Biological Chemistry ‘Just Accepted’ Papers Biological Chemistry ‘Just Accepted’ Papers are papers published online, in advance of appearing in the print journal. They have been peer-reviewed, accepted and are online published in manuscript form, but have not been copy edited, typeset, or proofread. Copy editing may lead to small differences between the Just Accepted version and the final version. There may also be differences in the quality of the graphics. When papers do appear in print, they will be removed from this feature and grouped with other papers in an issue.
Biol Chem ‘Just Accepted’ Papers are citable; the online publication date is indicated on the Table of Contents page, and the article’ s Digital Object Identifier (DOI), a unique identifier for intellectual property in the digital environment (e.g., 10.1515/hsz-2011-xxxx), is shown at the top margin of the title page. Once an article is published as Biol Chem ‘Just Accepted’ Paper (and before it is published in its final form), it should be cited in other articles by indicating author list, title and DOI.
After a paper is published in Biol Chem ‘Just Accepted’ Paper form, it proceeds through the normal production process, which includes copy editing, typesetting and proofreading. The edited paper is then published in its final form in a regular print and online issue of Biol Chem. At this time, the Biol Chem ‘Just Accepted’ Paper version is replaced on the journal Web site by the final version of the paper with the same DOI as the Biol Chem ‘Just Accepted’ Paper version.
Disclaimer Biol Chem ‘Just Accepted’ Papers have undergone the complete peer-review process. However, none of the additional editorial preparation, which includes copy editing, typesetting and proofreading, has been performed. Therefore, there may be errors in articles published as Biol Chem ‘Just Accepted’ Papers that will be corrected in the final print and online version of the Journal. Any use of these articles is subject to the explicit understanding that the papers have not yet gone through the full quality control process prior to advanced publication.
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2016-0107 Research Article
iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry Jeremy Ryan1, Joan Montero1, James Rocco2 and Anthony Letai1,*
1
Department of Medical Oncology, Dana Farber Cancer Institute, 450 Brookline Ave, M430, Boston, MA 02210, USA
2
Department of Otolaryngology, Ohio State University Wexner Medical Center, Columbus, OH 43212, USA
*Corresponding author e-mail: [email protected]
1 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Abstract Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell’s dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell’s response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell’s propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96 and 384 well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.
Keywords: apoptosis; cancer; chemotherapy; flow cytometry; mitochondria; BCL-2.
2 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Introduction Dysregulation of the mitochondrial pathway of apoptosis can result in degenerative disorders when excess apoptosis is present and diseases such as cancer and autoimmune disorders when apoptosis is insufficient. Because the apoptotic pathway mediates the cellular response to a number of toxic insults, including chemotherapy, understanding and predicting apoptotic response has therapeutic potential. However, the mitochondrial pathway of apoptosis is regulated by the BCL-2 family of proteins which has more than fifteen members (Brunelle et al., 2009), and splice variants as well as post-translational modification occur in many of them. Because of this, accurately modeling all of the possible interactions becomes extremely difficult, and the cellular input required is prohibitive in the case of most clinical samples. Rather than trying to quantify levels of the BCL-2 family and their interactions, we have developed a method for interrogating the mitochondrial apoptotic machinery as a whole by applying to mitochondria measured doses of synthetic peptides corresponding to the BH3 domains of the pro-apoptotic BH3-only proteins followed by measurement of mitochondrial outer membrane permeabilization (MOMP). MOMP occurs when sufficient death signaling causes the multi-domain pro-apoptotic proteins BAX and BAK to assume their activated conformations followed by homo-oligomerization to form pores in the mitochondrial outer membrane. Because we use BH3 domains as perturbagens, we refer to this method as BH3 profiling. BH3 profiling was first created as an ELISA-based method that measured the proportion of cytochrome c left within mitochondria in a heavy membrane preparation after exposure to a fixed dose of BH3 peptide at a set time (Certo et al., 2006). We defined the concept of “priming” experimentally as being related to the proportion of cytochrome c that was released following BH3 peptide exposure. The more cytochrome c that was released from mitochondria following BH3 peptide exposure, the more primed the cell was for apoptosis. While this method was effective, it is also labor intensive, expensive, and required as many as one hundred million cells per assay. When discussing priming, it is useful to understand that it is better used as a relative than an absolute term. One cell type can be described as more primed than another, though it is harder to decide categorically that one is primed and the other is not. In addition, it should be noted there can be important differences between responses to more selective sensitizer peptides,
3 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry differences which can be exploited to identify selective MCL-1 dependence, for instance. Therefore, one cell may appear to be more primed than another when the BAD BH3 peptide is used, but less when the NOXA BH3 peptide is used. For this reason, we usually restrict our use of the term “primed” to reflect response to the more promiscuously acting BH3 peptides, such as BIM, BID or PUMA, where the ranking of sensitivity between different cells is usually very similar. It should also be noted that one can imagine different combinations of BCL-2 proteins that will give the same level of priming, given the diversity of pro-and anti-apoptotic family members. Therefore, priming can hint at underlying molecular biology, but it generally cannot definitively by itself assign protein levels or interactions. Nonetheless, priming has proved for us to be the most useful measurable phenotype in performing the task of understanding and predicting cell death response to a wide variety of perturbations, including chemotherapy. The requirement for large numbers of cells was in part due to the inefficiency of heavy membrane preparations. We later improved our method by instead testing mitochondria in situ, using digitonin to gently permeabilize the plasma membrane and permit diffusion of BH3 peptides into cells. This simple change afforded a 100-fold reduction in the number of cells required per profile (Ryan et al., 2010, 2013). Digitonin permeabilization has been used in the past for enzymatic studies (Aragon et al., 1980; Liu et al., 1999; Cassany et al., 2009) and to better visualize cytoskeletons in cells (Fiskum et al., 1980). Digitonin permeabilizes membranes with an efficiency that is related to membrane cholesterol content. Since plasma membranes have a higher concentration of cholesterol than mitochondria, low doses of digitonin cause a rapid permeabilization of the plasma membrane, while mitochondria, remain intact. Sufficient structure persists in permeabilized cells to allow for cell surface labeling and flow cytometry, which in turn allows for the study of sub-populations within a single sample. Furthermore, the reduced demands on sample size made possible the study of a broader range of clinical samples. Included in the results of these studies was the ability to segregate AML patients as responders or non-responders based on their profile (Vo et al., 2012), and an insight into the gap in priming between disease and normal tissue that contributes to the reason a therapeutic window exists (Ni Chonghaile et al., 2011). At the time we added the technique of digitonin permeabilization, we also began to measure MOMP with potentiometric dyes such as JC-1 and TMRE instead of by loss of cytochrome c. These dyes afforded a rapid and inexpensive way to measure MOMP, and in conjunction with a plate reader could even allow real-time kinetic measurements of loss of mitochondrial 4 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry integrity. However, despite what has been accomplished with JC-1 based BH3 profiling, there remained room for further improvement, particularly as it applies to experiments using flow cytometry. A significant practical challenge for JC-1 based profiling is that the cells are respiring during all parts of the measurement including data acquisition. This means that the time spent acquiring one well or tube is added to those that have not yet been processed. It also means that cytometric data must be acquired immediately after peptide exposure is complete. Second, any infectious particles in the sample remain viable during handling and acquisition which makes them more difficult and dangerous to handle. Finally, staining certain intracellular targets such as p53 or Ki-67 is not possible with potentiometric methods due to the additional time needed for their staining. To address these issues, we have developed a new and simple method to perform BH3 profiling using formaldehyde fixation to terminate peptide exposure. Fixation preserves the cell's structure at the time of fixation, neutralizes infectious particles, allows for membrane and organelle bound proteins to be stained, and allows samples to be analyzed weeks after they are produced. BH3 profiling by intracellular staining (iBH3) measures cytochrome c retained by mitochondria as measured by fluorescently labeled antibodies against cytochrome c as a measure of MOMP instead of using potentiometric dyes. Profiling by iBH3 can be performed as a purely additive process in which each reagent is added to the reaction without centrifugation or wash steps. This process reduces cell loss and also makes it simple to use with liquid handling automation to increase throughput. We demonstrate here the basic principles governing iBH3 as well as how it may be applied to a number of primary tissue samples. By adding a drug pre-treatment to a sample, we can also show the difference in priming caused by the treatment with a method we call dynamic BH3 profiling, DBP, and we show that this can serve as a biomarker to predict chemotherapeutic response in vivo.
