Journal: JCI Insight

Article Title: Resolving the difference between left-sided and right-sided colorectal cancer by single-cell sequencing

doi: 10.1172/jci.insight.152616

Figure Lengend Snippet: ( A ) The t-SNE plot that showed the distribution of cancer cell lineages (yellow, n = 2196 cells) within the atlas. Cancer cell populations were reclustered into 9 subclusters (color coding). ( B ) Top 5 marker genes of 15 major cell types identified in this profile. ( C ) Annotation by left-sided and right-sided CRC cells. ( D ) The fraction of cells that originated from left-sided and right-sided CRC samples for 9 subgroups identified in this profile. ( E ) Kaplan-Meier survival curves of OS based on HSPA1A, GOLGB1, AGR3, MUC5AC, NTS, SPINK4, CLCA1, OLFM4, and PIGR expression using the online bioinformatics tool Kaplan-Meier Plotter. ( F ) Violin plots and immunochemistry display the distribution of expression of AGR2, AGR3, MUC5AC, SPINK4, TFF1, and TFF2 between left-sided and right-sided CRC. Scale bars: 100 µm.

Article Snippet: Anti-TFF1 antibody (13734-1-AP) and anti-TFF2 antibody (13681-1-AP) were purchased from Proteintech.

Techniques: Marker, Expressing

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A . Pie chart showing the distribution of cells in each cluster. B . Pseudotime ordering of cancer cells, coloring by cluster. C, D . Dot plot of the expression of the indicated genes in the different clusters. The size of each dot represents the percentage of cells within a given cluster that expresses the gene; the intensity of color indicates the level of mean expression. The order of the clusters matches the order of the clusters in the pseudotime trajectory. E . Representative RNA ISH of Fgfr2 (blue staining), Spdef (red staining), Foxa3 (red staining), Ern2 (blue staining) and immunohistochemical staining for Fgfr2 (red staining) and Agr2, Gcnt3, Clca1, Muc5ac, Gkn2, Gkn3, Tff1, Tff2, Epcam (brown staining) in KPC tumor sections.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A . Pie chart showing the distribution of cells in each cluster. B . Pseudotime ordering of cancer cells, coloring by cluster. C, D . Dot plot of the expression of the indicated genes in the different clusters. The size of each dot represents the percentage of cells within a given cluster that expresses the gene; the intensity of color indicates the level of mean expression. The order of the clusters matches the order of the clusters in the pseudotime trajectory. E . Representative RNA ISH of Fgfr2 (blue staining), Spdef (red staining), Foxa3 (red staining), Ern2 (blue staining) and immunohistochemical staining for Fgfr2 (red staining) and Agr2, Gcnt3, Clca1, Muc5ac, Gkn2, Gkn3, Tff1, Tff2, Epcam (brown staining) in KPC tumor sections.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A . Pie chart showing the distribution of cells in each cluster. B . Pseudotime ordering of cancer cells, coloring by cluster. C, D . Dot plot of the expression of the indicated genes in the different clusters. The size of each dot represents the percentage of cells within a given cluster that expresses the gene; the intensity of color indicates the level of mean expression. The order of the clusters matches the order of the clusters in the pseudotime trajectory. E . Representative RNA ISH of Fgfr2 (blue staining), Spdef (red staining), Foxa3 (red staining), Ern2 (blue staining) and immunohistochemical staining for Fgfr2 (red staining) and Agr2, Gcnt3, Clca1, Muc5ac, Gkn2, Gkn3, Tff1, Tff2, Epcam (brown staining) in KPC tumor sections.