Chemical probes and methods for single-cell detection and quantification of epichaperomes in hematologic malignancies

Abstract

Detection of protein connectivity dysfunctions in biological samples, i.e., informing on how protein-protein interactions change from a normal to a disease state, is important for both biomedical research and clinical development. The epichaperome is an executor of protein connectivity dysfunction in disease, and thus a surrogate for its detection. This chapter will detail on published methods for epichaperome detection and quantification that combine the advantages of multiparameter flow cytometry with those of the PU-FITC fluorescently labeled epichaperome detection probe. It will offer a comprehensive method description that includes the synthesis and characterization of an epichaperome detection probe and of the negative control probe, the preparation of the biospecimen for epichaperome analysis, the execution of the epichaperome detection and quantification assay and lastly, the data acquisition and analysis. The method provides, at single-cell level, the functional signature of cells, differentiating itself from other single-cell methods that provide a catalog of molecules.

1. Introduction

The potential of single-cell analysis in both basic research and clinical diagnostics has led to excitement in the scientific community. Reports on both the development of appropriate methods and their implementation in the inquiry of disease have exponentially increased over the past decade. Understanding cellular heterogeneity has been a major thrust of technological development, resulting in an increasingly powerful set of instrumentation, protocols, and methods for analyzing single cells at the DNA sequence, RNA expression and protein abundance levels (Hwang, Lee, & Bang, 2018).

Given the ease of accessibility of liquid tumor biopsies, studies in hematological malignancies have been at the forefront of single-cell analysis (Brierley & Mead, 2020). Single-cell technologies, such as flow cytometry or morphology, have historically been routine laboratory diagnostics in hematology, where predefined markers or cell types are investigated for diagnostic and prognostic purposes. Advances in single-cell RNA-sequencing that measure the expression of up to 10⁴ genes simultaneously within an individual cell, have increased insights into cell state and diversity. DNA, RNA, protein, DNA methylation status and chromatin accessibility at single-cell resolution are now feasible (Stuart & Satija, 2019). Numerous technical advances have made DNA and RNA analysis routine, yet protein analysis is far more challenging (Marx, 2019). The complexity of the proteome, lack of amplification methods and of specific high-affinity probes make protein analysis technically demanding.

Shifting the application of these methodologies from description of cellular heterogeneity to a deeper understanding of disease mechanism and identification of tractable disease targets is however a challenge for these techniques. Inherently, these methods catalog sets of molecules in cells, whether DNA, RNA or protein. The functional outcome of such changes defined by how groups of proteins organize into interconnected cell-wide proteins networks remains an unresolved technical challenge.

We here describe a method that provides a functional signature of the cell instead of cataloging molecules—it does so by informing on how protein-protein interactions (PPIs) change from a normal to a disease state. The method is based on recent advances in the biology of cell stress, whereby stressors create protein connectivity dysfunctions, at the proteome-wide level, executed by a restructuring of chaperones and co-chaperones, collectively called the chaperome, into new structures, termed epichaperomes (Inda et al., 2020; Joshi et al., 2018; Kishinevsky et al., 2018; Kourtis et al., 2018; Rodina et al., 2016; Wang et al., 2019). Unlike chaperone proteins, which as their name implies, safeguard how proteins are synthesized and ensure cellular activities are coordinated properly, epichaperomes change how proteins interact with each other. It causes them to improperly organize inside cells, aberrantly affecting cellular phenotypes. The presence of epichaperomes therefore signifies improper organization of proteins in PPI networks and a pathologic phenotype. Monitoring epichaperome levels offers an indirect read-out, and is a surrogate of, proteome-wide dysfunction in the context of disease, such as in cancer and neurodegenerative disorders including Alzheimer’s and Parkinson’s (Inda et al., 2020; Joshi et al., 2018; Kishinevsky et al., 2018; Kourtis et al., 2018; Rodina et al., 2016). Epichaperome expression is also a biomarker of response to certain therapies (Inda et al., 2020; Joshi et al., 2018; Kishinevsky et al., 2018; Kourtis et al., 2018; Rodina et al., 2016). There is therefore a real need to develop streamlined protocols for the detection and quantification of epichaperomes in biological specimens and in live patients.

In this chapter we provide protocols for the preparation of an epichaperome probe and for the implementation of such probe in epichaperome detection and quantitation. Specifically, we will detail on the preparation of a fluorescein isothiocyanate (FITC)-labeled small molecule epichaperome probe, we call PU-FITC (Rodina et al., 2016). We also provide protocols for the use of PU-FITC in single-cell detection of the epichaperome in liquid tumors using multiparameter flow cytometry (MPFC). The goal of this chapter is to expand on published methods (Rodina et al., 2016; Taldone et al., 2011; Zong et al., 2015) and combine the advantages of MPFC with those of the PU-FITC fluorescently labeled epichaperome detection probe. It will offer a comprehensive method description that includes the synthesis and characterization of an epichaperome detection probe and of the negative control probe, the preparation of the biospecimen for epichaperome analysis, the execution of the epichaperome detection and quantification assay and lastly, the data acquisition and analysis.

