Journal: bioRxiv

Article Title: β1 integrin regulates alveolar epithelial cell differentiation following injury

doi: 10.1101/2022.08.05.502988

Figure Lengend Snippet: (A) High-power images of thick frozen sections from day 7 LPS-treated β1 f/f and β1 rtTA lungs immunostained for pro-SP-C (green) with phalloidin F-actin probe (magenta), arrows indicate areas of actin-rich lateral protrusions. (B) Area of pro-SP-C+ AT2 cells from day 7 LPS-treated β1 f/f and β1 rtTA mice (48.4±1.8 μm 2 in β1 f/f lungs compared to 66.8±3.0 μm 2 in β1 rtTA lungs, ≥ 40 cells measured/ mouse imaged from 3 different sections, n = 6 mice/group, two-tailed t-test with p =0.0004). (C) High-power images of frozen sections prepared from day 7 injured β1 f/f and β1 rtTA lungs immunostained for pro-SP-C with JLA20 and phalloidin probes applied to detect G-actin and F-actin, respectively. Membrane localization of G-actin denoted by arrows and F-actin by arrowheads. (D) Quantification of JLA20 and phalloidin expression in pro-SP-C+ AT2 cells in β1 f/f and β1 rtTA lungs 7 days after LPS (10 sections/ mouse, n = 5-6 mice/ group, two-tailed t-test with p =0.0088 for JLA20 and p =0.0482 for phalloidin). (E) Representative low (left 2 panels) and high-power (right 2 panels) images from day 7 LPS-treated β1 f/f and β1 rtTA lungs immunostained ezrin (white) from β1 f/f and β1 rtTA mice crossed to mTmG Cre-recombinase reporter. Arrows indicate ezrin expression localized to lateral extensions in β1 f/f AT2 cells, absent in β1 rtTA AT2 cells. (F-H) GTPase activation assay performed on AT2 cell lysates collected from unchallenged and day 7 LPS-treated β1 f/f and β1 rtTA lungs (n=6-8 mice/ group for each assay; RhoA one-way ANOVA p <0.0001, F value 53.42, df=3; Cdc42 one-way ANOVA p <0.001, F value 17.46, df=3; Rac1 one-way ANOVA p <0.0001, F value 31.09, df=3). * p < 0.05. Scale bar = 5 μm for A and C, 100 μm for low power panels and 10 μm for high power panels in E.

Article Snippet: The following primary antibodies and probes were used: anti-pro-SP-C (Abcam ab90716), anti-T1α (podoplanin, Developmental Studies Hybridoma Bank 8.1.1), anti-ezrin (Cell Signaling Technologies 3145S), anti-cytokeratin 8 (Invitrogen PA5-24607), and anti-Ki67-FITC (eBioscience 11-5698-90), anti-CD68 (Abcam ab125212), anti-Ager (R and D AF1145), JLA20 (Developmental Studies Hybridoma Bank JLA20-s), and phalloidin (Invitrogen A12380).

Techniques: Two Tailed Test, Membrane, Expressing, Activation Assay

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: (a) Lung lobe sections 6m after Kras induction in AT1 cells (left) or 2m after induction in AT2 cells (middle) with non-solid (open arrowheads) and solid (closed arrowheads) tumors, and quantitation of tumor cell proliferation (right) (n=3 mice per group, five tumors per mouse) (b) Representative close-up H&E stain of an AT1-derived mixed histology adenoma and an AT2-derived solid adenoma with quantification of histology (n=3 mice per group, 9-18 tumors per mouse). (c) PAGA of scRNA-seq profiles of indicated populations with feature plots of AT1 and AT2 markers - 722 Hopx-CreER>KrasG12D, 305 Sftpc-CreER>KrasG12D, and 428 KrasWT cells. (d) Co-staining for LAMP3 (purple) and pERK (left, yellow) or pc-JUN (right, yellow) in AT1 (top) and AT2 (bottom) derived adenomas with quantitation. (n=4 mice for Sftpc-CreER, n=5 mice for Hopx-CreER; 2-5 tumors per mouse; 1-3m after tamoxifen for Sftpc-CreER and 4-6 months after tamoxifen for Hopx-CreER). (e) H&E stain of lungs with AT1 (top) and AT2 (bottom) derived tumors in controls (WT) or with concomitant activation of Wnt signaling (loxEx3) at 4m after Kras induction with quantitation of tumor size and histology (n=2 mice per genotype for Sftpc-CreER, n=11 mice per genotype for Hopx-CreER; 2-10 tumors per mouse; 2m or 4-6m after Kras induction for Sftpc-ER and Hopx-ER mice, respectively). Open arrowheads denote lepidic tumors, closed arrowheads denoted non-lepidic tumors. (f) Representative images and quantitation of proliferation (Ki67), pERK and pJUN in AT1 (top) and AT2 (bottom) derived tumors with (Ex3) and without (WT) activation of Wnt signaling (n= 2 littermate-paired mice per genotype, 5 tumors per mouse, total of 2,041-12,069 tumor cells per group, 2m and 4m after Kras induction for Sftpc-ER and Hopx-ER mice, respectively).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Quantitation Assay, Staining, Derivative Assay, Activation Assay

