001; ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Imaging, Staining, Generated, Flow Cytometry, Gene Expression, RNA Expression, Cell Culture, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Representative histograms of surface CD80 and CD86 expression on lung cDC2s from indicated groups. B. gMFI for surface CD80 and CD86 on lung cDC2s. C. Representative histograms of surface CD80 and CD86 expression on lung MCs. D. gMFI for CD80 and CD86 on lung MCs. E. Representative histograms of surface CD80 and CD86 expression on lung Foxp3 + Tregs. F. gMFI for CD80 and CD86 on lung Foxp3 + Tregs. Representative data shows individual mice with mean ± SEM from one of three independent experiments with n L=L4-5 mice per group. For statistical analysis, a two-tailed t test was performed for parametric data. *, p<0.05, **, p<0.01, ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Olfactomedin4 marks luminal progenitor cells that give rise to secretory cell lineage in the mouse cervix

doi: 10.64898/2026.04.28.721179

Figure Lengend Snippet: A. Schematic of lineage-tracing strategy using Krt8-Cre ERT2 ; Rosa26 tdTomato mice with tissues collected across nonpregnant and early-to late-pregnancy: gestation day (GD) 6, 12, 15, 18 and in labor (IL). B. RNA expression in the endocervix for tdTomato (red), Spdef (yellow), and Muc5b (pink) across diestrus, pregnancy stages and in labor. Spdef and Muc5b were co-stained in the same section, with each marker displayed in separate panels, whereas tdTomato was detected on a separate section. Epi-epithelial and Str- stromal regions are indicated. Images are representative of n=1 biological replicate per time point and scale bars =100 µm.

Article Snippet: The Krt8-Cre/ ERT2 (17Blpn/J) mice were sourced from Jackson Laboratories.

Techniques: RNA Expression, Staining, Marker

Journal: bioRxiv

Article Title: Olfactomedin4 marks luminal progenitor cells that give rise to secretory cell lineage in the mouse cervix

doi: 10.64898/2026.04.28.721179

Figure Lengend Snippet: A. Schematic of lineage tracing strategy using Olfm4-Cre ERT2 ; Rosa26 tdTomato mice. B. RNA expression of tdTomato+ (red), Spdef (cyan) and Muc5b (green) in endocervical tissue from nonpregnant (diestrus) and pregnancy (GD 6,12,15, 18 and IL). All three markers were detected in the same section, with each signal displayed in separate panels. Images are representative of n≥ 3 biological replicates per time point and scale bars =100 µm. C. Schematic of workflow used to generate organoid cultures from tdTomato negative and tdTomato positive epithelial populations from the cervix of nonpregnant diestrus Olfm4-Cre ERT2 ; Rosa26 tdTomato mice. D. Organoids generated from flow cytometry sorted tdTomato+ and tdTomato- epithelial populations. tdTomato+ derived organoids retained tdTomato signal, whereas tdTomato-progenitors formed organoids that did not express tdTomato. n = 3 biological replicates and scale bars =100 µm. E. Schematic of short-term and long-term lineage-tracing strategy using Olfm4 Cre ERT2 ; Rosa26 tdTomato mice (8-week-old females). Short-term tracing (top): Tamoxifen (Tmx) was administered at estrus, diestrus, or GD11, and tissues were collected 24-48 hrs later at the subsequent time points (estrus to diestrus, diestrus to estrus, GD11 to GD12). Long-term tracing (bottom): Tmx was administered at the NP stage, followed by a first pregnancy. After the first pregnancy, tissues were collected at two stages of the estrous cycle, or on GD15 of a second pregnancy. F. RNAscope images of endocervix from short-term tracing experiments. Panels indicate tdTomato⁺ (red), Spdef (cyan), Muc5b (green) and nuclei (blue). In short-term tracing, tdTomato⁺ cells are present and co-localize with goblet markers during diestrus (first column) and on GD12 (third column). All three markers were stained in the same section and are displayed in separate panels. Representative images from n = 2 biological replicates per condition. Scale bars = 100 µm. G. RNAscope images of endocervix from long-term tracing experiments. Panels indicate tdTomato⁺ (red), Spdef (cyan), Muc5b (green) and nuclei (blue). In long-term tracing, tdTomato⁺ cells persist and give rise to secretory goblet cells that express Spdef and Muc5b in diestrus (second column) and in a second pregnancy at GD15 (third column). All three markers were stained in the same section and are displayed in separate panels. Representative images from n = 2 biological replicates per condition. Scale bars = 100 µm.