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A, B, D, G, H, I . Quantification of weight of tumors derived from mT69a ( A, B, G, H ) and mT6 ( D, I ) orthotopically grafted organoid (OGO) models of Foxa3 knock-out (KO) ( A ), Spdef KO ( B, D ), Agr2 KO ( G ), Ern2 KO or partial inactivation DN ( H, I ) and mRosa26 clones in nu/nu mice. Results show mean of biological replicates. Unpaired Student’s t test. C . Kaplan–Meier survival curve of percent survival for mT69a OGO models of Spdef KO (n=5) and mRosa26 (n=5) clones in nu/nu mice. Log-rank (Mantel–Cox) test. E . Venn diagram of the differentially expressed genes identified by RNA-seq following Spdef KO and restoration by cDNA expression in mT69a and mT6 organoids (up-regulated genes: q-value < 0.05, log2 of fold change > 0; down-regulated genes: qvalue < 0.05, log2 of fold change < 0). F . Agr2 and Ern2 expression as determined by RT-qPCR in mT69a (triangle), mT6 (circle) and mT23 (square) organoids following Spdef KO and re-expression. Paired Student’s t test.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Derivative Assay, Knock-Out, Clone Assay, RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A . RT-qPCR analysis of Agr2 expression and PCR-based analysis of TSS deletion in Agr2 KO and mRosa26 clones in mT69a organoids. B, C . RT-qPCR analysis of Ern2 expression and PCR-based analysis of TSS deletion in Ern2 KO or partial inactivation DN and mRosa26 clones in mT69a ( B ), mT6 ( C ) organoids. D . Images of tumors derived from mT69a OGO models of Agr2 KO and mRosa26 clones in nu/nu mice on day 26 after transplantation. F, H . Images of tumors derived from mT69a ( F ) and mT6 ( H ) OGO models of Ern2 KO or partial inactivation DN and mRosa26 clones in nu/nu mice on day 26 ( F ) and 20 ( H ) after transplantation. E, G, I . Representative H&E stain of tumors from D, F, H. Scale bars, 100 μm. J . Representative RNA ISH of Spdef (red staining) and Ern2 (blue staining) and immunohistochemical staining for Agr2 in tumor sections derived from mT69a OGO models of mRosa26 clones.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Quantitative RT-PCR, Expressing, Clone Assay, Derivative Assay, Transplantation Assay, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A . Heatmap showing unbiased hierarchical clustering of RNA-seq data of LCM epithelium from patients with PDA for marker genes up-regulated in scRNA-seq cluster 1 (Table S1). The color scheme of the heatmap represents z-score of log 2 (normalized counts). Blue and red horizontal bar at the bottom indicates classical and basal-like subtype of PDA samples, respectively. Blue and red dots next to the gene name to the left of the heatmap indicate known classical and basal-like genes based on Moffit classification , respectively. B . SPDEF score of classical and basal-like PDA samples from . C . SPDEF score of Slow and Fast-progressing human organoid lines from . D, E . Heatmap showing unbiased hierarchical clustering of RNA-seq data from the PanCuRx Translational Research Initiative dataset of patients with locally advanced PDA for genes of GO signatures ‘glandular epithelial cell differentiation’ ( D ) and ‘secretory pathway’ ( E ). Samples are ranked based on their SPDEF score. The color scheme of the heatmap represents z-score of log 2 (TPM values). F . Representative RNA ISH of SPDEF and ERN2 and immunohistochemical staining for AGR2 in human PDA sections. G . RT-qPCR analysis of SPDEF expression in control tumors derived from transplant models of hF27, HPAF-II, CFPAC1, BxPC3, PATU8988S, YAPC, AsPC1 in NSG mice. Results show mean of biological replicates. H . Representative AB and nuclear red staining of control tumors derived from transplant models of hF27, HPAF-II, CFPAC1, BxPC3, PATU8988S, YAPC, AsPC1 in NSG mice.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: RNA Sequencing, Marker, Clinical Proteomics, Cell Differentiation, Immunohistochemical staining, Staining, Quantitative RT-PCR, Expressing, Control, Derivative Assay

