fetal bovine serum dialyzed  (corning life sciences)

Journal: bioRxiv

Article Title: Dissecting cell type-specific metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2020.03.09.984237

Figure Lengend Snippet: Pyruvate Carboxylase in Cancer Cells is Required for PDAC Tumor Growth In Vivo. (A) Western blot for PC expression levels in AL1376 murine sgPC PDAC cell lines compared to SgControl cell lines made using CRISPRi using β-actin as a control. (B) Fractional labeling of aspartate M+1 following 24 hours of 1- 13 C-pyruvate tracing in AL1376 murine sgPC PDAC cell lines compared to SgControl cell lines. The difference in labeling between AL1376 sgControl cells and sgPC-1 cells (p=0.1564) was not significant and the difference in labeling between AL1376 sgControl cells and sgPC-2 cells (p=0.0304) was significant based on unpaired, two-sided student’s t tests. Mean +/− SD is shown. (C) Aspartate M+1 isotopomer labeling from (B) was normalized to pyruvate M+1 labeling as a surrogate for pyruvate carboxylation activity following 24 hours of 1- 13 C-pyruvate tracing. The difference in pyruvate carboxylation activity between AL1376 sgControl cells and sgPC-1 cells (p=0.0782) was not significant and the difference in pyruvate carboxylation activity between AL1376 sgControl cells and sgPC-2 cells (p=0.0260) was significant based on unpaired, two-sided student’s t tests. Mean +/− SD is shown. (D) Proliferation rate of sgControl and sgPC AL1376 knockdown murine PDAC cell lines generated using CRISPRi over 3 days in DMEM with 10% FBS. Differences in proliferation between AL 1376 sgControl cells and sgPC-1 (p=0.0609) or sgPC-2 (p=0.7585) cells were not significant based on unpaired, two-sided student’s t tests. Mean +/− SD is shown. (E) Western blot analysis of PC expression levels in sgPC knockdown PDAC organoids compared to sgControl organoids made using CRISPRi using β-actin as a control. (F) Fluorescent images of sgControl or sgPC knockdown murine PDAC organoids cultured in DMEM-pyruvate with 10% dialyzed FBS alone (left) or with murine PSCs (right). (G) Quantification of tdTomato fluorescence from images in (F). sgControl organoids with PSCs trended towards higher tdTomato fluorescence compared to sgPC-1 organoids with PSCs (p=0.1166) and had significantly higher tdTomato fluorescence than sgPC-2 organoids with PSCs (p=0.0272) based on unpaired, two-tailed student’s t-tests. Mean +/− SD is shown. (H) Western blot for PC expression levels in AL1376 murine sgPC knockdown and PC overexpression PDAC cell lines compared to SgControl cell lines using β-actin as a control. (I) Fractional labeling of aspartate M+1 following 24 hours of 1- 13 C-pyruvate tracing in AL1376 murine sgPC and PC rescue PDAC cell lines compared to SgControl cell lines made using CRISPRi. Labeling is significantly increased in AL1376 SgControl + PC cells compared to sgControl + EV cells (p

Article Snippet: For organoid-PSC co-culture experiments, co-cultures were grown in DMEM without pyruvate (Corning 10-017-CV) supplemented with 10% dialyzed FBS and penicillin-streptomycin.

Techniques: In Vivo, Western Blot, Expressing, Labeling, Activity Assay, Generated, Cell Culture, Fluorescence, Two Tailed Test, Over Expression

Journal: bioRxiv

Article Title: Dissecting cell type-specific metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2020.03.09.984237

