Real-time monitoring of single circulating tumor cells with a fluorescently labeled deoxy-glucose by in vivo flow cytometry.

Circulating tumor cells (CTCs) play an essential role in metastasis and serve as an important prognostic biomarker. The technology of CTC labeling and detection in vivo can greatly improve the research of cancer metastasis and therapy. However, there is no in vivo technology to detect CTCs in clinic. In this study, we demonstrate that 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), a 2-deoxy-glucose analog, can work in vivo to indicate CTCs and metastases fluorescently by direct intravenous injection. During the development of an implanted tumor in mice, the spontaneous CTCs released from the primary tumor into blood vessels can be labeled by 2-NBDG due to the abnormal metabolism of CTCs. The green fluorescence of 2-NBDG from CTCs is then noninvasively detected by an in vivo flow cytometry system. Due to the high uptake of glucose by tumor cells, the CTCs in mice can maintain a high 2-NBDG level and thus be distinguished by 2-NBDG fluorescence in vivo efficiently, enabling tumor detection in vivo like positron emission tomography (PET) but at the single-cell resolution. Our results suggest 2-NBDG, a glucose analog with high biosafety, holds promising potential in clinical applications, similar to the widely-used contrast medium 2-F18 -fluorodeoxyglucose in PET.

Monitoring circulating prostate cancer cells by in vivo flow cytometry assesses androgen deprivation therapy on metastasis.

It remains controversial whether surgical castration prolongs survival rate and improves therapy prospects in patients suffering from prostate cancer. We used PC3 cell line to establish prostate tumor models. In vivo flow cytometry and ultrasonic imaging were used to monitor the process of prostate cancer growth, development and metastasis. We found out that the number of circulating tumor cells (CTCs) in orthotopic tumor model was higher than that in subcutaneous tumor model. The CTC number in orthotopic tumor model was due to burst growth, while CTC number in subcutaneous tumor model showed a gradual increase with tumor size. After androgen deprivation therapy (ADT) through testicular extraction, we constructed GFP-PC3 subcutaneous tumor models and orthotopic tumor models. We found dramatically decreased CTC number, relieved symptoms caused by the tumor, and significantly prolonged survival time after testicular extraction in orthotopically transplanted prostate tumor model, while the carcinogenesis process and metastases were little influenced by ADT in subcutaneous tumor model. ADT treatment can restrict tumor growth, decrease the CTC number significantly and inhibit distant invasion through inhibition of tumor proliferation and tumor angiogenesis in orthotopical prostate tumor model. © 2018 International Society for Advancement of Cytometry.

[Backscatter micro-spectra discrimination of liver cancer cell based on principal component analysis arithmetic and back propagation neural network].

In order to achieve the automatic identification of liver cancer cells in the blood, the present study adopted a principal component analysis (PCA) and back propagation (BP) algorithm of feedforward neural networks to identify white blood cells and red blood cells in mice and human liver cancer cells, HepG2. The present paper shows the process in which PCA was carried out after obtaining spectral data by fiber confocal back-scattering spectrograph, selecting the first two principal components as spectral features, and establishing a neural network pattern recognition model with two input layer nodes, eleven hidden layer nodes and three output nodes. In order to verify whether the model would give accurate identification of cells, we chose 195 object data to train the model with 150 sets of data as training set and 45 sets as test set. According to the results, the overall recognition accuracy of the three cells was above 90% with the average relative deviation only 4.36%. The results showed that PCA+BP algorithm could automatically identify liver cancer cells from erythrocyte and white blood cells, which will provide a useful tool for the study of metastasis and biological metabolism characteristics of liver cancer.

[Non-contacting photoacoustic tomography in biological samples].

In photoacoustic imaging the ultrasonic signals are usually detected by contacting transducers. For some applications, contact with the tissue should be avoided, e.g. in those of brain functional imaging. As alternatives to contacting transducers interferometric techniques can be used to acquire photoacoustic signals remotely. Here, a system for non-contact photoacoustic tomography imaging (NCPAT) has been established. This approach enables NCPAT not to exceed laser exposure safety limits. The stimulated source of NCPAT utilized a laser with center wavelength of 532 nm and output intensity of 17.5 mJ/cm 2, and a laser heterodyne interferometry was used to receive the photoacoustic signals. The NCPAT was used to implement on a rotational imaging geometry for photoacoustic tomography with a real-tissue phantom. The photoacoustic imaging was obtained by applying a reconstruction algorithm to the data acquired for NCPAT. Experiments results showed that the NCPAT system with detection 15 dB bandwidth of 2.25 MHz could resolve spherical optical inclusions with dimension of 500 µm and multi-layered structure with optical contrast in strongly scattering medium. The method could expand the scope of photoacoustic and ultrasonic technology to in-vivo biomedical applications where contact is impractical.

