Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.

Lymph Liquid Biopsy for Detection of Cancer Stem Cells.

Collection of a blood sample defined by the term "blood liquid biopsy" is commonly used to detect diagnostic, prognostic, and therapeutic decision-making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem-like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L-CTCs) was determined by spheroid-forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem-like L-CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre-metastatic disease, stem-like L-CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem-like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid-forming L-CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L-CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry.

In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease.

The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.

Optical clearing for photoacoustic lympho- and angiography beyond conventional depth limit in vivo.

Photoacoustic (PA) imaging (PAI) is an emerging powerful tool for noninvasive real-time mapping of blood and lymphatic vessels and lymph nodes in vivo to diagnose cancer, lymphedema and other diseases. Among different PAI instruments, commercially available raster-scanning optoacoustic mesoscopy (RSOM) (iThera Medical GmbH., Germany) is useful for high-resolution imaging of different tissues with high potential of clinical translation. However, skin light scattering prevents mapping vessels and nodes deeper than 1-2 mm, that limits diagnostic values of PAI including RSOM. Here we demonstrate that glycerol-based tissue optical clearing (TOC) overcomes this challenge by reducing light scattering that improves RSOM depth penetration. In preclinical model of mouse limb in vivo, the replacement of conventional acoustic coupling agents such as water on the mixture of 70 % glycerol and 30 % ultrasound (US) gel resulted in the increase of tissue imaging depth in 1.5-2 times with 3D visualization of vessels with diameter down to 20 µm. To distinguish blood and lymphatic networks, we integrated label-free PA angiography (i.e., imaging of blood vessels), which uses hemoglobin as endogenous contrast agent, with PA lymphography based on labeling of lymphatic vessels with exogenous PA contrast agents. Similar to well-established clinical lymphography, contrast agents were injected in tissue and taken up by lymphatic vessels within a few minutes that provided quick RSOM lymphography. Furthermore, co-injection of PA contrast dye and multilayer nanocomposites as potential low-toxic drug-cargo showed selective prolonged accumulation of nanocomposites in sentinel lymph nodes. Overall, our findings open perspectives for deep and high resolution 3D PA angio- and lymphography, and for PA-guided lymphatic drug delivery using new RSOM & TOC approach.

New Frontiers in Diagnosis and Therapy of Circulating Tumor Markers in Cerebrospinal Fluid In Vitro and In Vivo.

One of the greatest challenges in neuro-oncology is diagnosis and therapy (theranostics) of leptomeningeal metastasis (LM), brain metastasis (BM) and brain tumors (BT), which are associated with poor prognosis in patients. Retrospective analyses suggest that cerebrospinal fluid (CSF) is one of the promising diagnostic targets because CSF passes through central nervous system, harvests tumor-related markers from brain tissue and, then, delivers them into peripheral parts of the human body where CSF can be sampled using minimally invasive and routine clinical procedure. However, limited sensitivity of the established clinical diagnostic cytology in vitro and MRI in vivo together with minimal therapeutic options do not provide patient care at early, potentially treatable, stages of LM, BM and BT. Novel technologies are in demand. This review outlines the advantages, limitations and clinical utility of emerging liquid biopsy in vitro and photoacoustic flow cytometry (PAFC) in vivo for assessment of CSF markers including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA), proteins, exosomes and emboli. The integration of in vitro and in vivo methods, PAFC-guided theranostics of single CTCs and targeted drug delivery are discussed as future perspectives.

Amplification of photoacoustic effect in bimodal polymer particles by self-quenching of indocyanine green.

A new type of bimodal contrast agent was made that is based on the self-quenching of indocyanine green (ICG) encapsulated in a biocompatible and biodegradable polymer shell. The increasing of a local ICG concentration that is necessary for the obtaining of self-quenching effect was achieved by freezing-induced loading and layer-by-layer assembly. As a result, an efficient photoacoustic(optoacoustic)/fluorescent contrast agent based on composite indocyanine green/polymer particles was successfully prepared and was characterized by fluorescence and photoacoustic(optoacoustic) tomography in vitro. This type of contrast agent holds good promise for clinical application owing to its high efficiency and biosafety.

Photoswitchable Spasers with a Plasmonic Core and Photoswitchable Fluorescent Proteins.