2. Functional and biochemical characteristics of the epichaperome

2.1. The chaperome in disease

The chaperome is an assembly of chaperones and co-chaperones. While the first study to compile a list of the human chaperome was published in 2014 and reported on 147 bioinformatically predicted members, we now know that it includes over 300 members in human cells (Hadizadeh Esfahani, Sverchkova, Saez-Rodriguez, Schuppert, & Brehme, 2018). Their effects are executed through short-lived chaperome complexes and in a one-on-one, dynamic cyclic fashion, aiding protein folding, unfolding, degradation or disaggregation (Gidalevitz, Prahlad, & Morimoto, 2011). There are several major chaperone systems of the cell, such as of the heat shock protein 70 (HSP70), HSP90 and HSP60, which use the energy of ATP binding and hydrolysis to carry out their actions (Jayaraj, Hipp, & Hartl, 2020). Disease states are often associated with changes in the chaperome. Most commonly appreciated are those driven by alterations in the expression level of chaperome members. In cancer, an increase in expression may signify a surge in demand for protein folding and stabilization. Conversely, in neurodegenerative diseases, a decrease in chaperome subsets may signify a collapse in the folding capacity of the cell enabling for protein misfolding and aggregate formation (Brehme et al., 2014; Hartl, 2017).

2.2. The epichaperome in disease

Chronic stressors, of genetic, environmental or proteotoxic nature, resculpt a fraction of the chaperome from folding executors into scaffolding platforms (Joshi et al., 2018). Biochemically this is executed by increasing the interaction strength, number of interactions and the identity of interacting partners among individual chaperome proteins, to form ‘epichaperomes’. These long-lived heterooligomeric chaperome pools do not act in protein folding and degradation, but rather as multimolecular scaffolds that pathologically remodel proteome-wide, the activity of protein pathways and in turn, phenotypes (Inda et al., 2020). The chaperones HSP90 and heat shock cognate 70 (HSC70) nucleate (i.e., are at the epicenter of) these epichaperome scaffolds. It should be noted that whereas HSP90 and HSC70 are abundant proteins, averaging each 2–3% of the total protein mass, and found in all cells in the human body, the fraction of HSP90 and HSC70 incorporated into epichaperomes is in contrast minor and localized to diseased cells and tissues (Inda et al., 2020; Kishinevsky et al., 2018; Pillarsetty et al., 2019; Rodina et al., 2016). Thus, whereas chaperone proteins (i.e., HSP90, HSP70) enable individual proteins to be made and fold into a functional conformation, epichaperomes change how thousands of proteins in the cell interact with each other. It causes them to improperly organize, aberrantly affecting cellular phenotypes.

In the context of Alzheimer’s disease, epichaperome formation enables aberrant connectivity in synaptic protein pathways, and may be a surrogate for detecting synaptic dysfunction in this context (Inda et al., 2020). In cancer, MYC hyperactivation is associated with epichaperome formation (Kourtis et al., 2018; Rodina et al., 2016). In T-cell acute lymphoblastic leukemia, NOTCH, which acts as an upstream MYC activator, is an epichaperome inducer in this context. Blocking NOTCH activity, by preventing its cleavage at the cell surface with γ-secretase inhibitors, negatively modulated MYC, and in turn decreased epichaperome abundance (Kourtis et al., 2018). In leukemias, we found a direct and quantitative link between hyperactivated signaling pathways and epichaperome abundance (Zong et al., 2015). The presence of epichaperomes therefore signifies context-dependent cellular dysfunction and may be used as a surrogate for the presence of functionally aberrant cellular states.

2.3. Epichaperome abundance, a biomarker of vulnerability

In cancer, there is a positive correlation between epichaperome abundance and vulnerability of tumors to its inhibition - the more of the chaperome units in a cell are incorporated into epichaperomes, the more sensitive the cancer cell becomes to pharmacologic or genetic inhibitors of important epichaperome components (i.e., HSP110, HSP90, HOP, HSC70, AHA1) (Kourtis et al., 2018; Rodina et al., 2016; Taldone et al., 2020; Zong et al., 2015). Irrespective of tumor type, 50–60% of tumors express variable epichaperome levels and ~10–18% are high expressors indicating that a fraction of tumors may be amenable to single agent epichaperome inhibitor, highlighting the need to develop probes and assays to detect and quantify epichaperomes in tumors.