Article Snippet: The Krt8-Cre/ ERT2 (17Blpn/J) mice were sourced from Jackson Laboratories.

Techniques: RNA Expression, Generated, Flow Cytometry, Derivative Assay, RNAscope, Staining

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of CCR2-Cre ERT2 ;ZsGreen mouse. b , Representative confocal images showing lineage + and lineage - macrophages in the lungs of CCR2-Cre ERT2 ;ZsGreen mice after tumour organoid engraftment. DAPI (blue), ZsGreen (lineage-traced monocytes, green), CD64 (grey), and RFP (red, tumour). Scale bar, 100 μm. Insets (bottom) are enlargements of the dashed boxes (top). Scale bars, 50 μm. Images representative of n = 3 mice. c , Flow cytometry analysis of alveolar macrophages in control and clodronate liposome-treated lungs. d , Quantification of alveolar macrophages in control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. e , Representative confocal images of macrophages and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), F4/80 (grey) and RFP (red, tumour). Scale bar, 100 μm. Images representative of n = 3 mice. f , Dotplot of key chemokine receptors in neutrophils and γδ T cells. Each dot is coloured based on the average expression and sized based on the percentage of cells expressing that gene, as shown. g,h , Representative confocal images of neutrophils ( g ), γδ T cells ( h ) and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), Ly6G (green) and RFP (red) ( g ). DAPI (blue), GL3 (green) and RFP (red) ( h ). Scale bar, 100 μm. Images representative of n = 3 mice. i , Flow cytometry analysis of neutrophils from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. Ly6G (neutrophils), CD11B (leukocytes/granulocytes). j , Quantification of neutrophils from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. k , Flow cytometry analysis of γδ T cells from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. GL3 (TCR gamma/delta), CD3ε (T cells). l , Quantification of γδ T cells from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Flow Cytometry, Control, Two Tailed Test, Expressing, Activation Assay

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Representative confocal images confirming an absence of detectable Areg expression in homeostatic lungs ( Confetti ). Right panels are the magnifications of left panel. DAPI (blue), pro-SPC (green), Areg (grey), YFP (yellow) and RFP (red). Scale bar, 50 µm. Image representative of n = 2 mice. b , Violin plot showing the expression of Areg and EGFR across epithelia, mesenchymal and immune cell populations. c , Experimental scheme to investigate the effect of Areg on wildtype mesenchymal cells. d , Representative brightfield images showing morphological changes of lung mesenchymal cells treated with Areg after 7 days in 3D Matrigel culture. Images are representative of n = 3 independent experiments. Scale bar, 50 µm. e , Representative confocal images showing the expression of reprogrammed fibroblast markers in lung mesenchyme cultures in ( c ). Top panel: DAPI (blue) and Acta2 (red). Bottom panel: DAPI (blue) and Pdgfrβ (red). Scale bar, 50 µm. f , Experimental scheme to investigate the effect of Areg in AMs. g , Flow cytometry analysis of AMs numbers after Areg treatment. h , Flow cytometry analysis showing Msr1 expression in AMs from ( f ). i , Quantification of the MFI for Msr1 in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. j , Flow cytometry analysis showing MHCII expression on AMs from ( f ). k , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. l , Experimental scheme to investigate the effect of Areg on RFP + tumour cells isolated from Red2Kras lungs. m , Bright-field image from ( l ). n , Representative confocal image of RFP + tumour organoids from ( l ). DAPI (blue), Krt8 (grey), pro-SPC (green) and RFP (red). Images representative of n = 3 independent experiments. Scale bar, 100 µm. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ; tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing reprogrammed fibroblasts and lineage-labelled cells in ( o ). Right panels are magnifications of left panel for each condition. DAPI (blue), ZsGreen (green), Pdgfrβ (grey), and RFP (red). Scale bar, 50 µm. Images of representative n = 3 independent experiments.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Expressing, Flow Cytometry, Control, Two Tailed Test, Isolation