Photoswitchable fluorescent proteins (PFPs) that can change fluorescence color upon excitation have revolutionized many applications of light such as tracking protein movement, super-resolution imaging, identification of circulating cells, and optical data storage. Nevertheless, the relatively weak fluorescence of PFPs limits their applications in biomedical imaging due to strong tissue autofluorecence background. Conversely, plasmonic nanolasers, also called spasers, have demonstrated potential to generate super-bright stimulated emissions even inside single cells. Nevertheless, the development of photoswitchable spasers that can shift their stimulated emission color in response to light is challenging. Here, we introduce the novel concept of spasers using a PFP layer as the active medium surrounding a plasmonic core. The proof of principle was demonstrated by synthesizing a multilayer nanostructure on the surface of a spherical gold core, with a non-absorbing thin polymer shell and the PFP Dendra2 dispersed in the matrix of a biodegradable polymer. We have demonstrated photoswitching of spontaneous and stimulated emission in these spasers below and above the spasing threshold, respectively, at different spectral ranges. The plasmonic core of the spasers serves also as a photothermal (and potentially photoacoustic) contrast agent, allowing for photothermal imaging of the spasers. These results suggest that multimodal photoswitchable spasers could extend the traditional applications of spasers and PFPs in laser spectroscopy, multicolor cytometry, and theranostics with the potential to track, identify, and kill abnormal cells in circulation.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

In vivo liquid biopsy using Cytophone platform for photoacoustic detection of circulating tumor cells in patients with melanoma.

Most cancer deaths arise from metastases as a result of circulating tumor cells (CTCs) spreading from the primary tumor to vital organs. Despite progress in cancer prognosis, the role of CTCs in early disease diagnosis is unclear because of the low sensitivity of CTC assays. We demonstrate the high sensitivity of the Cytophone technology using an in vivo photoacoustic flow cytometry platform with a high pulse rate laser and focused ultrasound transducers for label-free detection of melanin-bearing CTCs in patients with melanoma. The transcutaneous delivery of laser pulses via intact skin to a blood vessel results in the generation of acoustic waves from CTCs, which are amplified by vapor nanobubbles around intrinsic melanin nanoclusters. The time-resolved detection of acoustic waves using fast signal processing algorithms makes photoacoustic data tolerant to skin pigmentation and motion. No CTC-associated signals within established thresholds were identified in 19 healthy volunteers, but 27 of 28 patients with melanoma displayed signals consistent with single, clustered, and likely rolling CTCs. The detection limit ranged down to 1 CTC/liter of blood, which is ~1000 times better than in preexisting assays. The Cytophone could detect individual CTCs at a concentration of ≥1 CTC/ml in 20 s and could also identify clots and CTC-clot emboli. The in vivo results were verified with six ex vivo methods. These data suggest the potential of in vivo blood testing with the Cytophone for early melanoma screening, assessment of disease recurrence, and monitoring of the physical destruction of CTCs through real-time CTC counting.

Bioinspired magnetic nanoparticles as multimodal photoacoustic, photothermal and photomechanical contrast agents.

Nanoparticles from magnetotactic bacteria have been used in conventional imaging, drug delivery, and magnetic manipulations. Here, we show that these natural nanoparticles and their bioinspired hybrids with near-infrared gold nanorods and folic acid can serve as molecular high-contrast photoacoustic probes for single-cell diagnostics and as photothermal agents for single-cell therapy using laser-induced vapor nanobubbles and magnetic field as significant signal and therapy amplifiers. These theranostics agents enable the detection and photomechanical killing of triple negative breast cancer cells that are resistant to conventional chemotherapy, with just one or a few low-energy laser pulses. In studies in vivo, we discovered that circulating tumor cells labeled with the nanohybrids generate transient ultrasharp photoacoustic resonances directly in the bloodstream as the basis for new super-resolution photoacoustic flow cytometry in vivo. These properties make natural and bioinspired magnetic nanoparticles promising biocompatible, multimodal, high-contrast, and clinically relevant cellular probes for many in vitro and in vivo biomedical applications.

Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry.

Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multicolor in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed. © 2018 International Society for Advancement of Cytometry.

Photoacoustic and fluorescent effects in multilayer plasmon-dye interfaces.

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near-infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold-ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low-toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near-infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.

Noninvasive label-free detection of circulating white and red blood clots in deep vessels with a focused photoacoustic probe.