2.4. Epichaperome probes

In biomedical research, and for diagnostics, antibodies are typically used to detect the presence or the absence of a protein in biological specimens. The epichaperome is however not a single protein but rather a conglomerate of tightly bound proteins. The nature of the epichaperome, therefore, which presents as stable, multi-partner complexes affected by strong interactions as opposed to other cellular forms of chaperomes present in dynamic complexes of weak interactions, provide opportunities for its detection through small molecules (Fig. 1A). For example, chemical probes may kinetically differentiate epichaperomes from the more abundant chaperomes (Rodina et al., 2016). [¹²⁴I]-PU-H71, a radiolabeled form of the inhibitor PU-H71, is such a probe as it dissociates from HSP90 incorporated into epichaperomes much more slowly (i.e., over days) than it does from other HSP90 pools (i.e., minutes to hours); this difference in the k_off (i.e., dissociation rate constant) provides it with epichaperome selectivity. This probe is used in clinic to assay and image epichaperomes in solid tumors though positron emission tomography (PET) and monitor target engagement by inhibitor in patients’ tumors at single-lesion resolution in real time (Pillarsetty et al., 2019). Similarly, a radiolabeled form of the inhibitor PU-AD showed proof-of-principle for epichaperome formation in relevant brain regions of Alzheimer’s disease patients (Indaetal., 2020). These probes therefore provided evidence in live patients that epichaperome-mediated pathologic rewiring of protein networks is both detectable and quantifiable through properly labeled epichaperome probes.

Fig. 1.

(A) Schematic showing the concept behind epichaperome, and the use of small molecule probes to select for and detect the epichaperome over the more abundant, and ubiquitously present, chaperome. (B) Chemical structures of the epichaperome probe PU-FITC and the control molecule PU-FITC9, and the synthetic scheme used for their synthesis.

3. Epichaperome probe preparation

In the context of hematologic malignancies and for detection by flow cytometry, a fluorescently labeled probe is optimal. Among various available dyes, FITC is detected in the FL1 channel on most instruments and is a good choice for flow cytometry (excitation and emission spectrum peak wavelengths of approximately 495nm/519nm). We developed the probe PU-FITC, which we validated for its ability to detect and quantify epichaperomes via flow cytometry and can be also utilized to evaluate cells by fluorescence microscopy. PU-FITC is based on PU-H71 and incorporates the attachment of FITC to the 9-(3-(isopropylamino)propyl) moiety of PU-H71 (Fig. 1B). We also developed a negative control, the probe PU-FITC9, a molecule that retains a part of the PU-H71 scaffold but lacks activity (Fig. 1B).

3.1. Equipment

1. 10mL single neck round-bottomed flask (Chemglass Life Sciences, Catalog no. CG150682)

2. Magnetic stir bar (Fisher Scientific, Catalog no. 22-127100)

3. Rubber septum (Fisher Scientific, Catalog no. K774261-0014)

4. Magnetic stir plate (VWR, Catalog no. 97018-486)

5. Aluminum foil (Fisherbrand, Catalog no. 01-213-101)

6. Rotary evaporator (Büchi® Rotavapor® R-215 evaporator, Mfr no. Büchi, 23111V000)

7. 9mL pressure tube (Fisher Scientific, Catalog no. 50-974-638)

8. Oil bath (Fisher Scientific, Catalog no. S159-500)

9. 0.45μm nylon syringe filter (Fisher Scientific, Catalog no. 03-050-485)

10. 7mL glass vials (Fisher Scientific, Catalog no. 03-340-128)

11. Preparatory TLC plates (Fisher Scientific, Catalog no. 50-465-365)

12. Genevac (GeneVac Personal Evaporator EZ-2 Plus HCl Compatible, Model: EZ-2 Mk2)

13. HPLC (Waters Autopurification system with Waters 2996 Photodiode Array Detector, Waters 2420 Evaporative Light Scattering Detector, Waters Micromass ZQ Detector), and a reversed phase column (Waters X-Bridge C18 analytical column, 4.6×150mm, 5μm or Waters X-Bridge C18 prep column, 19×150mm, 5μm)

14. HRMS (Waters Micromass LCT Premier system)

15. NMR (Bruker Avance III UltraShield Plus 500 NMR Spectrometer & Bruker Avance III UltraShield Plus 600 NMR Spectrometer)

3.2. Chemicals and solvents

1. PU-H71

2. FITC (Sigma-Aldrich, Catalog no. F7250)

3. Et3N (Sigma-Aldrich, Catalog no. 471283)

4. DMF (Sigma-Aldrich, Catalog no. 227056)

5. 2-Methoxyethanol (Sigma-Aldrich, Catalog no. 284467)

6. K₂CO₃ (Sigma-Aldrich, Catalog no. 209619)

7. CuI (Sigma-Aldrich, Catalog no. 03140)

8. Morpholine (Sigma-Aldrich, Catalog no. 252360)

9. MeOH (Fisher Scientific, Catalog no. A454-4)

10. Chloroform-d (Fisher Scientific, Catalog no. NC9754641)

11. Methanol-d₄ (Fisher Scientific, Catalog no. NC9917462)

3.3. Probe synthesis and characterization

PU-H71 was synthesized as described previously (He et al., 2006). The epichaperome detection probe PU-FITC was prepared in a single step from the reaction of PU-H71 with FITC. The control probe PU-FITC9, a molecule that retains a part of the PU-H71 scaffold but lacks activity, was prepared in two steps from PU-H71. In the first step, the reaction of PU-H71 with excess 2-methoxyethanol in the presence of K₂CO₃, CuI, and morpholine at 100 °C for 20 h yields 1. In the second step, 1 was reacted with FITC to give PU-FITC9 (Fig. 1B).