Results There are two major distinctions between the older JC-1 or TMRE based profiling and iBH3: fixation and method of measuring MOMP. Both methods begin with a single cell suspension that can originate from cell cultures or primary tissue samples (Figure 1). The cells can then be stained with fixable viability dyes followed by staining using antibodies directed against cell surface epitopes to later identify subpopulations within the sample. Next, the cells are exposed to digitonin in a mitochondrial buffer to provide direct access to mitochondria for
5 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry compounds or peptides. At this point, the methods diverge. JC-1 based profiling requires respiring mitochondria to sustain a potential across the inner mitochondrial membrane. When the peptides induce BAX and/or BAK oligomerization, MOMP follows, followed in turn (with a delay of roughly 30 minutes) by loss of the potential gradient across the inner mitochondrial membrane. It is this gradient (also known as ΔΨm) that is measured by JC-1 and TMRE. Because the cells must be respiring, and because the ΔΨm-sensitive dyes fix very poorly, chemical fixatives cannot be employed, and the peptide exposure continues even during data acquisition such that re-sampling a tube at the start and end of acquisition often yields different results. In addition, cells exposed to viruses including lentivirus and HIV will still bear live virus during analysis. Chemical fixation of cells after peptide exposure results in a controlled termination of peptide exposure and fixes any infectious agents in the sample rendering them safer to handle. Also, the time sensitive window for iBH3 is greatly reduced and no longer includes the period of data acquisition which reduces variability (Figure 1B). In addition, fixed cells can also be stained for intracellular targets including cytochrome c, which is the second point where the two methods diverge. The intracellular stained BH3 protocol (iBH3) measures the physical permeabilization event, rather than the secondary loss of ΔΨm, yielding a more direct measurement of MOMP. Not all proteins can be detected during the iBH3 protocol. In Figure 2A, where color indicates the presence of protein, the act of permeabilizing the plasma membrane with digitonin causes rapid release of all soluble cytosolic proteins while those inside of organelles or bound to membranes will remain, including cytochrome c. Cytosolic proteins, including non-membrane bound fluorescent proteins routinely used as selection markers, cannot be used to discriminate populations. However, fluorescent proteins, including GFP, targeted to the plasma membrane, nucleus, or other organelles will remain inside the cell after digitonin exposure. Note that since the plasma membrane is permeabilized, when the cytochrome c escapes the mitochondria, it also escapes the cell, so that it is total cellular cytochrome c that is measured in the cytometer in iBH3. Mitochondria that undergo MOMP lose cytochrome c and antibody staining while those that remain intact stain strongly for cytochrome c (Figure 2A). MOMP measured by iBH3 is BAX/BAK dependent as well as both peptide and time dependent. In figure 2B, wild type MEFs and BAX/BAX DKO MEFs show that even when a broad range of peptide types and concentration is used, MOMP does not occur in the absence of the BAX and BAK. This comparison also serves as an important control for new peptides 6 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry or compounds for use in iBH3 as any compound that can cause cytochrome c release from a BAX and BAK deficient cell, such as alamethicin, is not acting on target at the concentration tested. When BAX or BAK are present, the response of all other peptides are both time and dose dependent as shown for the potent BIM peptide (Figure 2C). Increasing peptide exposure time causes a shift in the EC50 of the peptide to lower concentrations. The BIM BH3 peptide is generally the most potent peptide used in any BH3 profiling method, and less potent peptides or those with more restricted activity, such a HRK which binds only to BCL-XL, may not show any activity at shorter exposure times. A period of 30-90 minutes is typically used with 60 minutes being the default exposure time. It is usually easier to reduce the peptide concentration rather than reducing the time as this reduces variance due to error in the stopping time as well as reduces cost in terms of reagent usage. The more sensitive mitochondria are to promiscuous peptides such as BIM, the more sensitive the cells are to apoptosis-inducing insults such as chemotherapy. This increased propensity to undergo apoptosis as shown by BH3 profiling is called priming. The more primed the cell is, the easier it usually is to kill. It is important to remember that when comparing priming between samples, you must compare the same peptide or compound at an equivalent dose. Comparing BIM to BAD is not informative while comparing BIM response in one cell to another allows you to rank them. Cells of different apoptotic dependencies can exist within a single sample. iBH3 is capable of measuring the dependence of each population simultaneously and independent of each other. For example, three cell lines were mixed together in equal proportions, distinguished with surface antibodies, and their profiles extracted by flow cytometric gating on each population (Figure 2D). The results show here are identical to those of the cell lines run in separate tubes (data not shown.) As long as the cells can be labeled with a mark that can be detected by flow cytometry, the number of populations that can be distinguished is limited mainly by the rarity of the population and the number of channels available for analysis on the cytometer. Human PBMC populations identified by their cell surface epitopes (Figure 3A) show that many of the circulating immune cells have a similar profile with a strong response to BAD indicating dependence on BCL-2, BCL-W, or BCL-XL. CD56 (NK T-cell) and CD14 (myeloid) populations, however, show an increased response to NOXAA, which binds to MCL1, and to HRK, which binds to BCL-XL, suggesting that they are more MCL1 and BCLXL dependent than the B- or T-cell populations profiled here.
