In the present study, we used motexafin lutetium as photosensitizer under conditions (10 mg/kg, 180-min drug–light interval) previously shown to lead to an acute, tumor-specific vascular response in treated tumors (22
). Other studies found that motexafin lutetium uptake in mouse tissue peaked after 180 min [measured in mouse foot (23
)], with its localization determined to be roughly equivalent in the tissue (SMT-F tumor) and plasma (24
). In the RIF tumor model, we found that an interval of 180 min led to higher motexafin lutetium concentrations in the plasma than in the tumor. Illumination at this time led to a spasmotic decrease in blood flow, confirming the presence of an acute vascular response with this drug–light interval. This prominent vascular response is not unlike that found in PDT with other photosensitizers, such as BPD, Tookad, hypericin and chlorin e6, if light is delivered when plasma concentrations of the drugs are high (25
). In the present study, the continuing progressive development of vascular damage over illumination and the time thereafter is seen in a decreased access of a perfusion marker to the tumor vessels and a significant increase in tumor hypoxia (pO2
of 1 mmHg at 180 min after PDT compared to 9 mmHg in controls). However, vascular damage was incomplete, because perfused vessels, although reduced in number compared to controls, were still detectable at the conclusion of PDT.
As found by others, we found that a lower fluence rate benefited PDT response to protocols that favor vascular damage (8
). We sought to understand the mechanisms responsible for the low-fluence-rate enhancement in this study, which was assessed through improvements in long-term tumor response to PDT at 25 compared to 75 mW/cm2
. A lower fluence rate can benefit PDT response by slowing the rate of photochemical oxygen consumption, thereby allowing for better maintenance of tumor oxygenation and increasing direct tumor cell killing (29
). Since the vasculature was the primary target under the conditions used, we evaluated fluence-rate effects on blood hemoglobin oxygen saturation (SO2
) in microvasculature. PDT created significant decreases in SO2
at both fluence rates used. The fact that decreases in SO2
were detectable in the minutes after PDT (when it was technically feasible to make the measurements) suggests that this oxygen depletion was secondary to vascular effects that had developed during treatment. Similarly, the lack of a fluence-rate effect on oxygenation suggests that oxygen depletion did not result from photochemical consumption but rather was a downstream consequence of vascular effects. Under conditions of substantial decreases in tumor perfusion such as those found during PDT at both fluence rates, resulting decreases in tissue oxygen tensions could lead to increased oxygen extraction from hemoglobin and therefore decreases in oxyhemoglobin. However, SO2
cannot be expected to track precisely with vascular shutdown because it measures the oxygenation of only perfused vessels.
Further evidence that the fluence rate did not affect oxygen tensions or the direct PDT effect in tumor cells is apparent in the results of the clonogenicity studies. At the lower fluence rate there was no significant advantage to cell killing measured immediately after PDT. Clonogenicity immediately after PDT at both fluence rates was lower than that found in light controls, which suggests the presence of a fluence-rate-independent component to direct cell killing for these treatment conditions. However, in this study, assessment of direct cell death immediately after the completion of PDT could be confounded by cytotoxicity secondary to vascular effects that developed early in PDT, especially during PDT at the lower 25 mW/cm2 fluence rate, which required over 120 min of illumination and thus provided ample time for any effects of early vascular damage to propagate. As a result, it is not possible to attribute cell death measured at the conclusion of PDT solely to direct cell killing; nevertheless, any contribution of direct killing to response was small and independent of fluence rate.
