The first study on therapeutic efficacies of a vascular disrupting agent CA4P among primary hepatocellular carcinomas with a full spectrum of differentiation and vascularity: Correlation of MRI‐microangiography‐histopathology in rats

Abstract

To better inform the next clinical trials of vascular disrupting agent combretastatin-A4-phosphate (CA4P) in patients with hepatic malignancies, this preclinical study aimed at evaluating CA4P therapeutic efficacy in rats with primary hepatocellular carcinomas (HCCs) of a full spectrum of differentiation and vascularity by magnetic resonance imaging (MRI), microangiography and histopathology. Ninety-six HCCs were raised in 25 rats by diethylnitrosamine gavage. Tumor growth was monitored by T2-/T1-weighted-MRI (T2WI, T1WI) using a 3.0 T scanner. Early vascular response and later intratumoral necrosis were detected by dynamic-contrast-enhanced (DCE) MRI and diffusion-weighted-imaging (DWI) before, 1 and 12 hr after CA4P iv-administration. In vivo MRI-findings were validated by postmortem-techniques. Multi-parametric MRI revealed rapid CA4P-induced tumor vascular shutdown within 1 hr, followed by variable intratumoral necrosis at 12 hr. Tumor volumes decreased by 10% at 1 hr (p < 0.05), but resumed at 12 hr. Correlations of semi-quantitative DCE parameter initial-area-under-the-gadolinium-curve (IAUGC30) with histopathology proved partial vascular closure and compensational reopening (p < 0.05). The higher grades of vascularity prevented those residual tumor tissues from CA4P-caused ischemic necrosis. By histopathology using a 4-scale cellular-differentiation criteria and a 4-grade tumor-vascularity classification, percentage of CA4P-induced necrosis negatively correlated with HCC differentiation (r = -0.404, p < 0.001) and tumor vascularity (r = -0.370, p < 0.001). Ordinal-logistic-regression helped to predict early tumor responses to CA4P in terms of tumoral differentiation and vascularity. Our study demonstrated that CA4P could induce vascular shutdown in primary HCCs within 1 hr, resulting in various degrees of tumor necrosis at 12 hr. MRI as a real-time imaging biomarker may help to define tumor vascularity and differentiation and further to predict CA4P therapeutic outcomes.

Predicting Clinical Efficacy of Vascular Disrupting Agents in Rodent Models of Primary and Secondary Liver Cancers: An Overview with Imaging-Histopathology Correlation

“…Compared with models with intrahepatically transplanted tumor, the model of primary liver cancer generated by administration of chemical carcinogens [43] is time consuming but more fruitful. More importantly, such a hepatic carcinogenetic model is of high clinical value for mimicking human primary liver cancers that are featured by heterogeneous tumor angiogenesis, diverse histological types, various degrees of cellular differentiation and different tumor progression stages [24,25,35,36,44]. By using a complete carcinogen diethylnitrosamine (DENA) in rats, multifocal hepatocellular carcinomas (HCCs) in a full spectrum of tumor vascularity and cellular differentiation superimposed on various degrees of liver cirrhosis could be induced [35,36,43].…”

“…In our studies, a modified DENA gavage feeding protocol was developed [25,26] (Figure 1). Briefly, DENA gavage was administrated daily in rats under instant gas anesthesia at 5-10 mg/kg/day via a prolonged flexible plastic esophagogastric tube (Fuchigami Kikai, Kyoto, Japan) [43] for eight to fourteen weeks ( Figure 1H).…”

“…Briefly, DENA gavage was administrated daily in rats under instant gas anesthesia at 5-10 mg/kg/day via a prolonged flexible plastic esophagogastric tube (Fuchigami Kikai, Kyoto, Japan) [43] for eight to fourteen weeks ( Figure 1H). The gavage tube was customized into this specific length of 16 cm for the purpose of precisely passing the cardia and, therefore, securely preventing liquid backflow and choking when rats were under gas anesthesia [25,26] ( Figure 1I). This length fits both smaller rats like Wistar albino Glaxo/Rijswijk (WAG/Rij) strain weighting around 200 g and larger strains such as Wistar and Sprague Dawley (SD) strains weighting ≥ 400 g. Next, by monitoring tumor growth weekly with MRI, liver nodules became recognizable on T2-weighted imaging (T2WI) after seven to twenty-three weeks, and were ready for CA4P therapy after 12-31 weeks until the maximum tumor diameter in each rat reached around 1.0 cm ( Figure 1J).…”

“…Chemically induced primary liver cancer model in rodents could histopathologically mimic human liver cancers at many aspects [25,26,34,35]. Increased clinical values from preclinical studies may also be realized through utilizing the similarities between animal models and cancer patients.…”

“…To closely mimic the diverse histopathological features of hepatocellular carcinomas (HCCs) in human patients [23] who may subject to VDAs treatment, serial studies on CA4P have been conducted on the basis of chemically induced primary HCCs in rodents [24][25][26][27]. Notably, heterogeneous responses to CA4P were discovered in relation to tumor size [24], vascular geography [25,26], and cellular differentiation [24][25][26][27], which distinctly differed from the uniform pronounced tumoricidal effect in transplanted tumor models with massive central tumor necrosis embraced by a peripheral viable rim [4,15,[28][29][30][31]. Therefore, this calls for noninvasive and effective evaluation methods both for proper HCC patient selection before CA4P administration, and for real-time monitoring of the drug efficacy featuring vascular shutdown in minutes to hours and tumor necrosis in hours to overnight after VDA treatment.…”