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Schematic illustrating 3D organoid co-cultures of wildtype AMs and mesenchymal cells with lineage-labelled RFP + cells isolated Red2Kras lungs. b , Representative flow cytometry plots showing the populations of macrophages (CD45 + EpCAM − ), tumour cells (CD45 − EpCAM + ), and mesenchymal cells (CD45 − EpCAM − ) gated from CD31 − populations and stablished in ( a ). c , Flow cytometry analysis of Msr1 expression in AMs from ( a ). d , e , Quantification of the MFI for Msr1 ( d ) and relative cell numbers ( e ) of AMs obtained from ( a ). Data are mean ± s.e.m. Each dot represents one independent experiment. 1 ( n = 4 ), 2 ( n = 4 ) and 3 ( n = 4 ). P values were calculated using two-tailed unpaired t-test. f , Experimental scheme to investigate the effect of Areg on AMs co-cultured with wildtype mesenchymal and AT2 cells. g , Flow cytometry analysis showing the proportion of AMs from ( f ). h , Quantification of frequency of AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. i , Flow cytometry analysis showing MHCII expression in AMs from ( f ). j , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. k , Experimental scheme to investigate the effect of Areg/Ereg on AMs co-cultured with wildtype mesenchymal and AT2 cells. l , Quantification of the relative cell numbers of AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg/Ereg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. m , Quantification of MFI for MHCII in AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. n , qPCR analysis of Arg1 , Ym-1 , and Tnfa expression in AMs co-cultured with wildtype mesenchyme and AT2 cells treated with PBS, Areg alone, or Areg/Ereg. Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ), Areg ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ;tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing lineage-labelled DATP-like mutant cells in 3D organoid co-cultures from ( o ). DAPI (blue), ZsGreen (green), Sox9 (grey), and RFP (red, tumour). Scale bar, 50 µm. q , Quantification of the percentage of Sox9 expressing cells within RFP + mutant organoids assessed in ( p ). Data are presented as mean ± s.e.m. Each dot represents one organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using Mann–Whitney test. r , Representative confocal images showing lineage-labelled cells in 3D organoid co-cultures with wild-type mesenchymal cells and treated with Gefitinib or DMSO. DAPI (blue), LpCat1 (green) and RFP (red, tumour). Scale bar, 100 µm. s , Quantification of the percentage of LpCat1 expressing cells within RFP + mutant organoids. Data are presented as mean ± s.e.m. Each dot represents one organoid from three independent experiments. DMSO ( n = 3 ), Gefitinib ( n = 3 ). P values were calculated using Mann–Whitney test. t , Schematic illustrating 3D organoid cultures of mutant AT2 cells isolated from Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. u , Representative confocal images showing the expression of DATP-like cell state marker in Red2Kras RFP + mutant organoids from ( t ). DAPI (blue), Sox9 (green) and RFP (red, tumour). Scale bar, 50 µm. v , Percentage of DATP-like cell states expressing Sox9 in Red2Kras RFP + mutant organoids from ( u ). Data are presented as mean ± s.e.m.; Each dot represents an individual organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Isolation, Flow Cytometry, Expressing, Two Tailed Test, Cell Culture, Control, Mutagenesis, MANN-WHITNEY, Marker

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a . Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of Pdgfra-Cre ERT2 ;ZsGreen;iDTR mice. b-d . Representative confocal images for fibrotic fibroblasts ( b ), macrophage ( c ) and mutant epithelial cells ( d ) in PBS-treated and DTR-treated lungs. DAPI (blue), Pdgfrβ (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( b ). DAPI (blue), Cd68 (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( c ). DAPI (blue), Sox9 (grey), and RFP (red, tumour) ( d ). Images are representative of n = 2 mice. Scale bar, 100 μm. e , Experimental design for EGFR tyrosine kinase inhibitor administration. Red2Kras mice received two doses of tamoxifen and were treated with DMSO or Gefitinib (80 mg/kg) at the indicated timepoints. Lung tissues were collected for histological analyses. f , Representative tile scans of entire lung lobe cross-sections from Red2Kras animals treated with DMSO or Gefitinib. DAPI (blue) and RFP (red, tumour). Scale bar, 1,000 µm. g , Percentage of RFP + mutant cell area relative to lobe area assessed in ( f ). Data are as mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ). h,i Representative confocal images for reprogrammed fibroblasts, macrophages and lineage-labelled cells in DMSO or Gefitinib-treated lungs. DAPI (blue), Tnc (grey) and RFP (red, tumour) ( h ). Images representative of n = 2 (DMSO) and n = 2 (Gefitinib) mice. DAPI (blue), F4/80 (grey) and RFP (red, tumour) ( i ). Images representative of n = 2 (DMSO) and n = 3 (Gefitinib) mice. Scale bar, 50 µm. j , Flow cytometry analysis for AMs from Red2Kras lungs treated with DMSO or Gefitinib. k , Quantification of the percentage of AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 3 ) and Gefitinib ( n = 3 ). P values were calculated using two-tailed unpaired t-test. l , Flow cytometry analysis for MHC-II expression on AMs from DMSO- or Gefitinib-treated Red2Kras lungs. m , Quantification of the MFI for MHC-II in AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ) and Gefitinib ( n = 3 ). n , Representative confocal images for neutrophils and lineage-labelled cells in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Ly6G (green) and RFP (red, tumour). Images representative of n = 3 biologically independent mice. Scale bar, 50 µm. o-q , Representative confocal images of the different epithelial states in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Sox9 (grey) and RFP (red, tumour) ( o ). DAPI (blue), Cd177 (grey) and RFP (red, tumour) ( p ). DAPI (blue), Ager (grey), LpCat1 (yellow) and RFP (red, tumour) ( q ). Scale bar, 50 µm. r , Quantification of the percentage of each cell state within the RFP + mutant cell population in DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ).

