Monitoring Cell Death in Regorafenib-Treated Experimental Colon Carcinomas Using Annexin-Based Optical Fluorescence Imaging Validated by Perfusion MRI

Abstract

Objective: To investigate annexin-based optical fluorescence imaging (OI) for monitoring regorafenib-induced early cell death in experimental colon carcinomas in rats, validated by perfusion MRI and multiparametric immunohistochemistry.

Materials and methods: Subcutaneous human colon carcinomas (HT-29) in athymic rats (n = 16) were imaged before and after a one-week therapy with regorafenib (n = 8) or placebo (n = 8) using annexin-based OI and perfusion MRI at 3 Tesla. Optical signal-to-noise ratio (SNR) and MRI tumor perfusion parameters (plasma flow PF, mL/100mL/min; plasma volume PV, %) were assessed. On day 7, tumors underwent immunohistochemical analysis for tumor cell apoptosis (TUNEL), proliferation (Ki-67), and microvascular density (CD31).

Results: Apoptosis-targeted OI demonstrated a tumor-specific probe accumulation with a significant increase of tumor SNR under therapy (mean Δ +7.78±2.95, control: -0.80±2.48, p = 0.021). MRI detected a significant reduction of tumor perfusion in the therapy group (mean ΔPF -8.17±2.32 mL/100 mL/min, control -0.11±3.36 mL/100 mL/min, p = 0.036). Immunohistochemistry showed significantly more apoptosis (TUNEL; 11392±1486 vs. 2921±334, p = 0.001), significantly less proliferation (Ki-67; 1754±184 vs. 2883±323, p = 0.012), and significantly lower microvascular density (CD31; 107±10 vs. 182±22, p = 0.006) in the therapy group.

Conclusions: Annexin-based OI allowed for the non-invasive monitoring of regorafenib-induced early cell death in experimental colon carcinomas, validated by perfusion MRI and multiparametric immunohistochemistry.

Fig 1. Cell death-related OI signal before and after treatment.

Fusion of color-coded fluorescence and anatomic white-light image of one animal before (A) and after (B) a one-week regorafenib monotherapy. Note the significant increase in cell death-related signal under therapy, predominantly in the center of the tumor. High signal intensity is also observed in the kidneys and the urinary bladder due to renal probe elimination. OI signal intensity (SI) is displayed in arbitrary units (a.u.).

Fig 2. Ex vivo OI analysis of explanted main organs.

Fusion of color-coded fluorescence and anatomic white-light image. In accordance with the in vivo results, highest OI signal is observed in the kidneys (1) and the tumor (2) (red arrows). As a blood pool organ, the spleen (3) demonstrates relatively high probe accumulation. Only subtle OI signal is detected in the gut (4), the skin (5), and the thigh muscle (6). OI signal intensity (SI) is displayed in arbitrary units (a.u.).

Fig 3. Development of individual OI and MR perfusion values.

Line graphs depicting development of individual optical fluorescence imaging (OI, SNR) and plasma flow (PF, mL/100 mL/min) values between baseline and follow-up. Note the unidirectional increase of OI SNR (A) and the unidirectional suppression of PF (C) under therapy. On the contrary, omnidirectional development of individual OI SNR (B) and PF (D) was observed in the control group.

Fig 4. Decrease of tumor perfusion following anti-angiogenic therapy.

Tumor perfusion (Plasma Flow, PF) before (A) and after (B) a one-week regorafenib treatment of a subcutaneous colon carcinoma xenografts over the left abdominal flank. Note the significantly reduced plasma flow after therapy (B vs. A).

Fig 5. Representative immunohistochemical stainings.

Tumor cell apoptosis (TUNEL, A and B), tumor microvascular density (CD31, C and D), and tumor cell proliferation (Ki-67, E and F) in the control (left column; A, C, and E) and the therapy group (right column; B, D, and F). Note the significantly higher apoptosis rate (B vs. A) as well as the significantly lower microvascular density (D vs. C) and proliferation (F vs. E) in the therapy group.