Clinical significance of immunohistochemically detected extracellular matrix proteins and their spatial distribution in primary cancer.

Our understanding of cancer has evolved mainly from results of studies utilizing experimental models. Simplification inherent to in vitro cell culture models enabled potential ways of cell behaviour in response to various external stimuli to be described, but it has led also to disappointments in clinical trials, presumably due to the lack of crucial tissue components, including extracellular matrix (ECM). ECM and its role in healthy and diseased tissues are being explored extensively and significance of ECM for cell behaviour has been evidenced experimentally. Part of the information gathered in such research that is relevant for natural conditions of a human body can be identified by carefully designed analyses of human tissue samples. This review summarizes published information on clinical significance of ECM in cancer and examines whether effects of ECM on cell behaviour evidenced in vitro, could be supported by clinically based data acquired from analysis of tissue samples. Based on current approaches of clinical immunohistochemical analyses, impact of ECM components on tumour cell behaviour is vague. Except of traditionally considered limitations, other reasons may include lack of stratification of analyzed cases based on clinicopathologic parameters, inclusion of patients treated postoperatively by different treatments or neglecting complexity of interactions among tumour constituents. Nevertheless, reliable immunohistochemical studies represent a source of crucial information for design of tumour models comprising ECM corresponding to real clinical situation. Knowledge gathered from such immunohistochemical studies combined with achievements in tissue engineering hold promise for reversal of the unfavourable trends in the current translational oncologic research.

Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro.

BACKGROUND: Extracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure. METHODS: Human T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy. RESULTS: Cell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A. CONCLUSION: 8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.

Hexaminolevulinate-mediated photodynamic purging of marrow grafts with murine breast carcinoma.

Photodynamic therapy (PDT) with porphyrin precursors is an established therapy for certain tumors. This study aimed to explore the use of hexaminolevulinate (HAL), a porphyrin precursor, for photodynamic purging of BM grafts contaminated with cells of the 4T1 breast carcinoma cell line. The optimal PDT dose was not effective in eradicating 4T1 cells when the tumor cells were mixed with murine marrow cells in vitro. However, the number of pulmonary metastases was reduced, and the survival of experimental animals was prolonged substantially when they were subjected to TBI followed by transplantation of syngeneic BM containing metastasized 4T1 cells that had been treated ex vivo by HAL-PDT. Despite the failure of in vitro experiments, HAL-based photodynamic purging could be a useful modality for treating animals bearing an experimental breast carcinoma.

Hexaminolevulinate-mediated photodynamic purging of leukemia cells from BM.

Photodynamic therapy (PDT) with porphyrin precursors has been established for tumor treatment. This study aimed at examining applicability of hexaminolevulinate (HAL) for photodynamic purging of leukemic cells from BM grafts and evaluating the clinical relevance of in vitro models. The PDT dose resulting in no colony formation by leukemic cells in vitro, in pure form or in a mixture with BM cells, was insufficient for complete killing of the leukemic cells ex vivo and for the treatment of the leukemia-bearing animals in vivo. The efficacy of HAL-PDT in cell lines in vitro should be verified in clinically relevant in vivo models.

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples.

OBJECTIVES: Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine. METHODS: Flow cytometry was utilized to detect protoporphyrin IX in an artificial model consisting of normal urinary bladder transitional epithelial cells (NBECs) from healthy volunteers' urine and an established human urinary bladder carcinoma cell line, TCCSUP, after incubation with hexaminolevulinate (HAL). In addition, urine samples from 19 patients with histopathologically confirmed superficial bladder cancer were examined. RESULTS: Incubation of NBECs or TCCSUP cells with HAL for 1 hour resulted in production of protoporphyrin IX only in the TCCSUP cells. Incubation of a mixture of NBECs and TCCSUP cells with HAL gave rise to a separated subpopulation of cells with protoporphyrin IX fluorescence. After cell sorting by flow cytometry the protoporphyrin IX-containing subpopulation of cells was confirmed as TCCSUP cells on cytological examination. It was possible to detect 5% TCCSUP cells in the mixture of NBECs/TCCSUP cells. To test the feasibility of the method in clinica diagnosis, urine samples from patients with bladder cancer were also measured with comparable, although preliminary and limited, results to those of cytological examination. CONCLUSIONS: The preliminary results show that the technique may be feasible for the detection of bladder cancer cells in urine with possible advantages of simplicity, reliability and objectivity.

