Abstract
Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1–10 CTC/mL) due to the small volume of blood samples (5–10 mL). Consequently, they can miss up to 103–104 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 102–103 times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-resolution PAFC beyond the diffraction and spectral limits.
Highlights
We demonstrate PA Flow Cytometry (PAFC)’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast circulating tumor cells (CTCs) and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis
Using label-free, real-time PAFC in a melanoma-bearing mouse model, we discovered that palpation, biopsy, and conventional and laser surgery (Figure 1, left) may either initiate release of CTCs into the blood, where they previously were not present, or dramatically increase (10–30-fold) CTC counts above the previous level; both of which can increase the risk of metastasis [29]
We have summarized recent advances in the development of a new concept of in vivo blood cancer test using the principle of in vivo flow cytometery with PA detection schematics (PAFC)
Summary
Up to 90% of cancer deaths are related to metastasis in the distant organs due to the hematogenous and lymphatics dissemination of circulating tumor cells (CTCs) shed from the primary tumor [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. Conventional flow cytometry (FC) in vitro, currently the most powerful analytic tool, can potentially provide multiplex (up to 6–8 markers) detection of CTCs using multicolor probes [31,32] In this technology, labeled cells in fast (a few m/s) flow with a high cell rate (up to 105 cells/s) are accurately positioned by hydrodynamical or acoustical focusing into a single file (5–10 μm in diameter) that passes through tightly focused laser beams used to detect scattering and fluorescent light from individual cells, as well as providing dynamic imaging at a rate of 102–103 cell/s. These problems can be solved by assessing a significantly larger volume of blood up to a patient’s entire blood volume with in vivo FC, whose principles were proposed in 2004 simultaneously by Zharov and other groups [33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49]
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