How to Integrate Cellular Metabolism Assays Into Your Research: Considerations and Challenges

Transcription

1 How to Integrate Cellular Metabolism Assays Into Your Research: Considerations and Challenges Donna Leippe Sr Research Scientist

2 Outline for Today s Webinar Introduction to Cell Metabolism Metabolite Detection Methods Sample Preparation Considerations and Strategies Bioluminescent Metabolite Detection Assays Applications o Cancer Cells in Culture Metabolites in Media Intracellular Metabolites Multiplexing with Viability Assays o T Cell Activation o Screening for Inhibitors 2

3 Cell Metabolism Critical for proper cell health and function Plays role in many diseases Active area of interest for many research fields Immunology Obesity Cancer Diabetes Inflammation Aging Neuroscience Cardiology 3

4 Cell Metabolism: Three Basic Needs of a Dividing Cell Biosynthesis ATP NADPH Carbon NADP + ADP ATP:ADP:AMP ROS Energy Requirements NAD + :NADH Lipids Nucleotides Carbohydrates Proteins NADPH ATP NADP + ADP GSH GSSG GSH Redox Control NADP + :NADPH GSSG Cairns, R.A, Harris, I.S. and Mak, T.W. (2011) Regulation of cancer cell metabolism. Nat. Rev. Cancer 11,

5 Metabolite Levels Provide Valuable Information Are cells functioning properly? How are cells responding to internal/external signals? Is metabolism functioning normally, or leading to aberrant growth? How is metabolism impacting the health or disease of an organism? 5

6 Detect Metabolic Changes By Measuring Metabolites Glycolysis and glutaminolysis are two major metabolic pathways o Glucose and lactate, or glutamine and glutamate, are key metabolites in these pathways, respectively Measurement of key metabolites can help you: o Monitor cell growth o Optimize cell culture conditions o Determine the effects of treatments (e.g. small compound inhibitors, toxins) o Study metabolic vulnerabilities of cancer cells o Understand fuel requirements for proper immune cell function 6

7 Metabolite Detection Methods Untargeted 100 s metabolites per sample Targeted Focus on Key Metabolites Analytical Methods Large amount of sample Organic extractions Limited sample number Specialized instrument Core Facility Coupled-Enzyme Reactions (H 2 O 2 or NADH) Plate-Based Absorbance/Fluorescence Applicable to 96-well plates Many sample types Larger amount of sample Cumbersome sample prep Plate reader Bioanalyzer Blood, culture media Lower sensitivity Limited sample number Specialized instrument Plate-Based Bioluminescence Applicable to 96-well plates Many sample types Smaller amount of sample Simpler sample prep Plate reader/luminometer 7

8 Key Considerations when Measuring Metabolites How will I quickly stop metabolism and recover metabolites from my samples? What is the metabolite concentration in my samples with respect to the sensitivity and linear range of the assay I am using? o o Determines how much sample you will need Impacts sample processing requirements Are interfering substances present? o Impacts sample processing requirements These questions are independent of the specific method used to measure metabolites 8

9 Sample Preparation

10 Requirements of Sample Preparation For good recovery of metabolites: Release metabolites from sample (lyse cells, homogenize tissues) Rapidly stop metabolism to freeze metabolite levels Reduce substances that interfere with assay (proteins, cell debris) Remove or inactivate endogenous enzymes Maintain stability of metabolites 10

11 Current Methods Require Protein Removal Cell Samples: Centrifuge after lysis to pellet precipitate, debris and proteins Treat cells Lyse Cells Treat with Acid Neutralize with Base Perchloric Acid (6N) Potassium Hydroxide (6N) Ready for assay Liquid Samples: (e.g. media, sera) Use spin columns to remove proteins 10KDa MW cut-off membrane 11

12 Simplified Sample Preparation: Goals Rapid and Simple: Reduce number of steps to simplify and shorten process time Avoid centrifuging and spin columns for pelleting precipitates after cell lysis, removing cell debris, and/or deproteinization Process multiple samples rapidly Process cells directly in wells without need for collecting cells Provide a common set of guidelines for processing multiple sample types 12

13 Simplified Sample Preparation: Strategies The sensitivity and linear range of the bioluminescent assays facilitate simpler sample preparation Greater Sensitivity = Need Less Sample Start with less sample (e.g. fewer cells, less tissue) Dilute sample to fit into linear range Reduces interfering enzymes and cell debris in samples When do you need to lyse/ homogenize or inactivate enzymes Use Acid Inactivation Solution Use HCl (no precipitates) Lyse, inactivate endogenous enzymes and degrade NAD(P)H Can process cells directly in well Removes the need to centrifuge cell debris and deproteinization 13

