Diagnostic targeting of colon cancer using a novel fluorescent somatostatin conjugate in a mouse xenograft model.

Colorectal carcinoma is one of the more prevalent, highly malignant human tumors, occurring in about 7% of the population. However, if diagnosed and treated in its early stages, colon cancer is curable. In our study, we used a mouse xenograft model to investigate the capability of a fluorescent conjugate of a novel synthetic somatostatin (SST) analog to improve detection of human colorectal tumors that are characterized by over-expressed SST receptors. Human HT-29 colon carcinomas were induced in nude mice. After administration of the fluorescent SST conjugate, in vivo low- and high-magnification fluorescence microscopy, as well as high-resolution spectrally resolved imaging were performed, and the time-dependent biodistribution was determined quantitatively (using fiber-optic spectroscopy). Administration of the conjugate (at concentrations of 6 mg/kg body weight) enabled targeting small (1-5 mm diameter) tumors with high sensitivity and selectivity. Toxicity studies at dosages up to 1,000 mg/kg body weight did not reveal any drug related abnormalities. In conclusion, the SST conjugate significantly enhanced the detection of HT-29 colon tumors by fluorescence imaging because of a 5- to 8-fold increase in the contrast between malignant and normal tissues.

Laser treatment of venous malformations.

Venous malformations (VM) are developmental errors comprised of enlarged dysplastic blood vessels. Clinically they manifest as either a faint blue patch or a soft blue vascular mass. Treatment options include a sclerosing agent (such as alcohol or ethiblock), surgery or laser therapy. A review of the literature concerning VM and laser treatment yielded a few sporadic reports describing series of 3 to 46 cases. In this retrospective study we reviewed the files of the relevant cases. The data included the extent of the lesion, the different anatomical sites, age of patient and the extent of clearance of the lesion as the treatment's end point. This study includes 56 cases which makes it the largest series presented. The success rate of laser treatment was 92.8%. The immediate complication rate was very low (approximately 3.57%) including minimal scarring and deformity. No long term complications were noted. Our study concludes that treatment of VM is a difficult task due to the nature of the lesions. Laser treatment of these lesions enables one to obtain good results with a very low incidence of complications. Surgery and other treatment modalities are not always satisfactory, yield similar or less efficient results and have a higher complication rate. Laser treatment can play an important role in the treatment of VM and in fact may be the treatment of choice in some settings.

Targeting small-cell lung cancer with novel fluorescent analogs of somatostatin.

Early, accurate detection of small-cell lung cancer (SCLC), before it becomes systemic, is essential for successful treatment. Fluorescence-based imaging provides safe, sensitive detection of malignancies. Targeted delivery of fluorophores increases sensitivity of endoscopic imaging. We synthesized novel somatostatin analogs, based on backbone cyclic peptides, and conjugated them with fluorescent agents. Nineteen conjugates differing in core peptide, length of alkyl linker and fluorescence moiety (rhodamine and fluorescein) were tested in vitro, using a receptor binding assay, and nine of the more promising conjugates were tested in vivo by fiber-optic spectrofluorimetry and quantitative spectral imaging, on an H69 human SCLC tumor mouse xenograft model. The lead compound showed exceptional tumor/normal tissue ratios, ranging from 9 to 90, and has potential for targeting SCLC overexpressing somatostatin receptors.

Influence of laser wavelength and pulse duration on gas bubble formation in blood filled glass capillaries.

