Phenotypic and functional characterization of a bortezomib-resistant multiple myeloma cell line by flow and mass cytometry

Abstract

Multiple myeloma (MM) is an incurable malignant plasma cell neoplasm. Proteasome inhibitors including Bortezomib (Bz) are used to treat MM, and treatment failure due to drug resistance occurs. Bz-sensitive and -resistant MM cells have distinct immunophenotypic signatures that correlate with clinical outcome. These changes can be identified by fluorescence-based cytometry (FBC), however, FBC is rarely used in predicting Bz resistance. Mass cytometry (MC) is a recently developed variation of flow cytometry that detects heavy metal-ion tagged antibodies using time-of-flight mass spectrometry allowing for detection of up to 38 epitopes simultaneously in a single cell, without significant overlap, exceeding the dimensionality of FBC 3-4-fold. Here, we compared FBC and MC in the immunophenotypic characterization of Bz-sensitive and -resistant human MM cell line U266. We show that Bz-resistant cells are associated with the loss of CD56 and CD66a adhesion molecules as well as an activation signature.

Keywords: Multiple myeloma; bortezomib; cytometry; drug resistance.

Figure 1. Scatter plots of multicolor flow cytometry (FBC) and mass cytometry (MC)

Panels A and C depict FBC, and panes B and D depict MC. In alternating rows we compared parental (P) with bortezomib/Velcade resistant (VR) cell lines. A) By FBC, both the P and VR cell lines have similar scatter characteristics. Viable cells are included by Boolean gating based on increased forward scatter (FSC) and low side scatter (SSC). Doublet discrimination is sequentially performed by including singlets based on FSC and SSC height (H) versus width (W) plots. B) In MC, live singlets are identified by DNA content (based on iridium intercalation, Ir193/195), and live cells are identified by cisplatin (PT195) exclusion. C–D) Cell surface staining characteristics of multiple myeloma markers by C) FBC and D) MC.

Figure 2. MC analysis reveals multidimensional corrections of cell surface markers that differ between parental and Bz resistant cells

Cell lines were stained with cell surface markers, intracellular signaling intermediates, and functional markers (including phosphor-specific antibodies) and analyzed by MC. The fold change in the expression levels between parental and Bz resistant populations of the tested antibodies was calculated from the signal intensity and plotted as a heatmap (Figure 2A) and scatter plots (Figure 2B). We gated on both the CD56hi and CD56lo (Figure 2C) populations in the parental and Bz resistant cells and asked whether particular markers associate with CD56 surface expression; each marker depicted in individual histograms (Figure 2C).

Figure 3. SPADE plots reveal immunophenotypic and functional discrepancies between parental (P) and bortezomib-resistant (Bz) multiple myeloma cells

Parental and Bz-resistant multiple myeloma cells were stained with a panel of 14 cell surface antibodies and 4 functional epitopes and measured by mass cytomtery. SPADE analysis was performed on this data set by clustering on the 14 cell surface markers. Rainbow color scale reflects expression values (arcsinh difference) of each epitope. The smallest node represents 7 cells. The largest node represents 708 cells.

Figure 4. viSNE analysis identifies a subpopulation of Bz resistant cells with correlates to a subpopulation in parental cells

Parental and Bz-resistant multiple myeloma cells were stained with a panel of 14 cell surface antibodies and 4 functional epitopes and measured by mass cytometry. viSNE analysis was performed on these data by using all epitopes that were measured. The same number of cells from each group were sampled for creation of the viSNE plot. Rainbow color scale reflects expression values (arcsinh difference) of each epitope. Each dot represents a single measured cell. The labels at the top of each plot indicate the epitope that is displayed.