Figure Lengend Snippet: Assessment of Pyruvate Carboxylase Activity in PDAC Cancer Cells and Fibroblasts. (A) Sections from tumors arising in LSLKrasG12D/+; p53 fl/fl; Pdx-1-Cre (KP −/− C) mice were stained with an antibody against pyruvate carboxylase. Scale bar represents 100µm. (B) Representative image from a human pancreatic tumor tissue microarray that was stained with an antibody against pyruvate carboxylase. Scale bar represents 100µm. (C) Pyruvate carboxylase expression was assessed by qPCR in whole PDAC tumors and different cell populations in sorted from PDAC tumors arising KP −/− C mice. The difference in expression between sorted cancer cells and the whole tumor (p=0.5485) or between sorted cancer cells and fibroblasts from tumors was not significant (p=0.0722) based on unpaired, two-tailed student’s t-tests. Mean +/− SEM is shown. 36B4 was used as a housekeeping gene control. (D-F) The fractional labeling of aspartate (D) or malate (E) in cultured murine PDAC cells derived from tumors in KP −/− C mice (black) and in cultured isolated pancreatic stellate cells (PSCs) (grey) was measured after exposure to U- 13 C-glucose for 24 hours. The M+2 and M+3 isotopomers are shown for each metabolite. Mean +/− SD is shown. (F) Fluorescent images of KP −/− C PDAC cancer cell organoids expressing TdTomato cultured in DMEM-pyruvate with 10% dialyzed FBS (top) or in the same media conditions with murine PSCs included in the culture (bottom). (G) Quantification of tdTomato fluorescence from images in (G). Organoid cultures with PSCs included had significantly higher tdTomato fluorescence than organoids cultured without PSCs (p=0.0014) based on an unpaired, two-tailed student’s t-test. Mean +/− SD is shown. (H) Expression of pyruvate carboxylase as assessed by qPCR in PDAC cancer cells or PSC cells sorted from organoid co-cultures (3D) or in standard 2D culture as indicated. The difference between cancer cells in 3D compared to PSCs in 3D (p=0.1071) was not significant, but the differences between cancer cells in 3D and cancer cells in 2D (p=0.0140), cancer cells in 3D compared to PSCs in 2D (p=0.0209), PSCs in 3D compared to cancer cells in 2D (p

Article Snippet: For organoid-PSC co-culture experiments, co-cultures were grown in DMEM without pyruvate (Corning 10-017-CV) supplemented with 10% dialyzed FBS and penicillin-streptomycin.

Techniques: Activity Assay, Mouse Assay, Staining, Microarray, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Labeling, Cell Culture, Derivative Assay, Isolation, Fluorescence

Journal: bioRxiv

Article Title: Dissecting cell type-specific metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2020.03.09.984237

Figure Lengend Snippet: Assessment of Pyruvate Carboxylase Activity in PDAC Cancer Cells and Fibroblasts. (A) Distribution of pyruvate carboxylase staining intensity scores from a tissue microarray containing sections from 90 human pancreatic tumors. (B) Representative TMA cores containing human pancreatic tumors showing pyruvate carboxylase staining intensity scored as 0, 1, or 2. Scale bars represent 100µm. (C) Distribution of the scores for percentage of cells positive for pyruvate carboxylase staining in each sample of a tissue microarray containing sections from 90 human pancreatic tumors. (D) Representative TMA cores containing human pancreatic tumors showing the percentage of cells positive for pyruvate carboxylase staining scored as 0-4. Scale bars represent 100µm. ( E) Images including bright field (left) and fluorescent (right) of murine PDAC organoids cultured from KP −/− C mice engineered such that the cancer cells express a fluorescent TdTomato allele. Cultures shown use standard PDAC organoid culture conditions ( Boj et al., 2015 ). (F) Fluorescent images of murine PDAC organoids from LSLKrasG12D/+; p53R172H/+; Pdx-1-Cre; (KPC) mice engineered such that the cancer cells express a fluorescent TdTomato allele cultured in DMEM-pyruvate with 10% dialyzed FBS alone (top) or with murine PSCs (bottom). (G) Quantification of tdTomato fluorescence from images in (E). Organoids with PSCs had significantly higher tdTomato fluorescence than organoids grown alone (p=0.0007) based on an unpaired, two-tailed student’s t-test. Mean +/− SD is shown. (H-K) The fractional labeling of aspartate (H) , malate (I), citrate (J), or α-ketoglutarate (αKG) (K) in murine PDAC organoid-PSC co-cultures was measured after 24 hours of tracing with U- 13 C-glucose. The full isotopomer distribution is shown for each metabolite. Mean +/− SD is shown. M+2 and M+3 labeling data from S2H-I is the same as that shown in Figure 2I-J .

