High-sensitivity detection of breast tumorsin vivoby use of a pH-sensitive near-infrared fluorescence probe

Abstract

Abstract. We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumordetection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe

“… 12 The transport of a CypHer5E-conjugated molecule from early endosomes (pH 6.0–6.5) to lysosomes (pH 4.5–5.5) could yield a fluorescence enhance factor (change in fluorescent emission) of about 2- to 4-fold depending on the D/P ratio of the CypHer5E-labeled molecule. 12 , 28 This difference in fluorescence intensity would still allow CypHer5E-labeled molecules to be detected with 8200 CDS or Celigo, although when internalized to early endosomes and not further routed to lysosomes. Nevertheless, routing to the lysosome would result in higher fluorescence intensity per internalized molecule and would thus favor the detection of antibodies that are indeed transported to the lysosome over antibodies that remain in early or recycling endosomes.…”

In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1

“…We have chosen NIRF imaging to assess the binding and biodistribution of mAb62 as this technique utilises non-ionising radiation and therefore has the important advantage of being harmless, in contrast to other functional imaging modalities such as PET or SPECT. In addition, it is a simple, quick and relatively cheap method, which delivers a variety of information such as on target expression, probe biodistribution and tissue characteristics such as pH and oxygen levels or enzymatic activity (Napp et al 2010 , 2011 ; Mathejczyk et al 2011 , 2012 ). Moreover, the use of NIR light allows for relatively deep tissue penetration of up to 2 cm, which means that even deeply located structures, such as a fluorescently labelled pancreatic tumour, can be easily detected within a living mouse (Napp et al 2010 ).…”

Spatial mapping of fluorophore quantum yield in diffusive media

“…There has been some work done in using fluorescence lifetime measurements to determine pH conditions in tissue, but this requires relatively complex and expensive pulsed laser systems and time-correlated single photon counting techniques. [24][25][26][27] Moreover, fluorophores which are sensitive to temperature, oxygen content, or protein binding can also be used with QYI, potentially allowing a variety of microenvironmental conditions to be determined noninvasively. Several assumptions were made in this study in order to simplify the processing required to generate QYI maps, and there are limitations to the use of this methodology for certain applications.…”