14 Measure Metabolites in Many Sample Types Cell Culture: Extracellular/ Media Cell Culture: Total = Cells + Media Tissues Many Sample Types One Metabolite Assay Cell Culture: Intracellular Plasma, Sera 3D Spheroids Microtissues Yeast Bacteria 14

15 Sample Preparation: Dilution Linear Range of Assays Concentrations in Cell Culture Medium Concentrations in Plasma and Serum 1µM 10µM 100µM 1mM 10mM Dilution For samples with <100µM to mm concentrations of metabolites Dilute into buffer (e.g. PBS) to be in linear range of assay (10 to 100-fold) Need small amounts of sample for dilution (e.g. medium or precious blood samples) Dilute out interfering substances; no other sample preparation is needed Can use dilution for measuring more than one metabolite 15

16 Sample Preparation: Acid Inactivation Cell Lysates Tissues Linear Range of Assays 1µM 10µM 100µM 1mM 10mM Sensitivity Can assay metabolites at lower concentrations At concentrations in cells plated in wells of 96-well plates ( ,000 cells per well) Do not need large amounts of precious samples (e.g. tissues) Acid Inactivation Solution (HCl) These samples contain enzymes that interfere with the assay (e.g. dehydrogenases) Inactivates enzymes during lysis/ homogenization step Provides additional lysis/homogenization power Degrades endogenous NAD(P)H (lowers background) 16

17 Bioluminescent Metabolite Detection Assays

18 Metabolite Detection Assays Use Bioluminescent NAD(P)H Detection Technology NAD(P)H = NADH or NADPH 18

19 Analogous Assay Principle Resazurin Diaphorase Resorufin FLUOR NADH NAD+ 560/590 nm 19

20 Different Performance Sensitivity, Linear Range, Signal Window NADH Titration Enzyme Titration Sensitivity: LOD ~5 50nM Wide Linear Range: ~1000-fold Good Signal Window: Max S/B ~500 Zhou, W. et al. (2014) ChemBioChem, 15:

21 Metabolite Detection Technology Glucose Lactate Glutamate Glutamine 21

22 Assay Protocol Steps: 1. Prepare Detection Reagent 2. Prepare Sample 3. Mix 1:1 in 96 or 384-well plate 4. Incubate for 1 hour at room temperature 5. Record luminescence 22

23 Performance of Glucose, Lactate, Glutamate Assays Linear Range: Glucose: up to 50µM Glutamate: up to 50µM Lactate: up to 200µM Sensitivity (L.O.D.): nm S/B max:

24 Measuring Glutamine and Glutamate Protocol: Glutamine Measurement No Glutamine Dehydrogenase Two-Step protocol 1. Convert glutamine to glutamate using Glutaminase enzyme (30 minute reaction at room temperature) 2. Add Glutamate Detection Reagent Two wells are needed to measure Glutamine (one well for Total glutamine + glutamate and one well for glutamate only) Efficient conversion of glutamine to glutamate RLU from glutamine ~ RLU from glutamate 24

25 Greater Sensitivity Enables Smaller Changes To Be Measured Lactate secretion by MCF7 breast cancer cells over time Luminescent and fluorescent methods Larger signals above background Easier to detect smaller changes Fluorescence Luminescence Red dotted line = background control 25

26 Broader Linear Range Provides Convenience Broad Linearity and Large Assay Window Analyze samples that have varying concentrations (e.g. different cell numbers, over time) Dilute media 10 to 100-fold One dilution can cover the entire range Can see larger changes Glucose consumption by A549 lung cancer cells 15,000 cells (dark bars) and 5000 cells (light bars) per well Red dashed lines are the lower and upper limits of the assays Fluorescence Luminescence Wider Assay Window 26

27 Metabolites in Many Sample Types Cell Culture: Extracellular/ Media Cell Culture: Total = Cells + Media Tissues Many Sample Types One Metabolite Assay Cell Culture: Intracellular Plasma, Sera 3D Spheroids Microtissues Yeast Bacteria 27

28 Application: Cancer Cells in Culture Metabolites in Media

29 Metabolites in Cell Culture Medium Monitoring metabolites in medium provides valuable information about cell growth, metabolic states and changes in metabolic pathways Growing cells consume glucose and glutamine and secrete lactate and glutamate Lactate production is used as an indicator of glycolysis pathway Glucose Glutamine Glycolysis Measure glucose and lactate Lactate CELL Glutamate Glutaminolysis Measure glutamine and glutamate 29

30 Considerations When Plating Cells Medium composition is important o Fuel requirements of cells o Assay requirements Media formulations o Glucose concentrations in culture media range from 5 25mM o Media also contain glutamine (2 4mM), pyruvate, amino acids, etc. o Start with defined concentrations of metabolite of interest Animal sera (FBS) contain mm concentrations of metabolites 30

