Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) Growth of syngeneic KPC tumors implanted subcutaneously and treated with vehicle control (Control) or DON (10mg/kg) for 2 weeks. Tumor volumes were measured with a digital caliper at the indicated timepoints. Data are expressed as mean ± SEM of n=4 mice/8 tumors per group. b) Tumor weights of KPC orthotopic tumors from animals treated with vehicle control (Ctrl) or DON (10mg/kg) for 10 days. Representative tumor images are shown. Data are expressed as mean ± SEM of n=8 or n=9 mice per group (Ctrl, DON). c) Tumor weights of PaTu 8988T orthotopic tumors from animals treated with vehicle control (Ctrl) or DON (10mg/kg) for 3 weeks. Representative tumor images are shown. Data are expressed as mean ± SEM of n=7 mice per group. d) Tumor weights of 779E orthotopic tumors from animals treated with vehicle control (Ctrl) or DON (10mg/kg) for 2 weeks. Representative tumor images are shown. Data are expressed as mean ± SEM of n=8 or n=7 mice per group (Ctrl, DON). e) Immunohistochemical staining of the proliferation markers phospho-Histone H3 (pHis-H3) and Ki-67 in KPC orthotopic tumors treated with vehicle (Ctrl) or DON (10mg/kg). Representative images are shown. Scale bar 100 μm. Quantification of pHis-H3- or Ki-67-positive nuclei/field is shown as mean ± SEM of n=4 tumors per group. f) Characterization of macrometastases in animals with KPC orthotopic tumors treated with vehicle or DON (10mg/kg). Representative images of macrometastases in different tissues are shown. The number of mice with metastases in each organ site was quantified as indicated in the table. n=13 or n=15 mice per group as indicated. Statistical significance was calculated using unpaired two-tailed Student’s t test (a-e) and by Fisher’s Exact test (f). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Control, Immunohistochemical staining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) Body weights of mice bearing subcutaneous or orthotopic PDAC tumors after treatment with vehicle (Ctrl) or DON. Data are expressed as mean ± SEM. KPC heterotopic (n=4), KPC orthotopic (n=8 and n=9), PaTu 8988T orthotopic (n=7), and 779E orthotopic (n=8 and n=7). b) Immunohistochemical staining of the apoptosis marker Cleaved Caspase 3 (CC3) in KPC and 779E orthotopic tumors treated with vehicle (Control) or DON (10mg/kg). Representative images are shown. Red scale bar 50 μm. c) Immunohistochemical staining of the proliferation marker pHis-H3 in 779E orthotopic tumors treated with vehicle (Control) or DON (10mg/kg). Representative images are shown. Scale bar 100 μm. Quantification of pHis-H3-positive nuclei/field is shown as mean ± SEM of n=4 tumors per group. Statistical significance was calculated using unpaired t-test in A and C. * P <0.05, ** P <0.01

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Immunohistochemical staining, Staining, Marker, Control