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A, B . Z-score of log 2 (normalized counts) of SPDEF, AGR2, ERN2, FOXA3 and MUC5AC in classical and basal-like PDA samples from ( A ) and in Slow and Fast-progressing human organoid lines from ( B ). C, D . Heatmap showing unbiased hierarchical clustering of RNA-seq data from the PanCuRx Translational Research Initiative dataset of patients with locally advanced PDA for genes of GO signatures ‘positive regulation of regulated secretory pathway’ ( C ) and ‘epithelial cell differentiation’ ( D ). Samples are ranked based on their SPDEF score. The color scheme of the heatmap represents z-score of log 2 (TPM values). E . Classification of PDA cell lines based on their gene expression profiles in the indicated studies. F . RT-qPCR analysis of SPDEF expression in hF27, HPAF-II, CFPAC1, BxPC3, PATU8988S, YAPC, AsPC1 cell lines. Results show mean ± SD of two biological replicates. G . Representative AB and nuclear red staining of HPAF-II, CFPAC1, BxPC3, PATU8988S, YAPC, AsPC1 cell lines.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: RNA Sequencing, Clinical Proteomics, Cell Differentiation, Gene Expression, Quantitative RT-PCR, Expressing, Staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: A, B . Quantification of weight of tumors derived from hF27 ( A ) and CFPAC1 ( B ) orthotopically grafted models of SPDEF KO and hRosa26 clones in NSG mice. Results show mean of biological replicates. Unpaired Student’s t test. C . Kaplan–Meier survival curve of percent survival for HPAF-II orthotopically grafted models of SPDEF KO (n=5) and hRosa26 clones (n=5) in NSG mice. Log-rank (Mantel–Cox) test. D-F . Quantification of weight of tumors derived from BxPC3 ( D ), PATU8988S ( E ) and YAPC ( F ) orthotopically grafted models of hRosa26 and SPDEF KO clones with (SPDEF) or without (Empty) restoration of SPDEF expression in NSG mice. Results show mean of biological replicates. Unpaired Student’s t test. G . Kaplan–Meier survival curve of percent survival for AsPC1 orthotopically grafted models of hRosa26 (n=4) and SPDEF KO clones with (SPDEF) (n=5) or without (Empty) (n=5) restoration of SPDEF expression in NSG mice. H . SPDEF, AGR2 and ERN2 expression as determined by RT-qPCR in tumors derived from hF27, HPAF-II and CFPAC1 orthotopically grafted models of SPDEF KO and hRosa26 clones in NSG mice. Results show mean of biological replicates. Unpaired Student’s t test. I . Representative AB and nuclear red staining of BxPC3, YAPC, AsPC1 and PATU8988S orthotopically grafted models, in NSG mice, of hRosa26 and SPDEF KO clones with (SPDEF) or without (Empty) restoration of SPDEF expression. Scale bars, 100 μm. Bottom, average percentage of AB positive area. Each dot indicates a biological replicate. Unpaired Student’s t test.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma

doi: 10.1101/2022.03.18.484951

Figure Lengend Snippet: SPDEF, SPDEF cDNA, AGR2, ERN2, FOXA3 and MUC5AC expression as determined by RT-qPCR in tumors derived from AsPC1, BxPC3, PATU8988S and YAPC orthotopically grafted models of hRosa26 and SPDEF KO clones with (SPDEF) or without (Empty) restoration of SPDEF expression in NSG mice. Results show mean of biological replicates. Unpaired Student’s t test.

Article Snippet: To perform IHC, endogenous peroxidase activity was quenched in 3% H 2 O 2 for 20 min. Tissues were blocked in 2.5% Normal Horse Serum blocking solution (Vector Laboratories) and subjected to staining with the following antibodies overnight at 4C: Fgfr2 (Abcam, ab58201 1:1000), Agr2 (CST, 13062 clone D9V2F 1:100), Gcnt3 (ThermoFisher, PA5-24455 1:100), Clca1 (Abcam, ab180851 1:100), Muc5ac (Abcam, ab3649 1:100), Gkn2 (LSBio, C294182 1:100), Gkn3 (LSBio, C294181 1:100), Tff1 (OriGene, TA322883 1:100), Tff2 (Proteintech, 13681-1-AP 1:100), Epcam (CST, 93790 clone E6V8Y 1:100).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Clone Assay