Signal Processing Approaches for Diffuse in Vivo Flow Cytometry

1. Introduction

Flow cytometry is a fundamental and versatile tool in cell quantification, biochemical analysis, cell sorting, etc. Flow cytometry, which is referred here as in vitro flow cytometry or conventional flow cytometry, has been widely used in both basic biomedical research and clinical practice (e.g., monitoring circulating tumor cells (CTCs) in cancer therapy). In conventional flow cytometry, cells of interest, sampled from patients or experimental animals and subsequently labeled with fluorescent tracers, are pumped into a sheath flow. When the labeled cells pass through the laser beam and get excited in the sheath flow, the emitted fluorescence from the cells are detected and analyzed to provide the physical and biochemical information.^1,2 Nevertheless, the conventional flow cytometry can only test cells in vitro and requires blood drawing from patients or animals.³ This blood drawing process might change the native biological environment of the cells. Moreover, the blood sample volume is limited. To overcome these limitations, the fluorescence-based in vivo flow cytometry (IVFC) was proposed and demonstrated. The basic principle of fluorescence-based IVFC is to use the natural blood flow as the sheath flow. As cells, e.g., CTCs, pass through a focused laser slit across a blood vessel of a living animal, exogenous labeling or endogenous chemical contrasts agents are excited. Then the obtained fluorescence signal could provide the information of cells.⁴ As compared with the in vitro flow cytometry, the fluorescence-based IVFC can realize real-time, noninvasive, and dynamic monitoring of cells. Since it was proposed, the fluorescence-based IVFC has been used in biological research, making it an extremely important tool for basic research and a potential candidate for clinical diagnosis and prognosis evaluation of tumor metastases.^5,6,7 In this work, we review our recent works on fluorescence-based IVFC and the utility of fluorescence-based IVFC for biological studies, e.g., monitoring CTCs.

2. Operation Principle of the Fluorescence-Based IVFC

Lin et al. from Harvard Medical School reported the first fluorescence-based IVFC in 2004.⁴ The basic operation principle is shown in Fig. 1. After optical transformation and shaping, the laser beam is focused onto a blood vessel (such as an ear artery of a mouse). Once a fluorescently labeled target cell (such as a CTC) in the circulatory system passes through the laser focusing point (window), it is excited to generate fluorescence signal, which is collected by the fluorescence receiving system. Then the received fluorescence signals are analyzed by the software, thereby the number of target cells passing through the window per unit time (for example, every minute) could be obtained. The overall structure of fluorescence-based IVFC is shown in Fig. 2. Compared with conventional in vitro flow cytometry, fluorescence-based IVFC can provide biological information of circulating cells without extracting blood from samples, reducing the impact of environment and blood separation processes on experimental results. Meanwhile, fluorescence-based IVFC can monitor fluorescently labeled cells in vivo continuously, which avoids blood drawing or sacrificing animals. Furthermore, experimental data obtained at different times from the same animal can effectively reduce experimental errors induced by individual differences in animals.

Fig. 1. The basic principle of the in vivo fluorescent flow cytometry.

Fig. 2. The overall structure of the first in vivo fluorescent flow cytometry. L1, condenser lens; OL, microscope objective lens (40 $\times$ , 0.6 numerical aperture, infinity corrected); BS1, BS2, dichroic beam splitters; AL1–AL3, achromats; CL, cylindrical lens; M1–M4, mirrors; NDF, neutral-density filter; BPF, bandpass filter; PMT, photomultiplier tube.

In 2004, Georgakoudi et al. used IVFC to perform real-time and dynamic monitoring of tumor cells in the circulatory system,⁸ which proved that fluorescence-labeled IVFC was a new powerful tool for tumor cell monitoring in metastasis and early diagnosis of cancer. In 2005, Lin et al. monitored leukemia cells in the bone marrow circulatory system using a fluorescence-labeled IVFC. The experiment revealed the role of CXCR4 in the spreading of leukemia cells into the bone marrow microenvironment.⁹ In 2007, Novak et al. designed a dual-channel IVFC,¹⁰ which were composed of a semiconductor laser (473 $$ nm) and a He–Ne laser (633 $$ nm). The two-channel IVFC was used to monitor two cell populations labeled with different markers. The system was also used to measure the blood flow velocity in the mouse directly, expanding the application scope of the technology. In 2011, Li et al. improved the analysis for the IVFC. The circulatory kinetics of tumor cells were studied. It was found out that the higher the metastatic potential in circulating cells of liver cancer, the shorter the time of CTCs in the circulatory system, explaining the mechanism of tumor cell metastatic potential.¹¹

As we know, the penetration depth of visible light in biological tissue is much smaller than that of near infrared (NIR) light. Thus, we proposed NIR fluorescence-based IVFC in 2015.¹² We changed the excitation source from the visible light band to the NIR band ( $\sim$ 785 $$ nm). The proposed IVFC might expand its biological applications (e.g., stem cell research, cancer diagnosis).