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) DON dose response curves for the indicated PDAC cell lines. KPC and MIA PaCa-2 cells were treated for 48h, PaTu 8988T for 24h and 779E for 72h. Relative cell number was quantified by crystal violet staining. Representative images of the crystal violet staining are shown for the KPC cells. Data are presented relative to the untreated control condition and are representative of 3 independent experiments, each performed with 3 replicates per condition. b) Crystal violet assays showing relative cell number of KPC cells treated with or without DON (5μM) and supplemented with the indicated metabolites for 24h. Representative images are shown. Data are presented relative to the untreated control (-DON) for each condition and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. Metabolites assessed were non-essential amino acids (NEAAs), α-ketoglutarate (α-KG), nucleosides (Nuc) and N-acetyl glucosamine (GlcNac). c) Crystal violet assays showing relative cell number of KPC cells treated with or without DON (5 μM) supplemented with either a cocktail of NEAAs or the indicated individual amino acids (0.1mM) for 24h. Representative images are shown. Data are presented relative to the untreated control (-DON) for each condition and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. d), e) Relative number of KPC cells (d) or PaTu 8988T cells (e) treated with DON at the indicated concentrations with or without asparagine (Asn, 0.1mM) supplementation. Data are presented relative to the untreated control condition (-Asn, 0μM DON) and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. f) Quantification of intracellular Asn levels in the indicated PDAC cell lines treated with vehicle control (Ctrl) or DON (10μM for KPC and PaTu 8988T, 1mM for 779E) for 24h. Data are presented relative to untreated control. Data are expressed as mean ± SEM of n=5 wells (KPC, 779E) or n=3 wells (PaTu 8988T). Statistical significance was calculated using One-way ANOVA followed by Dunnet’s multiple comparisons test (b,c) or unpaired two-tailed Student’s t test (d-f). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a), b) Relative number of 779E (a) or PaTu 8988T (b) cells treated with or without DON (1mM or 10μM, respectively) with supplementation of the indicated metabolites for 24h. Cells were stained with crystal violet and stained area was quantified. Representative images are shown. Data are presented relative to untreated control for each condition and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. c), d) Relative number of 779E (c) or PaTu 8988T (d) cells treated with or without DON (1mM or 10μM, respectively) supplemented with a cocktail of NEAAs or the indicated individual amino acids (0.1mM) for 24h. Representative images are shown. Data are presented relative to untreated control for each condition and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. e), f) Relative number of 779E cells (e) or MIA PaCa-2 cells (f) treated with DON at the indicated concentrations with or without 0.1mM Asn supplementation. Data are presented relative to untreated control for each condition and are representative of 3 independent experiments. Data are expressed as mean ± SEM of triplicate wells. g) Quantification of intracellular aspartate (Asp) levels in PaTu 8988T cells treated with or without DON at the indicated concentrations for 24h. Data are presented relative to untreated control. Data are expressed as mean ± SEM of n=3 samples. h-j) Quantification of intracellular levels of the indicated TCA cycle metabolites in KPC (h), 779E (i) or PaTu 8988T (j) cells treated with vehicle control or DON (10μM, 10μM, and 1mM, respectively) for 24h. Data are presented relative to untreated control. Data are expressed as mean ± SEM of n=5 samples (h,i) and n=3 samples (j). Statistical significance was calculated using One-way ANOVA followed by Dunnet’s multiple comparisons test (a-d,g) or unpaired two-tailed Student’s t test in (e,f,h-j). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) Immunoblot assessing Asns protein levels in KPC and PaTu 8988T cells treated with DON at the indicated concentrations for 24h. Tubulin was used as a loading control. The results are representative of 3 independent experiments. b) Immunoblot assessing Asns protein levels in KPC and PaTu 8988T cells treated with 10μM DON with or without Asn supplementation for 24h. Tubulin was used as a loading control. The results are representative of 3 independent experiments. c) Relative ASNS mRNA levels as assessed by qPCR in KPC or PaTu 8988T cells treated with 10μM DON with or without Asn supplementation for 24h. Data are presented relative to untreated control (Ctrl) and are representative of 3 independent experiments. Data are expressed as mean ± SEM of n=3 replicates. d) Immunoblot assessing Asns protein levels in KPC orthotopic tumors treated with vehicle (Control) or DON (10mg/kg) for 2 weeks. Tubulin was used as a loading control. e) Quantification of Asns protein relative to Tubulin in KPC orthotopic tumors from d). Data are expressed as mean ± SEM of n=5 or n=6 tumor samples per group. f) Immunohistochemical staining of Asns protein in KPC orthotopic tumors treated with vehicle control (Control) or DON (10mg/kg). Representative images of n=4 mice per group are shown. Scale bar, 100μm. g) Immunoblot assessing Asns protein levels in KPC cells transfected with non-targeting negative control siRNA (siNC) or two different hairpins targeting ASNS (siASNS#1 and siASNS#2) for 24h followed by 5μM DON treatment for 24h. h), i) Relative number of KPC cells after transfection with non-targeting siNC control, or siASNS#1 or siASNS#2 for 24h followed by 5μM DON treatment for 24h. Representative images of n=3 independent experiments (h). Quantification of relative cell number as assessed by crystal violet staining (i) where data is presented relative to untreated control for each hairpin and is representative of 3 independent experiments. Data are expressed ± SEM of n=3 replicate wells. j) Relative number of KPC cells after transfection with empty vector or an ASNS-expressing vector for 24h followed by DON treatment at the indicated concentrations. Quantification of crystal violet staining is shown relative to untreated control for each condition and is representative of 3 independent experiments. Data are expressed ± SEM of n=3 replicate wells. k) Immunoblot assessing Asns protein levels in KPC cells transfected with empty vector or ASNS-expressing vector for 48h. Tubulin was used as loading control. Results are representative of 3 independent experiments. Statistical significance was calculated using One-way ANOVA followed by Tukey’s multiple comparisons test (c) or unpaired two-tailed Student’s t test (e, i and j). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Western Blot, Control, Immunohistochemical staining, Staining, Transfection, Negative Control, Plasmid Preparation, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) Relative ASNS mRNA expression levels as assessed by qPCR in 779E cells treated with 0.5mM DON with or without 0.1mM Asn supplementation for 24h. Data are presented relative to untreated control and are representative of 3 independent experiments. Data are expressed as mean ± SEM of n=3 replicates. b) Immunoblot assessing Asns protein expression in 779E cells treated with 0.5mM DON with or without 0.1mM Asn supplementation for 24h. Tubulin was used as a loading control. c) Immunohistochemical staining of Asns protein in PaTu 8988T orthotopic tumors treated with vehicle control (Control) or DON (5mg/kg). Representative images of n=4 mice per group are shown. Scale bar, 100μm. d) Relative ASNS mRNA expression levels in KPC cells as assessed by qPCR after transfection with non-targeting negative control siRNA (siNC) or two different hairpins targeting ASNS (siASNS#1 and siASNS#2) for 24h. Data are presented relative to siNC control and are representative of 3 independent experiments. Data are expressed as mean ± SEM of n=3 replicates. Statistical significance was calculated using One-way ANOVA followed by Tukey’s multiple comparisons test. **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Expressing, Control, Western Blot, Immunohistochemical staining, Staining, Transfection, Negative Control