7 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry Note that any structure containing mitochondria can be subjected to iBH3. Platelets, despite their size, contain active mitochondria, and they can be profiled under similar conditions as cells. The response of platelets in iBH3 (Figure 3B) with a particularly strong response to BAD and HRK peptides and no response to NOXAA which corresponds to a strong BCL-XL dependence that platelets are known to possess. In this case, three BH3 mimetic small molecules were also included for direct mitochondrial exposure in the profile. Both ABT-263 and ABT-737 induce significant cytochrome c release in platelets while ABT-199 does not. This would suggest that ABT-263 and ABT-737 should have an on-target toxicity in platelets, and this is what is observed when platelets are incubated with these compounds and stained for annexin V as a measure of death(Mason et al., 2007; Zhang et al., 2007) (Figure 3C). As such, it is not surprising that thrombocytopenia emerged as a dose-limiting toxicity in clinical trials of ABT-263. It is also possible to perform iBH3 on solid tissues, but they must first be dissociated so that they can be stained and analyzed by flow cytometry. To demonstrate this, a human OPSCC tumor was enzymatically dissociated using collagenase and hyaluronidase to yield a single cell suspension. To remove necrotic cells, a fixable live/dead dye was added prior to surface labeling with CD45 for infiltrating leukocytes and CD44 and EpCAM to identify epithelial populations within the tumor. In Figure 4, cells are first separated into CD45+ and CD45– followed by further separation of the CD45– compartment based on CD44 vs EpCAM. In total, five populations are identified in this manner, and the lower side scatter CD45+ population, likely lymphocytes, general shows the greatest priming with almost all peptides shown meaning they are also the most easily killed. Many of the tumor population, including CD45– CD44+ EpCAM- and unstained cells, show a strong response to BIM and PUMA, which bind to all anti-apoptotic proteins, that is almost as great as the lymphocyte populations, and they should be also sensitive to treatment. Of note, the EpCAM+ population shows a reduced response to all peptides testing which means it is less primed than any of the others and represents a population what may be more difficult to clear kill with agents that rely on apoptosis for tumor cell clearance. The current examples thus far are all measures of upfront or baseline priming. There is another important variation on iBH3 that does not measure the baseline priming but rather the change of priming as a result of a short treatment. This measure of dynamic changes in priming induced by perturbations prior to BH3 profiling is known as dynamic BH3 or DBP. One can imagine two cells of equivalent baseline priming, but only one possesses the target of 8 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry a targeted agent, (e.g., an amplified or mutated tyrosine kinase). However, if the cells are both treated with a sub-lethal dose of the compound for 24 hours or less and then profiled relative to their untreated control, the responsive cells are likely to show an increase in priming long before they die while the non-responsive cells will show no change from baseline. By using DBP, one can rapidly determine whether or not a given treatment will kill, even if the killing would not occur until days later. An absolutely critical feature of the DBP approach is that it can be reliably applied to primary cancer cells, as ex vivo culture beyond 24 hours is not required. This distinction is of great practical importance, as the accuracy and utility of ex vivo functional approaches has historically been limited by their requirement for multi-day ex vivo culture, a great challenge for primary cancer cells. In DBP’s most general form (Figure 5A), a sample is taken and the cells isolated for analysis. This may be a cell culture, Ficoll gradient to prepare PBMCs or an enzymatic digest to produce adherent cells that can be seeded for overnight treatment. Once the cells are isolated, they are plated along with their untreated control, and the number of peptides and drug combinations possible will depend on the amount of sample available. Once the cells are treated, they are profiled using iBH3 in the same way baseline samples are processed, but the untreated profile is subtracted from all treated profiles to produce a delta priming, and a positive delta indicates a likelihood that the treatment that induced it will show a response in terms of cell death in vitro or in vivo. To demonstrate, cell lines PC9 and SK-MEL-5, were treated with a panel of drugs to predict which would be cytotoxic at later times. After 24 hours of drug exposure, DBP revealed that each cell line was predicted to respond to a different subset of drug as indicated by the large Δ priming values (Figure 5B and D). Viability by annexin V staining after 72 hours of drug exposure showed a close correlation between delta priming and cell death (Figure 5C and E) (Montero et al., 2015).
Discussion iBH3 is a robust phenotypic assay that provides both how prone a cell is to undergo apoptosis as well as the anti-apoptotic proteins being used to prevent apoptosis. In the case of BH3 mimetic compounds such as ABT-263 and ABT-199, iBH3 can determine whether these compounds will be effective in treatment. In addition, BH3 mimetic compounds can be used in the place of peptides during iBH3 to determine their specificity and activity in the context of intact mitochondria in order to assist in the creation and validation of new BH3 mimetic
9 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry compounds. Even unnatural BH3 peptides have been screened by and are now used in iBH3 including the MCL-1 specific peptide MS1 (Foight et al., 2014). Much of our attention has now turned towards predicting which treatment will succeed for any given patient, and this requires both the efficient use of primary samples and a means to screen numerous compounds swiftly enough that ex vivo culture does not exert the dominant force on cell survival. The long culture times needed to assess cell death in most cases yields a result that does not predict response in patients, but using dynamic BH3 profiling, we have successfully shortened the culture period and provided a means to detect death signaling well before the onset of frank cell death (Montero et al., 2015). At present, given sufficient sample, we can test hundreds of conditions against a tumor sample to determine which treatments have the best chance of success, and we feel this will become an invaluable tool in guiding patient therapy as well as drug development.
Materials and methods Mannitol, potassium chloride, EDTA, EGTA, HEPES free acid, glycine, potassium hydroxide, triton X-100, digitonin, and succinic acid (Sigma Aldrich), Protease-free BSA and standard BSA (Gemini Bioscience), Tris base (Fisher Scientific) were used as received. Anti-cytochrome C antibody clone 6H2.B4 conjugated to Alexa Fluor 488 or Alexa Fluor 647 (Biolegend) were obtained as 0.5 mg/ml solutions. 10× Perm/Wash (BD Bioscience) Tween20 (Amresco) Non-Binding 96-well clear plates (Corning) or FACS tubes (Corning Falcon) Zombie Aqua, CD45, CD44, and EpCAM (Biolegend).
Buffers MEB: 150 mM mannitol, 50 mM KCl, 5 mM succinate, 20 μM EDTA, 20 μM EGTA, 0.1% protease-free BSA, 10 mM HEPES, pH to 7.5 with KOH; Buffer N2: 1.7 M Tris base, 1.25 M glycine, pH 9.1; 10× Tween-20 intracellular staining buffer: 10% standard BSA, 2% Tween20 in PBS; 10× Triton X-100 intracellular staining buffer: 10% standard BSA, 1% Triton X100in PBS; 10× Saponin intracellular staining buffer: 10% standard BSA, 2% saponin in PBS.
10 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
BH3 Profiling by Intracellular staining (iBH3) To prepare iBH3 reaction in 96 -well plates, 50 μl of peptides or compounds in MEB / 20 µg/ml digitonin at 2 times their final concentration is added to each well. A cell suspension in MEB at 0.2 - 5×106 cells/ml is made and 50 µl of the cell suspension is added to each well. The plate is incubated at 25oC for 45-90 min followed by the addition of 40 µl 4% formaldehyde in PBS. Cells are fixed for 10 min at RT, quenched by the addition of 40 µl Buffer N2, and stained by adding 20 µl of 10× staining solution containing 125-250 ng/ml cytochrome c antibody overnight at 4oC. If Triton X-100 buffers are used, the samples must be washed once prior to analysis. Tween-20, BD Perm/Wash, and saponin based buffers can proceed directly to FACS without washing. Volumes can be scaled up for larger tubes or down for 384 well plates. Once time, peptide concentration, and cell density are chosen, they must remain the same between replicates to ensure the most accurate result. PUMA2A, an inert peptide, or no peptide defines full cytochrome c retention and a lack of MOMP while 25 μM alamethicin, which causes BAX/BAK independent MOMP, serves as a complete cytochrome c release control. Gating around the cells in the PUMA2A control using SSC vs. cytochrome c provides the % cytochrome c positive cells, and 100 minus this value defines % cytochrome c loss. Alternatively, the median fluorescence intensity (MFI) of the cytochrome c stain can be used to calculate % cytochrome c loss as follows:
With the exception of dose response curves, cytochrome c loss is generally preferred to retention for clarity by having peptides that cause MOMP show a gain of signal rather than loss.
