Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

[Photochemical internalisation (PCI): a further development of photodynamic therapy for the treatment of skin cancer]. / Photochemische Internalisierung (PCI): eine Weiterentwicklung der photodynamischen Therapie zur Behandlung von Hautkrebs.

Recently, several new and non-invasive methods have been introduced for the treatment of skin cancers. Topical creams using the immune modulator imiquimod or the COX inhibitor diclofenac (with hyaluronic acid) are now registered for use against neoplasms such as basal or squamous cell carcinoma. Another modern treatment option is photodynamic therapy (PDT). A refined version of PDT, namely photochemical internalisation, is currently subject to a first clinical trial in patients with osteosarcoma, squamous cell carcinoma, head and neck cancer as well as adenocarcinoma of the breast. Preliminary results from this trial suggest that PCI seems to be a promising treatment.

Enhanced photodynamic destruction of a transplantable fibrosarcoma using photochemical internalisation of gelonin.

Photochemical internalisation (PCI) is a technique for releasing biologically active macromolecules from endocytic vesicles by light activation of a photosensitiser localised in the same vesicles of targeted cells. This study investigated the PCI of the toxin gelonin as a way of enhancing the effect of photodynamic therapy (PDT) on a human malignant fibrous histiocytoma transplanted into nude mice using the photosensitiser disulphonated aluminium phthalocyanine (AlPcS(2a)). Pharmacokinetic studies after intraperitoneal administration showed that the serum level of AlPcS(2a) fitted a biexponential model (half-lives of 1.8 and 26.7 h). The tumour concentration was roughly constant up to 48 h, although fluorescence microscopy showed that the drug location was initially mainly vascular, but became intracellular by 48 h. To compare PDT with PCI, 48 h after intraperitoneal injection of 10 mg kg(-1) AlPcS(2a), and 6 h after direct intratumour injection of 50 microg gelonin (PCI) or a similar volume of phosphate-buffered saline (PDT controls), tumour-bearing animals were exposed to red light (150 J cm(-2)). Complete response was observed for more than 100 days in 50% of the PCI tumours but only 10% of the PDT tumours (P<0.01). In tumours examined histologically 4 days after light delivery, the depth of necrosis was 3-4 mm after PDT, but 7 mm after PCI. The deeper effect after PCI demonstrates that the light fluence needed to kill tumour is less than with PDT. We conclude that PCI with gelonin can markedly enhance the effect of PDT on this type of tumour and may have a role clinically as an adjunct to surgery to control localised disease.

Porphyrin-related photosensitizers for cancer imaging and therapeutic applications.

A photosensitizer is defined as a chemical entity, which upon absorption of light induces a chemical or physical alteration of another chemical entity. Some photosensitizers are utilized therapeutically such as in photodynamic therapy (PDT) and for diagnosis of cancer (fluorescence diagnosis, FD). PDT is approved for several cancer indications and FD has recently been approved for diagnosis of bladder cancer. The photosensitizers used are in most cases based on the porphyrin structure. These photosensitizers generally accumulate in cancer tissues to a higher extent than in the surrounding tissues and their fluorescing properties may be utilized for cancer detection. The photosensitizers may be chemically synthesized or induced endogenously by an intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) or 5-ALA esters. The therapeutic effect is based on the formation of reactive oxygen species (ROS) upon activation of the photosensitizer by light. Singlet oxygen is assumed to be the most important ROS for the therapeutic outcome. The fluorescing properties of the photosensitizers can be used to evaluate their intracellular localization and treatment effects. Some photosensitizers localize intracellularly in endocytic vesicles and upon light exposure induce a release of the contents of these vesicles, including externally added macromolecules, into the cytosol. This is the basis for a novel method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, gene-encoding plasmids, adenovirus, peptide-nucleic acids and the chemotherapeutic drug bleomycin. The background and present status of PDT, FD and PCI are reviewed.

Light directed gene transfer by photochemical internalisation.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

Photochemical disruption of endocytic vesicles before delivery of drugs: a new strategy for cancer therapy.

