Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability
- Massachusetts Institute of Technology, United States
- University of Massachusetts, United States
- Dana-Farber Cancer Institute, United States
- University of Chicago, United States
Figures
Figure 1 with 3 supplements
Stable Isotope dilution can be utilized to analyze the composition of TIF.
(A) Schematic of TIF isolation. (B) LDH activity assay measuring the amount of LDH present in whole tumors, plasma, and TIF from PDAC tumor bearing mice. LDH activity was calculated for the entire …
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Figure 1—source data 1
13C metabolite peak areas of metabolites suspended in plasma and water for matrix effect analysis in Figure 1C.
- https://doi.org/10.7554/eLife.44235.007
Figure 1—figure supplement 1
TIF isolation causes only minor increases in some metabolites known to be affected by ischemia in tumors.
LC/MS measurements of relative levels of 112 metabolites (see Figure 1—figure supplement 1—source data 1) were made in extracts from KP-/-C PDAC tumors (n = 5) that were dissected in half and where …
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Figure 1—figure supplement 1—source data 1
Relative levels of metabolites in paired PDAC tumors that were either dissected and flash frozen, or were frozen after TIF-isolation in Figure 1—figure supplement 1.
- https://doi.org/10.7554/eLife.44235.009
Figure 1—figure supplement 2
TIF metabolite quantification is reproducible, particularly when using stable isotope dilution.
Scatter plots of average LC/MS measurements of metabolite concentrations across two technical replicates of 6 PDAC TIF samples run on different days using either stable isotope dilution (left) or …
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Figure 1—figure supplement 2—source data 1
Metabolite concentrations in 6 PDAC TIF samples analyzed in two inter-day technical replicates in Figure 1—figure supplement 2.
- https://doi.org/10.7554/eLife.44235.008
Figure 1—figure supplement 3
Overview of sample preparation and data analysis.
(A) A schematic describing sample and standard preparation for LC/MS analysis. First, extraction mix with added isotopically labeled internal standards is prepared. This extraction mix is then used …
Figure 2 with 7 supplements
TIF metabolite levels are different than those in plasma.
(A) Hierarchical clustering of PDAC TIF and mouse plasma samples based on LC/MS measurements of 136 metabolite concentrations. (B) Principal component analysis of PDAC TIF samples and mouse plasma …
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Figure 2—source data 1
Concentrations of 136 metabolites determined by both external standard calibration and stable isotope dilution in all PDAC TIF and plasma samples in Figure 2.
- https://doi.org/10.7554/eLife.44235.018
Figure 2—figure supplement 1
Method of blood collection affects plasma metabolite levels.
Principal component analysis of mouse plasma samples based on LC/MS measurements of 117 metabolite concentrations from C57BL/6J mice bearing PDAC tumors. Blood was collected using either …
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Figure 2—figure supplement 1—source data 1
Concentrations of 117 metabolites determined by both external standard calibration and stable isotope dilution for all plasma samples isolated from C57BL/6J mice either by retro-orbital or cardiac puncture collection in Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.44235.019
Figure 2—figure supplement 2
TIF metabolite levels are different from those in plasma of paired mice.
(A) Hierarchical clustering of PDAC TIF and mouse plasma samples from paired mice based on LC/MS measurements of 136 metabolite concentrations. (B) Principal component analysis of PDAC TIF samples …
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Figure 2—figure supplement 2—source data 1
13C metabolite peak areas of metabolites suspended in plasma and TIF for matrix effect analysis in Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.44235.020
Figure 2—figure supplement 3
Loading plot presenting the contribution of individual metabolites to the PCA components in Figure 2B.
https://doi.org/10.7554/eLife.44235.013
Figure 2—figure supplement 4
Hierarchical clustering of PDAC TIF and mouse plasma samples based on LC/MS measurements of 136 metabolite concentrations.
https://doi.org/10.7554/eLife.44235.014
Figure 2—figure supplement 5
Plasma and TIF exhibit different matrix effects.
LC/MS measurement of equal concentrations of 70 13C chemical standards suspended in either mouse plasma or mouse PDAC TIF. Data are plotted as the log2 fold change between the peak area of the 13C …
Figure 2—figure supplement 6
PDAC TIF differs from plasma when comparing only those metabolites quantified using internal isotope-labeled standards.
Hierarchical clustering (A) and principal component analysis (B) of PDAC TIF and mouse plasma samples based on LC/MS measurements of 62 metabolite concentrations using internal standards. For all …
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Figure 2—figure supplement 6—source data 1
Concentrations of 62 metabolites determined only by stable isotope dilution in all PDAC TIF and plasma samples in Figure 2—figure supplement 6.
- https://doi.org/10.7554/eLife.44235.021
Figure 2—figure supplement 7
PDAC TIF metabolite levels are not significantly different between large and small tumors.
Principal component analysis of TIF samples from large (1.71–1.24 g) and small (1.22–0.78 g) PDAC tumors based on LC/MS measurements of 136 metabolite concentrations. n = 4 for large PDAC tumors and …
Figure 3 with 2 supplements
Tumor location dictates metabolic TIF composition.
