In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Abstract

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at Ϸ2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.folate conjugates ͉ in vivo imaging ͉ multiphoton microscopy ͉ metastasis ͉ cancer diagnosis

‘Go with the flow ’: A review of methods and advancements in blood flow imaging

“…Yet, despite these limiting factors, confocal and two-photon imaging have been used in a wide variety of applications: rodent cerebral microcirculation investigations [275][276][277][278]; used in conjunction with particle image velocimetry (see § 4.1) to investigate micro-channel blood flow [279]; as a quantitative method in the study of embryonic vasculature dynamics [280,281]; dynamic studies of Alzheimer's disease and epilepsy [274]; and in cytometric studies of the relationships between stem cells [282], tumour cells [283] and their tissue environments [284,285].…”

Detection of circulating tumor cells in non-small cell lung cancer

“…Fluorescent folate conjugates which bind FOLR1 with nanomolar affinity were characterized as tumor-specific ligands in combination with multiphoton fluorescence imaging (20 A kit to label and detect CTCs through FOLR1 expression in the peripheral blood of cancer patients was developed by Genosaber Biotech (CytoploRare ® , Shanghai, China). In brief, white blood cells are removed from 3 mL of blood with anti-CD45 and anti-CD14 immunomagnetic beads followed by labeling of remaining cells with an oligonucleotide-FOLR1 probe.…”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…These ligands can be regarded as a tumor-specific receptor to construct a "guided molecular missile" with different therapeutic warheads. In an effort to contribute to the development of optical imaging modalities, a variety of fluorescent dyes e.g., fluorescein, Texas Red, the rhodamines, the cyanines (indocyanine green, thiazole orange) [57][58][59], polymethine cyanine dyes (the Cy3, Cy5, Cy5.5, Cy7 series) and the Alexa Fluors have been conjugated to folic acid [60][61][62][63][64]. Nowadays, the development of fluorescent dyes with longer excitation wavelengths (λEx>500 nm) transparent to human tissues [65,66] shows great promise, because imaging of malignant cells by optical means is not hazardous for living organisms [67].…”