Diffusion-weighted MRI for imaging cell death after cytotoxic or apoptosis-inducing therapy

Abstract

Background: Non-invasive serial imaging is desirable to detect processes such as necrotic and apoptotic cell death in cancer patients undergoing treatment. This study investigated the use of diffusion-weighted (DW-) magnetic resonance imaging (MRI) for imaging cell death induced by either a cytotoxic drug (irinotecan), or the apoptosis-inducing agent birinapant, in human tumour xenografts in vivo.

Methods: Nude mice bearing human SW620 colon carcinoma xenografts were treated with vehicle, irinotecan (50 mg kg(-1)) or birinapant (30 mg kg(-1)) for up to 5 days. DW-MRI was performed prior to and on days 1, 3 and 5 during treatment. Assessment of tumour apoptosis and necrosis ex vivo was used to validate the imaging findings.

Results: Both irinotecan and birinapant induced significant tumour growth delay. Irinotecan induced a small increase in the tumour apparent diffusion coefficient (ADC) after 1 day, with a 20 and 30% increase at days 3 and 5 respectively. ADC was unchanged in the vehicle- and birinapant-treated tumours despite a growth delay in the latter. Histological analysis showed that irinotecan increased necrosis at days 3 and 5, and induced apoptosis after 1 day, compared with vehicle. Birinapant induced apoptosis after day 3, but had no effect on tumour necrosis.

Conclusions: Tumour ADC changes after irinotecan treatment were associated with the induction of a mixture of necrotic and apoptotic cell death, whereas induction of apoptosis alone with birinapant was not sufficient to induce changes in tissue microstructure that were detectable with DW-MRI. ADC is a useful non-invasive biomarker for early detection of response to cytotoxic drugs, but false negatives may arise while detecting apoptotic response to birinapant.

Figure 1

The anti-tumour effects of birinapant or irinotecan therapy in SW620 human colon adenocarcinoma xenografts. (A) Actual tumour volume changes and (B) tumour volume changes relative to baseline (day 0) over the course of treatment show an increase in tumour volume in the vehicle-treated cohort, while both drugs (birinapant and irinotecan) induced significant tumour growth delay. (C) Changes in tumour ADC relative to baseline in vehicle- and drug-treated cohorts over 5 days. Black arrows indicate the dosing schedule. Data are mean for tumour volumes or mean of median values for ADC ±1 s.e.m., *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2

Diffusion MRI is sensitive to early irinotecan treatment in SW620 xenografts. Representative T₂-weighted anatomic MRI images overlaid with parametric ADC maps acquired prior to (day 0) and at 1, 3 and 5 days following treatment with vehicle, irinotecan or birinapant in SW620 tumours. Scale bar 5 mm. A full color version of this figure is available at British Journal of Cancer journal online.

Figure 3

The effects of irinotecan or birinapant treatment on ADC in SW620 xenografts. Frequency scatter plots show the distribution of the tumour ADC values prior to and at days 1, 3 and 5 after treatment with vehicle, irinotecan or birinapant. ADC frequency values were calculated across three MRI slices for each mouse and data are the mean ±1 s.e.m. frequency values of all mice in each cohort at each time point.

Figure 4

Histological assessment of cell death in SW620 xenografts at day 5 after treatment with vehicle, irinotecan or birinapant. (A) Composite images of H&E-stained sections indicating necrotic regions (arrows). (B) Composite images from frozen tumour sections stained with the fluorescein in situ cell death detection kit. Areas with DNA fragmentation fluoresce green. (C) Composite images from frozen sections stained with the apoptotic marker cleaved caspase-3, detected using an Alexa-546-conjugated secondary antibody that fluoresces red. Scale bar 0.5 mm. A full color version of this figure is available at British Journal of Cancer journal online.

Figure 5

Ex vivo assessment and quantification of histological markers of cell death in SW620 xenografts treated with vehicle, irinotecan or birinapant. (A) Necrotic area, (B) cleaved caspase-3 positive area, and (C) TUNEL positive area. Results are means+1 s.e.m of two sections per tumour for n=4 per time point of each treatment, except for n=3 at day 5 in the irinotecan-treated cohort. (D) Western blot from tumours harvested at day 5 after treatment showing an increase in PARP cleavage in the irinotecan-treated group. (E) Densitometry of western blots corrected by using β-actin for tumours harvested at days 1, 3 and 5 after treatment showing an increase in PARP cleavage in irinotecan- or birinapant-treated compared to vehicle-treated tumours after day 3. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.