Prerequisites for effective adenovirus mediated gene therapy of colorectal liver metastases in the rat using an intracellular neutralizing antibody fragment to p21-Ras

Abstract

Ras mutations are present in 40-50% of colorectal cancers. Inactivating this oncogene may therefore reduce proliferation capacity. In order to target ras we studied the transduction efficacy and anti tumour activity of an adenoviral vector expressing an intracellular, neutralizing single chain antibody to p21-ras (Y28). In in vitro studies transfection levels of the K-ras mutated rat colon carcinoma cell line CC531 were studied using the LacZ marker gene. In our in vivo liver metastases model different routes of administration were evaluated to determine which regimen resulted in the best transfection levels and tumour responses: intravenous injection, intratumoural injection, isolated liver perfusion, or hepatic artery infusion. CC531 cells are readily transfected in vitro, resulting in significant inhibition of tumour cell proliferation by the Y28 construct. Intravenous injection did not result in any measurable transfection. Intratumoural injection resulted only in the transfection of tumour cells along the needle track. IHP as well as single HAI achieved low transfection levels of tumour tissue. Expression of Y28 was demonstrated in tumours after IT injection, HAI and IHP. Whereas, repeated HAI's clearly achieved expression in and around tumour associated vessels. Only five times repeated HAI's with Y28 resulted in a tumour response: in all animals tumour growth was inhibited, and in three rats out of eight a complete regression of the liver tumours was observed.

Figure 1

Schematic representation of (A) an isolated hepatic perfusion (IHP) and (B) a hepatic artery infusion (HAI).

Figure 2

Growth of CC531 colon carcinoma cells in vitro after exposure to increasing concentrations of AV.1.0CMV.Y28 (Y28) or AV1.0CMV (empty). Five independent assays were performed in duplicate for each point on the line. Mean values (±s.e.m.) are shown.

Figure 3

(A,B,C) Y28 fluorescence immunohistochemistry on cryosections of tumours collected 24 h after treatment in vivo with AV.1.0CMV.Y28 (Y28). (A) Tumour after IT, transfection around the needle track. (B) Foci of Y28 expression in tumour after IHP. (C) Expression in tumour after HAI. Original magnification: A, B and C: 16×, insert 40×. No staining was found in case of treatment with AV1.0CMV.

Figure 4

Two examples of X-gal stained and RECA stained cryosections of tumours of different animals after HAI treatment with AV.1.0CMV.LacZ. Tumour vessel (brown) and perivascular orientation of transfected cells (blue) are clearly visible. Original magnification: 40×. No staining was found in case of treatment with AV1.0CMV.

Figure 5

Tumour response of CC531 tumours in vivo after 5×HAI with AV.1.0CMV.Y28 (n=8) or AV1.0CMV (n=4) treated rats. On days 0, 2, 4, 6, and eight rats were infused (treatment schedule indicated by: ↑). Mean values (±s.e.m.) are shown (P<0.02 on day 12 and P<0.05 on day 16). At day 12–24 animals with progressive disease had to be sacrificed because of bulky tumour growth (indicated by: ×), so only the partial and complete responders are depicted after that time point.