D.E. Dolmans

The antivascular effects of photodynamic therapy (PDT) and their mechanisms are not clearly understood. Here, we examined the effects of PDT with a novel photosensitizer MV6401 on the microvasculature in a mammary tumor (MCaIV) grown in a murine dorsal skinfold chamber and in normal tissue controls. The mice were irradiated with light 15 min after i.v. administration of MV6401 when the drug was localized only in the vascular compartment, as shown by fluorescence microscopy and immunohistochemistry. PDT with MV6401 caused a dose-dependent biphasic blood flow stasis and vascular hyperpermeability, as determined by intravital microscopy. This biphasic response was classified into two components: (a) an acute response observed immediately after PDT; and (b) a long-term response observed at times greater than 3 h after PDT. The acute temporal vascular effects were characteristic of vasoconstriction but not of thrombus formation. However, the long-term vascular shutdown was mediated by thrombus formation, as evidenced by histological evaluation and inhibition with heparin. Minimal effects were observed in normal vessels after antivascular doses used against the tumor, but there was no long-term vascular damage. In concert with the stasis, a dose-dependent tumor growth delay was observed. This study provides mechanistic insights into antitumor vascular effects of PDT and suggests novel strategies for tumor treatment with PDT.

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

Photodynamic therapy (PDT) is a locally administered therapy currently being investigated in various clinical and preclinical settings. Tumor-host interaction is an important determinant of tumor biology and response to treatments. Here we report for the first time the effects of PDT on an orthotopic, murine mammary tumor model. PDT utilizes two individually nontoxic components: (a) the localization in the target site of a photosensitizing drug; and (b) the activation of the photosensitizer by light of an appropriate wavelength and energy. PDT after a single dose of the photosensitizer MV6401 induced drug dose-dependent, long-term blood flow shut down and tumor growth delay in the MCaIV tumor, grown in the mammary fat pad. The plasma half-life of MV6401 was approximately 20 min, and the drug was confined to the vascular compartment shortly after administration. However, it accumulated in the interstitial compartment at 2-6 h after the administration. Two equal MV6401 doses injected 4 h and 15 min before the light administration allowed the photosensitizer to localize in both vascular and tumor cell compartments. The fractionated drug dose PDT more effectively induced tumor growth delay than the same total dose given as a single dose either at 4 h or at 15 min before light administration. The long-term effect of the fractionated drug PDT on blood flow was also more extensive than single-dose PDT. Fractionated photosensitizer dosing PDT offers a new strategy to optimize PDT therapy.