Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus (JSRV) closely resembles lung cancer in sheep infected with JSRV

Abstract

Background: Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome.

Results: We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types.

Conclusion: Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for insertional mutagenesis, gene activation, viral replication, or expression of other viral gene products in sheep lung tumorigenesis, although these processes may play a role in other clinically less important sequelae of JSRV infection such as metastasis observed with variable frequency in sheep.

Figure 1

Mab staining of mouse and sheep lung tumors. Left panels are from a mouse exposed 2 months previously to an AAV6 vector encoding JSRV Env (ARJenv) [10], and right panels are from sheep 85RS14 (Table 1) that was experimentally infected with JSRV. Sections were stained with the Env Mab C9 and were counterstained with methyl green. Arrow in lower right panel indicates inflammatory cells that do not stain for Env expression.

Figure 2

Mab staining of JSRV-infected sheep lung tumors from around the world. Sheep numbers and countries of origin are: A, 96238 from South Africa; B, 95234 from South Africa; C, 92K3 from Kenya; D, 81R16 from Peru; E and F, 85RS1 from the USA (experimentally-infected); G, 84RS28 from the USA; and H, B-96/00 from Spain. Sections were stained with Mab B3, C9, or both. Scale bars indicate a distance of 100 μm.

Figure 3

Adenocarcinoma in a mouse 6 months after exposure to the ARJenv vector. Tissues were stained with hematoxylin and eosin. Left panel, adenoma (left) and adenocarcinoma (right). Right panel, close-up of an adenocarcinoma showing atypical nuclei.

Figure 4

Fibroblastic cell masses found in some JSRV-infected sheep. Left panels show Mab C9 staining and right panels show hematoxylin and eosin staining of sheep lung sections. Panels A and B show a round proliferative fibroblastic cell mass (myxomatous tissue) flanked at upper right and left by typical epithelial tumors from South African sheep 96238. Panels C and D show magnified views of the fibroblastic cell mass corresponding to the boxed areas in Panels A and B. Panel E shows Env staining of a large fibroblastic cell mass from naturally-infected United States sheep 85RS65. Panel F shows connective tissue containing fibroblasts at the edge of the lung from South African sheep 93141. These cells were all Env-negative (not shown).

Figure 5

Mab staining of ENTV Env. Top and middle panels show Env Mab staining of nasal adenocarcinoma from sheep 99RS39 infected with ENTV. Bottom panel shows Env Mab staining of lung tumor from mouse 5-3 exposed 4 months earlier to an AAV6 vector encoding ovine ENTV Env [12].

Figure 6

Hepatocellular tumors induced by intravenous injection of an AAV6 vector expressing JSRV Env. Panels A and B show low-magnification views of the same area of a liver stained with a mixture of the B3 and C9 Env Mab (light methyl green counterstain) (Panel A) or hematoxylin and eosin (Panel B). Panels C and D show a mixed tumor with adenomatous features in the upper right portion and adenocarcinomatous features in the lower left portion. Panel C shows staining with the Mab (hematoxylin counterstain) and Panel D shows staining with hematoxylin and eosin. Note the compression of liver tissue near the lower left side of the tumor. Panel E shows a high-magnification view of the same tumor in the panel above, with the division between adenoma and adenocarcinoma running from the top left to the bottom right of the panel. Note the linear staining between cells that likely represents Env in bile canaliculi. Panel F shows a tumor with a foamy appearance stained with the Env Mab (hematoxylin counterstain). Scale bars = 100 μm.

Figure 7

Hemangiomas and hemangiosarcomas induced by intravenous injection of the AAV6 vector expressing JSRV Env. Left panels show tumors stained with a mixture of the B3 and C9 Mab (light methyl green counterstain) and right panels show hematoxylin and eosin staining of the same areas shown in the left panels. Top panels show a large cavernous hemangiosarcoma arising in subcutaneous fat. Boxes indicate the areas shown in the middle panels (black boxes) and lower panels (white boxes). Middle panels show an area typical of hemangioma composed of cavernous blood vessels containing residual red blood cells (RBC) that are lined with a single layer of well differentiated, flattened endothelial cells. Note the intense Env Mab staining of endothelial cells but that the collagen and other cells between the vascular spaces do not stain with the Env Mab. Bottom panels show a high-magnification view of an area of hemangiosarcoma comprised of Env⁺ pleomorphic endothelial cells forming a solid cellular mass. Note examples of pleomorphic nuclei with prominent nucleoli (yellow arrows, lower right panel).