The development of methods for specific delivery of drugs is an important issue for many cancer therapy approaches. Most of macromolecular drugs are taken into the cell through endocytosis and, being unable to escape from endocytic vesicles, eventually are degraded there, which hinders their therapeutic usefulness. We have developed a method, called photochemical internalization, based on light-induced photochemical reactions, disrupting endocytic vesicles specifically within illuminated sites e.g. tumours. Here we present a new drug delivery concept based on photochemical internalization-principle -- photochemical disruption of endocytic vesicles before delivery of macromolecules, leading to an instant endosomal release instead of detrimental stay of the molecules in endocytic vesicles. Previously we have shown that illumination applied after the treatment with macromolecules substantially improved their biological effect both in vitro and in vivo. Here we demonstrate that exposure to light before delivery of protein toxin gelonin improves gelonin effect in vitro much more than light after. However, in vitro transfection with reporter genes delivered by non-viral and adenoviral vectors is increased more than 10- and six-fold, respectively, by both photochemical internalization strategies. The possible cellular mechanisms involved, and the potential of this new method for practical application of photochemical internalization concept in cancer therapy are discussed.

5-Aminolevulinic acid-based photochemical internalization of the immunotoxin MOC31-gelonin generates synergistic cytotoxic effects in vitro.

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.

In vivo documentation of photochemical internalization, a novel approach to site specific cancer therapy.

Photochemical internalization (PCI) is a unique procedure for site-specific delivery of several types of membrane-impermeable molecules to the cytosol of target cells. The technology is based on photochemical-induced release of endocytosed macromolecules from endosomes and lysosomes into the cytosol. The purpose of this study was to evaluate the therapeutic potential of PCI of the type I ribosomal-inactivating protein gelonin in an animal model. The photosensitizer aluminum phthalocyanine disulfonate (AlPcS(2a)) was injected intraperitoneally (10 mg/kg) into athymic female BALB/c (nu/nu) nude mice (8-9 mice per group) with subcutaneously growing human adenocarcinoma (WiDr) tumors 48 hr before exposure to 135 J/cm(2) of red light focused on the tumor. Six hours before light exposure a single dose of 50 microg gelonin was administrated intratumorally. Tumor growth was measured at least twice a week. After immunomagnetic separation of in vivo growing tumor cells the subcellular localization of the photosensitizer was evaluated by fluorescence microscopy. The photosensitizer localized in endocytic vesicles in in vivo growing WiDr cells. Furthermore, it was found that in vitro gelonin treatment of WiDr cells isolated from photosensitizer-treated mice potentiated a light-induced decrease of clonal survival. Complete remission in 6 of 9 (67%) of the treated mice were induced. Our findings indicate that photochemical treatment with the photosensitizer AlPcS(2a) activates the cytotoxic potential of gelonin in vivo. These results demonstrate that the synergistic effect of combining photoactivation of photosensitizer located in endocytic vesicles and gelonin is indeed a result of PCI of gelonin.

Release of gelonin from endosomes and lysosomes to cytosol by photochemical internalization.

Gelonin, a type I ribosome-inactivating plant toxin, executes N-glycosidase activity on eukaryotic ribosomes. However, on intact cells, gelonin is relatively non-toxic, due to an incapability to penetrate cell membranes. Recently, a novel method, photochemical internalization (PCI), was invented for the translocation of membrane-impermeable molecules including gelonin to the cytosol [K. Berg et al., Cancer Res. 59 (1999) 1180-1183]. The combination of gelonin and photoactivation of endosomal and lysosomal localizing photosensitizers gives strong synergistic cytotoxic effects. In this study, we have evaluated the intracellular transport and stability of gelonin. By fluorescence microscopy, it was shown that gelonin co-localizes with the endosomal and lysosomal localizing photosensitizer, aluminum phthalocyanine with two sulfonate groups on adjacent phenyl rings, and both molecules re-localized to cytosol subsequently to light exposure. Gelonin accumulated in endosomal compartments by incubation at 18 degrees C was released to cytosol by PCI with concomitant inhibition of protein synthesis indicating that PCI can be executed through rupture of endosomal vesicles. The cathepsin inhibitor L-trans-epoxysuccinyl-leucyl amido(4-guanido)butane increased the cytotoxic effect of gelonin after PCI when gelonin was provided as a 2 h pulse followed by 4 h chase before PCI. Thus, although gelonin can enter the cytosol from lysosomes, lysosomal degradation is a limiting factor for the outcome of PCI of gelonin.