3.3.1. Synthesis of PU-FITC

Note: PU-FITC and FITC are light sensitive and care should be taken to protect from light when handling these.

1. Add 16.7mg (0.0326mmol) of PU-H71, 14.0mg (0.0359mmol) of FITC, 0.1mL of Et₃N, 0.2mL of DMF and a magnetic stir bar to a 10mL round bottomed flask wrapped in aluminum foil and sealed with a rubber septum.

2. Stir the reaction mixture using a magnetic stir plate for 6h at room temperature.

3. Concentrate the reaction mixture under reduced pressure using a rotary evaporator to yield a residue.

4. Purify the residue by preparatory HPLC [(a) H₂O+0.1% TFA and (b) CH₃CN+0.1% TFA, 15–65% b over 20min at 20mL/min.] to give 21.2mg (72%) of PU-FITC.

Note: PU-FITC is light sensitive and should be protected from light.

1. Characterize the probe (Fig. 2). ¹H NMR (500MHz, CDCl₃) δ 8.15 (s, 1H), 7.86 (s, 1H), 7.77 (d, J = 7.9Hz, 1H), 7.34 (s, 1H), 7.09 (d, J = 7.9Hz, 1H), 7.01 (s, 1H), 6.63–6.71 (m, 4H), 6.51 (d, J = 7.3Hz, 2H), 6.02 (s, 2H), 5.53 (br s, 2H), 4.30 (br s, 2H), 3.64 (br s, 2H), 2.85 (br s, 1H), 2.27 (m, 2H), 1.23 (d, J = 6.2Hz, 6H); HRMS (ESI) m/z [M + H]⁺ calcd. for C₃₉H₃₃IN₇O₇S₂, 902.0928; found 902.0942; HPLC [(a) H₂O+0.1% TFA and (b) CH₃CN+0.1% TFA, 5 to 95% b over 13min. at 1.2mL/min.] R_t = 9.93min. (99%).

Fig. 2.

LC-MS chromatogram and MS of PU-FITC (A and B) and PU-FITC9 (C and D), respectively.

3.3.2. Synthesis of PU-FITC9

Step 1: Synthesis of 9-(3-(Isopropylamino)propyl)-8-(2-methoxyethoxy)-9H-purin-6-amine (1).

1. Add 30mg (0.0586mmol) of PU-H71,24mg (0.1757mmol) of K₂CO₃, 11 mg (0.0586mmol) of CuI, 1mL of 2-methoxyethanol, 10.2mg (10.2μL, 0.1171 mmol) of morpholine to a sealed pressure tube.

2. Heat the reaction mixture at 100 °C in an oil bath for 20h.

3. The reaction mixture was filtered through a 0.45μM nylon filter and then MeOH was passed through the filter to wash any remaining residue.

4. Concentrate the combined filtrate under reduced pressure using a rotary evaporator to yield a residue.

5. Purify the residue by preparatory TLC (CH₂Cl₂: MeOH-NH₃ (7N), 10:1) to give 17mg (94%) of 1 as a white solid.

6. Characterize the material. ¹H NMR (500 MHz, CDCl₃/MeOH-d₄) δ 8.15 (s, 1H), 4.64 (m, 2H), 4.21 (t, J = 6.5Hz, 2H), 3.83 (m, 2H), 3.44 (s, 3H), 3.35 (septet, J = 6.6Hz, 1H), 2.96 (t, J = 6.9 Hz, 2H), 2.39 (m, 2H), 1.43 (d, J = 6.6Hz, 6H); HRMS (ESI) m/z [M + H]⁺ calcd. For C₁₄H₂₅N₆O₂, 309.2039; found 309.2041.

Step 2: Synthesis of PU-FITC9.

Note: PU-FITC9 and FITC are light sensitive and care should be taken to protect from light when handling these.

1. Add 10.0mg (0.032mmol) of 1, 13.9mg (0.036mmol) of FITC (0.036mmol), 0.1mL of Et₃N, 0.3mL of DMF and a magnetic stir bar to a 10mL round bottomed flask wrapped in aluminum foil and sealed with a rubber septum.

2. Stir the reaction mixture using a magnetic stir plate for 6h at room temperature.

3. Concentrate the reaction mixture under reduced pressure using a rotary evaporator to yield a residue.

4. Purify the residue by HPLC [(a) H₂O+0.1% TFA and (b) CH₃CN + 0.1% TFA, 15–65% b over 20min at 20mL/min.] to give 18.3mg (82%) of PU-FITC9.

