Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A) Strategy applied to find candidate driver genes of the basal-like/squamous subtype in PDAC. Two cohorts (Bailey and Moffitt cohorts) ( , ) were interrogated to identify candidate driver genes of the basal-like/squamous subtype. The genes were narrowed down to those associated with patient survival in the NCI-UMD-German cohort. (B-D) SERPINB3 transcript and protein expression in PDAC tumors versus adjacent non-cancerous tissues showing upregulation of SERPINB3 in tumors in both the NCI-UMD-German cohort and a validation cohort (Moffitt cohort; GSE71729) (only mRNA). The lower panel shows Kaplan-Meier plots and the association of increased SERPINB3 with decreased PDAC patient survival. For the survival analysis in the validation cohort, patients in the upper and lower tertiles of SERPINB3 expression were compared. The panel to the right shows immunohistochemical detection of SERPINB3 in representative nontumor (score 0) and tumor tissues (scores 1-3). SERPINB3 protein was detected in both the nucleus and cytoplasm, as shown by the brown DAB-based immunostaining in the tumor cells. The images show the staining strength (score 0, nonstained; score 1, weak; score 2, moderate; score 3, strong). Scale bar is 20 μm. More details can be found in Materials and Methods. Data represent mean ± SD. Significance testing with unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.005

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Immunostaining, Staining

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A) The transcriptome of PDAC defines molecular subtypes. Earlier described gene signatures for molecular subtypes separate 175 patients of our NCI-UMD-German cohort into unclassified, basal-like/squamous, and classical/progenitor subtypes. Tumors defined as basal-like/squamous show upregulation of SERPINB3 mRNA expression (middle panel) and an association with inferior patient survival in the NCI-UMD-German cohort (right panel). (B-E) IPA (blue bars and tables) and GSEA highlight the activation of the similar pathways, including increased cellular movement, MYC activation, and induction of basal-like gene profile, in SERPINB3-high and basal-like/squamous PDAC tumors (B and C) and in human PDAC cell lines with SERPINB3 transgene overexpression (D and E). IPA; Ingenuity pathway analysis, GSEA; gene set enrichment analysis.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Expressing, Activation Assay, Over Expression

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A) Endogenous SERPINB3 protein levels in human PDAC cell lines. AsPC-1, MIA PaCa-2, and Panc 10.05 cells were selected to establish cell lines with SERPINB3 transgene expression. These cell lines have the classical PDAC subtype, as reported previously , and can be used to investigate the transition into the basal-like/squamous subtype after SERPINB3 overexpression. (B) Confirmation of SERPINB3 transgene overexpression at the protein level in AsPC-1, MIA PaCa-2, and Panc 10.05 cells.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Expressing, Over Expression

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: SERPINB3 transgene expressing PDAC cells were examined to define the function of SERPINB3. (A) Dose-response curves showing viability of gemcitabine-treated PDAC cell lines (CCK-8/WST-8 assay). SERPINB3 reduces gemcitabine sensitivity in Panc 10.05 but not in MIA PaCa-2 cells. (B) SERPINB3-overexpressing PDAC cells have an increased ability to invade. Cells that passed through a cell culture insert membrane coated with Matrigel were fixed after 48 hours and cells were counted. Data represent mean ± SD of 3 replicates with t-test. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Expressing, CCK-8 Assay, Cell Culture, Membrane

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: Vector control and SERPINB3-overexpressing AsPC-1 human PDAC cells were transplanted into the pancreas of immune-deficient (NOD-SCID) mice. The tumor xenograft-growing mice were sacrificed at 5 weeks and both tumor burden and lung metastasis were assessed. (A) Increased number of lung metastasis in mice transplanted with AsPC-1 cells carrying the SERPINB3 transgene (shown in graph to the right). Basophilic clusters indicate the metastatic lesions in sections of the lung (see arrows). (B) Upregulation of SERPINB3 does not increase the weight of the primary tumor xenografts in the pancreas. (C) Pathway enrichment analysis using IPA for the primary tumor xenografts from SERPINB3 overexpressing AsPC-1 versus vector control cells. IPA indicates the activation of a set of pathways, including “cellular movement” and “free radical scavenging” in SERPINB3-overexpressing AsPC-1, consistent with increased invasion and oxidative stress in these tumors. (D-E) Pathway enrichment analysis using IPA for the tumor stroma from xenografts of SERPINB3 overexpressing AsPC-1 versus vector control cells. The stroma in AsPC-1 SERPINB3 xenografts exhibits activation of pathways associated with “HIF1α” and “tumor microenvironment”, including “angiogenesis” and “metastasis/invasion/cell movement”. (F) The density of microvessels (angiogenesis) in the tumor xenografts examined by immunostaining for CD31. Microvessel density is increased in the stroma of SERPINB3-overexpressing AsPC-1 (arrows). Scale bar indicates 50 μm. (G) Immunostaining for 8-OHdG, a marker of oxidative stress, in the tumor xenografts. The percentage of the 8-OHdG positive cells as a measure for oxidative stress was assessed. The level of oxidative stress is increased in SERPINB3-overexpressing AsPC-1. Scale bar is 50 μm. Data represent mean ± SD. Significance testing with t-test. *p < 0.05, **p < 0.01, ***p < 0.005. IPA; Ingenuity pathway analysis.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Plasmid Preparation, Control, Activation Assay, Immunostaining, Marker

