Protoporphyrin IX Fluorescence Research Articles

Photodynamic anticancer activity evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates

5-Aminolevulinic acid (ALA) and its derivatives, serving as the endogenous precursor of the photosensitizer (PS) protoporphyrin IX (PpIX), successfully applied in tumor imaging and photodynamic therapy (PDT). ALA and its derivatives have been used to treat actinic keratosis (AK), basal cell carcinoma (BCC), and improve the detection of superficial bladder cancer. However, the high hydrophilicity of ALA and the conversion of PpIX to heme have limited the accumulation of PpIX, hindering the efficiency and potential application of ALA-PDT. This study aims to evaluate the PDT activity of three rationally designed series of ALA-HPO prodrugs, which were based on enhancing the lipophilicity of the prodrugs and reducing the labile iron pool (LIP) through HPO iron chelators to promote PpIX accumulation. Twenty-four ALA-HPO conjugates, incorporating amide, amino acid, and ester linkages, were synthesized. Most of the conjugates, exhibited no dark-toxicity to cells, according to bioactivity evaluation. Ester conjugates 19a-g showed promoted phototoxicity when tested on tumor cell lines, and this increased phototoxicity was strongly correlated with elevated PpIX levels. Among them, conjugate 19c emerged as the most promising (HeLa, IC50 = 24.25 ± 1.43 μM; MCF-7, IC50 = 43.30 ± 1.76 μM; A375, IC50 = 28.03 ± 1.00 μM), displaying superior photodynamic anticancer activity to ALA (IC50 > 100 μM). At a concentration of 80 μM, the fluorescence intensity of PpIX induced by compound 19c in HeLa, MCF-7, and A375 cells was 18.9, 5.3, and 2.8 times higher, respectively, than that induced by ALA. In conclusion, cellular phototoxicity showed a strong correlation with intracellular PpIX fluorescence levels, indicating the potential application of ALA-HPO conjugates in ALA-PDT.

Evaluation of the potential of Delta-aminolevulinic acid for simultaneous detection of bioburden and anti-microbial photodynamic therapy of MRSA infected wounds in Swiss albino mice

BackgroundThe dramatic increase of drug-resistant bacteria necessitates urgent development of platforms to simultaneously detect and inactivate bacteria causing wound infections, but are confronted with various challenges. Delta amino levulinic acid (ALA) induced protoporphyrin IX (PpIX) can be a promising modality for simultaneous bioburden diagnostics and therapeutics. Herein, we report utility of ALA induced protoporphyrin (PpIX) based simultaneous bioburden detection, photoinactivation and therapeutic outcome assessment in methicillin resistant Staphylococcus aureus (MRSA) infected wounds of mice. MethodsMRSA infected wounds treated with 10% ALA were imaged with help of a blue LED (∼405 nm) based, USB powered, hand held device integrated with a modular graphic user interface (GUI). Effect of ALA application time, bacteria load, post bacteria application time points on wound fluorescence studied. PpIX fluorescence observed after excitation with blue LEDs was used to detect bioburden, start red light mediated antimicrobial photodynamic therapy (aPDT), determine aPDT effectiveness and assess selectivity of the approach. ResultsALA-PpIX fluorescence of wound bed discriminates infected from uninfected wounds and detects clinically relevant load. While wound fluorescence pattern changes as a function of ALA incubation and post infection time, intra-wound inhomogeneity in fluorescence correlates with the Gram staining data on presence of biofilms foci. Lack of red fluorescence from wound granulation tissue treated with ALA suggests selectivity of the approach. Further, significant reduction (∼50%) in red fluorescence, quantified using the GUI, relates well with bacteria load reduction observed post topical aPDT. ConclusionThe potential of ALA induced PpIX for simultaneous detection of bioburden, photodynamic inactivation and “florescence-guided aPDT assessment” is demonstrated in MRSA infected wounds of mice.

Effectiveness of lapatinib for enhancing 5-aminolevulinic acid-mediated protoporphyrin IX fluorescence and photodynamic therapy in human cancer cell lines with varied ABCG2 activities.

5-Aminolevulinic acid (ALA) is a prodrug for protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) and fluorescence-guided tumor surgery. We previously reported that lapatinib, a repurposed ABCG2 inhibitor, enhanced ALA-induced PpIX fluorescence and PDT by blocking ABCG2-mediated PpIX efflux. In the present study, we evaluated how the variation in ABCG2 activities/protein levels affected tumor cell response to the enhancement of PpIX/PDT by lapatinib and Ko143, an ABCG2 tool inhibitor. ABCG2 activities and protein levels were determined in a panel of human cancer cell lines. Effects of lapatinib and Ko143 on enhancing ALA-PpIX fluorescence and PDT were evaluated and correlated with tumor cell ABCG2 activities. We found that both lapatinib and Ko143 enhanced ALA-PpIX fluorescence and PDT in a dose-dependent manner, although lapatinib exhibited lower efficacy and potency than Ko143 in nearly all cancer cell lines. The EC50 of ABCG2 inhibitors for enhancing ALA-PpIX and PDT had a positive correlation with tumor cell ABCG2 activities, indicating that tumor cell lines with lower ABCG2 activities were more sensitive to ABCG2 inhibitors for PpIX/PDT enhancement. Our results suggest that, for optimal therapeutic enhancement, the dose of ABCG2 inhibitors needs to be tailored based on the ABCG2 expression/activity in tumors.

Abstract B046: Irradiation of patient-derived high-grade gliomas reduces 5-ALA-induced fluorescence utility during re-resection

Abstract High grade gliomas HGGs are among the most aggressive primary brain tumors, yielding a median survival time of less than 2 years and a 5-year survival of 2.5%. There has been little in the way of treatments and novel approaches are needed to combat HGG’s poor prognosis. Recent studies have established that HGG cells exhibit metabolic reprogramming to adapt to diverse metabolic gradients within heterogenous tumor microenvironments. Using an unbiased metabolomics approach, we investigated metabolic changes both pre- and post-ionizing radiation across several patient-derived cell lines. Acute dosages of ionizing radiation resulted in significant changes in the synthesis of aminolevulinic acid (ALA), which is known to be converted into the fluorescent metabolite protoporphyrin IX (PpIX) in the porphyrin synthesis pathway. PpIX has been utilized by surgeons in fluorescence-guided tumor resection for clearer visualization of tumor boundaries in recent years. Fractionation of radiation therapy also resulted in dose-dependent changes in the pathway concerning heme synthesis within these cells, especially presenting decreased levels of ALA dehydratase (ALAD) and increased levels of ferrochelatase. As ALAD promotes PpIX accumulation and ferrochelatase catalyzes its conversion to heme, this indicates a decreased favorability for the accumulation of PpIX in the reprogrammed metabolism of HGG cells following radiation. Consequently, we propose that PpIX fluorescence is not a reliable visual aide in surgical re-resection of HGGs post-radiation therapy. Citation Format: Paras S. Minhas, Aarooran Durairaj, Melanie McReynolds, Katrin I. Andreasson, YooJin Jung, Joshua Rabinowitz. Irradiation of patient-derived high-grade gliomas reduces 5-ALA-induced fluorescence utility during re-resection [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B046.

Berberine as a potential enhancer for 5-ALA-mediated fluorescence in glioblastoma: increasing detectability of infiltrating glioma stem cells to optimize 5-ALA-guided surgery.

The prognosis of glioblastoma (GBM) correlates with residual tumor volume after surgery. In fluorescence-guided surgery, 5-aminolevulinic acid (ALA) has been used to maximize resection while avoiding neurological morbidity. However, not all tumor cells, particularly glioma stem cells (GSCs), display 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA fluorescence). The authors searched for repositioned drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA fluorescence. In this study, they investigated whether BBR can enhance 5-ALA fluorescence in GSCs and whether BBR can be applied to clinical practice as a 5-ALA fluorescence enhancer. The effects of BBR on 5-ALA fluorescence in glioma and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. As 5-ALA is metabolized for heme synthesis, the effects of BBR on mRNA expressions of 7 enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. Fluorescence microscopy revealed much more intense 5-ALA fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS showed that BBR greatly enhanced 5-ALA fluorescence compared with 5-ALA alone, and enhancement was much higher for GSCs than for glioma cells. Among the 7 enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. An in vivo study showed that 5-ALA fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue surrounding the tumor mass. BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to significantly augment fluorescence-guided surgery and photodynamic therapy.

Novel Iron-Chelating Prodrug Significantly Enhanced Fluorescence-Mediated Detection of Glioma Cells Experimentally In Vitro.

(1) Background: The protoporphyrin IX (PpIX)-mediated fluorescence-guided resection and interoperative photodynamic therapy (PDT) of remaining cells may be effective adjuvants to the resection of glioma. Both processes may be enhanced by increasing intracellular PpIX concentrations, which can be achieved through iron chelation. AP2-18 is a novel combinational drug, which ester-links a PpIX precursor (aminolaevulinic acid; ALA) to an iron-chelating agent (CP94). (2) Methods: Human glioma U-87 MG cells were cultured in 96-well plates for 24 h and incubated for 3 or 6 h with various test compound combinations: ALA (±) CP94, methyl aminolevulinate (MAL) (±) CP94 and AP2-18. PpIX fluorescence was measured at 0, 3 or 6 h with a Bio-tek Synergy HT plate reader, as well as immediately after irradiation with a 635 nm red light (Aktilite CL16 LED array), representing the PDT procedure. Cell viability post-irradiation was assessed using the neutral red assay. (3) Results: AP2-18 significantly increased PpIX fluorescence compared to all other test compounds. All treatment protocols effectively achieved PDT-induced cytotoxicity, with no significant difference between test compound combinations. (4) Conclusions: AP2-18 has potential to improve the efficacy of fluorescence-guided resection either with or without the subsequent intraoperative PDT of glioma. Future work should feature a more complex in vitro model of the glioma microenvironment.

Optimizing the administrated light dose during 5-ALA-mediated photodynamic therapy: Murine 4T1 breast cancer model.

Photodynamic therapy (PDT) is already used to treat many cancers, including breast cancer, the most common cancer in women worldwide. The destruction basis of this method is on produced singlet oxygen which is extremely reactive and is a major agent of tumor cell killing. The measurement of singlet oxygen produced within PDT is essential in predicting treatment outcomes and their optimization. This study aims to determine the optimal total light dose administered during PDT by calculating the singlet oxygen to facilitate the prediction of the treatment outcome in mice bearing 4T1 cell breast cancer. Monitoring the changes in photosensitizer fluorescence signals during PDT due to photobleaching can be one of the methods of determination of singlet oxygen generation in the PDT process. This study determined the oxygen singlet as a photodynamic dose from the three-dimensional Monte Carlo method and the photobleaching empirical dose constant. The photobleaching dose constant was established non-invasively by monitoring the invivo protoporphyrin IX (PpIX) fluorescence and photobleaching during PDT. The photobleaching dose constant (β) in J/cm2 was calculated using empirical fluorescence data. The invivo photobleaching dose constant of aminolevulinic acid was found to be 11.6 J/cm2 and based on this value, the optimal treatment light dose was estimated at 120 J/cm2 in mice bearing 4T1 breast cancer. It is concluded that information can be obtained regarding optimal treatment parameters by monitoring the invivo PpIX fluorescence and photobleaching during PDT.

TMET-11. BERBERINE AS A POTENTIAL ENHANCER FOR 5-ALA-MEDIATED FLUORESCENCE IN GLIOBLASTOMA: INCREASING DETECTABILITY OF INFILTRATING GLIOMA STEM CELLS TO OPTIMIZE 5-ALA-GUIDED SURGERY

Abstract OBJECTIVE The prognosis of glioblastoma multiforme correlates with residual tumor volume. In fluorescence-guided surgery, 5-aminolevulinic acid (5-ALA) has been used to maximize safe resection. We searched for repositioning drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA-fluorescence). Here, we investigated whether BBR can be applied to clinical practice as a 5-ALA-fluorescence enhancer. METHODS Effects of BBR on 5-ALA-fluorescence in glioma cells and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting; FACS) analysis. As 5-ALA is metabolized for heme synthesis, effects of BBR on mRNA expressions of seven enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. RESULTS Fluorescence microscopy revealed much more intense 5-ALA-fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS disclosed that BBR greatly enhanced 5-ALA-fluorescence compared to 5-ALA alone and enhancement was much higher for GSCs than for glioma cells. Among the seven enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. In vivo study showed that 5-ALA-fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue. CONCLUSIONS BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to augment fluorescence-guided surgery and photodynamic therapy.

Single-cell analysis of 5-aminolevulinic acid intraoperative labeling specificity for glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and resection is a key part of the standard of care. In fluorescence-guided surgery (FGS), fluorophores differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is believed to be specific to neoplastic glioma cells, but this specificity has not been examined at a single-cell level. The objective of this study was to determine the specificity with which 5-ALA labels the diversity of cell types in GBM. The authors performed single-cell optical phenotyping and expression sequencing-version 2 (SCOPE-seq2), a paired single-cell imaging and RNA sequencing method, of individual cells on human GBM surgical specimens with macroscopically visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allowed the authors to simultaneously image PpIX fluorescence and unambiguously identify neoplastic cells from single-cell RNA sequencing. Experiments were also conducted in cell culture and co-culture models of glioma and in acute slice cultures from a mouse glioma model to investigate cell- and tissue-specific uptake and secretion of 5-ALA and PpIX. SCOPE-seq2 analysis of human GBM surgical specimens revealed that 5-ALA treatment resulted in labeling that was not specific to neoplastic glioma cells. The cell culture further demonstrated that nonneoplastic cells could be labeled by 5-ALA directly or by PpIX secreted from surrounding neoplastic cells. Acute slice cultures from mouse glioma models showed that 5-ALA preferentially labeled GBM tumor tissue over nonneoplastic brain tissue with significant labeling in the tumor margins, and that this contrast was not due to blood-brain barrier disruption. Together, these findings support the use of 5-ALA as an indicator of GBM tissue but question the main advantage of 5-ALA for specific intracellular labeling of neoplastic glioma cells in FGS. Further studies are needed to systematically compare the performance of 5-ALA to that of potential alternatives for FGS.

Smartphone-based dual radiometric fluorescence and white-light imager for quantification of protoporphyrin IX in skin.

The quantification of protoporphyrin IX (PpIX) in skin can be used to study photodynamic therapy (PDT) treatments, understand porphyrin mechanisms, and enhance preoperative mapping of non-melanoma skin cancers. We aim to develop a smartphone-based imager for performing simultaneous radiometric fluorescence (FL) and white light (WL) imaging to study the baseline levels, accumulation, and photobleaching of PpIX in skin. A smartphone-based dual FL and WL imager (sDUO) is introduced alongside new radiometric calibration methods for providing SI-units of measurements in both pre-clinical and clinical settings. These radiometric measurements and corresponding PpIX concentration estimations are applied to clinical measurements to understand mechanistic differences between PDT treatments, accumulation differences between normal tissue and actinic keratosis lesions, and the correlation of photosensitizer concentrations to treatment outcomes. The sDUO alongside the developed methods provided radiometric FL measurements () with a demonstrated sub nanomolar PpIX sensitivity in 1% intralipid phantoms. Patients undergoing PDT treatment of actinic keratosis (AK) lesions were imaged, capturing the increase and subsequent decrease in FL associated with the incubation and irradiation timepoints of lamp-based PDT. Furthermore, the clinical measurements showed mechanistic differences in new daylight-based treatment modalities alongside the selective accumulation of PpIX within AK lesions. The use of the radiometric calibration enabled the reporting of detected PpIX FL in units of with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX. The phantom, pre-clinical, and clinical measurements demonstrated the capability of the sDUO to provide quantitative measurements of PpIX FL. The results demonstrate the use of the sDUO for the quantification of PpIX accumulation and photobleaching in a clinical setting, with implications for improving the diagnosis and treatment of various skin conditions.

The formation of complexes with albumin increases the stability of the protoporphyrin IX spectra.

Photodynamic therapy is a high-target, low-invasive treatment utilized to manage a variety of malignant diseases and precancerous lesions. Protoporphyrin IX (PpIX) is one of the most important photosensitizers used in photodynamic therapy, carried to the cancer tissue by serum albumin. Its delivery by transport protein is one of the major factors in determining the efficacy of photodynamic therapy. The distribution of the albumin-PpIX complexes to the target tissue enables the accomplishment of an optimal PDT effect. This study aimed to assess in vitro the stability of spectrofluorimetric spectra of albumin-PpIX complexes. The experiment used three chemicals: PpIX, human serum albumin (HSA), and bovine serum albumin (BSA). Spectral data was recorded using a Kontron SFM-25 Instrument AG, at two excitation wavelengths λex=280 nm and 295 nm. A concentration of 1x10-5M of PpIX, in combination with 1.25x10-6M of HSA and 4x10-7M of BSA, have been recorded repetitively for ten days and compared to the initial spectrum. The maximum of PpIX fluorescence changed significantly on the first day following sample preparation. The maximum of PpIX - serum albumin complex was stable 10 and 4 days for HSA and 5 and 2 days for BSA for λex=280 nm and 295 nm, respectively. The formation of a complex between PpIX and serum albumin was seen to extend the stability of the spectrofluorimetric spectrum. However, a less significant effect was observed in the case of BSA, which could most plausibly be attributed to the variations in primary structure between HSA and BSA, leading to discernible variations in spectroscopic measurements.

Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions ( diameter and depth) were systemically (in oral solution) administered three doses of ALA (total ). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total of 635nm red LED light). The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which -value was at the time of treatment were associated with successful outcomes. These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

Near-infrared phototheranostics of tumors with protoporphyrin IX and chlorin e6 photosensitizers

BackgroundThe study aims to develop a method for phototheranostics of tumors in the near-infrared (NIR) range using protoporphyrin IX (PpIX) and chlorin e6 (Ce6) photosensitizers (PSs) Materials and MethodsPhototheranostics includes spectral fluorescence diagnostics of PS distribution and photodynamic therapy (PDT) using a single laser in the red spectral range. PpIX and Ce6 fluorescence were registered in the NIR range. PpIX and Ce6 photobleaching was determined during PDT by the change in PS fluorescence. NIR phototheranostics with PpIX and Ce6 were performed on optical phantoms and tumors of patients with oral leukoplakia and basal cell carcinoma. ResultsNIR spectral fluorescence diagnostics of optical phantoms with PpIX or Ce6 is possible when fluorescence is excited by 635 or 660 nm lasers. Fluorescence intensity of PpIX and Ce6 was measured in the range of 725–780 nm. The highest values of signal-to-noise in the case of phantoms with PpIX were observed at λexc=635 nm, and for phantoms with Ce6 at λexc=660 nm. NIR phototheranostics provides the detection of tumor tissues with PpIX or Ce6 accumulation. The PSs photobleaching in the tumor during PDT occurs according to a bi-exponential law. ConclusionPhototheranostics of tumors containing PpIX or Ce6 allows fluorescent monitoring of PS distribution in the NIR range and measuring PSs photobleaching during light exposure that provides personalization of the photodynamic exposure duration to deeper tumors. Using a single laser for fluorescence diagnostics and PDT reduces patient treatment time.

Evaluation of the Effect of Photodynamic Therapy on CAM-Grown Sarcomas.

Resection margin adequacy plays a critical role in the local control of sarcomas. Fluorescence-guided surgery has increased complete resection rates and local recurrence-free survival in several oncological disciplines. The purpose of this study was to determine whether sarcomas exhibit sufficient tumor fluorescence (photodynamic diagnosis (PDD)) after administration of 5-aminolevulinic acid (5-ALA) and whether photodynamic therapy (PDT) has an impact on tumor vitality in vivo. Sixteen primary cell cultures were derived from patient samples of 12 different sarcoma subtypes and transplanted onto the chorio-allantoic membrane (CAM) of chick embryos to generate 3-dimensional cell-derived xenografts (CDXs). After treatment with 5-ALA, the CDXs were incubated for another 4 h. Subsequently accumulated protoporphyrin IX (PPIX) was excited by blue light and the intensity of tumor fluorescence was analyzed. A subset of CDXs was exposed to red light and morphological changes of both CAMs and tumors were documented. Twenty-four hours after PDT, the tumors were excised and examined histologically. High rates of cell-derived engraftments on the CAM were achieved in all sarcoma subtypes and an intense PPIX fluorescence was observed. PDT of CDXs resulted in a disruption of tumor-feeding vessels and 52.4% of CDXs presented as regressive after PDT treatment, whereas control CDXs remained vital in all cases. Therefore, 5-ALA mediated PDD and PDT appear to be promising tools in defining sarcoma resection margins (PDD) and adjuvant treatment of the tumor bed (PDT).

P07.07.A Fluorescence lifetime imaging for improved visualization of 5-ALA fluorescence during biopsies of suspected brain tumors

Abstract Background Stereotactic brain tumor biopsies represent a standard neurosurgical technique enabling a precise histopathological tumor diagnosis which is of utmost importance for optimal further treatment allocation. However, non-diagnostic biopsies are not uncommon in routine clinical practice. To overcome this shortcoming, serial biopsies or intraoperative histopathology are typically performed prolonging the operating times and potentially increasing the risk of complications. Recently, the value of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence for visualization of diagnostic tumor tissue was demonstrated in navigation-guided brain tumor biopsies. However, a not negligible proportion of brain tumors lack visible fluorescence. In some of these tumors, a subvisual concentration of PpIX could be detected by spectroscopic analysis. Thus, detection of such subvisual PpIX accumulation in tumor samples in biopsies by the new “Fluorescence Lifetime Imaging” (FLIM) technique might lead to further improvement of this approach. Material and Methods Patients that underwent navigation-guided biopsy of a suspected brain tumor were eligible for inclusion in this study. After administration of 5-ALA, the tissue samples were checked for visible fluorescence during the navigation-guided stereotactic biopsy with a sterile microscope. In each sample the observed fluorescence signal was documented. Additionally, the tissue samples were investigated ex-vivo by FLIM for detection of PpIX accumulation. In each case, a histopathological diagnosis was established according to the WHO classification of CNS tumors. Results A total number of 20 tissue samples could be retrieved in 16 patients with suspected brain tumors. The biopsy with assistance of 5-ALA was feasible in all patients and no relevant side effects occurred. Visible PpIX fluorescence could be observed in 75% of cases. In contrast, 25% of samples did not show visible fluorescence. In one specimen, no fluorescence signal could be evaluated as the sample was covered by too much blood. However, significant accumulation of PpIX could be detected by FLIM in all samples in which evaluation was possible (n=19). A final histopathological diagnosis was feasible in all patients. Conclusion Fluorescence Lifetime Imaging is a promising new technique for improved detection of brain tumor tissue during navigation-guided biopsies. Thus, this new technique might lead in future to reduced operating times and complication rate by minimizing the cases that need serial biopsies or intraoperative histopathology.

The Effects of 5% 5-Aminolevulinic Acid Gel and Red Light (ALAD-PDT) on Human Fibroblasts and Osteoblasts.

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT), consisting of 5% 5-aminolevulinic acid-gel and 630 nm-LED, already used for antibacterial effects in the treatment of periodontitis, on human gingival fibroblasts (HGF) and primary human osteoblasts (HOB). HGF and HOB were incubated with different ALAD concentrations for 45 min, and subsequently irradiated with 630 nm-LED for 7 min. Firstly, the cytotoxicity at 24 h and proliferation at 48 and 72 h were assessed. Then the intracellular content of the protoporphyrin IX (PpIX) of the ROS and the superoxide dismutase (SOD) activity were investigated at different times. Each result was compared with untreated and unirradiated cells as the control. Viable and metabolic active cells were revealed at any concentrations of ALAD-PDT, but only 100-ALAD-PDT significantly enhanced the proliferation rate. The PpIX fluorescence significantly increased after the addition of 100-ALAD, and decreased after the irradiation. Higher ROS generation was detected at 10 min in HGF, and at 30 min in HOB. The activity of the SOD enzyme augmented at 30 min in both cell types. In conclusion, ALAD-PDT not only showed no cytotoxic effects, but had pro-proliferative effects on HGF and HOB, probably via ROS generation.

5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence Imaging for Tumor Detection: Recent Advances and Challenges

5-Aminolevulinic acid (5-ALA) is a natural amino acid and a precursor of heme and chlorophyll. Exogenously administered 5-ALA is metabolized into protoporphyrin IX (PpIX). PpIX accumulates in cancer cells because of the low activity of ferrochelatase, an enzyme that metabolizes PpIX to heme. High expression of 5-ALA influx transporters, such as peptide transporters 1/2, in cancer cells also enhances PpIX production. Because PpIX radiates red fluorescence when excited with blue/violet light, 5-ALA has been used for the visualization of various tumors. 5-ALA photodynamic diagnosis (PDD) has been shown to improve the tumor removal rate in high-grade gliomas and non-muscular invasive bladder cancers. However, 5-ALA PDD remains a challenge as a diagnostic method because tissue autofluorescence interferes with PpIX signals in cases where tumors emit only weak signals, and non-tumorous lesions, such as inflammatory sites, tend to emit PpIX fluorescence. Here, we review the current outline of 5-ALA PDD and strategies for improving its diagnostic applicability for tumor detection, focusing on optical techniques and 5-ALA metabolic pathways in both viable and necrotic tumor tissues.

5-Aminolevulinic acid for enhanced surgical visualization of high-grade gliomas: a prospective, multicenter study.

Greater extent of resection (EOR) is associated with longer overall survival in patients with high-grade gliomas (HGGs). 5-Aminolevulinic acid (5-ALA) can increase EOR by improving intraoperative visualization of contrast-enhancing tumor during fluorescence-guided surgery (FGS). When administered orally, 5-ALA is converted by glioma cells into protoporphyrin IX (PPIX), which fluoresces under blue 400-nm light. 5-ALA has been available for use in Europe since 2010, but only recently gained FDA approval as an intraoperative imaging agent for HGG tissue. In this first-ever, to the authors' knowledge, multicenter 5-ALA FGS study conducted in the United States, the primary objectives were the following: 1) assess the diagnostic accuracy of 5-ALA-induced PPIX fluorescence for HGG histopathology across diverse centers and surgeons; and 2) assess the safety profile of 5-ALA FGS, with particular attention to neurological morbidity. This single-arm, multicenter, prospective study included adults aged 18-80 years with Karnofsky Performance Status (KPS) score > 60 and an MRI diagnosis of suspected new or recurrent resectable HGG. Intraoperatively, 3-5 samples per tumor were taken and their fluorescence status was recorded by the surgeon. Specimens were submitted for histopathological analysis. Patients were followed for 6 weeks postoperatively for adverse events, changes in the neurological exam, and KPS score. Multivariate analyses were performed of the outcomes of KPS decline, EOR, and residual enhancing tumor volume to identify predictive patient and intraoperative variables. Sixty-nine patients underwent 5-ALA FGS, providing 275 tumor samples for analysis. PPIX fluorescence had a sensitivity of 96.5%, specificity of 29.4%, positive predictive value (PPV) for HGG histopathology of 95.4%, and diagnostic accuracy of 92.4%. Drug-related adverse events occurred at a rate of 22%. Serious adverse events due to intraoperative neurological injury, which may have resulted from FGS, occurred at a rate of 4.3%. There were 2 deaths unrelated to FGS. Compared to preoperative KPS scores, postoperative KPS scores were significantly lower at 48 hours and 2 weeks but were not different at 6 weeks postoperatively. Complete resection of enhancing tumor occurred in 51.9% of patients. Smaller preoperative tumor volume and use of intraoperative MRI predicted lower residual tumor volume. PPIX fluorescence, as judged by the surgeon, has a high sensitivity and PPV for HGG. 5-ALA was well tolerated in terms of drug-related adverse events, and its application by trained surgeons in FGS for HGGs was not associated with any excess neurological morbidity.

Identification of excimer delayed fluorescence by Protoporphyrin IX: A novel access to local chromophore concentration?

Protoporphyrin IX (PpIX) is a molecule produced in the mitochondria following the administration of its approved precursor, aminolevulinic acid (ALA). Strong light absorber at different wavelengths in the visible range, PpIX is extensively used as a photosensitizer (PS) for Photodynamic Therapy (PDT). PpIX is also an ideal molecular probe for the quantification of the tissue oxygen partial pressure (pO2), as its delayed fluorescence (DF) is quenched by oxygen, creating a direct relationship between the DF lifetime and the pO2. A limitation of both techniques is the ignorance of the PpIX concentration in tissues when the pO2 is measured or during PDT. In this study, the prompt (PF) and delayed fluorescence of PpIX dissolved in DiMethylFormamide (DMF) were acquired, in absence of oxygen, at different PpIX concentrations. Measurements of the PpIX emission for different excitation energies and temperatures, as well as spectral considerations led to the conclusion that E-type (thermal) DF was the dominant DF mechanism at low PpIX excited states concentrations (density of absorbed energy Hε[PpIX]<1 μJ. cm-3, H:excitation radiant exposure per pulse, ε: molar extinction coefficient at excitation wavelength) while P-type (Triplet Triplet Annihilation) DF took place at higher excited states concentrations (Hε[PpIX]>10 μJ. cm-3). The gradual development of a strong, red-shifted structureless DF peak at 670nm, invisible in the PF and absorption spectra, strongly points towards the first observation of PpIX excimer DF (EDF). It appears that, similarly to other aromatic molecules, PpIX excimers can be formed either by the encounter of two molecules in the first excited triplet state T1, or by the reaction of an excited singlet S1 with a triplet T1. Excimer DF could be beneficially used to determine the local concentration of PpIX, as the initial DF intensity ratio I0670/I0630 is linearly correlated with the local PpIX concentration, and thus rises up to the challenge of PpIX based pO2 measurement and PDT. This work could also pave the way for a fine comprehension of the production, diffusion and catabolization of PpIX in biological tissues.

213 5-Aminolevulinic Acid for Enhanced Surgical Visualization of High-Grade Gliomas: A Prospective, Multicenter Study

INTRODUCTION: Greater extent of resection is associated with longer overall survival in patients with high grade gliomas (HGG). 5-aminolevulinic acid (5-ALA) can increase EOR by improving intraoperative visualization of contrast-enhancing tumor during fluorescence-guided surgery (FGS). 5-ALA is converted by glioma cells into protoporphyrin IX (PPIX), which fluoresces under blue light. 5-ALA has been available for use in Europe since 2010, but only recently gained FDA approval as an intraoperative agent for HGG tissue. METHODS: This single-arm, prospective study included adults aged 18-80 with Karnofsky performance status (KPS)&gt;60 and an MRI diagnosis of suspected new or recurrent resectable HGG. Intraoperatively, 3-5 samples per tumor were taken and their fluorescence status recorded by the surgeon. Patients were followed for six weeks postoperatively for adverse events, changes in the neurological exam, and KPS. We performed multivariate analyses of the outcomes of KPS decline, EOR, and residual enhancing tumor volume to identify predictive patient and intraoperative variables. RESULTS: Sixty-nine patients underwent 5-ALA FGS, providing 275 tumor samples for analysis. PPIX fluorescence had a sensitivity of 96.5%, specificity of 29.4%, positive predictive value (PPV) for HGG of 95.4%, and diagnostic accuracy of 92.4%. Drug-related adverse events occurred at a rate of 22%. Serious adverse events occurred at a rate of 4.3%. There were 2 unrelated deaths. KPS was significantly lower at 48 hours and 2 weeks, but was not different at 6 weeks postoperatively. Complete resection of enhancing tumor occurred in 51.9% of patients. Smaller preoperative tumor volume and use of intraoperative MRI predicted lower residual tumor volume. CONCLUSION: PPIX fluorescence, as judged by the surgeon, has a high sensitivity and PPV for HGG. 5-ALA was well-tolerated, and its application by trained surgeons in FGS for HGGs was not associated with any excess neurological morbidity.