Note: PU-FITC9 is light sensitive and should be protected from light.

1. Characterize the probe (Fig. 2). ¹H NMR (600MHz, MeOH-d₄) δ 8.33 (s, 1H), 7.92 (s, 1H), 7.66 (dd, J = 8.1, 1.8Hz, 1H),7.15 (d, J = 8.2Hz, 1H), 6.73–6.83 (m, 4H), 6.58–6.65 (m, 2H), 4.73–4.76 (m, 2H), 4.23 (t, J = 6.5Hz, 2H), 3.81–3.85 (m, 2H), 3.74–3.81 (m, 2H), 3.41 (s, 3H), 2.28–2.37 (m, 2H), 1.30 (d, J = 6.6Hz, 6H); HRMS (ESI) m/z [M + H]⁺ calcd. for C₃₅H₃₆N₇O₇S, 698.2397; found 698.2399; HPLC [(a) H₂O+0.1% TFA and (b) CH₃CN+0.1% TFA, 5 to 95% b over 13min at 1.2mL/min.] R_t = 9.18min. (99%).

3.4. Probe stock preparation and storage for biological use

• 1PU-FITC: 1mM (1000×) in dimethyl sulfoxide (DMSO), store at −20°C (1 μM final concentration for staining). It is recommended to dispense into single use (10μL) aliquots in amber Eppendorf tubes.

1.8 Mg of PU-FITC in an amber Eppendorf tube was dissolved in 200μL of DMSO [molecular biology grade from Sigma Aldrich] and vortexed to make 10mM PU-FITC solution. This 10mM PU-FITC solution was further diluted (10-fold) with DMSO to make 2mL of 1mM PU-FITC solution and stored as small aliquots of 50μL in 40 amber Eppendorf tubes at −20 °C.

• 2PU-FITC9 (negative control): 1mM (1000×) in DMSO, store at −20°C (1μM final concentration for staining). It is recommended to dispense into single use (10μL) aliquots in amber Eppendorf tubes.

1.4Mg of PU-FITC9 in an amber Eppendorf tube was dissolved in 200μL of DMSO [molecular biology grade from Sigma Aldrich] and vortexed to make 10mM PU-FITC9 solution. This 10mM PU-FITC9 solution was further diluted (10-fold) with DMSO to make 2mL of 1mM PU-FITC9 solution and stored as small aliquots of 50μL in 40 amber Eppendorf tubes at −20°C.

4. Epichaperome detection and quantification

Multiparameter flow cytometry (MPFC) is widely used in the diagnosis of hematologic malignancies. It identifies the malignant cell type by detection of cell surface proteins to provide information on its differentiation and maturation stage (Cools & Vandenberghe, 2009). Therefore, in addition to the epichaperome detection probe, the protocols for detection of epichaperome-positive cell populations need to also incorporate the use of antibodies that detect specific cell surface markers and dyes to discriminate dead cells. While useful for hematologic malignancies in general, the protocol for epichaperome detection that we detail on below is for acute myeloid leukemia (AML) samples (Fig. 3).

Fig. 3.

Schematic showing sample preparation, epichaperome detection and data analysis. AML, acute myeloid leukemia;PB, peripheral blood;BM, bone marrow, RBC, red blood cells.

Several controls are incorporated to ensure robust and reproducible results. Among these, compensation controls are used to correct for potential emission spectral overlap (Njemini et al., 2014). Calibration and cytometer set-up and tracking beads are also included for routine quality control of the assay and the cytometers. By using these particles, the performance of the instrument and the sensitivity and linearity of the system can be determined. Finally, epichaperome-positive and -negative cells (cell lines or primary specimens) are included as positive and negative controls, respectively. For the assessment of AML samples, we use MOLM13 or MV4-11 cell lines as positive controls, and peripheral blood from healthy donors as negative controls (Rodina et al., 2016).

4.1. Equipment

1. BD™ LSR-II Flow Cytometer (BD Biosciences, Cat. No. LSRII, with BD FACSDiva™ software)

2. Countess™ II FL Automated Cell Counter (ThermoFisher, Cat. No. AMQAF1000)

3. Allegra 6KR, Refrigerated Floor Model (For 15mL and 50mL Falcon conical tubes) (Beckman Coulter, Cat. No. 366830)

4. BD Sero-Fuge™ 2002 2-Speed Centrifuge (For FACS tubes) (Fisher Scientific, Cat. No. 05-100-3)

5. VWR Scientific Standard Mini Vortexer (VWR, Cat. No. 58816-121)

6. XP Portable Pipet-aid® (Drummond Scientific Company, Cat. No. 4-000-101)

7. Eppendorf™ Research plus™ Variable Adjustable Volume Pipettes: Single-Channel (p2.5, p10, p20, p200, and p1000) (Fisher Scientific, Cat. No. 13-690-025, 13-690-026, 13-690-028, 13-690-030, and 13-690-032)

