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The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve thespeed of PAFC by using a high-pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low-pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near-infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease and real-time monitoring of therapy efficiency by counting CTCs before, during, and after therapeutic intervention. Herewith, we also address sensitivity of label-free detection of melanoma CTCs and introduce in vivo CTC targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment. © 2011 International Society for Advancement of Cytometry
Cutaneous melanoma is the third most common type of skin cancer after basal and squamous cell carcinoma. It accounts for only 3% of all cases, but it leads to 65% of all deaths from skin cancer (1–7). The most alarming aspect of cutaneous melanoma is its potential to metastasize at a very early stage of disease. As a result, most of all the melanoma deaths arise from metastases (1, 2).
Appearance of circulating tumor cells (CTCs) has been suggested as an early marker of metastatic development, cancer recurrence, and therapeutic efficacy (2, 8–10). The comprehensive review of 209 articles involving 53 studies that collected data on 5,433 patients have been well supported by this data (6). Among advanced technologies for detection of CTCs ex vivo [e.g., scanning cytometry, immunomagnetic enrichment, size filtration, negative cell sorting, and microfluidic chips (8–10)] mostly reverse transcription-polymerase chain reaction (RT-PCR) combined with cell-enrichment techniques was broadly used for melanoma CTCs (1–7). Some difficulties in reproducing the results of the RT-PCR assay were associated with differences in sample processing and generation of false-positive signals due to contamination, amplification of pseudogenes, and illegitimate transcription (6). False-negative signals, in contrast, were related to the poor quality of source materials and the genomic instability of malignant cells. Furthermore, RT-PCR is an indirect method and cannot provide a direct evidence of the presence of intact CTCs in the blood.
In general, the ultimate sensitivity threshold of most ex vivo CTCs assays is limited by the small blood sample volume, typically a few milliliters, in which no less than one CTC can be detected. As a result, in the entire volume of blood (∼5 L in adult) existing tests with current threshold sensitivity of 1–10 cells/mL can miss up to 5,000–50,000 cells, which are sufficient for the rapid development of a barely treatable or already incurable metastasis (9, 10). Invasive extraction of blood at discrete time points from an organism may lead to changes in CTC properties (e.g., morphology or marker expression) and prevents the long-term study of CTCs and metastasis development in the native biological environment. Little or no ability exists to collect blood samples of sufficient volume from clinically relevant anatomical sites such as the primary tumor area, lymph nodes, or bones.
Most of these problems can be solved by assessing large blood volume in vivo using the principles of flow cytometry with photothermal (PT), photoacoustic (PA), or fluorescent detection schematics proposed in 2004 by us and other groups (11–20). This approach provides the possibility of monitoring significantly larger blood volumes and potentially the entire blood volume in 1–2 h [the time it will take for 5 L of blood at 100 mL/min flow rate to pass through 2–3-mm peripheral blood vessels (15–17)]. Unfortunately, translation to humans of fluorescence-based CTCs labeling and detection techniques faces multiple challenges such as fluorescent probes toxicity, wide emission spectra of fluorophores in near-infrared (NIR) tissue transparency window, high intensity of continuous wave lasers used [∼100 W/cm2 compared to safe level of 0.2–0.5 W/cm2 (21)], and assessment of only superficial (≤0.1–0.3 mm) microvessels having slow flow. For example, in a mouse model in 50-μm blood vessel having flow velocity of 5 mm/s, 2 days are required to continuously assess the whole 2-mL blood volume.
PT and, especially, PA methods are free of these limitations (13, 16–18). These methods are based on nonradiative relaxation of absorbed laser energy into heat and accompanying acoustic effects (Fig. 1A). For nonfluorescent samples (e.g., melanin is weakly fluorescent), these methods offer the highest absorption sensitivity at the single-cell level (absorption coefficient of 10−2 – 10−3 cm−1). This level of absorption sensitivity makes it possible to noninvasively [i.e., a short-term temperature elevation of ≤0.5°C (22, 23)] detect individual nanoparticles, quantum dots, dyes, and biomolecules at a threshold comparable with that of fluorescence methods (23–25). Several successful clinical trials on humans demonstrated a clinical significance and safety of PA technique including 1) continuous monitoring of blood oxygenation in the internal jugular vein (10–20 mm in diameter, 5–10 mm deep in the presence of strong light scattering, and attenuation in the 15–20 mm layer of overlaying tissue (26); 2) detection and imaging of breast tumor at a depth of 3 cm (27); and 3) monitoring of blood hemoglobin in the wrist area overlying the radial artery at a depth of 3 mm (28). PA technology is safe for human subjects as it requires a laser energy fluence of only 5–20 mJ/cm2, which is well within the laser safety standard (∼30–100 mJ/cm2) in NIR range (e.g., 800–1100 nm) (21). Applied to a melanoma study, PA techniques have demonstrated promise for the assessment of melanoma cells in vitro (8, 16, 29) and imaging of melanoma tumor in vivo in static conditions (30).
Figure 1. In vivo photoacoustic flow cytometry (PAFC). A: The principles of in vivo PA detection of melanoma CTCs. B: Scheme of the experimental setup. C: The anesthetized mouse on the microscope stage with ultrasound transducer. Inset: enlarged image of the ear and transducer. D: Laser beam in mouse ear blood vessel. E: Melanoma cell among red blood cells. F: Pulse PA signal from blood (left) and from single B16F10 melanoma cell in blood (middle); PA signal trace in the presence of a melanoma cell (right).