31 Considerations When Plating Cells Growing cells consume glucose and glutamine and secrete lactate and glutamate The concentrations of these metabolites can change >50X over time, decreasing from mm to µm levels or increasing from µm to mm levels Changes in metabolite concentrations can be detected with greater sensitivity when using low glucose (<7 mm) and dialyzed animal serum o o Detect smaller changes Detect changes sooner 5 mm Glucose 2 mm Glutamine Low Lactate Low Glutamate Growing Cells Glucose Glutamine Lactate Glutamate 31

32 Detecting Metabolites in Cell Culture Media Remove small volume of medium and dilute into PBS (i.e. 2µl into 98µl) no need for acid treatment Sample = Cell Media Defined Medium: 5 mm glucose, 2 mm glutamine, 10% dialyzed serum Metabolite levels at different time points can be measured from the same well Assay immediately or freeze at -20 C Only one dilution factor is needed for multiple cell densities and time points All 4 metabolites can be measured from one diluted sample Diluted cell media is ready to be assayed at 1:1 ratio with Detection Reagent Glucose Lactate Glutamine Glutamate 32

33 Metabolite Levels: Changes Over Time During Cell Growth A549 lung cancer cells were plated at two cell concentrations per well in 96-well plates Glucose Consumption Plated in 100µl DMEM with 5mM glucose, 2mM glutamine and 10% dialyzed serum At the indicated time points, 2.5µl of medium was removed and diluted in 97.5µl PBS Dilutions were stored frozen at 20 C until the assay was performed 15,000 (dark bars) or 5,000 (light bars) cells per well Red dashed line = medium control 33

34 Metabolite Levels: Changes Over Time During Cell Growth Glucose Consumption Lactate Secretion 34

35 Metabolite Levels: Changes Over Time During Cell Growth 35

36 Metabolite Levels: Differences Between Cancer Cell Lines Different Glutamine Metabolism in Two Ovarian Cancer Cell Lines Glucose Consumption Lactate Secretion OVCAR-3 cells (low invasive) vs SKOV-3 cells (high invasive) Glutamine Consumption Glutamate Secretion 7,500 cells in 80 µl medium plated in 384-well plates From Leippe D., et al. (2017) SLAS Discovery 22: Yang, L., et al. (2014) Mol. Syst. Biol. 10:728 Yang, L., et al. (2016) Cell Metab. 24:

37 Application: Cancer Cells in Culture Intracellular Metabolites

38 Measuring Intracellular Metabolites: Considerations and Guidelines Intracellular levels vary with cell line/type, growth conditions and can change during handling Cells are lysed with Acid Inactivation Solution Concentrations align well with the sensitivity and linear range of the detection assays ( µm)* Measurements can be done with cells plated in 96-well plates (no need to collect cells) Extracellular levels >> intracellular levels; therefore the medium has to be removed and the cells have to be washed 38

39 Protocol: Measuring Intracellular Metabolites in Lysates of Cultured Cells Sample = Cultured cells in 96-well plate Cell Lysate ready to be assayed Remove medium and wash with PBS Lyse cells with acidic Inactivation Solution Add Detection Reagent directly to well OR Assay multiple metabolites from the same cell lysate by transferring aliquots to new wells Neutralize Lactate Glutamate Glucose Glutamine Glutamate Cell Lysate ready to be assayed Add Detection Reagent 39

40 Intracellular Metabolites: Lactate and Glutamate A549 lung cancer cells in 100µl medium were plated in 96-well plates Lactate After 24 hours, the medium was removed and the cells were washed twice with 200µl PBS For spike recovery determinations, PBS contained lactate or glutamate controls Glutamate The cells were then processed with Acid Inactivation Solution 40

41 Metabolite Levels: Differences Between Cancer Cell Lines Different Glutamine Metabolism in Two Ovarian Cancer Cell Lines as indicated by different intracellular pools of metabolites OVCAR-3 cells (low invasive) vs SKOV-3 cells (high invasive) 2,500 cells in 384-well plates From Leippe D., et al. (2017) SLAS Discovery 22: Yang, L., et al. (2014) Mol. Syst. Biol. 10:728 Yang, L., et al. (2016) Cell Metab. 24:

42 Application: Cancer Cells in Culture Multiplexing with Viability Assays

43 Multiplexing with Cell Viability Assays Provides more information, viability data, from your sample Aids in interpretation and normalization of data Is the viability assay lytic or non-lytic? To measure extracellular metabolites, just remove the culture medium before adding viability reagent to cells To measure intracellular metabolites, read the viability signal first, then remove medium and wash cells 43