4.2. Materials

• 1Falcon^® 15mL polypropylene centrifuge tube (CORNING, Cat. No. 352196)
• 2Falcon^® 50mL polypropylene centrifuge tube (CORNING, Cat. No. 352070)
• 3Falcon^® 96-well clear round-bottom TC-treated cell culture microplate (CORNING, Cat. No. 3535077)
• 4Disposable pipette tips, TipOne RPT filter tips (p1000, p300, p20 and p10) (USA Scientific, Cat. No. 1182-1830, 1180-9850, 1180-1850, and 1180-3850)
• 5Serological Pipette (25, 10 and 5mL) (USA Scientific, Cat. No. 1072-5410, 1071-0810 and 1075-0110)
• 6Falcon® 5mL round-bottom polystyrene tube (FACS tubes) (CORNING, Cat. No. 352054)
• 7Patient sample (3cc of peripheral blood (PB) or 1 cc of bone marrow (BM) collected in an EDTA tube or a Heparin tube)
• 8DPBS (ThermoFisher Scientific, Cat. No. 14190144)
• 9Sterile distilled water (ThermoFisher Scientific, Cat. No. 15230162)
• 101× Red blood cell (RBC) lysis buffer (ammonium chloride potassium (ACK) lysing buffer)

• aPrepare 1× RBC Lysis Buffer by diluting 10× ACK in sterile distilled water.
• b
  10× ACK buffer:

  1. 1.5M ammonium chloride (MilliporeSigma, CAS number 12125-02-9, Cat. No. AX1270)
  2. 100mM potassium bicarbonate (MilliporeSigma, CAS number 298-14-6, Cat. No. 237205)
  3. 0.5M EDTA, pH 8.0 (ThermoFisher Scientific, Cat. No. 15575020)

• 114′,6-Diamidino-2-phenylindole (DAPI) 5mg/mL (ThermoFisher Scientific, Cat. No. D1306) or 7-aminoactinomycin D (7AAD) 50 μg/mL (ThermoFisher Scientific, Cat. No. A1310)
• 12Antibodies for cell surface antigens to distinguish populations of interest depending on the hematological malignancy

• cAll antibodies must be titrated and used according to protocols established to evaluate populations of interest. The number of antibodies may differ depending on the cytometer utilized.

Note: Antibodies conjugated to FITC and Alexa 488 should not be included in the surface staining panel as FITC is the fluorochrome to be evaluated for epichaperome detection.

• 13For the example listed here to evaluate AML samples using a six multicolor approach, the following panel is recommended: CD45 APC Cy7 (BioLegend, clone 2D1, Cat. No. 368516); CD3 APC (BioLegend, clone HIT3a, Cat. No. 300312); CD33 PE (BioLegend, clone P67.6, Cat. No. 366608); CD34 PE Cy5 (BD Bioscicences, clone 581, Cat. No. 555823)
• 14
  Compensation beads:

  • d
    EasyComp fluorescent particles, FITC (Spherotech, Cat.No. ECFP-F1)
  • e
    EasyComp fluorescent particles, blank (Spherotech, Cat.No. ECFP-B)
  • f
    OneComp eBeads Compensation Beads (ThermoFisher Scientific, Cat. No. 01-111-42)

• 15Calibration particles: SPHERO™ Easy calibration fluorescent particles FITC (Spherotech, Cat. No. ECFP-F1-5K)
• 16
  FACS+E buffer: DPBS/0.5%FBS/5mMEDTA (Sterile, store at 4°C)

  • g
    FBS (ThermoFisher Scientific, Cat. No. 10438026)
  • h
    EDTA (ThermoFisher Scientific, Cat. No. 15575020)

• 17Positive control: MV4-11 (ATCC, Cat. No. CRL-9591) or MOLM13 (DSMZ, Cat. No. ACC 554). Note: Cells should be cultured as per the providers’ recommended culture conditions. Cells should be authenticated using short tandem repeat profiling and tested for mycoplasma before use.
• 18Negative control: mononuclear cells (MNCs) from peripheral blood from healthy donors
• 19PU-FITC: 1mM (1000×) in DMSO, store at −20°C (1μM final concentration for staining). It is recommended to dispense into single use (10μL) aliquots in amber Eppendorf tubes. Take a single aliquot of 1mM and dilute in PBS to 20μM as working stock solution
• 20PU-FITC9 (negative control): 1mM (1000×) in DMSO, store at −20°C (1μM final concentration for staining). It is recommended to dispense into single use (10μL) aliquots in amber Eppendorf tubes. Take a single aliquot of 1mM and dilute in PBS to 20μM as working stock solution
• 21Trypan Blue Stain (0.4%) for use with the Countess™ Automated Cell Counter (ThermoFisher Scientific, Cat. No. T10282)
• 22Cytometer Setup &Tracking (CS&T) Research Beads (use with BD FACSDiva™ software v7 or later) (BD Biosciences, Cat. No. 655051)

