Effects of interstitial heating on the RIF-1 tumor using an Nd:YAG laser with multiple fibers.

BACKGROUND AND OBJECTIVE: Hyperthermia was induced in tumor-bearing C3H mice using a Nd:YAG laser emitting near-infrared radiation at 1,064 nm. The efficacy of multiple implanted fiberoptics in the control of the RIF-1 tumor was investigated. STUDY DESIGN/MATERIALS AND METHODS: RIF-1 tumors in the right hind leg were heated interstitially at 42, 44, or 46 degrees C for 30 or 60 minutes. Two, three, or four 400-microns quartz fibers terminating in a 1.0-cm cylindrical diffusor were inserted into each tumor, as were five microthermocouples to monitor temperature during treatment. Laser Doppler Flow (LDF) was also recorded pre- and post-treatment to determine changes in red blood cell flux in overlying skin (42, 44, or 46 degrees C) and the center of the tumor (46 degrees C). RESULTS: These experiments indicated that interstitial heating at 42, 44, and 46 degrees C resulted in tumor growth delay, although long-term control of tumors was not achieved. Treatment using four fibers resulted in the greatest tumor growth delay at 42 and 44 degrees C, increasing tumor doubling time by 50% or greater compared to control tumors; tumor growth delay following 46 degrees C treatments was seven times greater than that in control tumors. Significant changes (decreases) in LDF (P < .05) were seen in four treatment groups, using two fibers at 42 degrees C for 30 minutes, four fibers at 44 and 46 degrees C for 60 minutes on the overlying skin, and 46 degrees C for 60 minutes in the center of the tumor. CONCLUSIONS: Initial data indicate that interstitial heating with multiple fibers increases tumor growth delay compared to previous single fiber treatments, with tumor growth delay increasing with increasing treatment temperature; however, long-term tumor control was not achieved under the conditions investigated. Follow-up studies will explore the use of higher temperatures and/or longer treatment times in order to optimize tumor response.

Evaluation of changes in oxygen tension as indicators of RIF-1 tumor response to Nd:YAG laser heating.

Heat-induced oxygen tension changes in RIF-1 tumors in C3H male mice were analyzed in an attempt to correlate these changes with tumor response to Nd:YAG laser heating. A low power, microprocessor-controlled Nd:YAG laser was used to superficially heat 250-300 mm3 tumors to base temperatures of 44, 45, 46, or 48 degrees C for 30 minutes via a flexible 600 microns quartz fiberoptic with a terminating microlens. A glass, Clark-style microelectrode was inserted into the center of each tumor allowing real time measurement of the tumor's oxygenation status before, during, and after heating. Results showed that heating at 44 degrees C caused a greater than 2-fold increase in oxygen tension during heating, while a temperature of 48 degrees C caused a brief initial increase in oxygen tension followed by a decrease to below pretreatment values. There was a significant correlation (P < 0.05) between relative tumor oxygen tension during and post-heating and tumor growth delay. A significant correlation (P < 0.05) was also seen between tumor base temperature during heating and tumor growth delay. It appears from our initial data that single point oxygen tension measurements in small RIF-1 tumors may be useful indicators of this model's response to Nd:YAG laser heating. This result may allow for modification of heating parameters (temperature/time) during treatment to optimize thermal response.

Microprocessor-controlled Nd:YAG laser for hyperthermia induction in the RIF-1 tumor.

Near-infrared radiation from a Nd:YAG laser at 1,064 nm was used interstitially or superficially to induce hyperthermia in RIF-1 tumors in C3H male mice. A single 600-microns quartz fiber with a 0.5-cm cylindrical diffusor or a weakly diverging microlens at its distal end was used to deliver laser energy to tumors in the hind leg (mean volume = 100 mm3). Two thermocouples were inserted into each tumor. One thermocouple controlled a microprocessor-driven hyperthermia program (maximum output of 3.5 Watts) to maintain the desired temperature. Tumors were exposed to various temperature-time combinations (42-45 degrees C/30 min). Our initial results indicated that excellent temperature control to within 0.2 degrees C of the desired temperature at the feedback thermocouple was achievable during both superficial and interstitial heat treatments. Temperatures at the second thermocouple, however, were found to be lower by as much as 2.3 degrees C (using the cylindrical diffusor) or higher by up to 4.6 degrees C (using the microlens) when compared to the feedback thermocouple temperature. Several correlations were seen between total dose, tumor growth delay, percent skin necrosis, and temperature at the second thermocouple after several superficial and interstitial treatments. Statistically significant improvements in tumor growth delay (at 42 and 45 degrees C) and increased percent skin necrosis at all temperatures were observed after superficial versus interstitial treatment.