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Mutagenesis, Flow Cytometry, Two Tailed Test, Expressing

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of CCR2-Cre ERT2 ;ZsGreen mouse. b , Representative confocal images showing lineage + and lineage - macrophages in the lungs of CCR2-Cre ERT2 ;ZsGreen mice after tumour organoid engraftment. DAPI (blue), ZsGreen (lineage-traced monocytes, green), CD64 (grey), and RFP (red, tumour). Scale bar, 100 μm. Insets (bottom) are enlargements of the dashed boxes (top). Scale bars, 50 μm. Images representative of n = 3 mice. c , Flow cytometry analysis of alveolar macrophages in control and clodronate liposome-treated lungs. d , Quantification of alveolar macrophages in control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. e , Representative confocal images of macrophages and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), F4/80 (grey) and RFP (red, tumour). Scale bar, 100 μm. Images representative of n = 3 mice. f , Dotplot of key chemokine receptors in neutrophils and γδ T cells. Each dot is coloured based on the average expression and sized based on the percentage of cells expressing that gene, as shown. g,h , Representative confocal images of neutrophils ( g ), γδ T cells ( h ) and lineage-labelled AT2 cells in control and clodronate liposome-treated lungs. DAPI (blue), Ly6G (green) and RFP (red) ( g ). DAPI (blue), GL3 (green) and RFP (red) ( h ). Scale bar, 100 μm. Images representative of n = 3 mice. i , Flow cytometry analysis of neutrophils from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. Ly6G (neutrophils), CD11B (leukocytes/granulocytes). j , Quantification of neutrophils from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test. k , Flow cytometry analysis of γδ T cells from control and clodronate liposome-treated lungs, at 4 weeks following oncogenic activation. GL3 (TCR gamma/delta), CD3ε (T cells). l , Quantification of γδ T cells from control ( n = 3 ) and clodronate liposome-treated ( n = 3 ) lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Flow Cytometry, Control, Two Tailed Test, Expressing, Activation Assay