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: Immunostaining of AT1 phenotype cells (NKX2-1+/LAMP3−) in Hopx-CreER>KrasG12D and Hopx-CreER>KrasWT mice for phosphorylated JUN with quantification of pJUN+ (arrowhead) and pJUN− (open arrowhead) cells (n=2 WT mice and 4 KrasG12D mice, 3 25x fields per mouse).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Expressing, Immunostaining

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: (a) Single cell profiles over pseudotime reveal emergence of cell-to-cell molecular heterogeneity at Early tumor stage with progressive increase in ERK target genes, lung progenitor and gastric markers. (b) Feature plot shows strong enrichment of Mki67 at Late tumor stage. (c) ERK pathway modulation in Hopx-CreER>KrasG12D mice with representative H&E stains, pERK staining, and quantitation of tumor area, number and histology (n=3 mice per group, 3m post tamoxifen with continual chow provision, ten largest tumors per group for tumor area). (d) Co-staining shows a cluster of GKN2 expressing cells within a LAMP3+ AT1-derived adenoma (above) and AT1-derived tumor cells with normal (arrowhead), reduced (open arrowhead) and absent (asterisk) NKX2-1 expression all positive for CTSE. (e) Maintenance of Nkx2-1 with induction of distal lung embryonic progenitor markers Sox9 and Id2 at Late tumor stage. (f) Gene scores derived from marker genes for high plasticity state in advanced AT2-derived Kras LuAd and for AT2-to-AT1 transitional intermediates in lung injury show enrichment in the same cluster of cells bridging Early and Late AT1 tumor stages. (g) Large ERK-activated AT1 LuAd with loss of Lamp3 in clusters correlating with increased proliferation, with quantitation. (n=5 areas LAMP3−/+). (h) Representative ERK-activated AT1 LuAds with central necrosis (left) or mucinous transformation (right). (i) Co-staining shows MUC5AC in the cytoplasm of NKX2-1+ tumor cells (arrowheads) and filling a lumen (arrow).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Staining, Quantitation Assay, Expressing, Derivative Assay, Marker, Transformation Assay

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: (a) Representative images of human lepidic (top) and non-lepidic (bottom) LuAd stained for proliferation (Ki-67), pERK and pc-JUN (n=5 individuals per group). (b) GSEA evaluating BioCarta pathways of cDNA microarray data from Zabeck et al. of lepidic and non-lepidic LuAds shows enrichment of MAPK and ERK activity in non-lepidic tumors (n=6 lepidic and 21 non-lepidic LuAds) (left). GSEA plot of the BioCarta MAPK pathway (center). Volcano plot of differentially expressed genes in the lepidic and non-lepidic groups highlighting members of the peptidyl-tyrosine dephosphorylation GO, PTPRR, DUSP10, and DUSP7, as well as constituents of MAPK signaling MAP2K1, MAPKAP5, MAPK6. (c) GSEA evaluating for canonical Wnt signaling revealed enrichment in the non-lepidic group (left) while several Wnt inhibitors, including DKK2 and AMER2, were more highly differentially expressed in the lepidic group (right). (d) A comprehensive mutation analysis by Caso et al. into lepidic and non-lepidic tumors reveals a lack of Wnt augmenting mutations in lepidic tumors with a significant number of Wnt augmenting mutations in non-lepidic human LuAd. (e) Human lepidic mucinous (top) and non-mucinous (bottom) LuAd both contain NKX2-1+ cells co-expressing intestinal markers (CTSE, HNF4A, left side of image at top and open arrowhead on bottom panel). The non-mucinous tumor also contains NKX2-1+HNF4A− (solid arrowhead) and NKX2-1−HNF4A+ (arrow) cells. Mucinous (MUC5AC+) region is NKX2-1Lo (asterisk). (e) Co-staining for AT1 (AGER) and AT2 (MUC1, HT2-280) membrane antibodies shows co-exclusive marking of AT1 and AT2 cells (open arrowheads) in non-tumor human lung (top) but co-expression of AT1 and AT2 markers by squamous cells in human lepidic LuAd (bottom, closed arrowheads). (f) Co-staining of AT1 (AGER), AT2 (MUC1) and driver mutation (EGFR L858R) antibodies in human lepidic LuAd showing ubiquitous oncogene and AT2 marker staining of the tumor but also flat cells in the periphery positive for the driver mutation that co-express both AT1 and AT2 markers (arrowheads) (n=2 individuals).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Staining, Microarray, Activity Assay, De-Phosphorylation Assay, Mutagenesis, Expressing, Marker