BACKGROUND AND OBJECTIVES: Hypervascular skin lesions (HVSL) are treated with medical lasers characterized by a variety of parameters such as wavelength lambda, pulse duration t(p), and radiant exposure E that can be adjusted for different pathology and blood vessel size. Treatment parameters have been optimized assuming constant optical properties of blood during laser photocoagulation. However, recent studies suggest that this assumption may not always be true. Our objective was to quantify thermally induced changes in blood that occur during irradiation using standard laser parameters. STUDY DESIGN/MATERIALS AND METHODS: Glass capillary tubes (diameter D = 100, 200, and 337 microm) filled with fresh or hemolyzed rabbit blood were irradiated once at lambda = 585, 595, or 600 nm, t(p) = 1.5 milliseconds; and also at lambda = 585 nm, t(p) = 0.45 milliseconds. E was increased until blood ablation caused formation of permanent gas bubbles. In a corroborative study, human blood was heated at 50 degrees C and absorbance spectra were measured as a function of time. RESULTS: Threshold radiant exposure, E(thresh), for gas bubble formation was found not to depend on lambda, which might be surprising in view of the 10-fold lower absorption coefficient at 600 nm as compared to 585 nm. The spectroscopic study revealed heat-induced changes in blood constituent composition of hemoglobins (Hb) from initially 100% oxyhemoglobin (HbO2) to deoxyhemoglobin (HHb) and, ultimately, methemoglobin (metHb) as the major constituent. Model calculations of E(thresh)(lambda,D) based on changing constituent blood composition during heating with milliseconds lasers were found to correlate with experimental results. CONCLUSIONS: For laser treatment of HVSL it appears that lambda is of secondary importance and that the choice of t(p) is a more important factor.

Monitoring PDT-induced damage using spectrally resolved reflectance imaging of tissue oxygenation.

Photodynamic therapy (PDT) with chlorin e6 (Chl) was monitored in vivo using vital microscopy and Fourier transform spectral imaging (FT-SI). Mammary C26 colon carcinoma, implanted intradermally in a mouse, was irradiated at 650 nm with various radiant exposures, 3 h after administration of 5 mg/kg Chl. The photodynamic response (PDR) in the skin flap with tumor was expressed as microcirculation disturbances (thrombi formation, multiple embolizations, arteriolar occlusion and venous stasis) and, dependent on the radiant exposure, was transient or permanent. These biological manifestations were accompanied by a change in hemoglobin (Hb)/oxyhemoglobin (HbO2) absorption spectra obtained in vivo by FT-SI. False-color mapping of hemoglobin oxygen saturation (OS) visualized the alteration of tissue oxygenation. The results demonstrate, for the first time, that FT-SI can serve as a sensitive non-invasive tool for OS monitoring of PDT effects.

Combined photodynamic and photothermal induced injury enhances damage to in vivo model blood vessels.

BACKGROUND AND OBJECTIVES: The degree of port wine stain (PWS) blanching following pulsed dye laser (PDL) therapy remains variable and unpredictable. Because of the limitations of current PDL therapy, alternative treatment approaches should be explored. The objective was to evaluate a novel methodology for selective vascular damage, combined photodynamic (PDT) and photothermal (PDL) treatment, using the in vivo chick chorioallantoic membrane (CAM) model. STUDY DESIGN/MATERIALS AND METHODS: Thirty microliters of benzoporphyrin derivative monoacid ring A (BPD) solution was administered intraperitoneally into chick embryos at day 12 of development. Study groups were: (1) control (no BPD, no light); (2) BPD alone; (3) continuous wave irradiation (CW) alone (576 nm, 60 mW/cm2, 125 seconds); (4) CW + PDL; (5) BPD+PDL; (6) PDT (BPD+CW); (7) PDL alone (585 nm, 4 J/cm(2)); and (8) PDT+PDL (BPD + CW followed immediately by PDL). Vessels were videotaped prior to, and at 1 hour post-intervention and then assessed for damage based on the following scale: 0, no damage; 1, coagulation; 1.5, vasoconstriction; 2.0, coagulation+vasoconstriction; 2.5, angiostasis; 3.0, hemorrhage. Damage scores were weighted by vessel "order." RESULTS: PDT + PDL resulted in significantly (P < 0.01) more severe vascular damage than was observed in any other study group: 127% more than PDT, 47% more than PDL alone. CONCLUSIONS: PDT + PDL is a novel and promising approach for selective vascular damage and may offer a more effective method for treatment of PWS and other vascular skin lesions.

Increase of dermal blood volume fraction reduces the threshold for laser-induced purpura: implications for port wine stain laser treatment.