4.3. Sample preparation

For immunophenotyping of AML samples and for epichaperome detection and quantification using MPFC, red blood cells (RBCs) are removed using RBC lysis buffer to isolate leukocytes. The RBC lysis buffer contains ammonium chloride which generates an imbalance in osmotic pressure resulting in RBC lysing, with little effect on leucocyte integrity. In this protocol we use a two-step RBC lysis procedure to ensure efficient lysis of RBCs and improve data collection and analysis. After lysing RBCs, the enriched leukocyte fraction of cells is washed with FACS buffer containing EDTA (FACS-E) to prevent cell-to-cell adhesion, followed by immunophenotyped using a panel of antibodies to identify cell surface markers that allow for the identification of populations of interest (e.g., lymphocytes and blasts cells).

4.3.1. Red blood cell (RBC) lysis

1. Transfer the peripheral (PB) or bone marrow (BM) sample into a 50mL Falcon tube and add 5 volumes of 1× ACK buffer. For example, when starting with 3mL of PB, add 15mL of 1× ACK buffer.

2. Invert the tube 10 times to mix.

3. Incubate at RT for 5min. Note that the red color should turn translucid as an indication of the RBCs getting lysed.

4. Spin down using Allegra 6KR centrifuge at room temperature (RT) at 1600 rpm for 7min.

5. Remove supernatant by aspirating carefully without disturbing the pellet.

6. Resuspend the pellet in 10mL of 1× ACK buffer.

7. Invert the tube 10 times to mix.

8. Incubate at RT for 5min.

9. Remove supernatant by aspirating carefully without disturbing the pellet.

10. Wash the pellet by adding 10mL FACS-E buffer.

11. Resuspend pellet well.

12. Spin down at RT/1600rpm for 7min.

13. Remove supernatant by aspirating carefully without disturbing the pellet

14. Repeat washing steps (10–12) two times.

15. Resuspend cell pellet in 5mL of FACS buffer.

16. Count cell numbers, excluding dead cells with trypan blue, using a cell counter such as the Countess II FL.

4.4. PU-FITC binding

For epichaperome evaluation at the single-cell level using MPFC and the PU-FITC probe is necessary to include the following controls: PU-FITC9 as control for non-specific binding, and a fluorescence minus one (FMO; which is a control sample without FITC staining). The binding assay is performed in FACS-E buffer, because utilizing culture media results in high background fluorescence. We find an incubation time of 4h to be optimal for AML cells but this may need to be adjusted for other specimens. Selection of antibodies for cell surface markers may be modified according to the cell populations to be evaluated. To minimize the possibility of spectral overlap, publicly available spectral overlap analyzers should be used for assistance in choosing the correct fluorescent label. All antibodies should be utilized according to manufacturer’s instructions and titrated for appropriate use. The assay should be performed in triplicate.

1. Place 300K to 500K of processed cells in 190μL of FACS-E buffer in 96 well plates.

   1. 3 wells for FMO control

   2. 3 wells for PU-FITC

   3. 3 wells for PU-FITC9

   Controls:

   • 30K MV4-11 cells or MOLM13 cells where 3 wells contain FMO, 3 wells PU-FITC and 3 wells FITC9. The cell line controls do not need to be stained with cell surface markers.
   • 300K to 500K MNC from PB from healthy donor cells where 3 wells contain FMO, 3 wells PU-FITC and 3 wells FITC9. These cells will be stained with cell surface markers.
   • Unstained 300K to 500K MNC from PB from healthy donor cells for adjustment of cytometer parameters, see Section 5.
   • 300K to 500K MNC from PB from healthy donor cells to be stained with cell death-exclusion dye only, such as DAPI or 7AAD (see Section 5, setup and compensation control).

2. Add 10μL of 20μM solution of PU-FITC or PU-FITC9 to each well as necessary.

3. Mix thoroughly with the pipette

4. Incubate for 4h in a tissue culture incubator at 37 °C, 5% CO₂

5. After 4h of incubation, carefully transfer the cell suspension into a FACS tube.

6. Add 1mL of FACS-E buffer to wash

7. Spin down cells at 3500rpm for 5min using a BD Sero-Fuge™ 2002.

8. Remove supernatant by aspirating carefully without disturbing the pellet

9. Repeat washing steps (6–8) two times.

10. Remove supernatant without disturbing pellet.

11. Add 100μL of mastermix of cell surface antibodies (example shown in table below for AML)

Note: Prepare the volume of mastermix for the number of samples. Be sure to make a surplus (e.g., n+1).

