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. 2014 Jan 1;74(1):56-67.
doi: 10.1158/0008-5472.CAN-13-2397. Epub 2013 Dec 5.

A Preclinical Assay for Chemosensitivity in Multiple Myeloma

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Free PMC article

A Preclinical Assay for Chemosensitivity in Multiple Myeloma

Zayar P Khin et al. Cancer Res. .
Free PMC article

Abstract

Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma, an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating three-dimensional extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used to convert experimental data into dose-exposure-response "surfaces," which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical multiple myeloma treatments, in three multiple myeloma cell lines and seven patient-derived primary multiple myeloma cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology.

Conflict of interest statement

Conflict of Interest Disclosures:

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Schematic view of microfluidic assay used for in vitro reconstruction of bone marrow
(1) Each microfluidic chip contains three chambers, each of them composed of two side reservoirs, and one center observation chamber. Myeloma and stromal cells are loaded in the observation chamber simultaneously, resuspended in collagen. Overnight, the matrix gellifies, and stromal cells adhere to the bottom of the chamber and stretch. (2) One of the side reservoirs is filled with medium with a chemotherapeutic agent (left), while the other is filled with standard growth medium (right). The diffusion of the chemotherapeutic agent from one reservoir to the other creates a stable gradient across the observation chamber. (3) The observation channel with the human MM cell line NCI-H929 and adherent bone marrow derived stromal cell line HS-5 is shown in bright field under a gradient of the necrosis-inducing peptide HYD-1. Note that MM cells on the left (higher drug concentration) have died and became dark spots, while cells on the right (lower drug concentration) are still alive. (4) A gradient of the fluorescent conjugated peptide FAM-HYD1 was established, and fluorescence quantified across the channel during 18h. Normalization and re-scaling to the minimum and maximum concentration within the observation channel confirm the linear stable gradient during the 18h-window of experiment.
Figure 2
Figure 2. Quantification of sensitivity of the human myeloma cell line NCI-H929 to the proteasome inhibitor bortezomib
The microfluidic assay described in this project generates a series of measurements corresponding to cell viability at combination of exposure time and drug concentration. These data points in turn are fit to the mathematical expression of dose response, Equation 1. (A) Sensitivity of the human myeloma cell line to the proteasome inhibitor bortezomib. (B) Goodness of fit of the mathematical model to the 1,670 data points. (C) Comparison of viability measurements at 24h between the mathematical model and a standard ATP-based bioluminescent assay, with NCI-H929 cells in suspension in media or in collagen, using a standard 96-well plate.
Figure 3
Figure 3. Intrinsic chemoresistance to melphalan
The human MM cell lines 8226/LR5, selected by continuous exposure to melphalan, and NCI-H929 were exposed for a 24h continuous stable of gradient of melphalan in the microfluidic chamber. While the cell line NCI-H929 was fit to a single population, the 8226/LR5 cell line was better fit by a two-population curve, with approximately 70% of resistant cells and 30% of sensitive. This result indicates that the loss of chemoresistance of 8226/LR5 cells in absence of melphalan might be due to heterogeneity in this population.
Figure 4
Figure 4. Effect of Cell Adhesion Mediated Drug Resistance in the MM cell line NCI-H929 treated with melphalan
(Top) The co-culture of the NCI-H929 human MM cell line with the human bone marrow derived stromal cell line HS-5 confers increased resistance to melphalan. Melphalan concentration and exposure required in order to reduce viability in 50% (kR and kT, respectively) increase from 28 to 40µM and 12 to 15h, respectively. (Bottom) Linear regression of fit and actual experimental points for both experiments.
Figure 5
Figure 5. Primary MM cells in co-culture with patient stroma are significantly more resistant to melphalan
Patient 14 is a newly diagnosed patient. MM cells were sorted (CD138+) from bone marrow aspirate, and seeded into microfluidic chamber in single (SCX, 0h) or co-culture with patient stromal cells (CoCx, 0h). Digital image analysis identifies live cells and pseudo-colors them as green. A stable linear gradient of melphalan was established across observation channel: 25µM on the left, 0µM on the right, and cells were imaged every 5 minutes for 48h. After 48h, almost all MM cells are dead in single culture (SCX, 48h), while a significant number of MM cells are still alive in co-culture with stroma (CoCx, 48h). (A) Dose response surfaces built using measurements of viability in single (SCX) and co-culture (CoCx). (B) Goodness of fit of dose response surfaces (model) and actual data points for single culture, and (C) co-culture.
Figure 6
Figure 6. Quantification of bortezomib-induced EMDR circumvention in primary MM cells
Patient 14 is a newly diagnosed patient. MM cells were sorted (CD138+) from bone marrow aspirate, and seeded into microfluidic chamber in single and co-culture with adherent stromal cells (CD138−). (A) In single culture, MM cells are significantly more sensitive than in co-culture (B). A dose-response assay with bortezomib indicated that 1nM was the highest concentration that did not cause MM cell death (C) during the 24h-period. By combining a stable gradient of melphalan, with a uniform concentration of bortezomib, the chemosensitive phenotype is restored in co-culture (D).
Figure 7
Figure 7. In vitro response of primary MM cells to bortezomib in single culture 3D collagen matrix
Patient 11 was a smoldering/standard-risk patient, and thus never previously treated with bortezomib. Patient 12 was a relapsed/standard-risk patient previously treated with bortezomib-based regimens, and high-dose melphalan followed by bone marrow transplantation. Patient 12’s bortezomib-based induction regimen (bortezomib/lenalidomide/dexamethasone) occurred 3 years prior to the biopsy used for this in vitro assay. Patient 13 was a newly diagnosed/high-risk patient, while patient 17 was a smoldering myeloma patient.

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