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Fluorescence-based discrimination of breast cancer cells by direct exposure to 5-aminolevulinic acid.

ABSTRACT: Protoporphyrin IX-fluorescence measurement is a powerful in situ approach for cancer detection after oral/topical administration of 5-aminolevulinic acid. However, this approach has not been clinically established for breast cancer, probably due to insufficient delivery of 5-aminolevulinic acid to the mammary glands. In the present study, we directly exposed breast cancer cells to 5-aminolevulinic acid to assess their discrimination via protoporphyrin IX-fluorescence. Fluorescence intensity (FI) was measured in the human breast cancer cell lines MCF7 and MDA-MB-231 and breast epithelial cell line MCF10A by confocal microscopy and flow cytometry. After 5-aminolevulinic acid exposure for 2 hours, protoporphyrin IX-FI in MCF7 and MDA-MB-231 cells significantly increased with marked cell-to-cell variability, whereas that in MCF10A cells increased moderately. Combined exposure of the cancer cells to 5-aminolevulinic acid and Ko143, a specific inhibitor of ATP-binding cassette transporter G2, further increased protoporphyrin IX-FI and alleviated the cell-to-cell variability in MCF7 and MDA-MB-231 cells, indicating improvement in the reproducibility and accuracy for fluorescence-based cancer detection. The increased FI by combined administration of these two drugs was also demonstrated in cells obtained via fine needle aspiration from mouse xenograft models inoculated with MDA-MB-231 cells. Furthermore, a cutoff value for increased protoporphyrin IX-FI ratio, before and after exposure to these drugs, clearly discriminated between cancer and noncancer cells. Taken together, direct exposure to 5-aminolevulinic acid and Ko143 may be a promising strategy for efficient fluorescence-based detection of breast cancer cells ex vivo using fine needle aspiration.

SUBMITTER: Morita M

PROVIDER: S-EPMC6746108 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Fluorescence-based discrimination of breast cancer cells by direct exposure to 5-aminolevulinic acid.

Morita Midori M Tanaka Hideo H Kumamoto Yasuaki Y Nakamura Akihiro A Harada Yoshinori Y Ogata Takehiro T Sakaguchi Koichi K Taguchi Tetsuya T Takamatsu Tetsuro T

Cancer medicine 20190805 12

Protoporphyrin IX-fluorescence measurement is a powerful in situ approach for cancer detection after oral/topical administration of 5-aminolevulinic acid. However, this approach has not been clinically established for breast cancer, probably due to insufficient delivery of 5-aminolevulinic acid to the mammary glands. In the present study, we directly exposed breast cancer cells to 5-aminolevulinic acid to assess their discrimination via protoporphyrin IX-fluorescence. Fluorescence intensity (FI) ...[more]

PMID: 31385432

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Project description:BackgroundRe-excision due to positive margins following breast-conserving surgery (BCS) negatively affects patient outcomes and healthcare costs. The inability to visualize margin involvement is a significant challenge in BCS. 5-Aminolevulinic acid hydrochloride (5-ALA HCl), a non-fluorescent oral prodrug, causes intracellular accumulation of fluorescent porphyrins in cancer cells. This single-center Phase II randomized controlled trial evaluated the safety, feasibility, and diagnostic accuracy of a prototype handheld fluorescence imaging device plus 5-ALA for intraoperative visualization of invasive breast carcinomas during BCS.MethodsFifty-four patients were enrolled and randomized to receive no 5-ALA or oral 5-ALA HCl (15 or 30 mg/kg). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed, and biopsies were collected from within and outside the clinically demarcated tumor border of the gross specimen for blinded histopathology.ResultsIn the absence of 5-ALA, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Both 5-ALA doses caused bright red tumor fluorescence, with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA group, the positive predictive value (PPV) for detecting breast cancer inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively. In the 30 mg/kg 5-ALA group, the PPV was 100.0% and 50.0% inside and outside the demarcated tumor border, respectively. No adverse events were observed, and clinical feasibility of this imaging device-5-ALA combination approach was confirmed.ConclusionsThis is the first known clinical report of visualization of 5-ALA-induced fluorescence in invasive breast carcinoma using a real-time handheld intraoperative fluorescence imaging device.Trial registrationClinicaltrials.gov identifier NCT01837225 . Registered 23 April 2013.

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.

Dataset Information

Fluorescence-based discrimination of breast cancer cells by direct exposure to 5-aminolevulinic acid.

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Fluorescence-based discrimination of breast cancer cells by direct exposure to 5-aminolevulinic acid.

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