Primary humans samples De-identified platelet apheresis collars and discarded platelet units were obtained from the Kraft Family Blood Center, Dana-Farber / Bringham and Woman’s hospital, PBMCs were purified using a ficoll gradient, and were surface stained using CD3, CD19, CD20, CD14, and CD56. De-identified human OPSCC tumor from Massachusetts General Hospital was provided by Dr. James Rocco. The OPSCC tumor was dissociated with 300 U/mL collagenase 4, 100 U/ml hyaluronidase, 125 U/ml DNAse I in DMEM/F12 for 1 hour at 37oC with shaking, filtered through a 70 micron strainer, and suspended in HBSS at 107 cells/ml
11 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry followed by the addition of zombie aqua viability dye at RT for 15 minutes followed by pelleting and suspension in 2% FBS in HBSS with CD45, CD44, and EpCAM antibodies on ice for 30 minutes before pelleting and suspending of cells in MEB at 4 million cells/ml. Fifty µl of cell suspension were added to 50 µl of 2× peptides in MEB with 20 µg/ml digitonin for one hour prior to fixation.
Dynamic BH3 profiling (DBP) Prior to iBH3, cells are treated for 4-24 hours with a sub-lethal dose of one or more compounds with at least one untreated control. Death should not be apparent at the time of harvest, and dose or time should be reduced if it is observed, and multiple doses can be tested as well as combinations. After drug treatment, cells are harvested and suspended in MEB, and cells are processed using the standard iBH3 procedure above. For each peptide treatment, the cytochrome c release value of the non-drug treated cells are subtracted from the cytochrome release value of the drug-treated samples to generate the drug-induced change in priming, or delta priming. For Figure 5, cells were exposed for 24 hours for DBP and 72 hours for viability to compounds as follows: gefitinib 1 µM (EGFR inhibitor), lapatinib 1 µM (HER2.EGFR inhibitor), TAE-684 1 µM (ALK inhibitor), MK-2206 1 µM (AKt inhibitor), PLX-4032 10 µM (BRAF inhibitor), AZD-6244 1 µM (MEK Inhibitor), BEZ235 1 µM (PI3K / mTOR inhibitor)
12 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
References Aragon, J. J., J. E. Feliu, R. A. Frenkel and A. Sols (1980). Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes. Proc Natl Acad Sci USA 77, 6324-6328. Brunelle, J. K. and A. Letai (2009). Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 122, 437-441. Cassany, A. and L. Gerace (2009). Reconstitution of nuclear import in permeabilized cells. Methods Mol Biol 464, 181-205. Certo, M., V. Del Gaizo Moore, M. Nishino, G. Wei, S. Korsmeyer, S. A. Armstrong and A. Letai (2006). Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351-365. Fiskum, G., S. W. Craig, G. L. Decker and A. L. Lehninger (1980). The cytoskeleton of digitonin-treated rat hepatocytes. Proc Natl Acad Sci USA 77, 3430-3434. Foight, G. W., J. A. Ryan, S. V. Gulla, A. Letai and A. E. Keating (2014). Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells. ACS Chem Biol 9, 1962-1968. Liu, J., N. Xiao and D. B. DeFranco (1999). Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19, 403-409. Mason, K. D., Carpinelli, M.R., Fletcher, J.I., Collinge, J.E., Hilton, A.A., Ellis, S., Kelly, P.N., Ekert, P.G., Metcalf, D., Roberts, A.W. et al., (2007). Programmed anuclear cell death delimits platelet life span. Cell 128, 1173-1186. Montero, J., Sarosiek, K.A., DeAngelo, J.D., Maertens, O., Ryan, J., Ercan, D., Piao, H., Horowitz, N.S., Berkowitz, R.S., Matulonis, U. et al., (2015). Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 160, 977-989. Ni Chonghaile, T., Sarosiek, K.A., Vo, T.T., Ryan, J.A., Tammareddi, A., Moore, V. del G., Deng, J., Anderson, K.C., Richardson, P. et al., (2011). Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 334, 11291133. Ryan, J. and A. Letai (2013). BH3 profiling in whole cells by fluorimeter or FACS. Methods 61, 156-164. Ryan, J. A., J. K. Brunelle and A. Letai (2010). Heightened mitochondrial priming is the basis for apoptotic hypersensitivity of CD4+ CD8+ thymocytes. Proc Natl Acad Sci USA 107, 12895-12900. Vo, T. T., Ryan, .J, Carrasco, R., Neuberg, D., Rossi, D.J., Stone, R.M., Deangelo, D.J., Frattini, M.G. and Letai, A. (2012). Relative mitochondrial priming of myeloblasts and normal HSCs determines chemotherapeutic success in AML. Cell 151, 344-355. Zhang, H., Nimmer, P.M., Tahir, S.K., Chen, J., Fryer, R.M., Hahn, K.R., Iciek, L.A., Morgan, S.J., Nasarre, M.C., Nelson, R. et al., (2007). Bcl-2 family proteins are essential for platelet survival. Cell Death Differ 14, 943-951.
13 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Tables and figures
Figure 1
Comparison of BH3 profiling method for flow cytometry. Potentiometric JC-1
(A) and fixed cell with intracellular staining iBH3 (B).
Figure 2
Principles guiding iBH3 design (A) Schematic of cell level events during iBH3
showing loss of cytoplasm upon digitonin addition, movement of cytochrome c after MOMP, and cartoon of expected output. Color indicates the presence of protein. (B) Wild type and BAX/BAK DKO MEFs showing peptide induced MOMP requires BAX and BAK. (C) MOMP induced by BIM peptide is dependent on both exposure time and dose. (D) Mixed cell lines labeled with cell surface
14 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry markers produce distinguishable BH3 profiles upon gating. BH3-only protein names listed in B-D refer to the BH3 only peptides, not the full length or native proteins.
Figure 3
iBH3 profiles of human blood components (A) PBMC populations identified
by surface marking (B) Baseline profile of human platelets, peptide and drug concentrations in micromolar. (C) Annexin V staining of treated platelets after 24 hours showing sensitivity to compounds that bind BCL-XL.
Figure 4
Human OPSCC tumor profiles using iBH3.
(A) Gating of CD45+ and CD45– populations. (B) Subdivision of CD44 and EpCAM populations within CD45– (C). iBH3 profile of all five gated populations. From Montero et al. (2015).
15 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Figure 5
Dynamic BH3 Profiling is a predictive biomarker.
(A) General workflow for DBP. Priming is cytochrome c release for iBH3, but can refer to depolarization in JC-1 based methods of DBP. In both cases, Δ Priming is the difference between treated and untreated populations. (B) DBP profile at 24 h and (C) Annexin V viability at 72 h for cell line PC9. (D) DBP profile at 24 hours and (E) Annexin V viability at 72 hours for SK-MEL-5. All drugs were used at 1 µM except for PLX-4032 which was used at 10 µM.