Photodynamic therapy reduces metastasis of breast cancer by minimizing circulating tumor cells.

Cancer metastasis after traditional surgery introduces a high barrier to therapy efficacy. Photodynamic therapy (PDT) for cancer is based on a photochemical process of photosensitizers that concentrate in tumors and release oxidant species under light excitation to destroy cells. Compared with traditional surgery, PDT provides minimal invasion and targeted therapy. In this in vivo study, we monitor the real-time and long-term dynamics of circulating tumor cells (CTCs) after a single round of PDT and after surgical resection in a breast cancer animal model. The CTC level is low after PDT treatment, and the recurrence of the primary tumor is postponed in the PDT group compared with the resection group. We find that metastasis is correlated with the CTC level, and the PDT-treated mice show no metastasis in the lung or liver. Our results suggest PDT can effectively reduce metastasis by minimizing CTCs after treatment and is a great technology for breast cancer therapy.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells.

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Microglia modulation with 1070-nm light attenuates Aß burden and cognitive impairment in Alzheimer's disease mouse model.

Photobiomodulation, by utilizing low-power light in the visible and near-infrared spectra to trigger biological responses in cells and tissues, has been considered as a possible therapeutic strategy for Alzheimer's disease (AD), while its specific mechanisms have remained elusive. Here, we demonstrate that cognitive and memory impairment in an AD mouse model can be ameliorated by 1070-nm light via reducing cerebral ß-amyloid (Aß) burden, the hallmark of AD. The glial cells, including microglia and astrocytes, play important roles in Aß clearance. Our results show that 1070-nm light pulsed at 10 Hz triggers microglia rather than astrocyte responses in AD mice. The 1070-nm light-induced microglia responses with alteration in morphology and increased colocalization with Aß are sufficient to reduce Aß load in AD mice. Moreover, 1070-nm light pulsed at 10 Hz can reduce perivascular microglia and promote angiogenesis to further enhance Aß clearance. Our study confirms the important roles of microglia and cerebral vessels in the use of 1070-nm light for the treatment of AD mice and provides a framework for developing a novel therapeutic approach for AD.

Visualizing Interactions of Circulating Tumor Cell and Dendritic Cell in the Blood Circulation Using In Vivo Imaging Flow Cytometry.

OBJECTIVE: Visualizing cell interactions in blood circulation is of great importance in studies of anticancer immunotherapy or drugs. However, the lack of a suitable imaging system hampers progress in this field. METHODS: In this work, we built a dual-channel in vivo imaging flow cytometer to visualize the interactions of circulating tumor cells (CTCs) and dendritic cells (DCs) simultaneously in the bloodstream. Two artificial neural networks were trained to identify blood vessels and cells in the acquired images. RESULTS AND CONCLUSION: Using this technique, single CTCs and CTC clusters were readily distinguished by their morphology. Interactions of CTCs and DCs were identified, while their moving velocities were analyzed. The CTC-DC clusters moved at a slower velocity than that of single CTCs or DCs. This may provide new insights into tumor metastasis and blood rheology. SIGNIFICANCE: This in vivo imaging flow cytometry system holds great potential for assessing the efficiency of targeting CTCs with anticancer immune cells or drugs.

Noninvasive monitoring of nanoparticle clearance and aggregation in blood circulation by in vivo flow cytometry.

