Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects

Abstract

Early imaging or blood biomarkers of tumor response is needed to customize anti-tumor therapy on an individual basis. This study evaluates the sensitivity and relevance of five potential MRI biomarkers. Sixty nude rats were implanted with human glioma cells (U-87 MG) and randomized into three groups: one group received an anti-angiogenic treatment (Sorafenib), a second a cytotoxic drug [1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU (Carmustine)] and a third no treatment. The tumor volume, apparent diffusion coefficient (ADC) of water, blood volume fraction (BVf), microvessel diameter (vessel size index, VSI) and vessel wall integrity (contrast enhancement, CE) were monitored before and during treatment. Sorafenib reduced tumor CE as early as 1 day after treatment onset. By 4 days after treatment onset, tumor BVf was reduced and tumor VSI was increased. By 14 days after treatment onset, ADC was increased and the tumor growth rate was reduced. With BCNU, ADC was increased and the tumor growth rate was reduced 14 days after treatment onset. Thus, the estimated MRI parameters were sensitive to treatment at different times after treatment onset and in a treatment-dependent manner. This study suggests that multiparametric MR monitoring could allow the assessment of new anti-tumor drugs and the optimization of combined therapies.

Figure 1

a) Experimental protocol. At D0 60 rats were orthotopically implanted with 10⁵ U-87 MG glioma cells. Eleven days later (D11) animals were randomized and stratified into 3 groups (n=20 per group) with similar mean tumor volume (using anatomical images). BCNU group received two intravenous injections of BCNU the first and the 13^th day after treatment onset (D14_(T0) and D27_(T13)). SORA group received daily oral administration of Sorafenib between the treatment onset and the 14^th day after (D14_(T0) to D28_(T14)). Untreated group received no treatment. For each group, 4 rats, called “longitudinal” subgroup were imaged at each time point (D13_(T-1), D15_(T1), D18_(T4) and D28_(T14)). After the last MRI measurement, animals were euthanized for subsequent ex vivo experiments. Per group, the sixteen remaining rats, called “single-time” subgroup, were imaged once and then euthanized at the end of imaging session to enable ex-vivo studies (4 animals/time point). b) Diagram of the MRI experiments for tumor follow-up (the main experiment). c) Diagram of the control MRI experiment designed to evaluate the USPIO extravasations (realized on an additional set of animal).

Figure 2

a) Percent of animal survival as function of time. Open squares show the survival time of untreated rats. Cross and open circles represent the animals treated by BCNU and Sorafenib, respectively. (n = 6 per group). Increased lifespan was presented for BCNU and SORA groups (%). b) Tumor volumes for untreated, BCNU and SORA groups assessed by MRI as function of time. Mean ± SD. p<0.001: ***, untreated versus SORA group; p<0.001: $$$; untreated versus BCNU group. p<0.001: ¥¥¥; SORA versus BCNU group.

Figure 3

In vivo MRI estimated parameters. (a) Evolution of ADC, (b) microvessel diameter (VSI), (c) blood volume fraction (BVf) and (d) vessel permeability (CE-MRI) as function of time across each group. A representative map and the averaged value for group analysis are shown for each parameter and for each group at each time point. Dotted lines on MRI maps represent tumor ROI determined from anatomical images. Mean ± SD. p<0.05: *, p<0.01: **, p<0.001: ***, untreated versus SORA group; p<0.001: $$$; untreated versus BCNU group.

Figure 4

Ex-vivo study. a) Histological and immunohistological images at D28_(T14). Example of hematoxylin/eosine (HE) staining representative of contralateral and intratumoral tissue in the untreated, BCNU and SORA groups. Black arrows point to the hypostained intratumoral edema. Collagen IV and RecA stainings representative of contralateral striatum and of tumor region for each group. White arrows point to intratumoral vessel only stained by collagen IV in SORA group. The cells density (cells/mm²) and the percentage of vessels doubly stained have been added to the HE and to the merged images respectively. Scale bar = 100 μm. b–c–d) Quantitative analysis of the collagen IV staining. Evolution of (b) vessel diameter, (c) vascular area and (d) vessels density as function of time across each group. MeanSD. *: P<0.05; **:p<0.01