16 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
Biol Chem ‘Just Accepted’ Papers are citable; the online publication date is indicated on the Table of Contents page, and the article’ s Digital Object Identifier (DOI), a unique identifier for intellectual property in the digital environment (e.g., 10.1515/hsz-2011-xxxx), is shown at the top margin of the title page. Once an article is published as Biol Chem ‘Just Accepted’ Paper (and before it is published in its final form), it should be cited in other articles by indicating author list, title and DOI.
After a paper is published in Biol Chem ‘Just Accepted’ Paper form, it proceeds through the normal production process, which includes copy editing, typesetting and proofreading. The edited paper is then published in its final form in a regular print and online issue of Biol Chem. At this time, the Biol Chem ‘Just Accepted’ Paper version is replaced on the journal Web site by the final version of the paper with the same DOI as the Biol Chem ‘Just Accepted’ Paper version.
Disclaimer Biol Chem ‘Just Accepted’ Papers have undergone the complete peer-review process. However, none of the additional editorial preparation, which includes copy editing, typesetting and proofreading, has been performed. Therefore, there may be errors in articles published as Biol Chem ‘Just Accepted’ Papers that will be corrected in the final print and online version of the Journal. Any use of these articles is subject to the explicit understanding that the papers have not yet gone through the full quality control process prior to advanced publication.
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2016-0107 Research Article
iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry Jeremy Ryan1, Joan Montero1, James Rocco2 and Anthony Letai1,*
1
Department of Medical Oncology, Dana Farber Cancer Institute, 450 Brookline Ave, M430, Boston, MA 02210, USA
2
Department of Otolaryngology, Ohio State University Wexner Medical Center, Columbus, OH 43212, USA
*Corresponding author e-mail: [email protected]
1 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Abstract Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell’s dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell’s response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell’s propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96 and 384 well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.
Keywords: apoptosis; cancer; chemotherapy; flow cytometry; mitochondria; BCL-2.
2 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Introduction Dysregulation of the mitochondrial pathway of apoptosis can result in degenerative disorders when excess apoptosis is present and diseases such as cancer and autoimmune disorders when apoptosis is insufficient. Because the apoptotic pathway mediates the cellular response to a number of toxic insults, including chemotherapy, understanding and predicting apoptotic response has therapeutic potential. However, the mitochondrial pathway of apoptosis is regulated by the BCL-2 family of proteins which has more than fifteen members (Brunelle et al., 2009), and splice variants as well as post-translational modification occur in many of them. Because of this, accurately modeling all of the possible interactions becomes extremely difficult, and the cellular input required is prohibitive in the case of most clinical samples. Rather than trying to quantify levels of the BCL-2 family and their interactions, we have developed a method for interrogating the mitochondrial apoptotic machinery as a whole by applying to mitochondria measured doses of synthetic peptides corresponding to the BH3 domains of the pro-apoptotic BH3-only proteins followed by measurement of mitochondrial outer membrane permeabilization (MOMP). MOMP occurs when sufficient death signaling causes the multi-domain pro-apoptotic proteins BAX and BAK to assume their activated conformations followed by homo-oligomerization to form pores in the mitochondrial outer membrane. Because we use BH3 domains as perturbagens, we refer to this method as BH3 profiling. BH3 profiling was first created as an ELISA-based method that measured the proportion of cytochrome c left within mitochondria in a heavy membrane preparation after exposure to a fixed dose of BH3 peptide at a set time (Certo et al., 2006). We defined the concept of “priming” experimentally as being related to the proportion of cytochrome c that was released following BH3 peptide exposure. The more cytochrome c that was released from mitochondria following BH3 peptide exposure, the more primed the cell was for apoptosis. While this method was effective, it is also labor intensive, expensive, and required as many as one hundred million cells per assay. When discussing priming, it is useful to understand that it is better used as a relative than an absolute term. One cell type can be described as more primed than another, though it is harder to decide categorically that one is primed and the other is not. In addition, it should be noted there can be important differences between responses to more selective sensitizer peptides,
3 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry differences which can be exploited to identify selective MCL-1 dependence, for instance. Therefore, one cell may appear to be more primed than another when the BAD BH3 peptide is used, but less when the NOXA BH3 peptide is used. For this reason, we usually restrict our use of the term “primed” to reflect response to the more promiscuously acting BH3 peptides, such as BIM, BID or PUMA, where the ranking of sensitivity between different cells is usually very similar. It should also be noted that one can imagine different combinations of BCL-2 proteins that will give the same level of priming, given the diversity of pro-and anti-apoptotic family members. Therefore, priming can hint at underlying molecular biology, but it generally cannot definitively by itself assign protein levels or interactions. Nonetheless, priming has proved for us to be the most useful measurable phenotype in performing the task of understanding and predicting cell death response to a wide variety of perturbations, including chemotherapy. The requirement for large numbers of cells was in part due to the inefficiency of heavy membrane preparations. We later improved our method by instead testing mitochondria in situ, using digitonin to gently permeabilize the plasma membrane and permit diffusion of BH3 peptides into cells. This simple change afforded a 100-fold reduction in the number of cells required per profile (Ryan et al., 2010, 2013). Digitonin permeabilization has been used in the past for enzymatic studies (Aragon et al., 1980; Liu et al., 1999; Cassany et al., 2009) and to better visualize cytoskeletons in cells (Fiskum et al., 1980). Digitonin permeabilizes membranes with an efficiency that is related to membrane cholesterol content. Since plasma membranes have a higher concentration of cholesterol than mitochondria, low doses of digitonin cause a rapid permeabilization of the plasma membrane, while mitochondria, remain intact. Sufficient structure persists in permeabilized cells to allow for cell surface labeling and flow cytometry, which in turn allows for the study of sub-populations within a single sample. Furthermore, the reduced demands on sample size made possible the study of a broader range of clinical samples. Included in the results of these studies was the ability to segregate AML patients as responders or non-responders based on their profile (Vo et al., 2012), and an insight into the gap in priming between disease and normal tissue that contributes to the reason a therapeutic window exists (Ni Chonghaile et al., 2011). At the time we added the technique of digitonin permeabilization, we also began to measure MOMP with potentiometric dyes such as JC-1 and TMRE instead of by loss of cytochrome c. These dyes afforded a rapid and inexpensive way to measure MOMP, and in conjunction with a plate reader could even allow real-time kinetic measurements of loss of mitochondrial 4 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry integrity. However, despite what has been accomplished with JC-1 based BH3 profiling, there remained room for further improvement, particularly as it applies to experiments using flow cytometry. A significant practical challenge for JC-1 based profiling is that the cells are respiring during all parts of the measurement including data acquisition. This means that the time spent acquiring one well or tube is added to those that have not yet been processed. It also means that cytometric data must be acquired immediately after peptide exposure is complete. Second, any infectious particles in the sample remain viable during handling and acquisition which makes them more difficult and dangerous to handle. Finally, staining certain intracellular targets such as p53 or Ki-67 is not possible with potentiometric methods due to the additional time needed for their staining. To address these issues, we have developed a new and simple method to perform BH3 profiling using formaldehyde fixation to terminate peptide exposure. Fixation preserves the cell's structure at the time of fixation, neutralizes infectious particles, allows for membrane and organelle bound proteins to be stained, and allows samples to be analyzed weeks after they are produced. BH3 profiling by intracellular staining (iBH3) measures cytochrome c retained by mitochondria as measured by fluorescently labeled antibodies against cytochrome c as a measure of MOMP instead of using potentiometric dyes. Profiling by iBH3 can be performed as a purely additive process in which each reagent is added to the reaction without centrifugation or wash steps. This process reduces cell loss and also makes it simple to use with liquid handling automation to increase throughput. We demonstrate here the basic principles governing iBH3 as well as how it may be applied to a number of primary tissue samples. By adding a drug pre-treatment to a sample, we can also show the difference in priming caused by the treatment with a method we call dynamic BH3 profiling, DBP, and we show that this can serve as a biomarker to predict chemotherapeutic response in vivo.
