Orthotopic xenografts of RCC retain histological, immunophenotypic and genetic features of tumours in patients

Abstract

Renal cell carcinoma (RCC) is an aggressive malignancy with limited responsiveness to existing treatments. In vivo models of human cancer, including RCC, are critical for developing more effective therapies. Unfortunately, current RCC models do not accurately represent relevant properties of the human disease. The goal of this study was to develop clinically relevant animal models of RCC for preclinical investigations. We transplanted intact human tumour tissue fragments orthotopically in immunodeficient mice. The xenografts were validated by comparing the morphological, phenotypic and genetic characteristics of the kidney tumour tissues before and after implantation. Twenty kidney tumours were transplanted into mice. Successful tumour growth was detected in 19 cases (95%). The histopathological and immunophenotypic features of the xenografts and those of the original tumours largely overlapped in all cases. Evaluation of genetic alterations in a subset of 10 cases demonstrated that the grafts largely retained the genetic features of the pre-implantation RCC tissues. Indeed, primary tumours and corresponding grafts displayed identical VHL mutations. Moreover, an identical pattern of DNA copy amplification or loss was observed in 6/10 cases (60%). In summary, orthotopic engrafting of RCC tissue fragments can be successfully used to generate animal models that closely resemble RCC in patients. These models will be invaluable for in vivo preclinical drug testing and for deeper understanding of kidney carcinogenesis. The raw data of the SNP array analysis has been submitted to the GEO database (Accession No. GSE29062).

Figure 1. Macroscopic and histological features of RCC grafts

A. Representative gross images of orthotopic RCC grafts (X200 magnification). Examples of three cRCC cases (a, case # 5; c, case # 6; d, case # 3) and one pRCC case (b, case # 11) are shown. Blue arrows indicate the engrafted tumor; yellow arrowheads indicate murine kidney; black arrowheads indicate murine adrenal gland; and the black arrow indicates the uterus of the mouse host. B. Representative histological images of H&E stained slides of primary tumors (upper panels, X200 magnification) and corresponding grafts (lower panels, X200 magnification) are shown: (e, i) FNG 2 cRCC (case # 2); (f, j) FNG 3 cRCC (case # 6); (g, k) FNG 3 pRCC (case # 11); (h, l) FNG 4 cRCC (case # 3). Black arrows indicate the mouse kidney.

Figure 2. Immunophenotypic characteristics of RCC grafts

A. Representative microscopic images of primary cRCC (a, b, c, d) and matched orthotopic xenograft (e, f, g, h) tissues immunostained for various markers (X600 magnification). Ki67 (a and e, case # 2), CAIX (b and f, case # 2), CD10 (c and g, case # 12), and VEGF (d and h, case # 6) stains are shown. B. Examples of species specific labeling of CD34-positive endothelial cells in two representative xenograft cases (X200 magnification). Murine (i) but not residual human (j) endothelial cells are detected in a FNG 4 cRCC graft grown in the host for 2 months (case # 19). Both murine (k) and human (l) endothelial cells are present in a FNG 3 cRCC graft grown in the host for 8 months (case # 9). Black arrows indicate the kidney of the mouse host.

Figure 3. Copy-number profiles of primary RCCs and corresponding xenografts

Amplifications (red) and deletions (blue), determined by normalized signal intensities from 250K SNP arrays are displayed across the genome (chromosomes are indicated along the y axis) for 10 primary RCC cases (T) and the matched xenografts (X). All cases, with the exception of a papillary RCC case (T11/X11), display low signal intensities at chromosome 3p, reflecting copy loss in that region.

Figure 4. Metastatic spread of ccRCC graft to the host lungs

Microscopic image of metastatic nodules detected in the lung (a) and liver (b) of a mouse that had been carrying an orthotopic cRCC graft (case # 3) for a period of 2.5 months (magnification X200 in the right and left panel, and X400 in the insets). The back arrow indicates the collapsed lung parenchyma surrounding the neoplastic nodule, and yellow arrow indicates the mouse liver.