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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0487fje. |
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University of Louvain Medical School, Pharmacology and Therapeutics Unit (FATH 5349), B-1200 Brussels;
* University of Louvain Medical School, Biomedical Magnetic Resonance Unit and Medicinal Chemistry and Radiopharmacy Unit, B-1200 Brussels; and
University of Louvain Medical School, Radiobiology and Radioprotection Unit, B-1200 Brussels, Belgium
2Correspondence: UCL Medical School, Unit of Pharmacology and Therapeutics, FATH 5349, 53 Ave. E. Mounier, B-1200 Brussels, Belgium. E-mail: feron{at}mint.ucl.ac.be
SPECIFIC AIM
The ultimate goal of radiotherapy is to induce irreversible damages in genetically unstable, fast-growing cancer cells while minimizing the cytotoxic effects on host tissues. The aims of the present study were to characterize the effects of low-dose irradiation on host-derived tumor endothelial cells (EC), examine the influence of these effects on tumor sterilization, and evaluate their possible exploitation to improve cancer treatment.
PRINCIPAL FINDINGS
1. Irradiation oppositely regulates expression of eNOS and caveolin in cultured EC and tumor microvasculature
We found that EC exposure to irradiation induced a dose-dependent up-regulation of eNOS expression and a parallel down-regulation of caveolin-1 (a physiological inhibitor of eNOS), leading to an enhanced potential of surviving EC to produce NO. We also locally irradiated murine tumors (resulting from the implantation of hepatocarcinoma (TLT) cells into the hind limb). Control and irradiated tumor microvessels (100–250 µm diameter) were dissected and pooled to be processed in immunoblotting experiments. Twenty-four hours after a local 6 Gy irradiation, the tumor microvasculature exhibited a fourfold increase in eNOS abundance and a simultaneous decrease in caveolin-1 expression.
2. Irradiation restores the NO-dependent vasomotion in tumor afferent arterioles
We next evaluated whether irradiation could modulate NO-mediated tumor arteriole reactivity. Accordingly, a pressure myograph system was used to study the agonist response of saphenous arterioles isolated from healthy mice or TLT tumor-bearing mice that were locally irradiated or not. Precontracted vessels were challenged by acetylcholine, which induced a dose-dependent dilation of control arterioles (Fig. 1
a). This pharmacological response appeared to be entirely NO dependent since it was abolished in the presence of the NOS inhibitor N
-nitro-L-arginine (L-NA) (100 µM). In diameter-matched TLT tumor arterioles, whereas Ach (up to 10-4 M) failed to induce any effect (Fig. 1b
), the Ach-induced vasorelaxation appeared completely restored when the vessels were issued from irradiated tumors (Fig. 1c
). The Ach-induced relaxation in irradiated tumor arterioles (as in control arterioles) was abolished in the presence of L-NA (100 µM) (Fig. 1c
).
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3. Nitric oxide conditions the vascular-dependent increases in tumor blood flow and oxygenation after irradiation
We then examined whether the changes observed in NO-dependent vasomotion induced by irradiation accounted for in situ changes in tumor blood flow and oxygenation. We used laser Doppler imaging and EPR oximetry to evaluate blood flow and pO2, respectively, in irradiated TLT tumors from mice treated or not with the NOS inhibitor L-NAME (500 mg/L). A local 6 Gy irradiation significantly increased tumor blood flow (+36±3%; P<0.01, n=5) and tumor oxygenation (+75±29%; P<0.05, n=8) as measured 24 h after treatment. The effect was maximal at 48 h, then progressively faded to return to levels 3–4 days after irradiation. Conversely, no significant difference in tumor blood flow and oxygenation was induced by irradiation of animals receiving L-NAME (P>0.05; n=5). Importantly, we verified that the only source of NO in the tumor was vascular eNOS.
4. X-ray-induced NO production participates in the effectiveness of radiotherapy
As tumor oxygenation is a key factor conditioning the effectiveness of irradiation, we examined whether the NO-dependent increase in pO2 induced by a first irradiation was required to potentiate the effects of a second irradiation. We measured daily the tumor diameters of mice submitted (or not) to one or two X-ray exposure(s) and evaluated the effects of L-NAME treatment. Whereas the nonirradiated TLT tumors exhibited a fast linear growth rate, single (day 0) and double (days 0 and 2) local 6 Gy irradiations considerably delayed tumor development, increasing the mean regrowth delay from 5.0 to 9.4 days, respectively (see Fig. 2
a). When tumor-bearing mice were treated with L-NAME from day -1 to day 2, the benefit of the second irradiation was abolished: the retardation in tumor growth did not differ from mice exposed to a single 6 Gy dose (Fig. 2a
). These observations were verified in another tumor model, e.g., fibrosarcoma (Fsa-II) implanted in another mice strain.