Results There are two major distinctions between the older JC-1 or TMRE based profiling and iBH3: fixation and method of measuring MOMP. Both methods begin with a single cell suspension that can originate from cell cultures or primary tissue samples (Figure 1). The cells can then be stained with fixable viability dyes followed by staining using antibodies directed against cell surface epitopes to later identify subpopulations within the sample. Next, the cells are exposed to digitonin in a mitochondrial buffer to provide direct access to mitochondria for
5 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry compounds or peptides. At this point, the methods diverge. JC-1 based profiling requires respiring mitochondria to sustain a potential across the inner mitochondrial membrane. When the peptides induce BAX and/or BAK oligomerization, MOMP follows, followed in turn (with a delay of roughly 30 minutes) by loss of the potential gradient across the inner mitochondrial membrane. It is this gradient (also known as ΔΨm) that is measured by JC-1 and TMRE. Because the cells must be respiring, and because the ΔΨm-sensitive dyes fix very poorly, chemical fixatives cannot be employed, and the peptide exposure continues even during data acquisition such that re-sampling a tube at the start and end of acquisition often yields different results. In addition, cells exposed to viruses including lentivirus and HIV will still bear live virus during analysis. Chemical fixation of cells after peptide exposure results in a controlled termination of peptide exposure and fixes any infectious agents in the sample rendering them safer to handle. Also, the time sensitive window for iBH3 is greatly reduced and no longer includes the period of data acquisition which reduces variability (Figure 1B). In addition, fixed cells can also be stained for intracellular targets including cytochrome c, which is the second point where the two methods diverge. The intracellular stained BH3 protocol (iBH3) measures the physical permeabilization event, rather than the secondary loss of ΔΨm, yielding a more direct measurement of MOMP. Not all proteins can be detected during the iBH3 protocol. In Figure 2A, where color indicates the presence of protein, the act of permeabilizing the plasma membrane with digitonin causes rapid release of all soluble cytosolic proteins while those inside of organelles or bound to membranes will remain, including cytochrome c. Cytosolic proteins, including non-membrane bound fluorescent proteins routinely used as selection markers, cannot be used to discriminate populations. However, fluorescent proteins, including GFP, targeted to the plasma membrane, nucleus, or other organelles will remain inside the cell after digitonin exposure. Note that since the plasma membrane is permeabilized, when the cytochrome c escapes the mitochondria, it also escapes the cell, so that it is total cellular cytochrome c that is measured in the cytometer in iBH3. Mitochondria that undergo MOMP lose cytochrome c and antibody staining while those that remain intact stain strongly for cytochrome c (Figure 2A). MOMP measured by iBH3 is BAX/BAK dependent as well as both peptide and time dependent. In figure 2B, wild type MEFs and BAX/BAX DKO MEFs show that even when a broad range of peptide types and concentration is used, MOMP does not occur in the absence of the BAX and BAK. This comparison also serves as an important control for new peptides 6 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry or compounds for use in iBH3 as any compound that can cause cytochrome c release from a BAX and BAK deficient cell, such as alamethicin, is not acting on target at the concentration tested. When BAX or BAK are present, the response of all other peptides are both time and dose dependent as shown for the potent BIM peptide (Figure 2C). Increasing peptide exposure time causes a shift in the EC50 of the peptide to lower concentrations. The BIM BH3 peptide is generally the most potent peptide used in any BH3 profiling method, and less potent peptides or those with more restricted activity, such a HRK which binds only to BCL-XL, may not show any activity at shorter exposure times. A period of 30-90 minutes is typically used with 60 minutes being the default exposure time. It is usually easier to reduce the peptide concentration rather than reducing the time as this reduces variance due to error in the stopping time as well as reduces cost in terms of reagent usage. The more sensitive mitochondria are to promiscuous peptides such as BIM, the more sensitive the cells are to apoptosis-inducing insults such as chemotherapy. This increased propensity to undergo apoptosis as shown by BH3 profiling is called priming. The more primed the cell is, the easier it usually is to kill. It is important to remember that when comparing priming between samples, you must compare the same peptide or compound at an equivalent dose. Comparing BIM to BAD is not informative while comparing BIM response in one cell to another allows you to rank them. Cells of different apoptotic dependencies can exist within a single sample. iBH3 is capable of measuring the dependence of each population simultaneously and independent of each other. For example, three cell lines were mixed together in equal proportions, distinguished with surface antibodies, and their profiles extracted by flow cytometric gating on each population (Figure 2D). The results show here are identical to those of the cell lines run in separate tubes (data not shown.) As long as the cells can be labeled with a mark that can be detected by flow cytometry, the number of populations that can be distinguished is limited mainly by the rarity of the population and the number of channels available for analysis on the cytometer. Human PBMC populations identified by their cell surface epitopes (Figure 3A) show that many of the circulating immune cells have a similar profile with a strong response to BAD indicating dependence on BCL-2, BCL-W, or BCL-XL. CD56 (NK T-cell) and CD14 (myeloid) populations, however, show an increased response to NOXAA, which binds to MCL1, and to HRK, which binds to BCL-XL, suggesting that they are more MCL1 and BCLXL dependent than the B- or T-cell populations profiled here.