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: (a) Lung lobe sections 6m after Kras induction in AT1 cells (left) or 2m after induction in AT2 cells (middle) with non-solid (open arrowheads) and solid (closed arrowheads) tumors, and quantitation of tumor cell proliferation (right) (n=3 mice per group, five tumors per mouse) (b) Representative close-up H&E stain of an AT1-derived mixed histology adenoma and an AT2-derived solid adenoma with quantification of histology (n=3 mice per group, 9-18 tumors per mouse). (c) PAGA of scRNA-seq profiles of indicated populations with feature plots of AT1 and AT2 markers - 722 Hopx-CreER>KrasG12D, 305 Sftpc-CreER>KrasG12D, and 428 KrasWT cells. (d) Co-staining for LAMP3 (purple) and pERK (left, yellow) or pc-JUN (right, yellow) in AT1 (top) and AT2 (bottom) derived adenomas with quantitation. (n=4 mice for Sftpc-CreER, n=5 mice for Hopx-CreER; 2-5 tumors per mouse; 1-3m after tamoxifen for Sftpc-CreER and 4-6 months after tamoxifen for Hopx-CreER). (e) H&E stain of lungs with AT1 (top) and AT2 (bottom) derived tumors in controls (WT) or with concomitant activation of Wnt signaling (loxEx3) at 4m after Kras induction with quantitation of tumor size and histology (n=2 mice per genotype for Sftpc-CreER, n=11 mice per genotype for Hopx-CreER; 2-10 tumors per mouse; 2m or 4-6m after Kras induction for Sftpc-ER and Hopx-ER mice, respectively). Open arrowheads denote lepidic tumors, closed arrowheads denoted non-lepidic tumors. (f) Representative images and quantitation of proliferation (Ki67), pERK and pJUN in AT1 (top) and AT2 (bottom) derived tumors with (Ex3) and without (WT) activation of Wnt signaling (n= 2 littermate-paired mice per genotype, 5 tumors per mouse, total of 2,041-12,069 tumor cells per group, 2m and 4m after Kras induction for Sftpc-ER and Hopx-ER mice, respectively).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Quantitation Assay, Staining, Derivative Assay, Activation Assay

Journal: Nature

Article Title: Oncogenic Kras reverts differentiated cells back into alveolar stem cells that generate lepidic adenocarcinoma

doi: 10.1038/s41586-023-06324-w

Figure Lengend Snippet: (a) Single cell profiles over pseudotime reveal emergence of cell-to-cell molecular heterogeneity at Early tumor stage with progressive increase in ERK target genes, lung progenitor and gastric markers. (b) Feature plot shows strong enrichment of Mki67 at Late tumor stage. (c) ERK pathway modulation in Hopx-CreER>KrasG12D mice with representative H&E stains, pERK staining, and quantitation of tumor area, number and histology (n=3 mice per group, 3m post tamoxifen with continual chow provision, ten largest tumors per group for tumor area). (d) Co-staining shows a cluster of GKN2 expressing cells within a LAMP3+ AT1-derived adenoma (above) and AT1-derived tumor cells with normal (arrowhead), reduced (open arrowhead) and absent (asterisk) NKX2-1 expression all positive for CTSE. (e) Maintenance of Nkx2-1 with induction of distal lung embryonic progenitor markers Sox9 and Id2 at Late tumor stage. (f) Gene scores derived from marker genes for high plasticity state in advanced AT2-derived Kras LuAd and for AT2-to-AT1 transitional intermediates in lung injury show enrichment in the same cluster of cells bridging Early and Late AT1 tumor stages. (g) Large ERK-activated AT1 LuAd with loss of Lamp3 in clusters correlating with increased proliferation, with quantitation. (n=5 areas LAMP3−/+). (h) Representative ERK-activated AT1 LuAds with central necrosis (left) or mucinous transformation (right). (i) Co-staining shows MUC5AC in the cytoplasm of NKX2-1+ tumor cells (arrowheads) and filling a lumen (arrow).

Article Snippet: Primary antibodies were against the following antigens: AGER (rat, 1:500, R&D MAB1179), AGER (goat, 1:250, R&D AF1145), p-cJUN (rabbit, 1:100, Cell Signaling 9164), CTNNB1-FITC (mouse, 1:250, BD Transduction Laboratories 14), CTSE (goat, 1:250, R&D AF1130), EGFR L858R (rabbit, 1:100, Cell Signaling 43B2), p-ERK1/2 (rabbit, 1:100, Cell Signaling 9101), FITC-Alexa488 (goat, 1:250, Invitrogen A11096), GFP (chicken, 1:500, Abcam 13970), GKN2 (rabbit, 1:250, Abcam EPR15377B), HT2-280 (mouse, 1:500, Terrace Biotech), Ki67 (rabbit, 1:100, Abcam 16667), LAMP3 (guinea pig, 1:500, Synaptic Systems #391005), MUC1 (rabbit, 1:500, Novus NBP160046), NKX2-1 (rabbit, 1:100, Abcam EPR5955), NKX2-1 (mouse, 1:100, Leica Novocastra SPT24), PDPN (hamster, 2 μg/ml, DSHB 8.1.1), PDPN-biotin (hamster, 1:250, eBioScience 8.1.1), pro-SPB-biotin (mouse, 1:500, Labvision SPB01), pro-SPC (rabbit, 1:500, Millipore AB3786).

Techniques: Staining, Quantitation Assay, Expressing, Derivative Assay, Marker, Transformation Assay