Blood clotting is a serious clinical complication of many medical procedures and disorders including surgery, catheterization, transplantation, extracorporeal circuits, infections, and cancer. This complication leads to high patient morbidity and mortality due to clot-induced pulmonary embolism, stroke, and in some cases heart attack. Despite the clear medical significance, little progress has been made in developing the methods for detection of circulating blood clots (CBCs), also called emboli. We recently demonstrated the application of in vivo photoacoustic (PA) flow cytometry (PAFC) with unfocused ultrasound transducers for detection of CBCs in small vessels in a mouse model. In the current study, we extend applicability of PAFC for detection of CBCs in relatively large (1.5-2 mm) and deep (up to 5-6 mm) blood vessels in rat and rabbit models using a high pulse rate 1064 nm laser and focused ultrasound transducer with a central hole for an optic fiber. Employing phantoms and chemical activation of clotting, we demonstrated PA identification of white, red, and mixed CBCs producing negative, positive, and mixed PA contrast in blood background, respectively. We confirmed that PAFC can detect both red and white CBCs induced by microsurgical procedures, such as a needle or catheter insertion, as well as stroke modeled by injection of artificial clots. Our results show great potential for a PAFC diagnostic platform with a wearable PA fiber probe for diagnosis of thrombosis and embolism in vivo that is impossible with existing techniques.

Current status, pitfalls and future directions in the diagnosis and therapy of lymphatic malformation.

Lymphatic malformations are complex congenital vascular lesions composed of dilated, abnormal lymphatic channels of varying size that can result in significant esthetic and physical impairment due to relentless growth. Lymphatic malformations comprised of micro-lymphatic channels (microcystic) integrate and infiltrate normal soft tissue, leading to a locally invasive mass. Ultrasonography and magnetic resonance imaging assist in the diagnosis but are unable to detect microvasculature present in microcystic lymphatic malformations. In this review, we examine existing tools and elaborate on alternative diagnostic methods in assessing lymphatic malformations. In particular, photoacoustics, low-toxicity nanoparticles and optical clearing can overcome existing challenges in the examination of lymphatic channels in vivo. In combination with photothermal scanning and flow cytometry, Photoacoustic techniques may provide a versatile tool for lymphatic-related clinical applications, potentially leading to a single diagnostic and therapeutic platform to overcome limitations in current imaging techniques and permit targeted theranostics of microcystic lymphatic malformations.

High-speed microscopy for in vivo monitoring of lymph dynamics.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-µm microspheres as cell phantoms on slides and in 90-µm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Spaser as a biological probe.

Understanding cell biology greatly benefits from the development of advanced diagnostic probes. Here we introduce a 22-nm spaser (plasmonic nanolaser) with the ability to serve as a super-bright, water-soluble, biocompatible probe capable of generating stimulated emission directly inside living cells and animal tissues. We have demonstrated a lasing regime associated with the formation of a dynamic vapour nanobubble around the spaser that leads to giant spasing with emission intensity and spectral width >100 times brighter and 30-fold narrower, respectively, than for quantum dots. The absorption losses in the spaser enhance its multifunctionality, allowing for nanobubble-amplified photothermal and photoacoustic imaging and therapy. Furthermore, the silica spaser surface has been covalently functionalized with folic acid for molecular targeting of cancer cells. All these properties make a nanobubble spaser a promising multimodal, super-contrast, ultrafast cellular probe with a single-pulse nanosecond excitation for a variety of in vitro and in vivo biomedical applications.

In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes.

Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes.

Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

Preclinical photoacoustic models: application for ultrasensitive single cell malaria diagnosis in large vein and artery.

In vivo photoacoustic flow cytometry (PAFC) has demonstrated potential for early diagnosis of deadly diseases through detection of rare circulating tumor cells, pathogens, and clots in nearly the entire blood volume. Before clinical application, this promising diagnostic platform requires verification and optimization using adequate preclinical models. We show here that this can be addressed by examination of large mouse blood vessels which are similar in size, depth and flow velocity to human vessels used in PAFC. Using this model, we verified the capability of PAFC for ultrasensitive, noninvasive, label-free, rapid malaria diagnosis. The time-resolved detection of delayed PA signals from deep vessels provided complete elimination of background from strongly pigmented skin. We discovered that PAFC's sensitivity is higher during examination of infected cells in arteries compared to veins at similar flow rate. Our advanced PAFC platform integrating a 1060 nm laser with tunable pulse rate and width, a wearable probe with a focused transducer, and linear and nonlinear nanobubble-amplified signal processing demonstrated detection of parasitemia at the unprecedented level of 0.00000001% within 20 seconds and the potential to further improve the sensitivity 100-fold in humans, that is approximately 106 times better than in existing malaria tests.