Early Detection of Treatment-Induced Mitotic Arrest Using Temporal Diffusion Magnetic Resonance Spectroscopy

Abstract

Purpose: A novel quantitative magnetic resonance imaging (MRI) method, namely, temporal diffusion spectroscopy (TDS), was used to detect the response of tumor cells (notably, mitotic arrest) to a specific antimitotic treatment (Nab-paclitaxel) in culture and human ovarian xenografts and evaluated as an early imaging biomarker of tumor responsiveness.

Methods: TDS measures a series of apparent diffusion coefficients (ADCs) of tissue water over a range of effective diffusion times, which may correspond to diffusion distances ranging from subcellular to cellular levels (~3-20 μm). By fitting the measured ADC data to a tissue model, parameters reflecting structural properties such as restriction size in solid tumors can be extracted. Two types of human ovarian cell lines (OVCAR-8 as a responder to Nab-paclitaxel and NCI/ADR-RES as a resistant type) were treated with either vehicle (PBS) or Nab-paclitaxel, and treatment responses of both in vitro and in vivo cases were investigated using TDS.

Results: Acute cell size increases induced by Nab-paclitaxel in responding tumors were confirmed by flow cytometry and light microscopy in cell culture. Nab-paclitaxel-induced mitotic arrest in treated tumors/cells was quantified histologically by measuring the mitotic index in vivo using a mitosis-specific marker (anti-phosphohistone H3). Changes in the fitted restriction size, one of the parameters obtained from TDS, were able to detect and quantify increases in tumor cell sizes. All the MR results had a high degree of consistency with other flow, microscopy, and histological data. Moreover, with an appropriate analysis, the Nab-paclitaxel-responsive tumors in vivo could be easily distinguished from all the other vehicle-treated and Nab-paclitaxel-resistant tumors.

Conclusion: TDS detects increases in cell sizes associated with antimitotic-therapy-induced mitotic arrest in solid tumors in vivo which occur before changes in tissue cellularity or conventional diffusion MRI metrics. By quantifying changes in cell size, TDS has the potential to improve the specificity of MRI methods in the evaluation of therapeutic response and enable a mechanistic understanding of therapy-induced changes in tumors.

Figure 1

Cell size changes measured by flow cytometry and light microscopy and cell cycle analysis. (A) Box-and-whisker plots of the FSC-W for OVCAR-8 and NCI/ADR-RES treated with various concentrations of Nab-paclitaxel (0, 100, and 500 nM). The sample size of each cohort is 6100. (B) Box-and-whisker plots of the cell size measured by light microscopy for OVCAR-8 and NCI/ADR-RES treated with various concentrations of Nab-paclitaxel (0, 100, and 500 nM). The sample size of each cohort is 620. For all the box-and-whisker plots, the 25th to 75th percentiles are blocked by the boxp; the black and red bands inside the box are the median and mean, respectively; and the whiskers mark the SD. ****P < .0001 as measured by one-way ANOVA with an FDR posttest. (C) Histograms of the emission light intensity of PI-labeled NCI/ADR-RES and OVCAR-8 cells treated with Nab-paclitaxel at 0 (black line), 100 (blue line), and 500 nM (red line).

Figure 2

Cellularity (A), percentages of mitotic cells (B), caspase-3–activated area (C), and percentages of Ki-67–positive cells (D) for Nab-paclitaxel–/PBS-treated OVCAR-8 and NCI/ADR-RES tumors. *P < .05 as measured by one-way ANOVA with an FDR posttest.

Figure 3

Fitted d (A), D_inf (B), D₀ (C), and PGSE-derived ADC (D) for OVCAR-8 and NCI/ADR-RES cell pellets treated with Nab-paclitaxel at 0, 100, and 500 nM. *P < .05 as measured by one-way ANOVA with an FDR posttest.

Figure 4

(A) ADC maps of a representative slice through tumor and a water phantom at frequencies ranging from 0 to 350 Hz, overlaid on T2-weighted MR images. The water phantom was placed beneath the mouse to monitor the accuracy and consistency of ADC measurements. (B) Representative ADC values of an OVCAR-8 tumor at day 0 (nontreated) and day 4 (Nab-paclitaxel–treated) obtained using TDS and conventional PGSE acquisitions at frequencies ranging from 0 to 350 Hz. Solid lines represent the fits using Eqs. (1), (2), (3). The fitted parameters for nontreated (blue) and Nab-paclitaxel–treated (red) OVCAR-8 are d = 5.92 μm, D_inf = 1.58 μm²/ms, and D₀ = 0.47 μm²/ms and d = 9.22 μm, D_inf = 1.36 μm²/ms, and D₀ = 0.37 μm²/ms, respectively. It is noted that the ADC measured using PGSE with a diffusion time of 48 milliseconds is plotted as the ADC at a corresponding frequency of 5.2 Hz.

Figure 5

Percentage changes in d (A), D_inf (B), D₀ (C), and PGSE-derived ADC (D) for OVCAR-8 and NCI/ADR-RES tumors receiving a 4-day treatment of either Nab-paclitaxel or PBS at a dose of 15 mg/kg.

Figure 6

Scatter plot of the percentages of change in two spectral parameters (restriction size d and D₀) for all the treated tumors.