Hexaminolevulinate-Mediated Photodynamic Purging of Marrow Grafts with Murine Breast Carcinoma
Total Page：16
File Type：pdf, Size：1020Kb
Bone Marrow Transplantation (2011) 46, 1118–1127 & 2011 Macmillan Publishers Limited All rights reserved 0268-3369/11 www.nature.com/bmt ORIGINAL ARTICLE Hexaminolevulinate-mediated photodynamic purging of marrow grafts with murine breast carcinoma BCˇunderlı´kova´1,2, V Vasovicˇ1, F Sieber3, T Furre4, E Borgen1, JM Nesland1,5 and Q Peng1,6 1Department of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway; 2International Laser Centre, Bratislava, Slovakia; 3Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA; 4Department of Medical Physics, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway; 5Faculty Division, Norwegian Radium Hospital, Medical faculty, University of Oslo, Oslo, Norway and 6Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Fudan University, Shanghai, China Photodynamic therapy (PDT) with porphyrin precursors being reinfused to the patient. Various methods have is an established therapy for certain tumors. This study recently been developed to eradicate tumor cells from the aimed to explore the use of hexaminolevulinate (HAL), autograft, leaving the normal stem cells undamaged. They a porphyrin precursor, for photodynamic purging of BM include exposure to chemotherapeutic drugs, long-term grafts contaminated with cells of the 4T1 breast carcinoma marrow cultures, immunological manipulation with immuno- cell line. The optimal PDT dose was not effective in magnetic devices or ab/complement combination and gene eradicating 4T1 cells when the tumor cells were mixed therapy (including antisense therapy). According to available with murine marrow cells in vitro. However, the number of literature, marrow purging by mafosfamide has provided pulmonary metastases was reduced, and the survival of long-term remissions in certain populations of patients with experimental animals was prolonged substantially when leukemia.1 they were subjected to TBI followed by transplantation of The ability of malignant cells to selectively accumulate syngeneic BM containing metastasized 4T1 cells that had photosensitizers offers the possibility of using photody- been treated ex vivo by HAL-PDT. Despite the failure of namic therapy (PDT) in BM purging. PDT involves in vitro experiments, HAL-based photodynamic purging administration of a tumor-localizing photosensitizer and could be a useful modality for treating animals bearing an its subsequent activation by light of appropriate wave- experimental breast carcinoma. length corresponding to the absorbance band of the Bone Marrow Transplantation (2011) 46, 1118–1127; photosensitizer, resulting in photodamage to the tumor doi:10.1038/bmt.2010.277; published online 8 November 2010 cells.2 Several exogenous photosensitizers have been pro- Keywords: hexaminolevulinate; breast carcinoma; marrow posed as agents for the purging of tumor cells from purging; photodynamic autografts, for example, merocyanine 540 (MC 540), photofrin, benzoporphyrin derivative monoacid ring A and sulfonated chloroaluminum phthalocyanine. Their efficiency was examined experimentally, especially for the treatment of leukemia.3–12 Small cell lung,13–15 prostate15 Introduction and breast tumor15–17 cells were mostly investigated from solid tumor cancers, with uncertain results. Usually, the High doses of chemotherapy and/or radiation therapy in in vitro studies evaluating phototoxicity against different combination with allogeneic BMT are used for the cells were carried out in models of separated pure cell lines, treatment of a number of hematologic malignancies and although several authors utilized artificial cell mixture selected solid tumors. However, the application of allo- models to mimic in vivo conditions.3,4,18–20 geneic bone marrow transplantation is still largely limited As the exogenous photosensitizers generally have a limited by lack of an HLA-matched donor, advanced stages of a selective accumulation in malignant cells, and a limited disease, graft failure and GVHD. Autologous BMT lacking selective photodynamic destruction of tumor cells therefore GVHD complications has thus been explored. In contrast ensues, the possibility of using 5-aminolevulinic acid (ALA) to allografts, however, autografts may harbor residual or its esters to produce endogenous protoporphyrin occult malignant cells that can cause a tumor relapse after IX (PpIX) in a much more selective manner could be considered. ALA is formed from glycine and succinyl CoA in heme biosynthesis. The last step of this process is Correspondence: Dr Q Peng, Department of Pathology, Norwegian the incorporation of iron into PpIX under the action of Radium Hospital, Oslo University Hospital, Montebello, Oslo N-0310, the ferrochelatase enzyme. By adding exogenous ALA, the Norway. E-mail: [email protected] naturally occurring PpIX may accumulate because of the 21,22 Received 17 February 2010; revised 2 July 2010; accepted 13 August limited capacity and/or low activity of ferrochelatase. 