Modifications of DAMPs levels in extracellular environment induced by aminolevulinic acid-based photodynamic therapy of esophageal cancer cells.

PURPOSE: Immunogenic cell death plays an important role in anticancer treatment because it combines cell death with appearance of damage associated molecular patterns that have the potential to activate anticancer immunity. Effects of damage associated molecular patterns induced by aminolevulinic acid-based photodynamic therapy were studied mainly on dendritic cells. They have not been deeply studied on macrophages that constitute the essential component of the tumor microenvironment. The aim of this study was to analyze features of esophageal cancer cell death in relation to release capacity of damage associated molecular pattern species, and to test the effect of related extracellular environmental alterations on macrophages. MATERIAL AND METHODS: Esophageal Kyse 450 carcinoma cells were subjected to aminolevulinic acid-based photodynamic therapy at different concentrations of aminolevulinic acid. Resting, IFN/LPS and IL-4 macrophage subtypes were prepared from monocytic THP-1 cell line. Cell death features and macrophage modifications were analyzed by fluorescence-based live cell imaging. ATP and HMGB1 levels in cell culture media were determined by ELISA assays. The presence of lipid peroxidation products in culture media was assessed by spectrophotometric detection of thiobarbituric acid reactive substances. RESULTS: Aminolevulinic acid-based photodynamic therapy induced various death pathways in Kyse 450 cells that included features of apoptosis, necrosis and ferroptosis. ATP amounts in extracellular environment of treated Kyse 450 cells increased with increasing aminolevulinic acid concentration. Levels of HMGB1, detectable by ELISA assay in culture media, were decreased after the treatment. Aminolevulinic acid-based photodynamic therapy induced lipid peroxidation of cellular structures and increased levels of extracellular lipid peroxidation products. Incubation of resting and IL-4 macrophages in conditioned medium from Kyse 450 cells treated by aminolevulinic acid-based photodynamic therapy induced morphological changes in macrophages, however, comparable alterations were induced also by conditioned medium from untreated cancer cells. CONCLUSION: Aminolevulinic acid-based photodynamic therapy leads to alterations in local extracellular levels of damage associated molecular patterns, however, comprehensive studies are needed to find whether they can be responsible for macrophage phenotype modifications.

Characterisation of collagen type I matrices for pathophysiologically relevant spatial cancer cell cultures.

Specific cues provided to cells by the extracellular matrix (ECM) are determined by its composition. Except of collagens other naturally occurring ECM components should be considered in designing 3D models of diseases. We used spectrophotometric and rheological measurements and confocal imaging to characterise collagen matrices of human origin that can be modified by clinically relevant ECM components. pH of the neutralising solution, but not incubation of solidified collagen matrices in serum-free culture medium with pH 5.0-9.0 affected distribution of collagen fibres. Admixture of fibronectin or tenascin-C influenced assembly kinetics and resulted in slight increase in the Young's moduli of the matrices, indicating their incorporation into the collagen matrices. Co-localization of fibronectin with collagen fibres was confirmed by fluorescence imaging. Various cell types relevant for tumour tissue were able to proliferate within the matrices suggesting that they can be used to study role of ECM components in cancer in spatial models.

Suppression of resistance to aminolevulinic acid-based photodynamic therapy in esophageal cell lines by administration of iron chelators in collagen type I matrices.

PURPOSE: Photodynamic therapy (PDT) utilizes visible light to activate the cytotoxic effects of photosensitizing drugs. PDT protocols require optimization to overcome treatment resistance and induce a beneficial anti-tumor immune response. The aim of this study was to examine the possibility to suppress the resistance of esophageal cell lines to aminolevulinic acid (ALA)-PDT by administration of iron chelators to induce sufficient cell cytotoxicity under pathophysiologically relevant conditions, mimicking the advanced stages of cancer. MATERIALS AND METHODS: Effects of ALA-PDT in combination with iron chelators were compared in three esophageal cell lines in conventional monolayers and in 3 D cultures based on collagen type I. Modified colony assay and fluorescence-based live cell imaging, respectively were applied. The latter was used also to test the capability of pre-polarized macrophages to interact with cancer cells subjected to ALA-PDT with or without iron chelators. RESULTS: Iron chelators were effective in the enhancement of ALA-PDT in all cell lines under both culture conditions. Fluorescence evaluation of cell viability in 3 D cultures indicated the contribution of apoptotic cell death after ALA-PDT, both with and without iron chelators. Engulfment of remnants of dead cancer cells by macrophages in 2 D cultures was indicated, however, the interaction between macrophages and cancer cells in 3 D cultures subjected to ALA-PDT with or without iron chelators was not present. CONCLUSIONS: The potential of iron chelators to enhance ALA-PDT was maintained in 3 D collagen matrices. Although PDT dose (ALA concentration, light exposure time) required modification in a cell line-dependent manner to achieve a comparable effect of PDT alone in conventional monolayers and in collagen matrices, the potential of iron chelators to suppress the resistance of esophageal cells to ALA-PDT was not influenced by a fibrillar collagen matrix.

