A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm2) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2 x 10(7) PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.

Neutrophils have become recognised as important contributors to the effectiveness of tumour eradication by photodynamic therapy (PDT). In this study, we have used the mouse SCCVII squamous cell carcinoma model to investigate the activity of neutrophils in tumours treated by PDT. Tumour levels of neutrophilic myeloperoxidase (MPO) demonstrated not only a massive and sustained sequestration of these cells in PDT-treated tumours but also revealed their activated state evidenced by the presence of released MPO. Among the adhesion molecules expressed on tumour vascular endothelium, ICAM-1 appears to be of primary importance in the invasion of neutrophils into PDT-treated tumours, because its functional blocking with monoclonal antibodies reduced the tumour cure rate. A marked upregulation of its ligands CD11b/CD18 and CD11c/CD18 found on neutrophils associated with PDT-treated tumours supports this assumption. To evaluate the role of inflammatory cytokines regulating neutrophil activity, neutralising antibodies were given to mice before PDT treatment. The results suggest that IL-1beta activity is critical for the therapeutic outcome, since its neutralisation diminished the cure rates of PDT-treated tumours. No significant effect was observed with anti-IL-6 and anti-TNF-alpha treatment. Further flow cytometry-based examination of neutrophils round in PDT-treated tumours revealed that these cells express MHC class II molecules, which suggests their engagement as antigen-presenting cells and involvement in the development of antitumour immune response.

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cel...

Inflammation and immunity development are well recognized as responses to tumor treatment by photodynamic therapy (PDT). To demonstrate that another major host response effector process, acute phase response, may be also induced by this cancer treatment modality, the expression of serum amyloid P component (SAP) acknowledged as a hallmark acute phase reactant in the mouse was investigated following PDT of murine FsaR fibrosarcomas. The results reveal almost 150-fold increase in the expression of SAP gene in the liver of mice bearing tumors treated by Photofrin-mediated PDT, while serum SAP levels increased around 50-fold at the peak interval about 24 hr post PDT. The same tumor treatment induced also the liver gene upregulation and serum levels elevation of another established acute phase reactant, mannose-binding lectin A (MBL-A). Both SAP and MBL-A were found to accumulate in PDT-treated tumors, but this includes local production because their genes in these tumor tissues were upregulated as well. Gene encoding C-reactive protein (CRP) was also upregulated almost 7-fold in the same tumor tissues, suggesting a rare example of CRP participation in host response of the mouse. Interleukin-6 and glucocorticoid hormones were identified as major mediators promoting tumor PDT-induced upregulation of liver SAP gene. Moreover, glucocorticoids were found to act as critical inducers of SAP gene upregulation in PDT-treated tumors. The study definitely proves the occurrence of a strong acute phase response following tumor PDT, and reveals that glucocorticoid hormones released during this development impact the expression of host response-relevant genes in PDT-treated tumors. ' 2007 Wiley-Liss, Inc.

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8 þ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.