Rabbit VX2 Liver Tumor Model: A Review of Clinical, Biology, Histology, and Tumor Microenvironment Characteristics

Abstract

The rabbit VX2 is a large animal model of cancer used for decades by interventional radiologists to demonstrate the efficacy of various locoregional treatments against liver tumors. What do we know about this tumor in the new era of targeted therapy and immune-oncology? The present paper describes the current knowledge on the clinics, biology, histopathology, and tumor microenvironment of VX2 based on a literature review of 741 publications in the liver and in other organs. It reveals the resemblance with human cancer (anatomy, vascularity, angiogenic profile, drug sensitivity, immune microenvironment), the differences (etiology, growth rate, histology), and the questions still poorly explored (serum and tissue biomarkers, genomic alterations, immune checkpoint inhibitors efficacy).

Keywords: angiography; embolization; imaging; immune oncology; locoregional treatments; tumor microenvironment.

Figure 1

(A) Macroscopic aspect of the uninodular VX2 tumor into the left lobe of the liver and (B) multinodular tumors, explanted at D14 after grafting. (C) Cross section of the uninodular tumor into the left liver and (D) of the multinodular liver lobe fixed in formalin. (E) Digitized histology section of the liver left lobe bearing uninodular or multinodular tumors (F) of different sizes stained with hematein–eosin–saffron showing the tumor in liver parenchyma. T, tumor; LL, left lobe; ML, median lobe; RL, right lobe; CL, caudate lobe. From (16, 17).

Figure 2

Evolution of VX2 liver tumor size from different reports.

Figure 3

(A, B) Abdominal ultrasound examination 30 and 36 days after tumor inoculation showing abdominal peritoneal metastases and peritoneum surrounding the carcinosis (arrows). The metastases increased in size and became more invasive over time. (C, D) Cone beam CT at the thoracic level shows small pulmonary nodules (arrows) at 18 days that have increased in size one week later. pc, peritoneal carcinosis; l, liver; vc, vena cava; a, aorta. From (22).

Figure 4

(A) Angiography and (B) cone beam CT acquisitions of a VX2 tumor after 13 days of tumor development showing the main tumor feeding artery (arrow). (C) Ultrasound image in Power Doppler mode of VX2 tumor at 13 days showing vessels inside the tumor core. (D) The same examination of the same tumor at 21 days showing vessels at the periphery of the nodule. (E) The same tumor in B mode gray scale showing the tumor as a heterogenous mass with hyperechogenic (arrow heads) and hypoechogenic (stars) areas that correspond to viable and necrotic areas respectively, and hypoechogenic aspect of the tumor boundaries (arrows). (F–H) Coronal MRI view of a 21-day tumor with diffusion weight imaging (F), T1-weighted before (G) and after (H) intravenous Dotarem contrast injection. The vessels are enhanced by gadolinium injection at the tumor boundaries while the necrotic core of the tumor remains unenhanced (stars). (I) Axial slice of a cone beam CT abdominal acquisition of a rabbit showing enhanced VX2 liver tumor after intra-arterial contrast injection. (J) Maximum intensity projection of a micro-CT acquisition of a VX2 tumor injected intra-arterially with radiopaque beads. Beads are seen with high attenuation in the vessels inside and around the nodule. All figure parts are from (16, 22).

Figure 5

(A–E) Digitized histology section of a VX2 tumor in the liver at different magnification showing (A) normal liver and tumor, (B) areas of viable tumor, necrosis, cystic cavities and fibrous stroma, (C) large undifferentiated tumor cells with high nucleo-cytoplasmic ratio, eosinophilic/pale cytoplasm, and ill-defined borders. (D) Apoptotic cell debris (arrows) in the necrotic areas. (E) Compressed and normal liver in the vicinity of the tumor t, tumor; l, liver; n, necrosis; vt, viable tumor; c, cyst. From (22, 55).

Figure 6

Representative immunohistochemistry images of (A) Ki-67 labeling for proliferating cells at low magnification, high magnification, and automatic counting with ImmunoRatio plug-in for ImageJ. (B) TUNEL for apoptotic cells at low magnification, high magnification, and automatic counting with ImmunoRatio plug-in for ImageJ. (C) CD31 for endothelial cells (arrow heads), (D) hypoxia-inducible factor HIF-1α, and (E) vascular endothelial growth factor VEGF. From (7, 22).

Figure 7

Schematic representation of elements of tumor microenvironment of an untreated VX2 tumor. Viable tumor cells are found at the periphery of the nodule while hypoxic cells locate in the center. The following non-tumor cells have been found in tumor microenvironment: CD3+ T cells, tumor-associated macrophages, tumor-associated fibroblasts, and cancer stem cells. The mediators and receptors overexpressed by the tumor include markers of angiogenesis (VEGF-A, VEGF-C, EGFR, HIF-1α), immunologic checkpoints (PDL-1, CTLA-4, Gal-3, LAG-3, A2aR), extracellular matrix (fibronectin receptor, MMPs, CD147).

Figure 8

Schematic representation of elements of immune microenvironment in VX2 tumor treated by RFA with immunostimulation. RFA destroys the tumor and produces tumor-specific antigens. Areas at the periphery of the tumor may remain viable and show increased expression of pro-angiogenic factors. Adjuvant stimulation of dendritic cells with CpG promotes a tumor-specific Th1 and cytotoxic T-cell reaction, increases the number of lymphocytes infiltrating the tumor, and allows the generation of memory T cells.

Figure 9

Schematic representation of elements of the tumor microenvironment in VX2 tumor treated by TACE with/without hypoxia inhibitor. Left panel: TACE with tyrosine kinase inhibitors (TKI) induces ischemia plus inhibition of angiogenesis via blockade of TK receptor phosphorylation, leading to tumor cell death. Some areas of the tumor may remain viable under hypoxic conditions due to embolization and show overexpression of HIF-1α, markers for tumor progression and immunosuppression. Right panel: counteracting the hypoxia (with oxygen release mediated by catalase) downregulates the expression of PD-L1, allowing the activation and infiltration of CD8+ cytotoxic T cells.