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Representative confocal images confirming an absence of detectable Areg expression in homeostatic lungs ( Confetti ). Right panels are the magnifications of left panel. DAPI (blue), pro-SPC (green), Areg (grey), YFP (yellow) and RFP (red). Scale bar, 50 µm. Image representative of n = 2 mice. b , Violin plot showing the expression of Areg and EGFR across epithelia, mesenchymal and immune cell populations. c , Experimental scheme to investigate the effect of Areg on wildtype mesenchymal cells. d , Representative brightfield images showing morphological changes of lung mesenchymal cells treated with Areg after 7 days in 3D Matrigel culture. Images are representative of n = 3 independent experiments. Scale bar, 50 µm. e , Representative confocal images showing the expression of reprogrammed fibroblast markers in lung mesenchyme cultures in ( c ). Top panel: DAPI (blue) and Acta2 (red). Bottom panel: DAPI (blue) and Pdgfrβ (red). Scale bar, 50 µm. f , Experimental scheme to investigate the effect of Areg in AMs. g , Flow cytometry analysis of AMs numbers after Areg treatment. h , Flow cytometry analysis showing Msr1 expression in AMs from ( f ). i , Quantification of the MFI for Msr1 in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. j , Flow cytometry analysis showing MHCII expression on AMs from ( f ). k , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents an independent experiment. Control ( n = 6 ) and Areg ( n = 6 ). P values were calculated using two-tailed unpaired t-test. n.s., not significant. l , Experimental scheme to investigate the effect of Areg on RFP + tumour cells isolated from Red2Kras lungs. m , Bright-field image from ( l ). n , Representative confocal image of RFP + tumour organoids from ( l ). DAPI (blue), Krt8 (grey), pro-SPC (green) and RFP (red). Images representative of n = 3 independent experiments. Scale bar, 100 µm. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ; tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing reprogrammed fibroblasts and lineage-labelled cells in ( o ). Right panels are magnifications of left panel for each condition. DAPI (blue), ZsGreen (green), Pdgfrβ (grey), and RFP (red). Scale bar, 50 µm. Images of representative n = 3 independent experiments.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Expressing, Flow Cytometry, Control, Two Tailed Test, Isolation

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a , Schematic illustrating 3D organoid co-cultures of wildtype AMs and mesenchymal cells with lineage-labelled RFP + cells isolated Red2Kras lungs. b , Representative flow cytometry plots showing the populations of macrophages (CD45 + EpCAM − ), tumour cells (CD45 − EpCAM + ), and mesenchymal cells (CD45 − EpCAM − ) gated from CD31 − populations and stablished in ( a ). c , Flow cytometry analysis of Msr1 expression in AMs from ( a ). d , e , Quantification of the MFI for Msr1 ( d ) and relative cell numbers ( e ) of AMs obtained from ( a ). Data are mean ± s.e.m. Each dot represents one independent experiment. 1 ( n = 4 ), 2 ( n = 4 ) and 3 ( n = 4 ). P values were calculated using two-tailed unpaired t-test. f , Experimental scheme to investigate the effect of Areg on AMs co-cultured with wildtype mesenchymal and AT2 cells. g , Flow cytometry analysis showing the proportion of AMs from ( f ). h , Quantification of frequency of AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. i , Flow cytometry analysis showing MHCII expression in AMs from ( f ). j , Quantification of the MFI for MHCII in AMs from ( f ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. k , Experimental scheme to investigate the effect of Areg/Ereg on AMs co-cultured with wildtype mesenchymal and AT2 cells. l , Quantification of the relative cell numbers of AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 3 ) and Areg/Ereg ( n = 3 ). P values were calculated using two-tailed unpaired t-test. m , Quantification of MFI for MHCII in AMs from ( k ). Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. n , qPCR analysis of Arg1 , Ym-1 , and Tnfa expression in AMs co-cultured with wildtype mesenchyme and AT2 cells treated with PBS, Areg alone, or Areg/Ereg. Data are mean ± s.e.m. Each dot represents one independent experiment. Control ( n = 4 ), Areg ( n = 4 ) and Areg/Ereg ( n = 4 ). P values were calculated using two-tailed unpaired t-test. o , Schematic illustrating 3D organoid co-cultures of fibroblasts isolated from Pdgfra-Cre ERT2 ; ZsGreen lungs with lineage-labelled cells isolated from Sftpc-Cre ERT2 ;tdTomato or Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. p , Representative confocal images showing lineage-labelled DATP-like mutant cells in 3D organoid co-cultures from ( o ). DAPI (blue), ZsGreen (green), Sox9 (grey), and RFP (red, tumour). Scale bar, 50 µm. q , Quantification of the percentage of Sox9 expressing cells within RFP + mutant organoids assessed in ( p ). Data are presented as mean ± s.e.m. Each dot represents one organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using Mann–Whitney test. r , Representative confocal images showing lineage-labelled cells in 3D organoid co-cultures with wild-type mesenchymal cells and treated with Gefitinib or DMSO. DAPI (blue), LpCat1 (green) and RFP (red, tumour). Scale bar, 100 µm. s , Quantification of the percentage of LpCat1 expressing cells within RFP + mutant organoids. Data are presented as mean ± s.e.m. Each dot represents one organoid from three independent experiments. DMSO ( n = 3 ), Gefitinib ( n = 3 ). P values were calculated using Mann–Whitney test. t , Schematic illustrating 3D organoid cultures of mutant AT2 cells isolated from Red2Kras lungs. Gefitinib or DMSO was treated for the indicated duration. u , Representative confocal images showing the expression of DATP-like cell state marker in Red2Kras RFP + mutant organoids from ( t ). DAPI (blue), Sox9 (green) and RFP (red, tumour). Scale bar, 50 µm. v , Percentage of DATP-like cell states expressing Sox9 in Red2Kras RFP + mutant organoids from ( u ). Data are presented as mean ± s.e.m.; Each dot represents an individual organoid from four independent experiments. DMSO ( n = 4 ), Gefitinib ( n = 4 ). P values were calculated using two-tailed unpaired t-test.