2010; published online 8 November 2010 Moreover, the activity of porphobilinogen deaminase, Photodynamic purging of tumor cells from BM BCˇunderlı´kova´ et al 1119 another enzyme involved in heme biosynthesis, is higher in disinfection. Both ends of the bones were cut and the BM some tumors,21,22 so that PpIX accumulates with a high was flushed out with sterile serum-free IMDM using a degree of selectivity in these tumors. Unfortunately, ALA 3-mL syringe with a 25-gauge needle. The total number of does not easily penetrate through cellular membrane because cells was counted. The cells were diluted with serum-free of its hydrophilic property. ALA esters, on the other hand, IMDM to a density of 8 Â 105 cells/mL. are more lipophilic and pass more easily through biological For in vitro studies, the 4T1 tumor cells were mixed with membranes.23–25 To our best knowledge, none of the ALA BM cells to produce 5 and 50% contents of the tumor cells esters have so far been studied for the purposes of in a suspension. The total cell density was kept at 8 Â 105 photodynamic purging. cells/mL. In the present study, we have explored the applicability of hexyl-ester of ALA, hexaminolevulinate (HAL), for PDT purging of BM grafts from tumor cells in the murine HAL-induced PpIX production in cells and cell cycle 4T1 breast carcinoma model. We compared the results analysis from models of individual cell types and their artificial Flow cytometry. The aliquots of 1 mL of the cell suspen- cell mixtures in vitro with those from their corresponding sions per well were used in 12-well plates. The cells were ex vivo/in vivo models of BM containing metastasized incubated with or without HAL for 4 h in a humidified 1 4T1 cells. incubator at 37 C and 5% CO2. For the measurements of intracellular PpIX, following the incubation with HAL, the cells were first scraped off from the substratum and Materials and methods repeatedly resuspended, as some of the 4T1 cells were attached to the flasks during the 4-h incubation. The cell Chemicals suspensions were washed to remove the drug-containing HAL was kindly provided by PhotoCure ASA (Oslo, incubation medium and cell pellets were then resuspended Norway). The stock solution of HAL was prepared in a in ice-cold phosphate-buffered saline. Samples, protected mixture (1:9) of ethanol and serum-free Iscove’s modified from light, were stored on ice and analyzed within Dulbecco’s medium (IMDM; Gibco, Invitrogen, Oslo, 30–60 min by flow cytometry. For cell cycle distribution studies, the cells after staining, as described later, were Norway) to a concentration of 12 mM and kept frozen at collected into tubes and kept at room temperature. À70 1C until use. All the chemicals used were of the highest purity commercially available. The measurements of intracellular PpIX and cell cycle distributions were performed using a LSR II flow cyto- meter (Becton Dickinson, San Jose, CA, USA). An argon Tumor cell line laser operating at 488 nm and a 355 nm UV laser were used The murine 4T1 metastatic mammary adenocarcinoma cell for fluorescence excitation of PpIX, propidium iodide line, growing in monolayers, was purchased from ATCC (Sigma-Aldrich, Hamburg, Germany) and Hoechst 33342 (Manassas, VA, USA, ATCC number: CRL-2539). The (Sigma, St Louis, MO, USA), respectively. The rate of data cells were incubated in vitro in IMDM supplemented with acquisition was not higher than 500 counts/s. For each 10% fetal bovine serum (Gibco, Invitrogen), L-glutamine sample, 10 000 events were collected using Diva software. (Gibco, Invitrogen), penicillin and streptomycin (Gibco, Forward scatter, side scatter and either red fluorescence Invitrogen). Passages up to number 25 were used in the (PpIX, propidium iodide) passing a combination of 685 nm present study. For experiments, the cells were resuspended dichroic and 695/40 nm band-pass filters or blue fluores- several times after trypsinization using a 1-mL syringe and cence (Hoechst 33342) passing 450/50 band-pass filter, were a 23-gauge needle to minimize cell clustering. collected simultaneously. Forward scatter-area vs side The 4T1 cells for the in vitro experiments were, after scatter-area, side scatter-area vs side scatter-width and harvesting, washed twice with serum-free IMDM, resus- forward scatter-area vs red fluorescence-area dot plots were pended and diluted in the serum-free IMDM to a density of used to gate out individual populations of cells. Mean of 5 8 Â 10 cells/mL (if not specified). the red fluorescence-area histogram was used to evaluate PpIX production. Histograms of Hoechst 33342 fluores- Animals cence were used for cell cycle comparisons. Propidium Approval for protocols of this study was obtained from the iodide fluorescence was used to estimate the content of Norwegian National Animal Research Authority, and all dead cells in the samples not incubated with HAL. experiments of animals were conducted according to For sorting of BM cells, FACS Diva flow cytometer was the National Ethical Committee’s Guideline on Animal used (Becton Dickinson). Individual cell populations were Welfare. Female BALB/c mice, 6–8 weeks old at the sorted out using forward scatter-area vs side scatter-area dot beginning of the experiment, were purchased from Harlan plots.