BACKGROUND AND OBJECTIVES: The average success rate in achieving total blanching of port wine stain (PWS) lesions treated with laser-induced selective photothermolysis is below 25%, even after multiple treatments. This is because smaller diameter (5-20 microm) PWS blood vessels are difficult to destroy with selective photothermolysis since the volumetric heat generated by absorption of laser light is insufficient to adequately heat the entire vessel wall. The aim of this study was to investigate a potential technique for more efficient photocoagulation of small diameter PWS blood vessels in PWS that respond poorly to selective photothermolysis. STUDY DESIGN/MATERIALS AND METHODS: The blood volume fraction (BVF) in the upper dermis of the forearm of human volunteers was increased by placing an inflated blood pressure cuff on the upper arm. Applied pressures were in the range of 80-100 mm Hg for up to 5 minutes. The increased BVF was determined by matching reflectance spectra measured with that computed using a diffusion model. The impact of increased BVF on purpura formation induced by a 0.45 milliseconds pulsed dye laser (PDL) at 585 nm wavelength was investigated in normal and in PWS skin. RESULTS: In the presence of a 100 mm Hg pressure cuff, the BVF, as determined from the diffusion model, increased by a factor of 3 in the forearm and by 6 in the hand. Increasing BVF by a factor of 3 corresponds to an increase in blood vessel diameters by a factor of radical 3 approximately 1.7. BVF increased at 1-3 minutes after application of the pressure cuff, remained constant at 3-5 minutes, and returned to baseline values at 3 minutes after removal of the pressure cuff. Approximately 40% less radiant exposure was needed to induce the same amount of purpura after PDL irradiation when the blood pressure cuff was used. Applying an 80 mm Hg pressure cuff reduced the required radiant exposure for purpura formation by 30%. Heating of blood vessels was calculated as a function of vessel diameter and of radiant exposure (at 585 nm and at 0.5 and 1.5 milliseconds pulse duration). CONCLUSIONS: Enlarging the vessel lumen, for example, by obstructing venous return, can significantly reduce the "small-vessel-limitation" in PDL treatment of PWS. Dilation of PWS blood vessels enables a more efficient destruction of smaller vessels without increasing the probability of epidermal damage.

Influence of wavelength on response to laser photothermolysis of blood vessels: implications for port wine stain laser therapy.

BACKGROUND AND OBJECTIVE: Treatment of port wine stains (PWS) by photothermolysis can be improved by optimizing laser parameters. We have studied the all-important role of wavelength (lambda) by performing pulsed laser photothermolysis in the vasculature of the chick chorioallantoic membrane (CAM) assay. STUDY DESIGN/MATERIALS AND METHODS: The CAM contains an extensive microvascular network ranging from capillaries with diameter D < 20 microm to blood vessels of D approximately 120 microm. The CAM assay enables observation and real-time video documentation of blood flow in pre-capillary arterioles (A) and post-capillary venules (V). The ScleroPlus trade mark laser (Candela Corp., Wayland, MA) has a smooth output over its fixed 1.5 ms pulse duration and allows the operator to vary several treatment parameters such as wavelength, spot size, and energy. Blood vessels in the CAM were irradiated at two clinically relevant wavelengths, lambda = 587 or 597 nm, constant spot size (7 mm), and at different exposures (E = 5-12 J/cm(2)). Threshold exposure (E(th)) (at which non-reversible damage occurred) were 5 J/cm(2) at 587 nm and 8 J/cm(2) at 597 nm. Mathematical modeling was developed to interpret initial (within 30 seconds) injury of arterioles and venules at both wavelengths as a function of D when varying E. RESULTS: Vessel injury was graded off-line from videotapes. For all combinations of lambda and E, measurable injury was evident within 30 seconds of irradiation and maximal damage was reached in less than 10 minutes. Vessel damage was found to depend strongly on lambda. Damage decreased with increasing vessel size. For all D, damage of arterioles was greater than for corresponding venules. CONCLUSIONS: Selection of the correct wavelength is crucial for successful laser therapy of hypervascular lesions and, ideally, should be based on knowledge of vessel diameters for a specific PWS lesion and of optical penetration depths in blood. As a general statement, smaller blood vessels (D = 10-60 microm) should be treated using shorter wavelengths such as 585 nm. To ensure deposition of sufficient laser energy, irradiation at 585 nm, characterized by lower optical penetration depth in blood, may be preferable for PWS therapy.

Spectra from 2.5-15 microm of tissue phantom materials, optical clearing agents and ex vivo human skin: implications for depth profiling of human skin.