Nanoparticles have been widely used in biomedical research as drug carriers or imaging agents for living animals. Blood circulation is crucial for the delivery of nanoparticles, which enter the bloodstream through injection, inhalation, or dermal exposure. However, the clearance kinetics of nanoparticles in blood circulation has been poorly studied, mainly because of the limitations of conventional detection methods, such as insufficient blood sample volumes or low spatial-temporal resolution. In addition, formation of nanoparticle aggregates is a key determinant for biocompatibility and drug delivery efficiency. Aggregation behavior of nanoparticles in blood is studied using dynamic light scattering in serum or serum protein solutions, which is still very different from in vivo condition. In this work, we monitored the dynamics of nanoparticle concentration and formation of nanoparticle aggregates in the bloodstream in live animals using in vivo flow cytometry (IVFC). The results indicated that nanoparticles in smaller size could stay longer in the bloodstream. Polyethylene glycol (PEG)-modification could prolong circulating time and reduce the formation of aggregates in the blood circulation. Our work shows that IVFC can be a powerful tool for pharmacokinetic studies of nanoparticles and other drug carriers, assessing cell-targeting efficiency, as well as potentially measuring cardiac output and hepatic function in vivo.

Systemically Infused Mesenchymal Stem Cells Show Different Homing Profiles in Healthy and Tumor Mouse Models.

Bone marrow-derived mesenchymal stem cells (MSCs) can localize in injured, inflamed, and cancerous tissues after systemic infusion. However, the dynamic homing profile of MSCs in the peripheral blood is not well characterized. Here, using in vivo flow cytometry to noninvasively monitor the dynamics of fluorescence-labeled cells, we found different clearance kinetics of systemically infused MSCs between healthy and tumor mouse models. The circulation times of MSCs in healthy mice and mice with subcutaneous tumors, orthotopically transplanted liver tumors, or metastatic lung tumors were 30, 24, 18, and 12 hours, respectively, suggesting that MSCs actively home to tumor environments. MSCs infiltrated into hepatocellular carcinoma (HCC) sites and preferentially engrafted to micrometastatic regions both in vivo and in vitro. The expression of epidermal growth factor, CXCL9, CCL25, and matrix metalloproteinases-9 by HCC cells differed between primary tumor sites and metastatic regions. By characterizing the homing profiles of systemically perfused MSCs under physiological and cancerous conditions, these findings increase our understanding of the migration of MSCs from the circulation to tumor sites and constitute a basis for developing MSC-based anti-cancer therapeutic strategies. Stem Cells Translational Medicine 2017;6:1120-1131.

Imaging acidosis in tumors using a pH-activated near-infrared fluorescence probe.

A novel pH activatable near-infrared fluorescence probe was developed, which successfully visualized acidosis in tumors. This probe holds promise to non-invasively predict the tumor metastasis potential and evaluate the therapeutic response.

Real-time monitoring of rare circulating hepatocellular carcinoma cells in an orthotopic model by in vivo flow cytometry assesses resection on metastasis.

The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.

Two-order targeted brain tumor imaging by using an optical/paramagnetic nanoprobe across the blood brain barrier.

Surgical resection is a mainstay of brain tumor treatments. However, the completed excision of malignant brain tumor is challenged by its infiltrative nature. Contrast enhanced magnetic resonance imaging is widely used for defining brain tumor in clinic. However its ability in tumor visualization is hindered by the transient circulation lifetime, nontargeting specificity, and poor blood brain barrier (BBB) permeability of the commercially available MR contrast agents. In this work, we developed a two-order targeted nanoprobe in which MR/optical imaging reporters, tumor vasculature targeted cyclic [RGDyK] peptides, and BBB-permeable Angiopep-2 peptides are labeled on the PAMAM-G5 dendrimer. This nanoprobe is supposed to first target the α(V)ß(3) integrin on tumor vasculatures. Increased local concentration of nanoprobe facilitates the association between BBB-permeable peptides and the low-density lipoprotein receptor-related protein (LRP) receptors on the vascular endothelial cells, which further accelerates BBB transverse of the nanoprobe via LRP receptor-mediated endocytosis. The nanoprobes that have penetrated the BBB secondly target the brain tumor because both α(V)ß(3) integrin and LRP receptor are highly expressed on the tumor cells. In vivo imaging studies demonstrated that this nanoprobe not only efficiently crossed intact BBB in normal mice, but also precisely delineated the boundary of the orthotropic U87MG human glioblastoma xenograft with high target to background signal ratio. Overall, this two-order targeted nanoprobe holds the promise to noninvasively visualize brain tumors with uncompromised BBB and provides the possibility for real-time optical-image-guided brain tumor resection during surgery.