7 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry Note that any structure containing mitochondria can be subjected to iBH3. Platelets, despite their size, contain active mitochondria, and they can be profiled under similar conditions as cells. The response of platelets in iBH3 (Figure 3B) with a particularly strong response to BAD and HRK peptides and no response to NOXAA which corresponds to a strong BCL-XL dependence that platelets are known to possess. In this case, three BH3 mimetic small molecules were also included for direct mitochondrial exposure in the profile. Both ABT-263 and ABT-737 induce significant cytochrome c release in platelets while ABT-199 does not. This would suggest that ABT-263 and ABT-737 should have an on-target toxicity in platelets, and this is what is observed when platelets are incubated with these compounds and stained for annexin V as a measure of death(Mason et al., 2007; Zhang et al., 2007) (Figure 3C). As such, it is not surprising that thrombocytopenia emerged as a dose-limiting toxicity in clinical trials of ABT-263. It is also possible to perform iBH3 on solid tissues, but they must first be dissociated so that they can be stained and analyzed by flow cytometry. To demonstrate this, a human OPSCC tumor was enzymatically dissociated using collagenase and hyaluronidase to yield a single cell suspension. To remove necrotic cells, a fixable live/dead dye was added prior to surface labeling with CD45 for infiltrating leukocytes and CD44 and EpCAM to identify epithelial populations within the tumor. In Figure 4, cells are first separated into CD45+ and CD45– followed by further separation of the CD45– compartment based on CD44 vs EpCAM. In total, five populations are identified in this manner, and the lower side scatter CD45+ population, likely lymphocytes, general shows the greatest priming with almost all peptides shown meaning they are also the most easily killed. Many of the tumor population, including CD45– CD44+ EpCAM- and unstained cells, show a strong response to BIM and PUMA, which bind to all anti-apoptotic proteins, that is almost as great as the lymphocyte populations, and they should be also sensitive to treatment. Of note, the EpCAM+ population shows a reduced response to all peptides testing which means it is less primed than any of the others and represents a population what may be more difficult to clear kill with agents that rely on apoptosis for tumor cell clearance. The current examples thus far are all measures of upfront or baseline priming. There is another important variation on iBH3 that does not measure the baseline priming but rather the change of priming as a result of a short treatment. This measure of dynamic changes in priming induced by perturbations prior to BH3 profiling is known as dynamic BH3 or DBP. One can imagine two cells of equivalent baseline priming, but only one possesses the target of 8 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry a targeted agent, (e.g., an amplified or mutated tyrosine kinase). However, if the cells are both treated with a sub-lethal dose of the compound for 24 hours or less and then profiled relative to their untreated control, the responsive cells are likely to show an increase in priming long before they die while the non-responsive cells will show no change from baseline. By using DBP, one can rapidly determine whether or not a given treatment will kill, even if the killing would not occur until days later. An absolutely critical feature of the DBP approach is that it can be reliably applied to primary cancer cells, as ex vivo culture beyond 24 hours is not required. This distinction is of great practical importance, as the accuracy and utility of ex vivo functional approaches has historically been limited by their requirement for multi-day ex vivo culture, a great challenge for primary cancer cells. In DBP’s most general form (Figure 5A), a sample is taken and the cells isolated for analysis. This may be a cell culture, Ficoll gradient to prepare PBMCs or an enzymatic digest to produce adherent cells that can be seeded for overnight treatment. Once the cells are isolated, they are plated along with their untreated control, and the number of peptides and drug combinations possible will depend on the amount of sample available. Once the cells are treated, they are profiled using iBH3 in the same way baseline samples are processed, but the untreated profile is subtracted from all treated profiles to produce a delta priming, and a positive delta indicates a likelihood that the treatment that induced it will show a response in terms of cell death in vitro or in vivo. To demonstrate, cell lines PC9 and SK-MEL-5, were treated with a panel of drugs to predict which would be cytotoxic at later times. After 24 hours of drug exposure, DBP revealed that each cell line was predicted to respond to a different subset of drug as indicated by the large Δ priming values (Figure 5B and D). Viability by annexin V staining after 72 hours of drug exposure showed a close correlation between delta priming and cell death (Figure 5C and E) (Montero et al., 2015).
Discussion iBH3 is a robust phenotypic assay that provides both how prone a cell is to undergo apoptosis as well as the anti-apoptotic proteins being used to prevent apoptosis. In the case of BH3 mimetic compounds such as ABT-263 and ABT-199, iBH3 can determine whether these compounds will be effective in treatment. In addition, BH3 mimetic compounds can be used in the place of peptides during iBH3 to determine their specificity and activity in the context of intact mitochondria in order to assist in the creation and validation of new BH3 mimetic
9 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry compounds. Even unnatural BH3 peptides have been screened by and are now used in iBH3 including the MCL-1 specific peptide MS1 (Foight et al., 2014). Much of our attention has now turned towards predicting which treatment will succeed for any given patient, and this requires both the efficient use of primary samples and a means to screen numerous compounds swiftly enough that ex vivo culture does not exert the dominant force on cell survival. The long culture times needed to assess cell death in most cases yields a result that does not predict response in patients, but using dynamic BH3 profiling, we have successfully shortened the culture period and provided a means to detect death signaling well before the onset of frank cell death (Montero et al., 2015). At present, given sufficient sample, we can test hundreds of conditions against a tumor sample to determine which treatments have the best chance of success, and we feel this will become an invaluable tool in guiding patient therapy as well as drug development.
Materials and methods Mannitol, potassium chloride, EDTA, EGTA, HEPES free acid, glycine, potassium hydroxide, triton X-100, digitonin, and succinic acid (Sigma Aldrich), Protease-free BSA and standard BSA (Gemini Bioscience), Tris base (Fisher Scientific) were used as received. Anti-cytochrome C antibody clone 6H2.B4 conjugated to Alexa Fluor 488 or Alexa Fluor 647 (Biolegend) were obtained as 0.5 mg/ml solutions. 10× Perm/Wash (BD Bioscience) Tween20 (Amresco) Non-Binding 96-well clear plates (Corning) or FACS tubes (Corning Falcon) Zombie Aqua, CD45, CD44, and EpCAM (Biolegend).
Buffers MEB: 150 mM mannitol, 50 mM KCl, 5 mM succinate, 20 μM EDTA, 20 μM EGTA, 0.1% protease-free BSA, 10 mM HEPES, pH to 7.5 with KOH; Buffer N2: 1.7 M Tris base, 1.25 M glycine, pH 9.1; 10× Tween-20 intracellular staining buffer: 10% standard BSA, 2% Tween20 in PBS; 10× Triton X-100 intracellular staining buffer: 10% standard BSA, 1% Triton X100in PBS; 10× Saponin intracellular staining buffer: 10% standard BSA, 2% saponin in PBS.
10 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
BH3 Profiling by Intracellular staining (iBH3) To prepare iBH3 reaction in 96 -well plates, 50 μl of peptides or compounds in MEB / 20 µg/ml digitonin at 2 times their final concentration is added to each well. A cell suspension in MEB at 0.2 - 5×106 cells/ml is made and 50 µl of the cell suspension is added to each well. The plate is incubated at 25oC for 45-90 min followed by the addition of 40 µl 4% formaldehyde in PBS. Cells are fixed for 10 min at RT, quenched by the addition of 40 µl Buffer N2, and stained by adding 20 µl of 10× staining solution containing 125-250 ng/ml cytochrome c antibody overnight at 4oC. If Triton X-100 buffers are used, the samples must be washed once prior to analysis. Tween-20, BD Perm/Wash, and saponin based buffers can proceed directly to FACS without washing. Volumes can be scaled up for larger tubes or down for 384 well plates. Once time, peptide concentration, and cell density are chosen, they must remain the same between replicates to ensure the most accurate result. PUMA2A, an inert peptide, or no peptide defines full cytochrome c retention and a lack of MOMP while 25 μM alamethicin, which causes BAX/BAK independent MOMP, serves as a complete cytochrome c release control. Gating around the cells in the PUMA2A control using SSC vs. cytochrome c provides the % cytochrome c positive cells, and 100 minus this value defines % cytochrome c loss. Alternatively, the median fluorescence intensity (MFI) of the cytochrome c stain can be used to calculate % cytochrome c loss as follows:
With the exception of dose response curves, cytochrome c loss is generally preferred to retention for clarity by having peptides that cause MOMP show a gain of signal rather than loss.