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5. Irradiation promotes liposomal gene delivery into the tumor
We also examined whether the increase in blood flow induced by ionizing radiation could be exploited to increase gene delivery to the tumor. We evaluated the efficacy of cationic lipid-based transfection of a reporter protein-encoding plasmid DNA in tumors. TLT tumor-bearing mice were locally irradiated and after 24 h injected i.v. with the plasmid–lipid complex. Tumors were collected 60 h later and lysates were immunoprecipitated. Whereas the HA-tagged reporter protein was barely detectable in nonirradiated tumors, the transgene was robustly expressed in tumors preexposed to a single 6 Gy dose (Fig. 2b
). This process of irradiation-induced DNA/liposome addressing the tumor was quasi-exclusively dependent on nitric oxide since the administration of L-NAME almost completely prevented the reporter protein expression in TLT tumors (Fig. 2b
). When these experiments were repeated using eNOS null mice bearing the Lewis lung carcinoma (LLC) tumor, irradiation failed to induce gene delivery into the tumor, whereas a net increase in reporter gene expression was observed in backcrossed control mice bearing LLC tumor.
CONCLUSIONS
In this study, we report that functional NO-mediated changes induced in the tumor vasculature by irradiation directly influence tumor response. This tumoricidal effect does not arise from induced vascular cytotoxicity/apoptosis as previously reported with large doses of ionizing radiation (Fig. 3
, left), but instead is observed with clinically more relevant, lower doses (Fig. 3
, right). Indeed, we show that limited doses of ionizing irradiation (2–6 Gy) can dramatically increase eNOS abundance in cultured EC as well as in the tumor microvasculature. This was associated with a reduction in the endogenous eNOS inhibitor caveolin, thereby reinforcing the stimulatory effect of irradiation on eNOS activity. The functional relevance of the expressional up-regulation of the NO pathway was verified by administering L-NAME to tumor-bearing mice: the NOS inhibitor abrogated the irradiation-induced increases in tumor blood flow, pO2, and radiosensitivity. Mice received L-NAME only during the interval between two irradiations, indicating that the tumor growth retardation observed after the second irradiation was strictly dependent on the increase in NO production induced by the first irradiation (see Fig. 2a
). Mechanistically, our data document that local exposure of the tumor to ionizing radiations can rescue the function of the arterioles perfusing the tumor. Indeed, the NO-dependent vasorelaxation observed in the arterioles of healthy animals was lost when the arterioles are located within the tumor (see Fig. 1b
). As suggested by the up-regulation of eNOS and down-regulation of caveolin, irradiation was able to reestablish the NO-mediated response (see Fig. 1c
).
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Pharmacological interventions aiming to increase blood flow and O2 are the subject of many experimental studies using either vasoconstrictors to increase perfusion pressure or vasodilators to decrease vascular resistance to flow. Positive and negative results have been observed with both approaches. The lack of specificity for the tumor vs. the host vessels indeed led to vasomotor effects mostly dependent on the overall arrangement of these vessels. Here we show that local delivery of ionizing radiation may bypass the specificity problem and selectively modulate flow resistance by targeting mature tumor vessels (i.e., those having the ability to vasorespond). An obvious correlate is the increase in tumor oxygenation and radiosensitization we have documented (see Fig. 2a
and Fig. 3
, right). It suggests that the frequency of exposure to X-rays could be fine-tuned in order to clinically exploit the best window of reoxygenation and/or increase in perfusion. Predictive assays aiming to evaluate tumor pO2/blood flow are under constant development and it should be easier in the future to adapt the fractionated scheme of irradiation to a given tumor in a given individual.
Finally, our data indicate that besides the strict context of tumor radiosterilization, low-dose radiotherapy could act as an adjuvant for a more selective delivery to the tumor (see Fig. 2b
and Fig. 3
, right). Here we have documented that cationic lipid DNA complex can be more efficiently targeted to locally irradiated tumors. Cationic liposomes are known to possess high tropism for tumors and are thought to be taken up by virtue of angiogenic EC endocytosis or extravasation from the leaky tumor vasculature. In the current study, the process leading to plasmid delivery into the tumor appears exquisitely dependent on the induction of NO production since the transgene expression was barely detectable when mice were treated with L-NAME at the time of the irradiation or when eNOS null mice were used. Therefore, besides the well-known intrinsic limitations in the size of the cargo–liposome complexes and the tumor pores, our observations emphasize the importance of NO-regulated blood flow, and probably permeability, for efficient delivery and expression into the tumors.
In conclusion, we report that low-dose ionizing radiations profoundly alter the function of EC lining tumor arterioles. These data unravel part of the rationale behind the effectiveness of the fractionated scheme radiotherapy and offer new perspectives for individual profiling of anti-cancer treatments. More generally, the current study provides bases for future investigations aiming to exploit the vascular effects of ionizing radiation as an adjuvant to promote selective delivery of genes and/or drugs to tumors.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0487fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0487fje 
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) and in mice irradiated at a single 6 Gy dose at day 0 (
) or irradiated twice (6 Gy at day 0 and 6 Gy at day 2) with (
) or without (
) L-NAME treatment from day -1 to day 2 (n=6–10). b) TLT tumor-bearing mice receiving or not L-NAME in drinking water were irradiated at the 6 Gy dose at day 0. The cationic lipid complex containing the HA-tagged reporter gene was injected in the tail vein at day 1 and tumors were collected on day 4. Corresponding lysates were immunoprecipitated with rabbit anti-HA antibodies (IgG) and immunoblotted with mouse anti-HA antibody (HA-tag). The transgene expression is consistently detected in untreated, irradiated tumors whereas longer film exposure is required to detect the reporter protein in the other conditions (arrowheads).