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We have developed in vivo time-resolved PA flow cytometry (PAFC) (13, 14) and demonstrated its potential for label-free counting of single CTCs (B16F10) in blood and lymph flow in melanoma-bearing mouse (16, 31). In particular, the association between disseminated melanoma cells count in prenodal lymphatics and metatastatic status of sentinel lymph nodes supports the presence of melanoma cells in lymphatics as a novel prognostic marker of metastasis (18). We also developed advanced PAFC schematic with a high-pulse repetition rate diode laser at 904 nm and demonstrated an ultrasensitive, label-free, PA enumeration of melanoma CTCs in the mouse blood microvessels with diameter of 50–300 μm (16). However, relatively low pulse energy of diode lasers (few microjoules) makes generation of detectable PA signals from individual CTCs in larger vessels challengeable, for example, in 2–3 mm hand veins and arteries at depths of 1–3 mm (our primary clinical target), or, potentially, in ∼1-cm jugular vein (26), where a probability to detect rare CTCs is higher due to higher flow velocity. To overcome these limitations, we recently developed the PAFC with high-pulse repetition rate Yb-doped fiberlaser having relatively high pulse energy (up to 50 μJ) at 1064 nm and demonstrated its application for the detection ofcirculating nanoparticles (32). In this work, we extend applications of the high-speed PAFC with high-pulse repetition NIR lasers to in vivo real-time detection of human melanoma CTCs.
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Here, we have presented the unique experimental data confirming the possibility of label-free detection of both human and mouse melanoma cells in fast blood circulation. The high-speed PAFC reduces the rate of false-negative errors for fast moving CTCs in large blood vessels. Multiple PA signals received from the same cell allow increasing detection sensitivity through noise reduction. Eventually, high-speed PAFC with ability to assess large deep vessels should be able to drastically decrease time required for monitoring the whole blood volume, thus decreasing detection limits down to desired threshold (9): ultimately, a few CTCs per whole blood volume (i.e, 5 L). We emphasize that with the use of melanin overexpression in melanoma cells as intrinsic cell marker in vivo label-free high-speed PAFC eliminates some potential problems of existing assays (e.g., low sensitivity due to assessment of small blood volume). This may provide precedent for a noninvasive in vivo blood cancer testing and, potentially purging (18) that focuses on melanoma, a highly aggressive, epidemic malignancy that is often widely metastatic at an early stage. Indeed, we demonstrated detection of B16F10 CTCs in blood circulation on the fourth day after subcutaneous inoculation of tumor cells.
Intrinsic PA contrast of melanin can be used in melanoma related research fields: 1) monitoring of melanoma cells dissemination in blood and in lymph as demonstrated previously (16, 31); 2) study of cell pigmentation and melanin synthesis in vitro and in vivo; 3) correlating CTCs count to primary tumor and sentinel lymph node status (14, 18). Clinical scenarios may include 1) blood screening for early CTCs before development of metastases; 2) testing for cancer recurrence; 3) individualized assessment of therapy efficiency (e.g., chemo, radiation, or immunotherapy) through real-time CTC count; and 4) potential metastasis prevention by well-timed therapy including in vivo noninvasive laser PT killing of melanoma CTCs (16).
In vivo label-free PAFC of low pigmented C8161 human melanoma cells demonstrated increased false negativity rate of about 60–80% compared to 12% for strongly pigmented B16F10 mouse melanoma cells (16). As soon as both high and low pigmented cells can be simultaneously presented in melanoma patients, we assume that PAFC can miss up to 50–60% of cells in a label-free mode. Nevertheless, despite the high-false-negative rate of label-free PAFC of human melanoma, it may have tremendous clinical significance in blood testing for early melanoma diagnosis, for CTCs counting at parallel progression of primary tumor and metastasis and for diagnosis of melanoma recurrence. In these cases, the fact that CTCs are present in blood flow may have higher importance than accurate CTCs count. The missing of low-pigmented melanoma CTCs should be more crucial for monitoring therapy efficiency. We believe that novel robust diagnostic platform using high-pulse rate lasers and label-free noninvasive approach can be more quickly translated to humans. Indeed, noninvasive PA methods already demonstrated higher resolution, sensitivity, and the penetration depth (up to 3 cm) compared to other optical modalities. According to our data, a high-speed PAFC makes is possible to distinguish individual melanoma cells in blood circulation at low levels of laser fluence (∼0.1 J/cm2 for 1064 nm), which satisfy laser safety standard (21). The clinical prototype could represent a portable fiber-based device that overlies the hand vessels [e.g., see prototype in the supplementary information in ref. (16)].
Herewith, we also address the problem of a high-false-negative rate of a label-free PAFC via molecular CTCs targeting by magnetic nanoparticles and their magnetic enrichment. Still, more studies are required to bring this approach to the bedside. Several other methods could be used to increase PA contrast of cells: 1) 5–10-fold increase in PA contrast by drug-induced activation of melanin production in melanoma cells (16); 2) melanogenesis activation in melanoma and nonmelanoma (e.g., breast cancer) cells via transfection with tyrosinase-activating plasmids (16); 3) in vitro incubation of cells with melanin nanoparticles (Fig. 2); and 4) reduction of blood background PA signal by the change in blood oxygenation, the hematocrit, and osmolarity within physiological norms (16).