Effect of Fluosol-DA 20% and oxygen on response of C57BL/6 mice to whole-body irradiation.

Normal tissue effects in mice due to combinations of a perfluorochemical emulsion, Fluosol-DA 20%, 100% oxygen, and whole-body irradiation were investigated. Eight-to-10-week-old C57BL/6 male mice were injected via the tail vein with 10 ml/kg of Fluosol-DA with and without subsequent exposure to oxygen for 60 minutes. Animals then received graded doses of whole-body radiation (4 MV photons) at a dose rate of 2.85 +/- .015 Gy/minute. Using linear regression analysis, the lethal doses of radiation to 50% and 10% of the animals within 30 days in the absence of Fluosol-DA and oxygen were 8.35 Gy (95% c.l.:7.77-8.93 Gy) and 6.73 Gy (95% cl.:6.21-7.25 Gy), respectively, and were unaffected by Fluosol-DA and/or oxygen pre-treatment. However, Fluosol-DA given alone or in combination with oxygen produced increased balding and decreased graying incidence in mice within 60 days, and resulted in depressed weight gain 15 to 60 days post-treatment. Normal tissue effects due to administration of Fluosol-DA and oxygen in combination with whole-body irradiation have been demonstrated but appear minimal compared to other anti-tumor modalities currently under investigation.

Nd:YAG laser-induced hyperthermia in a mouse tumor model.

Hyperthermic tumor response induced by 1,064-nm radiation from an Nd:YAG laser was investigated in DBA/2J female mice bearing the SMT-F mammary carcinoma. The measured temperature-depth profiles indicate that hyperthermic temperatures can be achieved in tumors ranging from 3 to 8 mm thick at power inputs on the order of 1 W. For small tumors, a 5-week complete response rate exceeding 50% required 45 minutes at 45.0 degrees C. Control of large tumors (6-8 mm thick) was not achieved. The observed tumor response rates are consistent with semiempirical time-temperature relations based on other heating modalities.

Hyperthermic potentiation of photodynamic therapy employing Photofrin I and II: comparison of results using three animal tumor models.

Hyperthermia induced by a microwave source (2,450 MHz) was used alone and in combination with photodynamic therapy (PDT) to treat the SMT-F, EMT-6, and RIF animal tumors in vivo. PDT was administered using either Photofrin I or II as the photosensitizer and an argon-pumped tunable dye laser (630 nm) as the light source. Greater than additive increases in long-term tumor control were achieved when hyperthermia was given immediately post-PDT in the SMT-F and RIF tumor systems. Only additive (or independent) increases in tumor control were achieved when hyperthermia was given immediately before PDT in all these tumor systems and when heat was applied post-PDT using the EMT-6 tumor. In a series of experiments using the SMT-F tumor, it was observed that decreases in PDT drug or light doses could be offset (in terms of tumor control) by the addition of a subsequent heat treatment. This result, along with others presented, indicates the clinical potential of PDT and hyperthermia as adjuvant cancer modalities.

Photodynamic therapy for treatment of malignant cutaneous lesions.

Photodynamic therapy, employing either hematoporphyrin derivative or dihematoporphyrin ether as the photosensitizer and an argon-pumped tunable dye laser as the activating light source, was used to treat ten patients who had primary or recurrent basal or squamous cell carcinoma or infiltrating ductal cell carcinoma lesions metastatic to the skin. Of the 155 sites that were treated, various degrees of edema, erythema, and necrosis, sometimes accompanied by blistering, were evident in all areas within 24 to 48 hours of light treatment. While our follow-up periods are short, four patients are free of disease at eight months or more posttreatment, two patients have had recurrent and/or persistent disease within four months, and four patients died within nine months from metastatic disease or unrelated disorders. Continued investigation into the use of photodynamic therapy for treatment of malignant lesions appears warranted, based on these preliminary clinical results.

Porphyrin fluorescence and photosensitization in head and neck cancer.