(A) Diagram of experimental models used to test the effect of tumor location on TIF metabolite levels. Principal component analysis (B) and hierarchical clustering (C) of PDAC TIF and PDAC …
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Figure 3—source data 1
Concentrations of 123 metabolites determined by both external standard calibration and stable isotope dilution in all autochthonous and subcutaneous PDAC TIF samples in Figure 3.
- https://doi.org/10.7554/eLife.44235.025
Figure 3—figure supplement 1
Loading plot presenting the contribution of individual metabolites to the PCA components in Figure 3B.
https://doi.org/10.7554/eLife.44235.023
Figure 3—figure supplement 2
Hierarchical clustering of PDAC TIF and PDAC subcutaneous allograft TIF samples based on LC/MS measurements of 123 metabolite concentrations.
https://doi.org/10.7554/eLife.44235.024
Figure 4 with 2 supplements
Dietary changes alter TIF composition.
(A) Schematic of experimental models used to test the effect of diet on TIF metabolite levels. Principal component analysis (B) and hierarchical clustering (C) of subcutaneous PDAC allograft TIF …
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Figure 4—source data 1
Concentrations of 123 metabolites determined by both external standard calibration and stable isotope dilution in all plasma and subcutaneous PDAC TIF samples in Figure 4.
- https://doi.org/10.7554/eLife.44235.029
Figure 4—figure supplement 1
Loading plot presenting the contribution of individual metabolites to the PCA components in Figure 4B.
https://doi.org/10.7554/eLife.44235.027
Figure 4—figure supplement 2
Hierarchical clustering of subcutaneous PDAC allograft TIF samples from mice fed standard mouse chow versus mice fed a defined diet based on LC/MS measurements of 123 metabolite concentrations.
https://doi.org/10.7554/eLife.44235.028
Figure 5 with 3 supplements
Tumor tissue of origin influences TIF composition independent of tumor location.
(A) Diagram of experimental models used to test the effect of tumor tissue of origin on TIF metabolite levels. Principal component analysis (B) and hierarchical clustering (C) of PDAC subcutaneous …
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Figure 5—source data 1
Concentrations of 104 metabolites determined by both external standard calibration and stable isotope dilution in all subcutaneous PDAC and LUAD TIF samples in Figure 5.
- https://doi.org/10.7554/eLife.44235.034
Figure 5—figure supplement 1
Tumor tissue of origin influences TIF when measured using internal standards only.
(A) Diagram of experimental models used to test the effect of tumor tissue of origin on TIF metabolite levels. Principal component analysis (B) and hierarchical clustering (C) of PDAC subcutaneous …
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Figure 5—figure supplement 1—source data 1
Concentrations of 66 metabolites determined by only stable isotope dilution in all subcutaneous PDAC and LUAD TIF samples in Figure 5—figure supplement 1.
- https://doi.org/10.7554/eLife.44235.035
Figure 5—figure supplement 2
Loading plot presenting the contribution of individual metabolites to the PCA components in Figure 5B.
https://doi.org/10.7554/eLife.44235.032
Figure 5—figure supplement 3
Hierarchical clustering of PDAC subcutaneous allograft TIF and LUAD subcutaneous allograft TIF samples based on LC/MS measurements of 104 metabolite concentrations.
https://doi.org/10.7554/eLife.44235.033
Figure 6 with 2 supplements
Genetic Keap1 status is not a major determinant of TIF composition in subcutaneous LUAD allograft tumors.
(A) Schematic of experimental models used to test the effect of genetic loss of Keap1 on TIF metabolite levels. Principal component analysis (B) and hierarchical clustering (C) of Keap1 wild-type …
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Figure 6—source data 1
Concentrations of 131 metabolites determined by both external standard calibration and stable isotope dilution in all sgControl and sgKeap1 LUAD TIF samples in Figure 6.
- https://doi.org/10.7554/eLife.44235.039
Figure 6—figure supplement 1
Loading plot presenting the contribution of individual metabolites to the PCA components in Figure 6B.
https://doi.org/10.7554/eLife.44235.037
Figure 6—figure supplement 2
Hierarchical clustering of Keap1 wild-type (sgControl) and Keap1 null (sgKeap1) subcutaneous LUAD allograft TIF samples based on LC/MS measurements of 131 metabolite concentrations.
https://doi.org/10.7554/eLife.44235.038Additional files
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Supplementary file 1
Information on metabolites analyzed in this study including metabolite name, organization of metabolite pools, concentration of each metabolite in each metabolite pool, polarity in which the metabolite was best detected, m/z, limit of detection, and method of quantification.
- https://doi.org/10.7554/eLife.44235.040
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Supplementary file 2
Composition of animal diets used in this study.
- https://doi.org/10.7554/eLife.44235.041
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Transparent reporting form
- https://doi.org/10.7554/eLife.44235.042