Article Snippet: For organoid-PSC co-culture experiments, co-cultures were grown in DMEM without pyruvate (Corning 10-017-CV) supplemented with 10% dialyzed FBS and penicillin-streptomycin.

Techniques: Activity Assay, Staining, Microarray, Cell Culture, Mouse Assay, Fluorescence, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: Dissecting cell type-specific metabolism in pancreatic ductal adenocarcinoma

doi: 10.1101/2020.03.09.984237

Figure Lengend Snippet: Pyruvate Carboxylase in Cancer Cells is Required for PDAC Tumor Growth In Vivo. (A) Proliferation rate of AL1376 murine PDAC cells without (sgControl) or with (sgPC) deletion in standard 2D culture. Mean +/− SD is shown. (B) Fluorescent images of murine PDAC cancer cell organoids expressing TdTomato without (sgControl) or with (sgPC) deletion cultured in DMEM-pyruvate with 10% dialyzed FBS alone (top) or with murine PSCs (bottom). (C) Quantification of tdTomato fluorescence from images in (B). Control organoids with PSCs had significantly higher tdTomato fluorescence than sgPC organoids grown with PSCs (p=0.0171) based on an unpaired, two-tailed student’s t-test. Mean +/− SD is shown. (D) Growth of sgControl (black) and sgPC (blue) AL1376 murine PDAC cells as tumors following subcutaneous transplantation into syngeneic B6 mice. The final tumor volume is significantly greater in sgControl AL1376 cells compared to sgPC cells based on unpaired, two-tailed student’s t-tests (p

Article Snippet: For organoid-PSC co-culture experiments, co-cultures were grown in DMEM without pyruvate (Corning 10-017-CV) supplemented with 10% dialyzed FBS and penicillin-streptomycin.

Techniques: In Vivo, Expressing, Cell Culture, Fluorescence, Two Tailed Test, Transplantation Assay, Mouse Assay

Journal: bioRxiv

Article Title: Methionine synthase is essential for cancer cell proliferation in physiological folate environments

doi: 10.1101/2020.06.12.149005

Figure Lengend Snippet: Characterization of cells cultured in 5-methyl THF. (A) Histogram of MTR mRNA expression levels in cell lines as reported in The Cancer Genome Atlas 23 . (B) Proliferation rate of T.T cells in media with the indicated folate before (no pre-starvation) or after 3 days of culture with no folic acid (pre-starvation). (C) LC/MS measurement of folic acid and 5-methyl THF concentrations present in the dialyzed fetal bovine serum used to prepare culture medium for this study (batch 1; see Methods). (D) LC/MS measurement of intracellular methionine, lysine, and serine levels in A549 and T.T cells cultured for the indicated number of days in media lacking any added folates. LC/MS measurement of intracellular SAM and SAH levels (E) and nucleotide levels (F) in A549 and T.T cells cultured for the indicated number of days in media lacking any added folates. (G) Proliferation rate of A549 or T.T cells cultured in media lacking any added folates for the indicated number of days. Mean +/- SD is displayed for all panels. p values indicated on all panels are derived from two-tailed, unpaired Welch’s t tests. For all LC/MS measurements, data are normalized to cell number and to an internal standard.

Article Snippet: For experiments, cells were grown in RPMI-1640 without phenol red with 10% FBS that has been dialyzed to remove small molecules (Gibco, 26400044; batch 1: Lot #1841165, batch 2: Lot #2093857).

Techniques: Cell Culture, Expressing, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Two Tailed Test