In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser

Abstract

The circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. However, its clinical utility for metastasis prevention remains unclear, because metastases may already be present at the time of initial diagnosis with existing assays. Their sensitivity ex vivo is limited by a small blood sample volume, whereas in vivo examination of larger blood volumes may be clinically restricted by the toxicity of labels used for targeting of CTCs. We introduce a method for in vivo photoacoustic blood cancer testing with a high-pulse-repetition-rate diode laser that, when applied to melanoma, is free of this limitation. It uses the overexpression of melanin clusters as intrinsic, spectrally-specific cancer markers and signal amplifiers, thus providing higher photoacoustic contrast of melanoma cells compared with a blood background. In tumor-bearing mouse models and melanoma-spiked human blood samples, we showed a sensitivity level of 1 CTC/mL with the potential to improve this sensitivity 10(3)-fold in humans in vivo, which is impossible with existing assays. Additional advances of this platform include decreased background signals from blood through changes in its oxygenation, osmolarity, and hematocrit within physiologic norms, assessment of CTCs in deep vessels, in vivo CTC enrichment, and photoacoustic-guided photothermal ablation of CTCs in the bloodstream. These advances make feasible the early diagnosis of melanoma during the initial parallel progression of primary tumor and CTCs, and laser blood purging using noninvasive or hemodialysis-like schematics for the prevention of metastasis.

Fig. 1

A. PAFC schematics with acquisition algorithm of PA signals. Laser parameters: 905 nm, 30 mJ/cm². B. Animal models (from left to right): microscopic schematic, diode laser beam after passing of mouse ear, pigmented mouse NIH-BG-NU-XID, and minimally-invasive fiber delivery of laser radiation into blood vessel.

Fig. 2

A. High resolution (×100) TDM images of single B16F10 cells with low and high melanin content and single B16F10 cell surrounded by mouse RBCs (first row from left to right, respectively). PT images of B16F10 cells with low and high melanin content and single mouse RBC (second row, respectively). PT signals from single low and high pigmented melanoma cells and single RBC (third row, respectively); amplitude/time scale/laser wavelength/fluence: (20mV/div)/(2μsec/div)/580nm/(60mJ/cm²), (100mV/div)/(4μsec/div)/580nm/(0.3 J/cm²), and (10mV/div)/(4μsec/div)/580 nm/(100mJ/cm²), respectively. B. Heterogeneity of the PT signal amplitudes for melanoma cells (850nm; 0.1 J/cm²). The inset shows distribution details for relatively low signals. C. PA signals from tumor-derived B16F16 cells in suspension (10⁷ cell/mL) and mouse RBCs in microscopic slide with attached transducer as function of laser fluence (wavelength, 800 nm; beam diameter, 100 μm, ~60 melanoma cells and ~4×10⁴ RBCs in the irradiated volume).

Fig. 3

A. PA spectra of ~50 μm diameter veins in mouse ear (empty circles); the average standard deviation for each wavelength is 20%. The absorption spectra of the B16F10 cells (dashed curves) were normalized to PA signals from single melanoma cells with strong (black circle) and weak (dark square) pigmentation; open square indicates signal from melanoma cell with GNRs; fragment of solid curves shows absorption for 100% of Hb and HbO_2. B. Two-wavelengths (λ1=639 nm; λ2=865 nm) identification of melanoma cells and RBCs.

Fig. 4

A. Clearance rate of B16F10 cells in ~50 μm-diameter ear vein in mice with low and high skin melanin pigmentation and 100 nm melanin nanoparticles (2 μg/mL, 50 μl in PBS) as a function of time after injection. Laser parameters: 905 nm, 30 mJ/cm². B. Melanoma tumor growth in mouse ear (top) and skin (middle) and CTC rate in ~150 μm skin vessels as a function of time after B16F10 tumor cell inoculation in the ear (red empty circle) and skin (blue empty square); dark red circle and blue square are averaged data (905 nm, 30 mJ/cm²).

Fig. 5

A. 2-D scanning PT cytometry of single melanoma cells in thin layer (120 μm) of RBCs (top), TDM image (colored central part) of single melanoma cells among WBCs after RBC lysis (middle), and one scan with appearance of one strong PT signal from single melanoma cell in background of low signals from WBCs (850 nm, 100 mJ/cm²). B. 1-D scanning PA capillary cytometry: PA detection of single B16F10 cells in 0.25-mm diameter of glass tube filled with mouse blood (850 nm, 100 mJ/cm²). C. PA signals from human blood samples from one donor at different number of melanoma cells in the irradiated volume (850 nm, 150 mJ/cm²⁾.

Fig. 6

A. In vivo capturing of relatively large CTCs by gentle mechanical squeezing of the bloodvessel in mouse ear. B. Image of a blood vessel area during squeezing with a pilot red (633 nm) laser beam. C. Fluorescent control of squeezing (excitation with diode laser, 805 nm, 0.5 W; emission, 830 nm) showing the accumulation of i.v. (injected in tail vein) Indocyanine Green (ICG) dye in blood vessel before the area is squeezed (top, right) and a decrease in the ICG concentration after the area is squeezed (bottom, left).