Mazen A. Juratli

Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances

Nanotechnology has been extensively explored for cancer diagnostics. However, the specificity of current methods to identify simultaneously several cancer biomarkers is limited due to color overlapping of bio-conjugated nanoparticles. Here, we present a technique to increase both the molecular and spectral specificity of cancer diagnosis by using tunable silver-gold nanorods with narrow surface-enhanced Raman scattering (SERS) and high photothermal contrast. The silver-gold nanorods were functionalized with four Raman-active molecules and four antibodies specific to breast cancer markers and with leukocyte-specific CD45 marker. More than two orders of magnitude of SERS signal enhancement was observed from these hybrid nanosystems compared to conventional gold nanorods. Using an antibody rainbow cocktail, we demonstrated highly specific detection of single breast cancer cells in unprocessed human blood. By integrating multiplex targeting, multicolor coding, and multimodal detection, our approach has the potential to improve multispectral imaging of individual tumor cells in complex biological environments.

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits.

Real-time monitoring of circulating tumor cell release during tumor manipulation using in vivo photoacoustic and fluorescent flow cytometry

Circulating tumor cells (CTCs) form metastases in distant organs. The purpose of this research was to determine if tumor manipulation could enhance cancer cell release from the primary tumor into the circulatory system.

Optical clearing in photoacoustic flow cytometry.

Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins.

Nonlinear photoacoustic signal amplification from single targets in absorption background.

Photoacoustic (PA) detection of single absorbing targets such as nanoparticles or cells can be limited by absorption background. We show here that this problem can be overcome by using the nonlinear photoacoustics based on the differences in PA signal dependences on the laser energy from targets and background. Among different nonlinear phenomena, we focused on laser generation of nanobubbles as more efficient PA signal amplifiers from strongly absorbing, highly localized targets in the presence of spatially homogenous absorption background generating linear signals only. This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo. Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background. Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5–20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

In vivo acoustic and photoacoustic focusing of circulating cells.

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models.

Dynamic Fluctuation of Circulating Tumor Cells during Cancer Progression

Circulating tumor cells (CTCs) are a promising diagnostic and prognostic biomarker for metastatic tumors. We demonstrate that CTCs’ diagnostic value might be increased through real-time monitoring of CTC dynamics. Using preclinical animal models of breast cancer and melanoma and in vivo flow cytometry with photoacoustic and fluorescence detection schematics, we show that CTC count does not always correlate with the primary tumor size. Individual analysis elucidated many cases where the highest level of CTCs was detected before the primary tumor starts progressing. This phenomenon could be attributed to aggressive tumors developing from cancer stem cells. Furthermore, real-time continuous monitoring of CTCs reveals that they occur at highly variable rates in a detection point over a period of time (e.g., a range of 0–54 CTCs per 5 min). These same fluctuations in CTC numbers were observed in vivo in epithelial and non-epithelial metastatic tumors, in different stages of tumor progression, and in different vessels. These temporal CTC fluctuations can explain false negative results of a one-time snapshot test in humans. Indeed, we observed wide variations in the number of CTCs in subsequent blood samples taken from the same metastatic melanoma patient, with some samples being CTC-free. If these phenomena are confirmed in our ongoing in vivo clinical trials, this could support a personalized strategy of CTC monitoring for cancer patients.

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions.

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.

Real-Time Label-Free Embolus Detection Using In Vivo Photoacoustic Flow Cytometry.

Thromboembolic events are one of the world’s leading causes of death among patients. Embolus or clot formations have several etiologies including paraneoplastic, post-surgery, cauterization, transplantation, or extracorporeal circuits. Despite its medical significance, little progress has been made in early embolus detection, screening and control. The aim of our study is to test the utility of the in vivo photoacoustic (PA) flow cytometry (PAFC) technique for non-invasive embolus detection in real-time. Using in vivo PAFC, emboli were non-invasively monitored in the bloodstream of two different mouse models. The tumor-free mouse model consisted of two groups, one in which the limbs were clamped to produce vessel stasis (7 procedures), and one where the mice underwent surgery (7 procedures). The melanoma-bearing mouse model also consisted of two groups, one in which the implanted tumor underwent compression (8 procedures), and one where a surgical excision of the implanted tumor was performed (8 procedures). We demonstrated that the PAFC can detect a single embolus, and has the ability to distinguish between erythrocyte–rich (red) and leukocyte/platelet-rich (white) emboli in small vessels. We show that, in tumor-bearing mice, the level of circulating emboli was increased compared to tumor-free mice (p = 0.0013). The number of circulating emboli temporarily increased in the tumor-free control mice during vessel stasis (p = 0.033) and after surgical excisions (signed-rank p = 0.031). Similar observations were noted during tumor compression (p = 0.013) and after tumor excisions (p = 0.012). For the first time, it was possible to detect unlabeled emboli in vivo non-invasively, and to confirm the presence of pigmented tumor cells within circulating emboli. The insight on embolus dynamics during cancer progression and medical procedures highlight the clinical potential of PAFC for early detection of cancer and surgery-induced emboli to prevent the fatal thromboembolic complications by well-timed therapy.

TERT Promoter Mutation Detection in Cell-Free Tumor-Derived DNA in Patients with IDH Wild-Type Glioblastomas: A Pilot Prospective Study

Purpose: We conducted a pilot study to assess the feasibility and the potential implications of detecting TERT promoter (TERTp)–mutant cell-free tumor-derived DNA (tDNA) in the cerebrospinal fluid (CSF) and plasma of glioblastoma patients. Experimental Design: Matched CSF and plasma samples were collected in 60 patients with glial tumors. The CSF collection was obtained during surgery, before any surgical manipulation of the tumor. The extracted tDNA and corresponding tumor DNA samples were analyzed for TERTp and isocitrate dehydrogenase (IDH) hotspot mutations. In addition, the variant allele frequency (VAF) of TERTp mutation in the CSF-tDNA was correlated with tumor features and patients’ outcome. Results: Thirty-eight patients had TERTp-mutant/IDH wild-type glioblastomas. The matched TERTp mutation in the CSF-tDNA was successfully detected with 100% specificity (95% CI, 87.6–100%) and 92.1% sensitivity (95% CI, 78.6–98.3%) (n = 35/38). In contrast, the sensitivity in the plasma-tDNA was far lower [n = 3/38, 7.9% (95% CI, 1.6–21.4%)]. We concordantly observed a longer overall survival of patients with low VAF in the CSF-tDNA when compared with patients with high VAF, irrespective of using the lower quartile VAF [11.45%; 14.0 mo. (95% confidence interval, CI, 10.3–17.6) vs. 8.6 mo. (95% CI, 4.1–13.2), P = 0.035], the lower third VAF [13%; 15.4 mo. (95% CI, 11.6–19.2) vs. 8.3 mo. (95% CI, 2.3–14.4), P = 0.008], or the median VAF [20.3%; 14.0 mo. (95% CI, 9.2–18.7) vs. 8.6 mo. (95% CI, 7.5–9.8), P = 0.062] to dichotomize the patients. Conclusions: This pilot study highlights the value of CSF-tDNA for an accurate and reliable detection of TERTp mutations. Furthermore, our findings suggest that high TERTp mutation VAF levels in the CSF-tDNA may represent a suitable predictor of poor survival in glioblastoma patients. Further studies are needed to complement the findings of our exploratory analysis. Clin Cancer Res; 24(21); 5282–91. ©2018 AACR.