Infrared measurements have been used to profile or image biological tissue, including human skin. Usually, analysis of such measurements has assumed that infrared absorption is due to water and collagen. Such an assumption may be reasonable for soft tissue, but introduction of exogenous agents into skin or the measurement of tissue phantoms has raised the question of their infrared absorption spectrum. We used Fourier transform infrared spectroscopy in attenuated total reflection mode to measure the infrared absorption spectra, in the range of 2-15 microm, of water, polyacrylamide, Intralipid, collagen gels, four hyperosmotic clearing agents (glycerol, 1,3-butylene glycol, trimethylolpropane, Topicare), and ex vivo human stratum corneum and dermis. The absorption spectra of the phantom materials were similar to that of water, although additional structure was noted in the range of 6-10 microm. The absorption spectra of the clearing agents were more complex, with molecular absorption bands dominating between 6 and 12 microm. Dermis was similar to water, with collagen structure evident in the 6-10 microm range. Stratum corneum had a significantly lower absorption than dermis due to a lower content of water. These results suggest that the assumption of water-dominated absorption in the 2.5-6 microm range is valid. At longer wavelengths, clearing agent absorption spectra differ significantly from the water spectrum. This spectral information can be used in pulsed photothermal radiometry or utilized in the interpretation of reconstructions in which a constant mu(ir) is used. In such cases, overestimating mu(ir) will underestimate chromophore depth and vice versa, although the effect is dependent on actual chromophore depth.

Effects of mass flow rate and droplet velocity on surface heat flux during cryogen spray cooling.

Cryogen spray cooling (CSC) is used to protect the epidermis during dermatologic laser surgery. To date, the relative influence of the fundamental spray parameters on surface cooling remains incompletely understood. This study explores the effects of mass flow rate and average droplet velocity on the surface heat flux during CSC. It is shown that the effect of mass flow rate on the surface heat flux is much more important compared to that of droplet velocity. However, for fully atomized sprays with small flow rates, droplet velocity can make a substantial difference in the surface heat flux.

Laparoscopic photodynamic diagnosis of ovarian cancer using 5-aminolevulinic acid in a rat model.

OBJECTIVE: The objective of this study was to determine the efficacy and sensitivity of laparoscopic photodynamic diagnosis to detect 5-aminolevulinic acid (ALA)-induced fluorescent tumors in an animal model. METHODS: Cancer cells were injected into the peritoneum of rats to induce peritoneal carcinomatosis. After 3-4 weeks, ALA was administered to establish fluorescence in tumor nodules. All intraperitoneal surfaces were inspected using fluorescence and white light laparoscopy. Suspicious lesions were then biopsied in vivo under either fluorescence or white light laparoscopic guidance. Fluorescence intensities of the cancerous lesions compared to normal tissues were determined. A pathologist blinded to our clinical impression analyzed all biopsied specimens. We compared the sensitivity of fluorescence and white light laparoscopic-guided detection of cancerous lesions and determined the clinical utility of fluorescent photodynamic diagnosis in detecting metastatic ovarian cancer. RESULTS: Forty-three biopsies were performed in vivo under laparoscopic fluorescent guidance and 42 biopsies were taken using white light in various regions of the peritoneal surface from nine rats. Ten biopsies were also removed from nonfluorescent regions as nontumor controls. Cancerous lesions showed significantly higher fluorescent intensity compared to noncancerous lesions. Cancerous lesions that were difficult to differentiate from normal surrounding tissue under white light conditions were clearly detected by ALA-induced fluorescence. The average size of these metastatic lesions biopsied under fluorescent light was 1.0 mm (range: 0.3-2.5) compared to 1.5 mm (range: 0.5-2.9) with white light illumination (P < 0.05). CONCLUSIONS: Fluorescent laparoscopic detection of micrometastatic ovarian cancer using ALA is significantly more sensitive than white-light laparoscopy in detecting smaller cancerous lesions in an ovarian cancer rat model. Human trials are indicated.

Wavelength-dependent properties of photodynamic therapy using hypericin in vitro and in an animal model.