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a), b) Relative number of KPC cells (a) or PaTu 8988T cells (b) treated with the indicated doses of DON in combination with L-asparaginase (ASNase, 0.5U/ml) for 24h. Cells were stained with crystal violet, representative images are shown. Quantification of crystal violet staining is shown relative to the untreated control and is representative of 3 independent experiments. The coefficient of drug interaction (CI) was calculated for each DON concentration (shown in graph). Data are expressed ± SEM of n=3 replicate wells. c) Cell viability was assessed in 6 PDAC patient-derived organoids (PDOs) treated with vehicle (Ctrl), 50μM DON, 0.3 U/ml ASNase, or combination of DON + ASNase. The CI is indicated in the graphs. Data are presented relative to untreated control and are representative of 2 independent experiments, with 5 replicates each. Data are expressed as mean ± SEM of n=5 replicate wells. d) Heatmap analysis of normalized gene expression (RNA-seq) of known DON-targeting enzymes in PDAC patient-derived organoids (PDO) used in c. e) Correlation analysis between normalized gene expression of the indicated DON-targeting enzymes and DON viability ratio, used as measurement of PDO responsiveness to DON treatment. Statistical significance was calculated using unpaired two-tailed Student’s t test (a,b) or Spearman correlation coefficient (e). ** P <0.01, **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Staining, Control, Concentration Assay, Derivative Assay, Expressing, RNA Sequencing Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a) Tumor weights of PaTu 8988T orthotopic tumors from animals treated with vehicle (Ctrl), ASNase (60U/mouse), DON (5-10mg/kg), or combination of DON + ASNase for 3 weeks. Data are expressed as mean ± SEM of n=13, n=8, n=12, or n=11 mice combined from 2 different cohorts. b) Asn levels quantified by GC-MS in plasma of mice bearing PaTu 8988T orthotopic tumors treated as indicated. Data are expressed as mean ± SEM of n=6, n=4, n=5, or n=6 mice per group. c) Quantification of intratumoral Asn levels in PaTu 8988T orthotopic tumors treated as indicated. Data are presented relative to no treatment control and quantification was calculated as amount of Asn per mg of tumor tissue. Data are expressed as mean ± SEM of n=6, n=4, n=6, or n=6 mice per group. d) Quantification of the percentage of mice with PaTu 8988T-derived primary orthotopic tumors that presented with macrometastases. n=13, n=8, n=12, or n=11 mice per group. e) Characterization of macrometastases in animals with PaTu 8988T orthotopic tumors treated as indicated. Representative images of macrometastases in the different tissues are shown. The white arrows point to macrometastases. The number of mice with macrometastases in each organ site was quantified as indicated in the table. n=13, n=8, n=12, or n=11 mice per group. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons (a-c) or Fisher’s exact test (e). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Gas Chromatography-Mass Spectrometry, Control, Derivative Assay

Journal: bioRxiv

Article Title: Glutamine mimicry suppresses tumor progression through asparagine metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2022.10.31.514261

Figure Lengend Snippet: a), b) Immunohistochemical staining of phospho-Histone H3 (pHis-H3; a) or cleaved Caspase 3 (CC3; b) in PaTu 8988T orthotopic tumors treated with vehicle (Control), ASNase (60U/mice), DON (5-10mg/kg), or a combination of DON + ASNase for 3 weeks. Representative images are shown. Scale bar 100 μm. c) Hematoxylin and Eosin (H&E) staining of liver sections from PaTu 8988T tumor-bearing mice treated with vehicle (Control), DON (5-10mg/kg), or a combination of DON + ASNase for 3 weeks. Metastases are indicated (M). Representative images are shown. Scale bar 200 μm.

Article Snippet: MIA PaCa-2 cells were obtained from the American Type Culture Collection (ATCC); PaTu 8988T cells were purchased from Elabscience.

Techniques: Immunohistochemical staining, Staining, Control