Phenotypical modifications of immune cells are enhanced by extracellular matrix.

Immune cells not only constitute tumour microenvironment but they may even affect disease prognosis as a result of dual functional roles that they may play in tumour tissues. Two frequently used established immune cell lines (lymphocytic Jurkat and monocytic THP-1) were used to test whether microenvironmental factors, especially molecular components of extracellular matrix, can shape the phenotype of immune cells. Proliferation, morphological and phenotypical analyses were applied to compare behaviour of the immune cells, typically cultured as suspensions in culture medium, with their behaviour in collagen type I-based and Matrigel-based 3D cultures. Density of both immune cell types in routine suspension cultures affected their subsequent proliferation in extracellular matrices. THP-1 cells appeared to be more sensitive to their surrounding microenvironment as judged from extracellular matrix type-dependent changes in their cell doubling times and from slight increase in their diameters in both extracellular matrix-containing cell cultures. Moreover, even chemically uninduced monocytic THP-1 cells were present in a minor fraction as CD68 positive cell population in collagen type I matrix indicating their partial differentiation to macrophages. Observed modifications of immune cells by microenvironmental factors may have profound implications for their roles in healthy and pathological tissues.

Modulation of aminolevulinic acid-based photoinactivation efficacy by iron in vitro is cell type dependent.

Iron availability to cells may be modified in the tumour microenvironment, which may be involved in treatment response. Iron availability affects the conversion of protoporphyrin IX to heme, which likely determines the efficacy of aminolevulinic acid-based photodynamic therapy (ALA-based PDT). We compared photoinactivation efficacy in three oesophageal cell lines in culture media differing in iron content, DMEM and RPMI 1640, and in RPMI 1640 supplemented with iron to understand the importance of iron presence for ALA-based PDT outcome. ALA-based PDT was more efficacious in DMEM than in RPMI 1640 in all tested cell lines. Consistently, the highest protoporphyrin IX fluorescence signals, indicating the highest level of protoporphyrin IX production, were detected from cell colonies incubated in DMEM compared to those incubated in RPMI 1640 irrespective of iron presence. Components in the culture media other than iron ions are likely to be responsible for the observed differences in two culture media. Nevertheless, iron supplementation to RPMI 1640 showed that the presence of ferric ions in the concentration range 0-8 mg/l affected ALA-based PDT efficacy in a cell type-dependent manner. In poorly differentiated carcinoma cells, the increased efficacy of ALA-induced photoinactivation in the presence of 0.1 mg/l of supplemented iron was found. At the same iron concentration, the slightly different mitochondrial potential at no modifications of the iron labile pool was observed. The efficacy of ALA-based PDT in vitro depends on the choice of culture medium and the presence of iron ions in culture medium depending on intrinsic properties of cells.

Extracellular matrix affects different aspects of cell behaviour potentially involved in response to aminolevulinic acid-based photoinactivation.

Two-dimensional cell cultures do not seem to be reliable models for anticancer drug discovery and validation. Numerous in vitro tumour models of different complexity have been evaluated with the aim to enhance anticancer drug development, but whether all these models could be considered as physiologically relevant is a question. Even type of the extracellular matrix may markedly influence experimental results and supposedly also clinical treatment outcome. By using three human oesophageal cell lines and three-dimensional cultures based on collagen type I, abundant component of stromal tissue, and Matrigel, a surrogate of basement membrane, we tested the impact of extracellular matrix on different aspects of cell behaviour. We applied live cell fluorescence confocal microscopy in combination with image analysis and supplemented it with immunohistochemical analysis of differentiation markers in fixed samples. We found that cell morphogenesis, differentiation, extracellular vesicle formation, protoporphyrin IX production from aminolevulinic acid and response to subsequent photodynamic intervention induced by red light may be affected by the type of extracellular matrix and these modifications occur in a cell-type dependent manner. Our results demonstrate that the choice of the correct extracellular matrix for in vitro tumour models is crucial for gathering clinically relevant information from in vitro experimental studies.

Potassium hydroxide treatment of UV-curable polysiloxane-type polymer for reproducible enhancement of cell adhesion and survival.