Photochemical internalisation increases the cytotoxic effect of the immunotoxin MOC31-gelonin.

Photochemical internalisation (PCI) was recently demonstrated as a unique procedure for site-specific delivery of several types of membrane impermeable macromolecules from endocytotic vesicles to the cytosol (Berg et al., 1999). The technology is based on the cytosolic release of endocytosed macromolecules from endosomes and lysosomes upon exposure of cells to photosensitising compounds, which became localised to these vesicles, and light. In our study the possibility to increase the cytotoxic effect of the immunotoxin MOC31-gelonin by PCI was examined. The type I ribosome-inactivating protein gelonin was covalently linked to the monoclonal IgG1 antibody MOC31, directed against epithelial glycoprotein-2 (EGP-2), an antigen expressed on most carcinoma cells. Five different cell lines, of which 4 expressed EGP-2, were treated with MOC31-gelonin and endosomal and lysosomal localising photosensitisers, followed by exposure to light. Insignificant cytotoxicity of the MOC31-gelonin was observed when the cells were incubated with the immunotoxin alone. However, in combination with endosomal and lysosomal localising photosensitizers, we demonstrate synergistic toxic effect of the MOC31-gelonin conjugate in a light-dependent manner. Our results indicate that PCI is a promising tool for increasing the cytotoxicity of immunotoxins, which is important for further improvement of the PCI concept towards possible use in cancer therapy.

Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Use of 5-aminolevulinic acid esters to improve photodynamic therapy on cells in culture.

Human tumor cells of the lines WiDr (adenocarcinoma of the rectosigmoid colon), NHIK 3025 (carcinoma of the cervix), and V79 Chinese hamster fibroblasts were treated with 5-aminolevulinic acid (ALA) and ALA esterified to C1-C3 and C6-C8 chained aliphatic alcohols (ALA-esters). In the human cell lines, esterification of ALA with the long-chain (C6-C8) alcohols was found to reduce 30-150-fold the amount of ALA needed to reach the same level of protoporphyrin IX (PpIX) accumulation as with non-esterified ALA. The long-chained ALA-esters were less efficient in stimulating PpIX formation in V79 cells, i.e., the same amount of PpIX was formed by a 1-2.6-fold lower concentration of long-chained ALA-esters than with ALA. Short-chained ALA-esters (C1-C3) induced 5 to 10 times lower PpIX accumulation than ALA in all of the cell lines. High-performance liquid chromatography and fluorescence microscopic studies indicated that esterification of ALA has neither impact on the fluorescing porphyrin species formed nor impact on their intracellular localization. The PpIX formed from ALA-esters and ALA was found to be equally efficient in sensitizing cells to photoinactivation. The present results indicate that esterified ALAs are new and promising drugs for use in photochemotherapy of cancer.

Extracellular phospholipase A2 expression in sarcoidosis.

This study was conducted in order to focus upon the Ca2- dependent secretory non-pancreatic phospholipase A2 (npPLA2) enzyme and its possible role in the pathophysiology of sarcoidosis. Serum samples were taken from 24 patients with sarcoidosis to determine the levels of npPLA2. Moreover, in another group of patients with active chest x-ray stage II and III sarcoidosis, bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial lung biopsies (TBL) were taken. Highly significant increase of npPLA2 in serum was found in patients compared to controls (p < 0.001). Furthermore, those patients with stable and inactive disease and those who were under treatment with corticosteroids, tended to have lower values than those with active disease and those who were untreated. An intense accumulation of npPLA2 was found in smooth muscle tissue in lung biopsy specimens, in close connection with fibroblast accumulation and deposition of collagen. These cells also stained positive for alpha-smooth muscle actin (alpha-SMA). In addition, when using the technique of in situ hybridization, expression of npPLA2-mRNA was found in the fibroblast layer surrounding the epitheloid cell granulomas. These fibroblasts did not stain positive for alpha-SMA. Our data suggest that npPLA2 is actively involved, and has an important role, in the pathophysiology of sarcoidosis.