The IVFC could count the specific cells in circulatory system in vivo. To further capture the morphological features of circulating cells, Lee et al. developed an image flow cytometer that visualizes and counts flowing cells rapidly in circulatory system.^{13,14,15,16,17} Wei et al. developed an image flow cytometer in combination with artificial intelligence technology for the counting and imaging of specific cell populations in the blood circulation, such as CTCs and immune cells. Compared with conventional IVFC, it can provide the morphology and structure information of cells, blood vessels and surrounding tissues, which is helpful for studying the hydrodynamic and behavioral characteristics of cells in blood.¹⁸

3. Applications of Fluorescence-Based IVFC

3.1. CTCs detection

As mentioned above, fluorescence-based IVFC could monitor circulating cells with high sensitivity and specificity in real time both noninvasively and continuously. These inherent characteristics make fluorescence-based IVFC a promising tool for biological research, especially for early diagnosis of diseases such as cancer.

In 2009, Wei et al. monitored liver tumor cells in circulatory system using fluorescence-based IVFC to assess the spread of liver tumor cells, endowing IVFC a new function, i.e., assessing the effectiveness of potential therapeutic interventions.¹⁹ The mice are first anesthetized, and the fluorescently labeled tumor cells were injected by intravenous injection. When the labeled tumor cell passed the laser slit, it was excited to generate fluorescence, which was then received by the fluorescence receiving system through a photomultiplier tube (PMT). The received optical signal is converted into an electrical signal. Finally, the number of cells passing through the window per unit time can be obtained and quantitatively analyzed. As shown in Fig. 3. The experiment focused on monitoring liver tumor cells in the circulatory system of small animal model for cancer diagnosis, characterizing pathological malignancies, assessing disease prognosis, predicting and measuring response to treatment, thereby providing valuable information for better treatment. The experiment combines IVFC technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics.

Fig. 3. Fluorescence signal from labeled circulating cells was recorded by IVFC and the detected trace of labeled circulating liver tumor cells. The peak within the trace indicates that a labeled cell passes through the slit of light and thus gives a burst of fluorescence.

In 2011, the same group measured circulation dynamics of metastatic tumor cells using IVFC and studied the relationship between circulating dynamics and metastatic potential. It was found out that the higher the tumor metastatic potential in liver cancer, the shorter the existing time in circulatory system, which explained the mechanism of tumor cell metastatic potential.¹¹ In 2012, they studied liver tumor metastasis using IVFC. The positive correlation between the number of CTCs and the size of tumor volume was found out for the first time. In addition, the results demonstrated that the resection of suspected tumors could effectively reduce the number of CTCs, as well as the proximal and distal metastasis of the tumor as shown in Figs. 4 and 5.²⁰

Fig. 4. The comparison of CTC dynamics and tumor growth between a subcutaneous and an orthotopic metastatic tumor model: CTC counts of the subcutaneous and the orthotopic metastatic tumor model measured by IVFC during eight weeks after tumor implantation. The measurement was carried out once a week and last at least 1 h each time.

Fig. 5. The comparison of CTC dynamics and tumor growth between a subcutaneous and an orthotopic metastatic tumor model: tumor growth in the subcutaneous and the orthotopic metastatic tumor model. Ultrasound imaging was used to determine the diameters of tumors.

In 2018, Pang et al. used fluorescence-based IVFC to achieve real-time dynamic monitoring of CTCs in circulatory system of mouse prostate cancer subcutaneous and orthotopic tumor models. It was found out that the number of CTCs in orthotopic tumor model was much higher than that in subcutaneous tumor model as shown in Fig. 6. With increment of time, the volume of subcutaneous tumor increased gradually, while it increased explosively in orthotopic tumor as shown in Figs. 7(a) and 7(b). Furthermore, survival rate of subcutaneous tumor model mice was also higher than that of orthotopic tumor model mice as shown in Figs. 8(a) and 8(b). As compared with that of subcutaneous tumor model, tumor progression stage of orthotopic tumor model was closer to clinical stage, indicating that orthotopic tumor model is more suitable for studying cancer metastasis.²² Clearly, the site of the tumor affects the CTC levels and the response to treatment. One would speculate that subcutaneous models of cancer are not valid in prostate cancer. More work needs to be done in both prostate cancer and other tumors to validate these results.