Wavelength effects in photodynamic therapy (PDT) with hypericin (HY) were examined in a C26 colon carcinoma model both in vitro and in vivo. Irradiation of HY-sensitized cells in vitro with either 550 or 590 nm caused the loss of cell viability in a drug- and light-dose-dependent manner. The calculated ratio of HY-based PDT (HY-PDT) efficiencies at these two wavelengths was found to correlate with the numerical ratio of absorbed photons at each wavelength. In vivo irradiation of C26-derived tumors, 6 h after intraperitoneal administration of HY (5 mg/kg), caused extensive vascular damage and tumor necrosis. The depth of tumor necrosis (d) was more pronounced at 590 than at 550 nm and increased when the light dose was raised from 60 to 120 J/cm2. The maximal depths of tumor necrosis (at 120 J/cm2) were 7.5+/-1.5 mm at 550 nm and 9.9+/-0.8 mm at 590 nm. Both values are rather high in view of the limited penetration of green-yellow light into the tissue. Moreover, the depth ratio, d590/d550 = 1.3 (P < 0.001), is smaller than expected considering the 2.2-fold lower HY absorbance and the 1.7-fold lower tissue penetration of radiation at 550 than at 590 nm. This finding indicates that in vivo the depth at which HY-PDT elicits tumor necrosis is not only determined by photophysical considerations (light penetration, number of absorbed photons) but is also influenced significantly by other mechanisms such as vascular effects. Therefore, despite the relatively short-wavelength peaks of absorption, our observations suggest that HY is an effective photodynamic agent that can be useful in the treatment of tumors with depths in the range of 1 cm.

Vascular response to laser photothermolysis as a function of pulse duration, vessel type, and diameter: implications for port wine stain laser therapy.

BACKGROUND AND OBJECTIVE: Treatment of port wine stains (PWS) by photothermolysis can be improved by optimizing laser parameters on an individual patient basis. We have studied the critical role of pulse duration (t(p)) on the treatment efficacy. STUDY DESIGN/MATERIALS AND METHODS: The V-beam laser (Candela) allowed changing t(p) over user-specified discrete values between 1.5 and 40 milliseconds by delivering a series of 100 microsecond spikes. For the 1.5 and 3 millisecond pulses, three spikes were observed at intervals t(p)/2 and for t(p)> or =6 milliseconds, four spikes separated by t(p)/3. The ScleroPlus laser (Candela) has a smooth output over its fixed 1.5 milliseconds duration. Blood vessels in the chick chorioallantoic membrane (CAM) were irradiated at fixed wavelength (595 nm), spot size (7 mm), radiant exposure (15 Jcm(-2)), and at variable t(p). The CAM contains an extensive microvascular network ranging from capillaries with diameter D<30 microm to blood vessels of D approximately 120 microm. The CAM assay allows real-time video documentation, and observation of blood flow in pre-capillary arterioles (A) and post-capillary venules (V). Vessel injury was graded from recorded videotapes. Mathematical modeling was developed to interpret results of vessel injury when varying t(p) and D. A modified thermal relaxation time was introduced to calculate vessel wall temperature following laser exposure. RESULTS: Arterioles. For increasing t(p), overall damage was found to decrease. For fixed t(p), damage decreased with vessel size. Venules. For all D, damage was smaller than for corresponding arterioles. There was no dependence of damage on t(p). For given t(p), no variation of damage with D was observed. Photothermolysis due to spiked (V-beam) vs. smooth (Scleroplus) delivery of laser energy at fixed t(p) (1.5 milliseconds), showed similar vessel injuries for al values of D (P>0.05). CONCLUSIONS: The difference between initial arteriole and venule damage could be explained by the threefold higher absorption coefficient at 595 nm in (oxygen-poor!) arterioles. In human patients, PWS consist of ectatic venules (characterized by higher absorption), so that these considerations favor the use of 595-nm irradiation for laser photothermolysis. For optimal treatment of PWS it is proposed that t(p) be between 0.1 and 1.5 milliseconds. This is based on a modified relaxation time tau'(d), defined as the time required for heat conduction into the full thickness of the vessel wall, which is assumed to have a thickness DeltaD approximately 0.1D. The corresponding tau'(d) will be a factor of about six smaller than given in the literature. For vessels with D between 30 and 300 mum, tau'(d) ranges from 0.1 to 1.5 milliseconds.