Primary humans samples De-identified platelet apheresis collars and discarded platelet units were obtained from the Kraft Family Blood Center, Dana-Farber / Bringham and Woman’s hospital, PBMCs were purified using a ficoll gradient, and were surface stained using CD3, CD19, CD20, CD14, and CD56. De-identified human OPSCC tumor from Massachusetts General Hospital was provided by Dr. James Rocco. The OPSCC tumor was dissociated with 300 U/mL collagenase 4, 100 U/ml hyaluronidase, 125 U/ml DNAse I in DMEM/F12 for 1 hour at 37oC with shaking, filtered through a 70 micron strainer, and suspended in HBSS at 107 cells/ml
11 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry followed by the addition of zombie aqua viability dye at RT for 15 minutes followed by pelleting and suspension in 2% FBS in HBSS with CD45, CD44, and EpCAM antibodies on ice for 30 minutes before pelleting and suspending of cells in MEB at 4 million cells/ml. Fifty µl of cell suspension were added to 50 µl of 2× peptides in MEB with 20 µg/ml digitonin for one hour prior to fixation.
Dynamic BH3 profiling (DBP) Prior to iBH3, cells are treated for 4-24 hours with a sub-lethal dose of one or more compounds with at least one untreated control. Death should not be apparent at the time of harvest, and dose or time should be reduced if it is observed, and multiple doses can be tested as well as combinations. After drug treatment, cells are harvested and suspended in MEB, and cells are processed using the standard iBH3 procedure above. For each peptide treatment, the cytochrome c release value of the non-drug treated cells are subtracted from the cytochrome release value of the drug-treated samples to generate the drug-induced change in priming, or delta priming. For Figure 5, cells were exposed for 24 hours for DBP and 72 hours for viability to compounds as follows: gefitinib 1 µM (EGFR inhibitor), lapatinib 1 µM (HER2.EGFR inhibitor), TAE-684 1 µM (ALK inhibitor), MK-2206 1 µM (AKt inhibitor), PLX-4032 10 µM (BRAF inhibitor), AZD-6244 1 µM (MEK Inhibitor), BEZ235 1 µM (PI3K / mTOR inhibitor)
12 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
References Aragon, J. J., J. E. Feliu, R. A. Frenkel and A. Sols (1980). Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes. Proc Natl Acad Sci USA 77, 6324-6328. Brunelle, J. K. and A. Letai (2009). Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 122, 437-441. Cassany, A. and L. Gerace (2009). Reconstitution of nuclear import in permeabilized cells. Methods Mol Biol 464, 181-205. Certo, M., V. Del Gaizo Moore, M. Nishino, G. Wei, S. Korsmeyer, S. A. Armstrong and A. Letai (2006). Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351-365. Fiskum, G., S. W. Craig, G. L. Decker and A. L. Lehninger (1980). The cytoskeleton of digitonin-treated rat hepatocytes. Proc Natl Acad Sci USA 77, 3430-3434. Foight, G. W., J. A. Ryan, S. V. Gulla, A. Letai and A. E. Keating (2014). Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells. ACS Chem Biol 9, 1962-1968. Liu, J., N. Xiao and D. B. DeFranco (1999). Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19, 403-409. Mason, K. D., Carpinelli, M.R., Fletcher, J.I., Collinge, J.E., Hilton, A.A., Ellis, S., Kelly, P.N., Ekert, P.G., Metcalf, D., Roberts, A.W. et al., (2007). Programmed anuclear cell death delimits platelet life span. Cell 128, 1173-1186. Montero, J., Sarosiek, K.A., DeAngelo, J.D., Maertens, O., Ryan, J., Ercan, D., Piao, H., Horowitz, N.S., Berkowitz, R.S., Matulonis, U. et al., (2015). Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 160, 977-989. Ni Chonghaile, T., Sarosiek, K.A., Vo, T.T., Ryan, J.A., Tammareddi, A., Moore, V. del G., Deng, J., Anderson, K.C., Richardson, P. et al., (2011). Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 334, 11291133. Ryan, J. and A. Letai (2013). BH3 profiling in whole cells by fluorimeter or FACS. Methods 61, 156-164. Ryan, J. A., J. K. Brunelle and A. Letai (2010). Heightened mitochondrial priming is the basis for apoptotic hypersensitivity of CD4+ CD8+ thymocytes. Proc Natl Acad Sci USA 107, 12895-12900. Vo, T. T., Ryan, .J, Carrasco, R., Neuberg, D., Rossi, D.J., Stone, R.M., Deangelo, D.J., Frattini, M.G. and Letai, A. (2012). Relative mitochondrial priming of myeloblasts and normal HSCs determines chemotherapeutic success in AML. Cell 151, 344-355. Zhang, H., Nimmer, P.M., Tahir, S.K., Chen, J., Fryer, R.M., Hahn, K.R., Iciek, L.A., Morgan, S.J., Nasarre, M.C., Nelson, R. et al., (2007). Bcl-2 family proteins are essential for platelet survival. Cell Death Differ 14, 943-951.
13 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Tables and figures
Figure 1
Comparison of BH3 profiling method for flow cytometry. Potentiometric JC-1
(A) and fixed cell with intracellular staining iBH3 (B).
Figure 2
Principles guiding iBH3 design (A) Schematic of cell level events during iBH3
showing loss of cytoplasm upon digitonin addition, movement of cytochrome c after MOMP, and cartoon of expected output. Color indicates the presence of protein. (B) Wild type and BAX/BAK DKO MEFs showing peptide induced MOMP requires BAX and BAK. (C) MOMP induced by BIM peptide is dependent on both exposure time and dose. (D) Mixed cell lines labeled with cell surface
14 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry markers produce distinguishable BH3 profiles upon gating. BH3-only protein names listed in B-D refer to the BH3 only peptides, not the full length or native proteins.
Figure 3
iBH3 profiles of human blood components (A) PBMC populations identified
by surface marking (B) Baseline profile of human platelets, peptide and drug concentrations in micromolar. (C) Annexin V staining of treated platelets after 24 hours showing sensitivity to compounds that bind BCL-XL.
Figure 4
Human OPSCC tumor profiles using iBH3.
(A) Gating of CD45+ and CD45– populations. (B) Subdivision of CD44 and EpCAM populations within CD45– (C). iBH3 profile of all five gated populations. From Montero et al. (2015).
15 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
iBH3: Fixable BH3 profiling for flow cytometry
Figure 5
Dynamic BH3 Profiling is a predictive biomarker.
(A) General workflow for DBP. Priming is cytochrome c release for iBH3, but can refer to depolarization in JC-1 based methods of DBP. In both cases, Δ Priming is the difference between treated and untreated populations. (B) DBP profile at 24 h and (C) Annexin V viability at 72 h for cell line PC9. (D) DBP profile at 24 hours and (E) Annexin V viability at 72 hours for SK-MEL-5. All drugs were used at 1 µM except for PLX-4032 which was used at 10 µM.
16 / 16
Brought to you by | Washington University in St. Louis Authenticated Download Date | 3/1/16 11:39 AM