Fig. 6. The prostate tumor progression had different profiles between subcutaneous tumor model and orthotopic tumor model. There was a difference in CTC counts between subcutaneous tumor model and orthotopic tumor model measured by IVFC.

Fig. 7. The tumor volume of s.c. tumor models (a) and orthotopic tumor model (b). $n=10$ in each group.

Fig. 8. The survival rate of two types of tumor models was significant difference. The median survival time is 27.5 $$ d in s.c. tumor models (a), and 15 $$ d in orthotopic tumor models (b). $p<0.0001$ , $n=10$ in each group.

3.2. CTC clusters detection

CTCs have been an emerging marker for cancer metastasis. However, the composition of a CTC event was elusive over a long period due to rare quantity of CTCs in blood and lack of quantificational detection methods. Several studies found out that a small portion of CTC events included CTC clusters, which had two or more CTC cells together.

Their metastatic potential was much higher than single CTC.^22,23 Previously, we applied IVFC to detect CTCs, without distinguishing CTC clusters from single CTCs.²⁰ In 2017, we proposed a method to distinguish clusters from single CTC in xenograft tumor models. We found out that IVFC signal of a CTC cluster has multipeaks while that of a single CTC has only one peak as shown in Fig. 9.²⁴ With this criterion, we could distinguish CTC clusters from single CTC in liver cancer model and prostate cancer model. The number and proportion of CTC clusters in total CTC events kept increasing during cancer metastasis, up to $\sim$ 30% in late cancer stage. The obtained results may help us understand the mechanism of cancer metastasis.

Fig. 9. Representative IVFC profiles of a CTC cluster (a) and single CTC (b).

Source: Adapted from Ref. 11, © 2016 International Society for Advancement of Cytometry.

3.3. Homing profiles detection of stem cells

Bone marrow mesenchymal stem cells (MSCs) are a rare population of nonhematopoietic stromal cells with tropism to damaged or tumor sites. In 2017, Xie et al. constructed a number of mouse tumor models, including subcutaneous tumor model, orthotopic tumor model and lung metastasis model of high metastatic liver cancer cells expressing green fluorescent, to study MSCs.²⁵ Bone marrow MSCs of the mice were isolated and cultured by primary culture, and then MSCs with uniform morphology and high purity were obtained. After injection of fluorescently labeled MSCs via tail vein, continuous monitoring by IVFC was performed. The results revealed that MSCs had a longer circulation time in the body and a significant rebound peak appeared at certain time. By primary isolation of HCCLM3 liver tumor cells in in situ solid tumors and metastases, gene expression levels before and after co-culture with MSCs were detected. Their results showed that hepatoma cells from metastases had higher expression levels of EGF, CXCL9, CCL25 and MMP-9 than solid tumor cells, which may result in differences in the ability to recruit MSCs both in vivo and in vitro. Combined with further in vivo imaging experiments, their results demonstrated homing dynamics and mechanism of MSCs in tumor environment and the interaction with hepatocellular carcinoma, providing the possibility to utilize MSCs as a drug carrier for anti-tumor therapy.

3.4. Nanoparticles monitoring

With the development of nanotechnology, many nanomedicines have been proposed and synthesized for biomedical applications. After entering the human body, the nanoparticles usually reach the target tissue through blood circulation. The state of nanoparticles in the blood circulation affects the efficiency of the drug. Therefore, studying the behavior of nanoparticles in the blood circulation has important biomedical significance. In order to increase the dose of the nanodrug to the target organ or tissue and subsequently therapy efficiency, some nanodrug carriers are designed to prolong the residence time of the nanodrug in the blood circulation. Thus, the concentration of nanodrug in the blood is an important indicator. In 2018, Wei et al. utilized IVFC to analyze the clearance process and particle aggregation behavior of two nanoparticles functionalized with PEG-3K-polylactic acid (PLA) and PEG-5K-PLA block copolymers. Then the nanoparticles (PEG-5K nanoparticles and PEG-3K nanoparticles) were injected into the blood circulation of mice through the tail vein, which was followed by in vivo testing by IVFC every 10 min. The baseline signal intensity produced by both nanoparticles decreased over time, meaning the clearance of nanoparticles during blood circulation (Fig. 10(a). In addition, the clearance kinetics of the two nanoparticles are different. The baseline signal intensity of the PEG-5K nanoparticles was higher than the baseline signal intensity of the PEG-3K nanoparticles at all time points. Therefore, the concentration of PEG-5K nanoparticles in the blood is higher than that of PEG-3K nanoparticles throughout the detection process (Fig. 10(b).