Cell surface marker stain			Per 100μL (per stain)	Per sample (n+1)
CD45	APC Cy7	BioLegend, clone 2D1	1	1×(n+1)
CD3	APC	BioLegend, clone HIT3a	0.5	0.5×(n+1)
CD33	PE	BioLegend, clone P67.6	0.05	0.05×(n+1)
CD34	PE Cy5	BD Biosciences, clone 581	2	2×(n+1)

1. Incubate for 15min at RT protected from light.

2. Wash with 1mL of FACS-E Buffer

3. Spin down cells at 3500 rpm for 5min

4. Remove supernatant by aspirating carefully without disturbing the pellet.

5. Resuspend in 200μL of FACS Buffer and add 200μL of 2× dead exclusion dye (For the example listed above use DAPI.)

6. Analyze by flow cytometry using a BD LSR-II Flow Cytometer.

5. Data acquisition and analysis

Before starting the acquisition of data, ensure that the cytometer is set up for optimal performance by running CS&T beads. It is recommended to check the cytometer’s configuration prior to the experiment’s design to ensure it has the appropriate photomultiplier (PMT) set-up to detect the fluorochromes selected. Use CS&T settings, and prior the running the test tubes, run the single-color controls to adjust PMTs for optimal detection of populations. Briefly, unstained PB control will be used to adjusts the voltages for forward scatter (FSC) and site scatter (SSC). Cell death-exclusion dye control will be used to adjust the PMTs. MV4-11 stained PU-FITC control will be used to adjust the PMT for FITC. For compensations, compensation beads will be included to be stained with antibody-fluorochrome conjugates used in the panel. For example, for the panel above we will use CD45-APC-Cy7, CD3-APC, CD33-PE and CD34 PE-Cy5, beads conjugated with the FITC, and cell death-exclusion dye stained PB cells. As an additional control, SPHERO™ easy calibration fluorescent particles FITC beads should be utilized in every experiment.

5.1. Preparation of compensation beads

1. Vortex beads bottle before using the beads.

2. Add 2 drops of positive beads plus 2 drops of negative beads into a 1mL FACS buffer in a FACS tube.

3. Aliquot 100μL bead mixture per antibody in a FACS tube.

4. Add antibody to bead mix.

5. Incubate for 10min at RT under dark conditions.

6. Wash beads 1× with 1mL FACS buffer.

7. Resuspend with 300μL FACS buffer.

5.2. Sample acquisition

1. PMT set-up for FITC.

• Run MV4-11 stained with PU-FITC before running compensation to determine the appropriate voltage for the FITC channel in the cytometer. Adjust the voltage such as the highest peak is between 10⁴ and 10⁵.
• If below or above, adjust the voltage of the FITC channel to have the brightest peak at 10⁵. Otherwise do not change the voltage.

• 3Run compensation tubes.
• 4In a FACS tube add one drop each from five calibration curve bead tubes in 200 μL FACS buffer.
• 5Run the FITC CALIBRATION CURVE as a sample under specimen.
• 6Run Samples, acquire at least 50k events.
• 7The acquired data by the cytometer is saved into a flow cytometry standard file (FCS) to be later analyzed using Flow Jo software.

5.3. Data analysis example

Begin analysis by plotting forward scatter-area (FSC-A) versus side scatter-area (SSC-A), and gate the population of interest (main population), excluding debris based on low SSC and FSC levels (Fig. 4A). As doublets may lead to inaccurate data analysis, select single cells using forward scatter height (FSC-H) vs. FSC-A density plot (Fig. 4B). A side scatter height (SSC-H) vs SSC-A plot can be additionally used to exclude doublets (Fig. 4C).

Fig. 4.

Data analysis example for epichaperome detection in a primary AML sample.

Gate viable cells by excluding dead cells by drawing a gate on DAPI (or 7AAD) negative cells (Fig. 4D). By plotting SSC-A vs CD45 in a density plot, gate on the leukocytes (CD45+ cells) (Fig. 4E). The example in Fig. 4 shows the next steps to gate an AML sample that has been stained with the panel listed above. Leukocytes can be further separated into lymphocytes (CD45^hi SSC^low) and blasts (CD45^dim) (Fig. 4FG). A two-parameter density blot for CD34 vs CD3 can be used to distinguish T cells, as they are not typically involved in the disease, by further gating the lymphocytes into CD3+ (Fig. 4G top). The blast gate may be further gated into CD34+ (Fig. 4G top) using the CD34 vs CD3 density plot. The FITC histogram is then generated to visualize the mean fluorescence intensity (MFI) of CD3+lymphocytes and CD34+blasts by overlaying PU-FITC9- and PU-FITC-treated samples (Fig. 4H). The MFI values should be recorded to calculate the epichaperome abundance. Epichaperome abundance is then calculated by combining the net binding ratio of blasts (PU-FITC/PU-FITC9) multiplied by the MFInet of the blasts over lymphocytes.

6. Concluding remarks

This is a relatively fast and cost-efficient protocol for epichaperome detection and quantification in biological specimens amenable for analysis by flow cytometry. Protocols outlined in this book chapter were adapted for use in patient selection and for monitoring target engagement in clinic.