44 Three Cell Viability Assays: Three Chemistries CellTiter-Glo Luminescent Cell Viability Assay (ATP) RealTime-Glo MT Cell Viability Assay CellTiter-Fluor Cell Viability Assay 44

45 Intracellular Metabolite Detection Multiplexed with Viability Assays Non-Lytic Viability (before lysis) Lytic ATP (after lysis) Intracellular Lactate in A549 Cells (after lysis) 45

46 Applications: T Cell Activation

47 Immunometabolism: T Cell Activation Intersection of Immunology and Metabolism: Immune cell metabolism drives immune cell function Cancer Immunotherapy: o Signals in the tumor cell environment o Competition for nutrients T Cell Activation Review Articles for further reading: Buck, M.D., et al. (2017) Cell, 169: O Neill, L.A.J., et al. (2016) Nat. Rev. Immunol. 16: Requires large amounts of energy and biomass to support growth, proliferation and effector function Naïve State (resting) o Quiescent, oxidative phosphorylation Effector State (activated) o Induced glycolysis, required for effector functions Monitor induced glycolysis by lactate production 47

48 T Cell Activation: Signal and Fuel Requirements Signal requirements: Two signals from both αcd3 and αcd28 are needed Fuel Requirements: Glutamine is needed; no activation occurs without it Human Peripheral Blood T Cells 250,000 cells in 100µl medium per well 48

49 T Cell Activation: Consumption of Glucose and Glutamine Glucose Consumption Glutamine Consumption Extensive consumption only by cells incubated with both signals in the presence of glutamine 49

50 Application: Screening for Inhibitors

51 Cells in Culture: Reconfiguration for Screening Applications Rapid Analysis Format For Lactate or Glutamate production Add Detection Reagent to well of cells o No medium removal o No medium dilution o No cell washing o Better suited to HTS Measure changes in total lactate or total glutamate levels Short incubations (within ~1 2 hrs; while levels are in linear range of the assays) Monitor effects of compounds Sample = Cells in Medium Add Acid Inactivation Solution Wait 5 minutes Add Detection Reagent 51

52 Rapid Analysis of Lactate Production Lactate levels increase with time Can detect increase within 30 minutes A549 lung cancer cells were plated in 96-well plates Cells were washed and given fresh 5mM glucose at the start of the time course, t = 0 minutes Acid Inactivation Solution was added to the cells at each time point to stop metabolism 52

53 Rapid Evaluation of Compounds that Inhibit or Activate Glycolysis Monitor glycolysis using lactate production as an indicator Observe effects of compounds that inhibit glycolysis or mitochondria function A549 lung cancer cells were treated with compounds for 1 hour at 37 C before assaying for lactate production 53

54 Rapid Analysis of Glutamate Production Glutamate produced by A549 cells Level increases with time Glutaminase inhibitor, BPTES, decreases glutamate production by SKOV-3 cells after 1 hr at 37 C Controls: No effects on Viability No direct effects on Detection System From Leippe D., et al. (2017) SLAS Discovery 22:

55 Evaluation for HTS: Acceptable Zʹ Values Can we meet criteria needed for HTS? Miniaturization/Lower volumes Automated Dispensing Acceptable Z value Experiment: o 6 µl of cells + medium o In 384-well low-volume plates o 1000 SKOV-3 cells or medium only per well o 60 wells of each per plate; two plates o 1 hour incubation at 37 C o Assay one plate for lactate production o Assay second for glutamate production From Leippe D., et al. (2017) SLAS Discovery 22: ; Z Factor: Zhang, J.H., et al. (1999) J. Biomol. Screen. 4:

56 Summary

57 Bioluminescent NAD(P)H Detection Technology: Assays for Cell Metabolism Research Glucose-Glo Assay Lactate-Glo Glutamate-Glo Glutamine/Glutamate-Glo Core NADH/NADPH Detection Technology NAD(P)H-Glo Assay NAD/NADH-Glo NADP/NADPH-Glo Glucose Uptake-Glo Assay 57

58 Summary Bioluminescent metabolite detection assays can be used to study glycolysis and glutamine metabolism Key metabolites glucose, lactate, glutamine and glutamate can be measured Greater sensitivity and wider linear ranges provide advantages for simpler sample preparation strategies Compatible with multiple sample types Applicable to studies of Cancer Cell Metabolism and Immunometabolism o o Cancer Cell Culture: culture medium, intracellular T Cell Activation: induction of glycolysis monitored through lactate production Effects of small molecule inhibitors/activators of pathways can be monitored Amenable to HTS formats and protocols 58

59 Thank you!