In general, fluence rate has been found to affect many aspects of the PDT response, including not only direct cytotoxicity to tumor cells and vascular damage but also contributions from an immune response (5
), which may be important when suboptimal damage to the tumor vasculature permits an influx of immune cells after PDT. The presence of an immune system component to tumor response of the present study was not explicitly examined and thus cannot be ruled out. Neither of the fluence rates studied in the present investigation produced complete shutdown of blood vessels immediately after PDT, which would allow for accessibility to host immune cells. Conditions that increased vascular damage (lower fluence rate) were associated with better long-term efficacy; thus, as found by others, the presence of a strong vascular response may supersede contributions from an immune response (5
Another characteristic of low-fluence-rate illumination is its association with longer illumination times for treatment to the same total dose (fluence) as that used at a higher fluence rate. In this study, PDT was significantly more effective when delivered to equivalent fluences at 25 mW/cm2
than at 75 mW/cm2
. However, delivery of 200 J/cm2
required 45 min at 75 mW/cm2
and 135 min at 25 mW/cm2
. Seshadri et al.
) showed that the additional treatment time at lower fluence rate could be an independent factor favoring the vascular component of tumor response to HPPH-PDT. That study was performed under conditions that favored plasma clearance of the drug prior to illumination (33
), as opposed to our present study, in which illumination was intentionally performed when plasma levels are high (24
). Nevertheless, the HPPH PDT data suggest that treatment duration may also play an important role in fluence-rate effects for PDT of tumor blood vessels. In agreement, our data show that longer treatment times are needed for the lower fluence rate to demonstrate a benefit.
Although greater vascular damage was found in this study at a lower fluence rate of 25 mW/cm2
compared to 75 mW/cm2
, further lowering the fluence rate to 8.4 mW/cm2
provided no additional tumor response. This suggests the presence of a threshold beyond which increasing the length of PDT and concurrently the length of time over which low tumor blood flow is maintained will provide no cytotoxic advantage. This finding is consistent with a hypothesis that an increase in vascular damage from low-fluence-rate PDT was a consequence of prolonged ischemia that led to vessel fragility; it is supported by the work of others documenting a relationship between the duration of ischemia and the development of detrimental biological effects (34
). These results are also consistent with other PDT studies in which lowering the fluence rate below 7 mW/cm2
[HPPH PDT (13
)] or 18 mW/cm2
)] reduced biological responses. At the very low fluence rates used in these and our studies there is the opportunity for greater repair of oxidative damage during the slow rate of light delivery (36
), which could contribute to a threshold effect, as could the limitations imposed by penetration of low-fluence-rate light in solid tissues.
Evidence of PDT-induced vascular effects can be detected in this study as increases in THC, which are a consequence of increases in the concentration of deoxyhemoglobin, but not oxyhemoglobin. These findings are suggestive of incomplete vascular shutdown and vascular leakage after PDT, which could lead to pooling of deoxygenated blood. Similar findings have been reported by others after both vascular- and cell-targeting PDT regimens. For example, a threefold increase in blood volume within the treated field on mouse skin was reported at 60 min after PDT with BPD with either a 15-min (vascular-targeting) or a 180-min (cell-targeting) drug–light interval (37
). Chen and colleagues found that BPD-PDT with a 15-min drug–light interval increased tumor vascular permeability over the hour after PDT (38
); moreover, they noted that greater vascular extravasation occurs in the tumor periphery compared to its center (39
). This latter observation is particularly relevant to the results of our study. We note that incomplete vascular shutdown after PDT is characterized by maintenance of blood flow within a tumor’s periphery and more widespread vascular showdown in the tumor center. Others have also documented that PDT can lead to sparing of vessels in the tumor periphery for a variety of different tumor models and photosensitizing conditions (26
In conclusion, this study establishes that a lower fluence rate can improve PDT response under conditions in which treatment is designed to have predominantly vascular effects. The improvement in response cannot be associated with improvements in vascular oxygenation at the lower fluence rate but instead appears to be related to the prolongation of ischemia during treatment with the lower fluence rate. More widespread vascular damage occurs at the lower fluence rate, which is detectable as smaller increases in blood volume and less accumulation of deoxygenated hemoglobin within the treated tumor. Increases in cytotoxicity at the lower fluence rate follow the vascular effects. These data indicate that further studies of low fluence rate in PDT regimens that predominantly damage the tumor vasculature are needed.