Radiosensitization mediated by a transfected anti-erbB-2 single-chain antibody in vitro and in vivo.
OBJECTIVE
The erbB-2 receptor is overexpressed in several human cancers, including ovarian, prostate, and breast. We have developed plasmid and adenoviral vectors expressing an anti-erbB-2 single chain antibody (sFv), directed to the endoplasmic reticulum (ER) of target cells, that is cytotoxic to tumor cells overexpressing erbB-2 through induction of apoptosis. The anti-erbB-2 sFv also sensitizes erbB-2 overexpressing cells to the cytotoxic effects of cisplatin. On this basis, we hypothesized that human ovarian cancer cells expressing anti-erbB-2 sFv with downregulated erbB-2 product, p185erbB-2, also would be sensitized to ionizing radiation. Therefore, we designed experiments to test the ability of the anti-erbB-2 sFv to radiosensitize human ovarian cancer cells in vitro and in vivo.


METHODS
To test our hypothesis, we established subcutaneous (s.c.) tumors in the flanks of nude mice with SKOV3.ip1 human ovarian cancer cells and SKOV3 cells stably expressing the ER directed anti-erbB-2 sFv (SKOV3/pGT21). The tumors were treated with 10 Gy 60Co, or received no radiation. We then determined the regression rate, delay in regrowth, and time to tumor doubling of the tumors treated with radiation in the transfected group and controls. In addition, SKOV3.ip1 and SKOV3/pGT21 tumors were dissected from the irradiated animals and assayed for differences in p185erbB-2 expression at 12 weeks after irradiation by immunohistochemistry. Further, in vitro clonogenic survival assays were performed on the parental SKOV3.ip1 and SKOV3/pGT21 cell lines.


RESULTS
A statistical analysis of the combined data was done for two in vivo experiments. The analysis of the combined data showed that animals with irradiated tumor SKOV3/pGT21 had a significantly higher regression rate (p = 0.0055), longer delay in regrowth (p = 0.0001) and time to tumor doubling (p = 0.0004), than those animals with tumor SKOV3.ip1 that received radiation. We observed a similar significant effect for the same parameters in the unirradiated tumor SKOV3/pGT21 compared to unirradiated tumor SKOV3.ip1. Immunohistochemical analysis of the SKOV3/pGT21 tumor cells demonstrated focal accumulation of p185erbB-2 in scattered clumps of cells and less p185erbB-2 membrane expression than cells of SKOV3.ip1 tumors. However, SKOV3.ip1 and SKOV3/pGT21 cells had similar in vitro sensitivity to radiation.


CONCLUSIONS
These data support the hypothesis that tumors with reduced p185erbB-2 expression mediated by the anti-erbB-2 sFv are rendered more susceptible in vivo to the cytotoxic effects of ionizing radiation than tumors that maintain their normal expression of p185erbB-2. However, a similar effect was not observed with the same tumor cells in vitro. Thus, as has been described by others (1, 2), in vitro and in vivo results do not always correlate. Therefore, appropriate assays to assess clinical relevance need to be determined for each particular system studied.
Stackhouse
M A
MA
Department of Radiation Oncology, University of Alabama at Birmingham, 35233-6832, USA.
Buchsbaum
D J
DJ
Grizzle
W E
WE
Bright
S J
SJ
Olsen
C C
CC
Kancharla
S
S
Mayo
M S
MS
Curiel
D T
DT
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000627:therapeutic use*; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D009368:Neoplasm Transplantation; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000532:radiotherapy*; D011836:Radiation Tolerance / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous; D014410:Tumor Stem Cell Assay
D000906:Antibodies; D009363:Neoplasm Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases