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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

ABSTRACT: Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy.

SUBMITTER: Kantere D

PROVIDER: S-EPMC3732385 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

Kantere Despina D Guldbrand Stina S Paoli John J Goksör Mattias M Hanstorp Dag D Wennberg Ann-Marie AM Smedh Maria M Ericson Marica B MB

Journal of biophotonics 20120919 5

Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed ...[more]

PMID: 22997024

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Project description:Simple Summary 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used in neurosurgery for intraoperative identification of high-grade glioma tissue. In this paper, using a fluorescence microscopy analysis on human tumor specimens, we assessed the actual number of fluorescence-positive tumor cells both in low-grade and high-grade glioma, and the ability of 5-ALA to cross the blood–brain barrier (BBB). We found that in high-grade gliomas, 32.7–75.5 percent of cells display 5-ALA induced PpIX fluorescence, whereas in low-grade gliomas the tumor cells did not fluoresce following 5-ALA. Immunofluorescence for BBB components suggested that 5-ALA does not cross the un-breached BBB. These findings are of crucial importance in planning neurosurgical resection of gliomas. Abstract 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used by neurosurgeons to identify the tumor cells of high-grade gliomas during operation. However, the issue of whether 5-ALA-induced PpIX fluorescence consistently stains all the tumor cells is still debated. Here, we assessed the cytoplasmatic signal of 5-ALA by fluorescence microscopy in a series of human gliomas. As tumor markers, we used antibodies against collapsin response-mediated protein 5 (CRMP5), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and anti-isocitrate dehydrogenase 1 (IDH1). In grade III–IV gliomas, the signal induced by 5-ALA was detected in 32.7–75.5 percent of CRMP5-expressing tumor cells. In low-grade gliomas (WHO grade II), the CRMP5-expressing tumor cells did not fluoresce following 5-ALA. Immunofluorescence with antibodies that stain various components of the blood–brain barrier (BBB) suggested that 5-ALA does not cross the un-breached BBB, in spite of its small dimension. To conclude, 5-ALA-induced PpIX fluorescence has an established role in high-grade glioma surgery, but it has limited usefulness in surgery for low-grade glioma, especially when the BBB is preserved.

Project description:SignificanceThe 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.AimWe 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.ApproachA 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.ResultsThe sDUO alongside the developed methods provided radiometric FL measurements (nW/cm2) 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 nW/cm2 with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX.ConclusionsThe 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.

Project description:Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells. Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted. Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model. Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Dataset Information

Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

Publications

Anti-Stokes fluorescence from endogenously formed protoporphyrin IX--implications for clinical multiphoton diagnostics.

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