Indistinct margin demarcation and autofluorescence are problems in fluorescence delineation of porphyrin-containing head and neck squamous cell cancer (HNSCC). We studied the time course and in vivo localization of porphyrin fluorescence in a squamous cell cancer hamster model and in human HNSCC. After intravenous injection, a gradient in fluorescence intensity developed rapidly until tumors fluoresced above a lower-intensity mucosal background. Hamster tumor and ulcerated HNSCC without porphyrin injection demonstrated autofluorescence grossly indistinguishable from fluorescence in porphyrin-injected tumors. However, fluorescence microscopy revealed autofluorescence to be a surface phenomenon and showed injected porphyrin localized in tumor stroma. We conclude that autofluorescence must be considered when interpreting porphyrin fluorescence. In addition, empirically designed photodynamic therapy can be effective in selected HNSCC. Data from animal experiments provide useful guidelines for the delivery of this therapy.

Interaction of photodynamic therapy and hyperthermia: tumor response and cell survival studies after treatment of mice in vivo.

The interaction of photodynamic therapy (PDT) and hyperthermia was studied in the radiation-induced-fibrosarcoma experimental mouse tumor system by tumor regrowth experiments as well as in vivo to in vitro cloning assays. In vivo, PDT (Photofrin II, 10 mg/kg i.p.), followed 24 h later by light (135 J/cm2, 630 nm) and/or heat (44 degrees C, 30 min) caused severe vascular damage (congestion of tumor vessels and hemorrhage) and subsequent disappearance of palpable tumor mass. While heat-treated tumors always started to regrow within 2 days of treatment, regrowth if it occurred was delayed to 4-5 days after PDT and 6-7 days following combined treatments. Only PDT followed by heat cured a considerable number of animals (45%), while PDT alone and heat followed by PDT cured less than 10% of animals, and heat alone caused no tumor cures. The various treatments differed in their immediate as well as their delayed effects on tumor clonogenicity when observed over a 24-h period. Tumors treated with PDT showed no immediate changes in clonogenicity, but progressive delayed cell death occurred if tumors remained in situ. Heat alone led to an immediate reduction in the number of clonogenic tumor cells, followed by some additional cell death for 4 h and subsequent recovery of clonogenicity. PDT followed by heat caused markedly potentiated immediate reduction in cell survival which may be the result of direct interaction of heat and PDT damage affecting the tumor cells. Some tumors rapidly progressed to total eradication, whereas others showed delayed survival values similar to those for tumor having received PDT only. In the reverse sequence, heat before PDT, the tumor cell survival kinetics resembled those following heat treatment alone. The comparative lack of effectiveness of this treatment regimen can be explained by the severe tumor hemorrhage caused by the initial heat treatment which reduces the transmission of light essential for the subsequent PDT treatment. This study shows that despite pronounced similarities in the microscopic and macroscopic appearance shortly after treatment by PDT or hyperthermia, these two modalities lead to tumor destruction by different mechanisms. Furthermore the combination of these two modalities in the proper sequence leads to potentiated cytocidal effects on the tumor cells in vivo.

Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy.

The effect of photodynamic therapy (PDT) on tumor growth as well as on tumor cell survival in vitro and in vivo was studied in the EMT-6 and RIF experimental mouse tumor systems. In vitro, RIF cells were more sensitive towards PDT than were EMT-6 cells when incubated with porphyrin (25 micrograms/ml, dihematoporphyrin ether) and subsequently given graded doses of light. In vivo, both tumor types responded to PDT (EMT-6, dihematoporphyrin ether, 7.5 mg/kg; RIF, dihematoporphyrin ether, 10 mg/kg; both followed 24 hr later by 135 J of light at 630 nm/sq cm) with severe vascular disruption and subsequent disappearance of tumor bulk. However, whereas the cure rate for EMT-6 tumors was 90%, it was 0% for RIF tumors. Raising the light dose to 200 J/sq cm resulted in 100% cures for EMT-6 tumors accompanied by damage to the surrounding tissues and 13% cures for RIF tumors. Tumor cell clonogenicity following PDT in vivo was assessed using the in vitro colony formation assay. In both tumors, it was found to be nearly unaffected by PDT if the tumor tissue was excised and explanted immediately following completion of treatment. This indicates that the effect of PDT on tumor cells directly was not sufficient to decrease tumor clonogenicity even at doses which led to total macroscopic tumor destruction. Where the tumors remained in situ following PDT and explantation was delayed for varying lengths of time (1 to 24 hr), tumor cell death occurred rapidly and progressively, indicating that tumor cell damage was expressed only if the cells remained exposed to the in situ environment after treatment. The kinetics and extent of tumor cell death were very similar for both tumor types despite their difference in cure rates. The reduction in tumor clonogenicity at 4 hr post-PDT closely matched that of tumor deprived of oxygen for the same period of time, implying that one of the major factors contributing to tumor destruction may be damage of the tumor circulation and the consequences of treatment-induced changes in tumor physiology.