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: Levels of reactive oxygen species in the SERPINB3-overexpressing PDAC cells and the control PDAC cells, showing that SERPINB3 promotes oxidative stress levels. Data represent mean ± SD with t-test. *p < 0.05, **p < 0.01, ***p < 0.005. Reactive oxygen species level was measured with the CellRox Deep Red fluorogenic reagent.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Control

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A-B) Heatmaps show metabolite patterns in human PDAC (n = 88) with high and low SERPINB3 expression level (A) or by tumor subtype (B). Red color shows upregulation of metabolites, blue indicates low abundance. In A, 32 metabolites were significantly increased and 3 were decreased in SERPINB3-high tumors, when compared with SERPINB3-low tumors (p < 0.05). In B, 15 metabolites were significantly increased and 2 were decreased in the basal-like/squamous subtype, when compared with the classical/progenitor subtype (p < 0.05). Acylcarnitines/carnitine, amino acids, and carbohydrates are increased in SERPINB3-high PDAC tumors and also in basal-like/squamous PDAC tumors. (C-D) Abundance patterns for individual acylcarnitine (C) and amino acid (D) by tumor SERPINB3 status and subtype. The lower panel in C shows Kaplan-Meier plots depicting the association of each acylcarnitine with PDAC survival. A higher level of 2-methylbutylcarnitine, butyrylcarnitine and palmitoylcarnitine associated with poor patient survival. Data represent mean ± SD. Significance testing with t-test. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Expressing

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A-B) Total carnitine is elevated in both SERPINB3-high and basal-like/squamous PDAC in the NCI-UMD-German cohort. (C) Kaplan-Meier plot showing that high total carnitine associates with poor survival of PDAC patients (the upper 25% quartile vs the lower 75%; Total carnitine low < 30.8, high ≥ 30.8). (D) L-carnitine has no effect on proliferation of PDAC cell lines (CCK-8/WST-8 assay). (E) L-carnitine increases invasiveness of PDAC cell lines. Cells that passed through a cell culture insert membrane coated with Matrigel were fixed after 48 hours of incubation. (F) Increased mitochondrial membrane potential in PDAC cell lines cultured with 1 mM of L-carnitine for 48 hours. Mitochondrial membrane potential was quantified in cells using MitoTracker Red FM and flow cytometry. Data represent mean ± SD. Student t-test was used to determine the significance in difference. *p < 0.05, **p < 0.01, ***p < 0.005. Total carnitine is the sum of all acylcarnitines/carnitine in the metabolome dataset: 2-methylbutyrylcarnitine (C5), acetylcarnitine, butyrylcarnitine, carnitine, decanoylcarnitine, deoxycarnitine, hexanoylcarnitine, isobutyrylcarnitine, isovalerylcarnitine, octanoylcarnitine, oleoylcarnitine, palmitoylcarnitine, propionylcarnitine, stearoylcarnitine, succinylcarnitine, valerylcarnitine

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: CCK-8 Assay, Cell Culture, Membrane, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: Increased mitochondrial membrane potential in SERPINB3-overexpressing PDAC cells. The mitochondrial membrane potential was quantified in cells using MitoTracker Red FM and flow cytometry. Data represent mean ± SD with t-test. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: Membrane, Flow Cytometry

Journal: bioRxiv

Article Title: SERPINB3 induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer

doi: 10.1101/2023.03.29.534766

Figure Lengend Snippet: (A) Increased hydroxyproline level in SERPINB3-overexpressing PDAC cells. (B) Hydroxyproline added to the culture medium has no effect on proliferation (CCK-8/WST-8 assay). (C) Hydroxyproline increases invasiveness of PDAC cell lines. Cells that passed through a cell culture insert membrane coated with Matrigel were fixed after 48 hours and counted. Data represent mean ± SD with t-test. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: To obtain stable cell lines overexpressing SERPINB3, PDAC cells (AsPC-1, MIA PaCa-2, and Panc 10.05) were infected with lentiviral particles produced by transfecting 293T cells using the lentiviral expression vectors and the Lenti-Pac TM HIV Expression Packaging system from Genecopoeia.

Techniques: CCK-8 Assay, Cell Culture, Membrane