Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity

Abstract

Conventional anticancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter antitumor drug activity. To address this limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (for example, myeloma, leukemia and solid tumors) stably expressing luciferase are cultured with nonmalignant accessory cells (for example, stromal cells) for selective quantification of tumor cell viability, in presence versus absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib resistance in leukemic cells. A stroma-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-kappaB, HIF-1alpha, myc, hTERT and IRF4; for biological aggressiveness; and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, shows more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anticancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stroma interactions.

FIGURE 1. Stromal cells modify the response of diverse tumor cell types to various agents

(a) CS-BLI based viability measurement was performed on Luc⁺ tumor cell lines plated in the presence and absence of HS-5 BMSCs (10,000 per well) at increasing numbers of tumor cells. (b) Tumor cells were plated in the presence or absence of BMSCs and treated with increasing doses of drug. The log₁₀ EC50 (+/− 95% Confidence Intervals of log₁₀ of EC50) of each drug and cell line in the presence or absence of BMSCs is displayed. Experiments with increased EC50 value in the presence of stromal cells are shown in red (stroma-induced drug resistance) and those with similar EC50 value in the presence vs. absence of stromal cells are shown in black (full dose-response curves are shown in Supplemental Figs 2–4), (c). Viability of MM.1S-GFP/luc cells treated with Doxo in the presence or absence of different BMSC lines was measured by CS-BLI. (d) Viability of MM.1S-GFP/luc cells treated with various anti-MM agents in the presences or absence of primary BMSCs from individuals with MM were analyzed (full response curves shown in Supplementary Fig 5).

FIGURE 2. Effect of blocking IL-6 and IL-6 Receptor on MM cell co-cultures with BMSCs

Viability of MM.1S-GFP/luc cells treated with Doxo for 48 h, in the presence or absence of HS-5 stromal cells, in the presence or absence of (a) IL-6 blocking antibody or (b) IL-6 Receptor blocking Ab was measured by CS-BLI. Values are normalized to each respective Doxo-free control. There is a significant difference in MM cell survival in the presence of stroma which is attenuated by IL-6 blocking antibody (2-way ANOVA; Drug P<0.0001; Ab P<0.0001; Interaction P<0.0001) and by the IL-6R blocking antibody (2-way ANOVA; Drug P<0.0001; Ab P<0.0001; Interaction P<0.0001).

FIGURE 3. Mechanisms of drug sensitivity modulation in the context of tumor-stromal interactions

GFP⁺ tumor cell lines were FACS-sorted following culture in the presence and absence of GFP⁻ HS-5 stromal cells for 24 h. RNA was isolated and cDNA generated for analysis on U133 2.0 Plus Affymetrix chips. (a) We compared select gene signatures in stroma-responsive MM cells in the presence vs. absence of stromal cells. (b) Comparison of average absolute signal (+/− SEM) in the stromal responsive cells lines in the presence vs. absence of stromal cells for NF-κB, Ras, and Akt transcriptional signatures (P<0.05). (c) A transcriptional signature of genes induced in MM cells by their interaction with stromal cells was used to classify Bortezomib- or Dex-treated patients with relapsed and relapsed/refractory MM in the randomized phase III APEX trial as having high vs. low expression of stroma-responsive genes. Patients with high expression of stroma-responsive genes had significantly shorter overall survival compared to those with low expression levels in the Dex arm (P=0.017, log-rank test). (e) In contrast, no significant difference was observed in the Bortezomib treatment arm between patients with high vs low transcriptional signature of stroma responsive genes (P=0.159, log-rank test).

Figure 4. Enhanced activity of reversine in the presence of stromal cells

(a) A library of ~1,000 small molecule inhibitors was screened for activity against MM.1S-GFP/Luc cells in the presence vs. absence of BMSCs. MM viability is shown for each compound (average ± SEM) in absence (black) and presence of stroma (red) and ranked along the X-axis in descending order of anti-MM activity in the absence of stroma. (b) MM.1S-GFP/luc viability was evaluated following reversine treatment in the presence and absence of HS-5 BMSCs by CS-BLI. (c–e) We compared the in vivo activity of reversine against MM.1S-GFP/luc cells in a diffuse bone lesion model vs. subcutaneous model in which tumor cells do not interact with BMSCs. Mice were inoculated with MM.1S-GFP/luc cells i.v. (c, e) or subcutaneously (d, f). Tumor burden was assessed weekly (IVIS imaging) and following engraftment mice were treated with reversine (1 mg kg⁻¹) or vehicle control. Average tumor burden is plotted on logarithmic scale (log₁₀ of average ± log₁₀ of SEM) for the i.v. (c) or subcutaneous (d) model. Mouse tumor burden was significantly reduced in the i.v. model in treated mice at day 27, 33 and 41 (Mann-Whitney 2-tailed test; Day 27 P=0.008, Day 33 P=0.008, Day 41 P=0.032), but was not significantly altered in the subcutaneous model at any time point. (e, f). Tumor burden for each mouse is also plotted for the i.v. (e) and s.c. (f) model. All mice eventually died of tumor burden, indicating that tumor engraftment occurred in all mice.