Potentiation of photodynamic therapy by heat: effect of sequence and time interval between treatments in vivo.

Photodynamic therapy (PDT) utilizing hematoporphyrin derivative (Hpd) as photosensitizer and an argon-dye laser as the light source was used alone and in combination with a localized microwave hyperthermia treatment to treat the SMT-F mammary carcinoma in mice. A 30-min heat treatment at 44.5 degrees C was applied 0-8 hr before or after a standard photodynamic treatment (67.5 or 135 J/cm2, given 24 hr post-7.5 mg/kg Hpd). Potentiation of PDT by heat was found to be related to the sequence of the treatments and the time interval between them. When 44.5 degrees C for 30 min was applied immediately after a 15-min PDT treatment, significant potentiation was seen (58% long-term tumor control vs 3 and 10%, respectively, for PDT and heat alone). This potentiation decreased with increasing time between PDT and heat, with tumor control values decreasing to 36, 20, and 14%, when 2, 4, and 8 hr, respectively, were allowed between treatments. Only additive effects of the independent therapies were found when this heat treatment was applied 0-8 hr before PDT. In other experiments, mice were treated with single or fractionated 30-min PDT treatments (two 15-min treatments separated by 0-, 2-, 4- or 8-hr intervals). Decreases in tumor control were seen with increasing time interval; only minor differences in tumor control were seen when 4-8 hr was allowed between treatments compared to a single 15-min treatment.

Interaction of hyperthermia and photoradiation therapy.

Local microwave hyperthermia (2450 MHz) was applied to axillary implants of the SMT-F mammary carcinoma in mice in combination with photoradiation therapy (PRT) in an attempt to determine if the two modalities interact. When 40.5 degrees C was applied for 30 min immediately prior to or immediately following PRT (630-nm light, 30 min, at 75 mW/cm2, 20-24 hr post 7.5 mg/kg hematoporphyrin derivative), enhancement of tumor response over that of PRT alone was seen as judged by lack of tumor regrowth (35 days or longer after treatment). A temperature of 41.5 degrees C applied for 30 min immediately following the 30-min PRT treatment produced a result slightly greater than that seen at 40.5 degrees C. When a temperature of 44.5 degrees C for 30 min was applied immediately following PRT, a substantial enhancement of tumor control at 35 days post-treatment was found (53% versus 19 and 4%, respectively, for hyperthermia and PRT alone). These results suggest that tumor response to PRT is enhanced by both a sublethal hyperthermic treatment (40.5 degrees C, 41.5 degrees C) and a moderately lethal heat treatment (44.5 degrees C) given for a short duration, when applied immediately before or after photoradiation.

Enhanced tumor control following sequential treatments of photodynamic therapy (PDT) and localized microwave hyperthermia in vivo.

Photodynamic therapy (PDT), or photoradiation therapy (PRT), utilizing hematoporphyrin derivative (HPD) as photosensitizer and an argon-dye laser system as the light source, was used alone and in combination with localized microwave hyperthermia (2450 MHz) to treat axillary tumors of the SMT-F mammary carcinoma in mice. Thirty-minute heat treatments were applied either immediately before or immediately after a standard PDT treatment of 630 nm light at 75 mW/cm2 for 30 min (135 J/cm2) given 24 hr post-7.5 mg/kg HPD, intraperitoneally (i.p.). Tumor control as judged by lack of tumor regrowth 35 days or longer after the combined treatments was compared to that following each treatment when given alone. Little or no enhancement of tumor control was seen when sublethal temperatures of 37.5, 38.5, and 39.5 degrees C were applied for 30 min immediately following the PDT treatment. However, increasing levels of enhancement were seen when heat treatments of 40.5 and 41.5 degrees C or 44.5 degrees C, given for 30 min, were applied immediately before or after the photodynamic treatment.