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Isolation, Flow Cytometry, Expressing, Two Tailed Test, Cell Culture, Control, Mutagenesis, MANN-WHITNEY, Marker

Journal: Nature

Article Title: Early fibrotic niches establish tumour-permissive microenvironments

doi: 10.1038/s41586-026-10399-6

Figure Lengend Snippet: a . Experiment scheme of orthotopic engraftment of tumour organoids into the lungs of Pdgfra-Cre ERT2 ;ZsGreen;iDTR mice. b-d . Representative confocal images for fibrotic fibroblasts ( b ), macrophage ( c ) and mutant epithelial cells ( d ) in PBS-treated and DTR-treated lungs. DAPI (blue), Pdgfrβ (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( b ). DAPI (blue), Cd68 (grey), ZsGreen (green, lineage-traced fibroblasts) and RFP (red, tumour) ( c ). DAPI (blue), Sox9 (grey), and RFP (red, tumour) ( d ). Images are representative of n = 2 mice. Scale bar, 100 μm. e , Experimental design for EGFR tyrosine kinase inhibitor administration. Red2Kras mice received two doses of tamoxifen and were treated with DMSO or Gefitinib (80 mg/kg) at the indicated timepoints. Lung tissues were collected for histological analyses. f , Representative tile scans of entire lung lobe cross-sections from Red2Kras animals treated with DMSO or Gefitinib. DAPI (blue) and RFP (red, tumour). Scale bar, 1,000 µm. g , Percentage of RFP + mutant cell area relative to lobe area assessed in ( f ). Data are as mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ). h,i Representative confocal images for reprogrammed fibroblasts, macrophages and lineage-labelled cells in DMSO or Gefitinib-treated lungs. DAPI (blue), Tnc (grey) and RFP (red, tumour) ( h ). Images representative of n = 2 (DMSO) and n = 2 (Gefitinib) mice. DAPI (blue), F4/80 (grey) and RFP (red, tumour) ( i ). Images representative of n = 2 (DMSO) and n = 3 (Gefitinib) mice. Scale bar, 50 µm. j , Flow cytometry analysis for AMs from Red2Kras lungs treated with DMSO or Gefitinib. k , Quantification of the percentage of AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 3 ) and Gefitinib ( n = 3 ). P values were calculated using two-tailed unpaired t-test. l , Flow cytometry analysis for MHC-II expression on AMs from DMSO- or Gefitinib-treated Red2Kras lungs. m , Quantification of the MFI for MHC-II in AMs from DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ) and Gefitinib ( n = 3 ). n , Representative confocal images for neutrophils and lineage-labelled cells in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Ly6G (green) and RFP (red, tumour). Images representative of n = 3 biologically independent mice. Scale bar, 50 µm. o-q , Representative confocal images of the different epithelial states in DMSO- or Gefitinib-treated Red2Kras lungs. DAPI (blue), Sox9 (grey) and RFP (red, tumour) ( o ). DAPI (blue), Cd177 (grey) and RFP (red, tumour) ( p ). DAPI (blue), Ager (grey), LpCat1 (yellow) and RFP (red, tumour) ( q ). Scale bar, 50 µm. r , Quantification of the percentage of each cell state within the RFP + mutant cell population in DMSO- or Gefitinib-treated Red2Kras lungs. Data are mean ± s.e.m. Each dot represents a biologically independent mouse replicate. DMSO ( n = 2 ), Gefitinib ( n = 3 ).

Article Snippet: Sftpc–Cre ERT2 (028054), R26R–Confetti (013731), Pdgfr α–Cre ERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory.

Techniques: Mutagenesis, Flow Cytometry, Two Tailed Test, Expressing