Mark G. Petersen

Treatment of Murine Cutaneous Melanoma with Near Infrared Light

Treatment of cutaneous melanoma (M‐3 and B16‐F10 implanted in mice) with rapidly‐scanned, tightly‐focused near infrared light elicits selective destruction of tumor tissue. A single laser treatment yielded complete eradication in >90% of B16‐F10 tumors with thicknesses of approximately 3 mm; amelanotic M‐3 tumors proved less responsive (ca 25% clearance rate). In addition to local tumor destruction, laser treatment of B16‐F10 tumors in immunocompetent mice stimulated enhanced cytokine levels (interleukin‐2 and interleukin‐10) within treated tumor tissues and rejection of tumor cells upon a subsequent challenge dose. Such an antitumor immune response may lead to improved outcomes at both the treatment site and at sites of distant metastasis.

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: Evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial NeoR gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 x 10(3) to 1.5 x 10(5) cells/cm2) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 micrograms/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the NeoR gene sequence at 2 and 3 wk. In contrast to transduced ECs, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.

A centering balloon for photodynamic therapy of esophageal cancer tested in a canine model.

Delivery of uniform circumferential light is desirable during photodynamic therapy of early or advanced esophageal cancer in human beings. Studies were performed in the canine esophagus to investigate whether use of a centering balloon would improve circumferential illumination of esophageal mucosa for photodynamic therapy of esophageal cancer. When the centering balloon was used, photodynamic therapy produced uniform and circumferential injury. With the cylindrical diffuser used in human studies, non-uniform and focal esophageal injury occurred. Placement of isotropic probes on the balloon wall allowed measurement and verification of relatively uniform light doses delivered to esophageal mucosa during balloon photodynamic therapy. The centering balloon has a potential role in improving light dosimetry during esophageal photodynamic therapy.

Photodynamic Therapy with Ultrafast Lasers

The photodynamic properties of several photosensitive compounds have been evaluated in vivo using simultaneous two-photon excitation (TPE) and multi-photon excitation (MPE). TPE and MPE are effected using a mode-locked laser, such as the mode-locked titanium:sapphire or Nd:YLF laser, the near infrared output of which allows direct promotion of various non-resonant transitions. Such lasers are exceptionally well suited for non-linear activation of exogenous or endogenous PDT agents in biological systems due to their extremely short pulse width, modest pulse energy, and high repetition rate; these features combine to effect efficient PDT activation with minimal potential for non- specific biological damage, improved spatial localization of activation, and enhanced depth of penetration. Results in several murine models are presented.

Photodynamic therapy using Verteporfin and 630-nm laser light in canine esophagus

Objective: The goal ofthis study was to determine a relationship between light dose and the delay time between administration of Verteporfin and light to ablate canine esophageal mucosa. Materials and Methods: Verteporfin was administered IV (0.75 mg/lg). 630-nm light from KTP/Dye laser was delivered using a 9-cm diffuser inside a specially designed reflective 7-cm windowed balloon. Initially, animals were treated at doses of 160 J/cm, 180 J/cm, 200 J/cm, and 220 J/cm 2-3 hours after Verteporfin injection. Based on the acute response in these animals and the plasma Verteporfin clearance, other treatments were devised and tested at 60 J/cm (at 1 5 minutes), 80 J/cm (22 minutes), 100 J/cm (30 minutes), and 145 J/cm (60 minutes). Results: In initial animals, 200 J/cm at 2 hours induced acceptable mucosal ablation. Using this light dose and the plasma drug clearance, subsequent treatments were tested resulting in similar injuries using 60 3/cm (15 minutes), 80 3/cm (22 minutes), 100 3/cm (30 minutes), and 145 J/cm (60 minutes). Conclusions: A relationship was established relating the light dose and the delay time between administration oflight and Verteporfin for ablation ofnormal canine esophageal mucosa.

Balloon photodynamic therapy of esophageal cancer: effect of increasing balloon size

We have found that an esophageal centering balloon improves light delivery to esophageal mucosa during photodynamic therapy (PDT). However, balloon pressure on esophageal mucosa could possibly reduce mucosal blood flow and oxygenation, therefore reducing the photodynamic therapy effect. Studies were performed in the canine esophagus of 10 animals to investigate whether increasing the size of the centering balloon and hence the pressure on esophageal mucosa would alter the tissue effect of PDT. It was concluded that increasing balloon size resulted in reduced tissue damage when mucosal equivalent light dose was administered during photodynamic therapy. Proper sizing of centering balloons will be necessary for balloon PDT of esophageal mucosal dysplasia or cancer.

Comparison between pulsed and continuous-wave lasers for photodynamic therapy

A study was conducted in the normal canine esophagus to compare continuous wave (argon- pumped dye-laser) and pulsed (KTP/532-pumped dye-laser) laser light for photodynamic therapy with PHOTOFRINR. 48 hours post injection, 630 nm laser light was delivered using a 24 mm diameter cylindrical esophageal PDT balloon positioned at either distal or proximal esophagus. A 1.0 cm cylindrical diffuser placed in the center of the balloon delivered 300 J/cm of light at an intensity of 400 mW/cm. Three dogs received continuous wave (CW) light proximally and pulsed light distally. Four dogs received CW light distally and pulsed light proximally. The light dose delivered to the esophageal mucosa was measured using three isotropic probes placed on the balloon wall. Similar mucosal light doses were verified for sites receiving pulsed or CW laser light. Two days after PDT, the severity of the esophageal lesions were evaluated endoscopically, grossly and histologically. While some response variability was observed among different animals, endoscopic examination of the lesions revealed comparable injury from CW and pulsed light in each subject. Based on the gross and histological examination of the lesions, the CW and pulsed laser-induced injuries could not be distinguished.