In vivo fluorescence diagnostics and photodynamic therapy of gastrointestinal superficial polyps with aminolevulinic acid. A clinical and spectroscopic study.

In the present study initial results of clinical study related to the treatment of patients having different types of precancerous lesions in the area of esophagus, stomach and intestine by photodynamic therapy (PDT) based on aminolevulinic acid (ALA-PDT) are reported. The procedure was performed by laser fibre system with the light guides introduced through biopsy channel of an endoscope. In addition, in vivo fluorescent diagnostics and spectral analyses of biopsies were performed. Each patient had a positive response to therapy. In two cases there was a total response and in other five cases more than sixty percent of suspected area was removed. Additionally, sigilocellular carcinoma of stomach was revealed in one case. It appears from the results of this study, that the treatment of precancerous lesions with ALA-PDT could be successful treatment modality.

pH-dependent spectral properties of HpIX, TPPS2a, mTHPP and mTHPC.

Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0-8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values < 5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.

Increased binding of chlorin e6 to lipoproteins at low pH values.

It is well known that the extracellular pH in tumors is lower than that of normal tissue. This has been proposed to be one of the reasons for the tumor selective uptake of several photosensitizers. Photosensitizers like chlorin e(6) are bound to blood components and delivered to different sites in the organism. Thus, the effect of pH on their interaction with human plasma needs to be studied in order to understand a possible role of the acidic microenvironment in tumors for the drug distribution. Increasing amounts of human plasma in the sample resulted in a gradual red shift of the fluorescence emission maxima of chlorin e(6), indicating binding of the drug to some of the plasma components. Titration showed that the drug-plasma interaction was pH-dependent. The titration curve had an inflection point at 7.4+/-0.1. The relative distribution of the drug among plasma components, as found after ultracentrifugation of chlorin e(6)-doped plasma in a salt gradient, showed more binding of the drug to nonlipoproteins than to lipoprotein classes at both pH values studied (6.5 and 7.4). A decrease in the pH was connected with a significant increase in drug-lipoprotein binding. The pH of the environment affects chlorin e(6)-plasma interaction and the distribution of the drug among different plasma components. The results of this study indicate a possible role of the acidic microenvironment in tumors for the preferential uptake and retention of several photosensitiziers.

Acid-base properties of chlorin e6: relation to cellular uptake.

Chlorins are attractive compounds for photodynamic therapy because of their high absorption in the red spectral region. In this study, the absorbance, fluorescence excitation and fluorescence emission spectra of chlorin e6 have been recorded as functions of pH in phosphate-buffered saline (PBS) solution with and without fetal calf serum (FCS). For pure PBS solutions, variation of the pH of the solution results in a shift of both the absorption and the fluorescence spectrum as well as in a decrease of the fluorescence intensity. Spectrophotometric and fluorimetric titration curves, based on observed changes, have been plotted. There is an indication of aggregate formation at low pH values (pH < 5). The presence of 5% FCS results in a shift of the titration curve, from an inflection point at about 6.5 to one at about 7.6. Pronounced spectral changes of the fluorescence emission spectra of protein-bound chlorin e6 (change of spectral shape, decrease of peak intensity) are also observed. The partition coefficients in the 1-octanol-water system increase with decreasing pH. Thus, relatively more of the drug is incorporated in the octanol phase at low pH. Cellular uptake of chlorin e6 in the presence of serum is significantly higher at pH 6.7 as compared with that at 7.3 and 7.6. We conclude that a change in the pH value of the surrounding medium leads to a change in the lipophilicity of chlorin e6. Such a change is likely to influence its binding to the serum proteins as well as its interaction with the plasma membrane of cells and may also be related to the selective tumor uptake of the drug.