Polysiloxanes have shown exquisite properties for fabrication of microstructures for various biomedical and biotechnological applications. Nevertheless, their biocompatibility in terms of cell adhesion and survival ability is controversial. A simple polysiloxane modifying procedure that reproducibly enhances cell adhesion was proposed. Sonication of the hybrid organic-inorganic polymer of polysiloxane type, Ormocomp, in potassium hydroxide (KOH)/ethanol solution enhanced adhesion and subsequent survival of a panel of four cell lines. Characterization of surface properties of untreated and KOH-treated Ormocomp coatings has revealed considerable negative charge of Ormocomp substrates based on measurements of zeta potentials. KOH treatment did not modify surface morphology as visualized by scanning electron microscopy, but it resulted in alteration in both chemical composition according to SIMS analysis and hydrophilicity evaluated by static water contact angles. The results suggest that the failure of the adherent cells to survive on Ormocomp coatings is related to cell adhesion. The negative surface charge of Ormocomp substrates may be one of the influencing factors; however, the modification of surface chemistry mediated by KOH and the resulting increase in hydrophilicity accompanied by modification of protein adsorption are more likely responsible for enhanced cell adhesion and survival on Ormocomp coatings. KOH treatment thus may serve as a simple, cost-effective procedure modifying polysiloxane-type polymers that leads to reproducible enhancement of cell adhesion.

Extracellular Matrix Containing in vitro Three-dimensional Tumor Models in Photodynamic Therapy-related Research.

Three-dimensional (3D) tumor models have been intensively evaluated for their use in cancer research, and there is a strong rationale behind using 3D cell cultures in photodynamic therapy (PDT)-related experimentation. In this contribution, it is explained why 3D cell cultures containing extracellular matrix (ECM) are preferred for this purpose. Results of experimental studies utilizing ECM-containing 3D cellular models in PDT research are summarized. Finally, the design of in vitro 3D models that would provide clinically relevant information is discussed.

Cell-type dependent response to photodynamic treatment in 3D collagen cell cultures.

Photodynamic therapy (PDT) can induce direct tumor cell destruction, indirect tumor cell inactivation due to vascular occlusion as well as immune response. Evidence suggests that the PDT-induced cell death is dependent on both PDT protocol-related as well as microenvironmental factors, and its mode is also decisive for the type of immune response. This suggests potential interrelationship among PDT-induced tumor cell destruction, immune response and microenvironmental factors. In the present study we analyzed the effect of a microenvironmental factor - extracellular matrix on the cellular response to photodynamic treatment in vitro. By using conventional proliferation and modified cell survival assays as well as fluorescence imaging, we compared efficacy of aminolevulinic acid (ALA)-PDT to inactivate three esophageal cell lines in two- and three-dimensional formats. Modified cell colony assay indicated comparable PDT doses leading to death of both Kyse 450 and Het-1A cells on plastic, whereas Kyse 70 cells were only partially responsive. In 3D collagen matrices, we were able to induce only death of Kyse 450 cells by ALA-PDT, if analyzed 24h after treatment. Consistently, only Kyse 450 cells were able to produce detectable amounts of PpIX after incubation of their 3D collagen cultures with ALA. Our results demonstrate that cellular response to ALA-PDT is cell-type dependent both in two- and three-dimensional formats and indicate that the extracellular matrix might modify it.

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture.

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

Issues to be considered when studying cancer in vitro.

Various cancer treatment approaches have shown promising results when tested preclinically. The results of clinical trials, however, are often disappointing. While searching for the reasons responsible for their failures, the relevance of experimental and preclinical models has to be taken into account. Possible factors that should be considered, including cell modifications during in vitro cultivation, lack of both the relevant interactions and the structural context in vitro have been summarized in the present review.

Factors implicated in the assessment of aminolevulinic acid-induced protoporphyrin IX fluorescence.

BACKGROUND: Photodynamic therapy and photodiagnosis of cancer requires preferential accumulation of fluorescent photosensitizers in tumors. Clinical evidence documents feasibility of ALA-based photodiagnosis for tumor detection. However, false positive results and large variations in fluorescence intensities are also reported. Furthermore, selective accumulation of fluorescent species of photosensitizers in tumor cell lines, as compared to normal ones, when cultured in vitro, is not always observed. To understand this discrepancy we analyzed the impact of various factors on the intensity of detected PpIX fluorescence. METHODS: Impacts of cell type, mitochondrial potential, cell-cell interactions and relocalization of PpIX among different cell types in co-cultures of different cell lines were analyzed by confocal microscopy and flow cytometry. Fluorescence spectroscopy was used to estimate absolute amounts of ALA-induced PpIX in individual cell lines. Immunofluorescence staining was applied to evaluate the ability of cell lines to produce collagen. RESULTS: Higher ALA-induced PpIX fluorescence in cancer cell lines as compared to normal ones was not detected by all the methods used. Mitochondrial activity was heterogeneous throughout the cell monolayers and could not be clearly correlated with PpIX fluorescence. Positive collagen staining was detected in all cell lines tested. CONCLUSIONS: Contrary to in vivo situation, ALA-induced PpIX production by cell lines in vitro may not result in higher PpIX fluorescence signals in tumor cells than in normal ones. We suggest that a combination of several properties of tumor tissue, instead of tumor cells only, is responsible for increased ALA-induced PpIX fluorescence in solid tumors. GENERAL SIGNIFICANCE: Understanding the reasons of increased ALA-induced PpIX fluorescence in tumors is necessary for reliable ALA-based photodiagnosis, which is used in various oncological fields.

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells.

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.

Subcellular localization pattern of protoporphyrin IX is an important determinant for its photodynamic efficiency of human carcinoma and normal cell lines.

Photodynamic therapy (PDT) is a combination of light with a lesion-localizing photosensitizer or its precursor to destroy the lesion tissue. PDT has recently become an established modality for several malignant and non-malignant conditions, but it can be further improved through a better understanding of the determinants affecting its therapeutic efficiency. In the present investigation, protoporphyrin IX (PpIX), an efficient photosensitizer either endogenously induced by 5-aminolevulinic acid (ALA) or exogenously administered, was used to correlate its subcellular localization pattern with photodynamic efficiency of human oesophageal carcinoma (KYSE-450, KYSE-70) and normal (Het-1A) cell lines. By means of fluorescence microscopy ALA-induced PpIX was initially localized in the mitochondria, whereas exogenous PpIX was mainly distributed in cell membranes. At a similar amount of cellular PpIX PDT with ALA was significantly more efficient than photodynamic treatment with exogenous PpIX at killing all the 3 cell lines. Measurements of mitochondrial membrane potential and intracellular ATP content, and electron microscopy showed that the mitochondria were initially targeted by ALA-PDT, consistent with intracellular localization pattern of ALA-induced endogenous PpIX. This indicates that subcellular localization pattern of PpIX is an important determinant for its PDT efficiency in the 3 cell lines. Our finding suggests that future new photosensitizers with mitochondrially localizing properties may be designed for effective PDT.

pH dependent uptake of porphyrin-type photosensitizers by solid tumor cells in vitro is not induced by modification of transmembrane potential.

The uptake of HpIX, TPPS2a and mTHPC by WiDr, THX cells and skin fibroblasts at pH 7.4 and 6.8 was compared. In the absence of serum, the uptake of HpIX was higher at lower pH. The difference was significant in WiDr cells (P < 0.01) and skin fibroblasts (P < 0.05). TPPS2a nor mTHPC showed any pH dependent uptake. Lowering the extracellular pH resulted in a significant depolarization (3-8 mV) of the cells. Application of tetraethylammonium chloride did not affect the cellular uptake of any of the photosensitizers. We conclude that the pH dependent uptake of photosensitizers is not mainly related to altered transmembrane potential.

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy.

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2 +/- 0.8 degrees - 108 charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4 +/- 1.1 x 108 charges per cell. This is significantly higher (p < 0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about -35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.

pH-dependent modification of lipophilicity of porphyrin-type photosensitizers.

Structural modifications of photosensitizers (changes in protonation, ionic state and aggregation state) under different environmental conditions should be precisely determined to understand the interaction of the photosensitizers with biological systems. In the present study partition coefficients of hematoporphyrin IX (HpIX), disulfonated meso-tetraphenylporphine, meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin in the 1-octanol-phosphate buffer system were determined in the pH region 4.0-8.0. Only the partition coefficients of HpIX and mTHPP were found to be pH dependent. Computer processing of fluorimetric titration data was applied to estimate pKa values of the imino nitrogens of mTHPP. Monoprotonated species of mTHPP seem to be unstable or nonexistent. The possibility that both imino nitrogens of this dye are protonated according to a common pKa is proposed. The pKa value of the imino nitrogens of mTHPP was found to be 2.99 +/- 0.04 after the application of a model taking aggregation of the drug into account. The contributions of various aqueous ionic species of mTHPP as functions of pH were calculated and compared with partition coefficients.

pH effects on the cellular uptake of four photosensitizing drugs evaluated for use in photodynamic therapy of cancer.

The difference in extracellular pH in malignant as compared to normal healthy tissues has been proposed to contribute to selective uptake of photosensitizers in tumors. Hematoporphyrin IX (HpIX), disulfonated meso-tetraphenylporphine (TPPS(2a)), meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin (mTHPC) were chosen to examine the pH dependence of their cellular drug uptake. The study was performed in the pH range 6.5-8.0 and showed that significantly higher amounts of the drug are taken up by T-47D cells at low pH values only in the case of HpIX. The pH value of the incubation medium did not influence the cellular uptake of mTHPP, mTHPC and TPPS(2a) significantly. The present work indicates that tumor selectivity of dyes, which get more lipophilic with decreasing pH value, may be related to the low extracellular pH value.

pH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures.

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.