Fig. 10. In vivo monitoring of nanoparticle concentration by IVFC validated by HPLC. (a) Nanoparticle concentration in blood circulation measured by IVFC $(n=3)$ . (b) Nanoparticle concentration in blood samples drawn at various time points was measured by HPLC in vitro $(n=3)$ . The error bars represent standard error of the mean. A.U., arbitrary unit.

Source: Adapted from Ref. 21, © 2018 Elsevier B.V. All rights reserved.

To further characterize the aggregates produced by PEG-3K and PEG-5K nanoparticles in vivo, peak height and peak width information were extracted as shown in Figs. 11 and 12. The aggregations of PEG-3K nanoparticles had slightly higher peak height than that of PEG-5K nanoparticles. This was due to the shorter PEG chain length, making it easier to aggregate and form larger aggregates. The peak width dynamics, as shown in Fig. 12, also supported this explanation. The peak widths of PEG-3 K aggregates were larger than those of PEG-5 K aggregates.

Fig. 11. Dynamics of peak height for PEG-3 K and PEG-5 K nanoparticle aggregates measured by IVFC $(n=3)$ .

Source: Adapted from Ref. 21, © 2018 Elsevier B.V. All rights reserved.

Fig. 12. Dynamics of peak width for PEG-3 K and PEG-5 K nanoparticle aggregates measured by IVFC $(n=3)$ .

Source: Adapted from Ref. 21, © 2018 Elsevier B.V. All rights reserved.

The proposed method based on IVFC is promising in the kinetics study of nanoparticles clearance in the blood circulation system. The concentration, aggregates, size of nanoparticles can be deduced from the detected baseline signal intensity, number of peak signals, the peak height and peak width, respectively, making the proposed methods meaningful in vivo pharmacokinetic studies. Briefly, in order to in vivo characterize the aggregation of nanoparticles using IVFC, fluorescent microspheres with different diameters were used in the experiments. The fluorescent microspheres with diameters of 0.5, 1 and 2 $$ $\mu$ m were injected via vein tail, respectively, and the eight and width of signal were extracted for analysis. The height and width of the extracted signal indicated the concentration of the fluorescent microspheres and action time between the microsphere and laser slit, respectively. Meanwhile, the fluorescent microspheres with different diameters would produce different distribution of eight and width, but there is a good linear relationship between them. It means that the concentrations and diameters of the aggregated particles could be induced from the eight and width of signal, respectively.

Meanwhile, the emerging photoacoustic flow cytometry (PAFC) can also be used for kinetics study,^{25,26,27,28,29} and the PAFC can assess the targeted efficiency of nanodrugs to CTCs.^30,31 The fluorescence IVFC provides a powerful tool for the circulating cells detection. It not only realizes real-time and dynamic noninvasive monitoring, but also provides explanation of the occurrence and development of tumors and the molecular mechanisms of metastasis.^{10,11,12,13,14,15}

4. Perspective

Fluorescence-based IVFC can collect information of circulating cells in vivo without drawing blood from animals. It can monitor fluorescently labeled cells continuously in the same living animal for a long time, enabling early diagnosis of diseases such as tumors and immunoassay of blood cells. Thus, fluorescence-based IVFC paves a new avenue for mechanistic study of tumor metastasis and evaluation of tumor treatment.

However, fluorescence-based IVFC requires in vitro labeling of tumor cells injected into the animals or genetic labeling in vivo, while the toxicity of many fluorescent dyes is still not clear.³² Therefore, the IVFC technology is still limited to laboratory research currently.

Zharov et al. found out that the photoacoustic signal of melanoma tumor cells was much higher than that of normal cells under the irradiation with specific wavelength. And then they proposed a new in vivo detection scheme: label-free IVFC based on photoacoustic effect (in vivo PAFC).^{5,28,29,33,34,35,36,37,38,39,40,41} Currently, PAFC has been used to study RBCs, WBCs,^33,34 CTC,³⁵ plant leaf vasculature,³⁶ dysentery³⁷ and nanoparticles.²⁹ For melanoma cells, PAFC used the detected number of circulating melanoma cells to assess tumor development^46,47 or recovery after surgery.³⁹ Due to the low melanin secretion of some melanoma cells, Zharov group used nanoparticle labeled cells to enhance the light absorption, and subsequently the intensity of photoacoustic signals.³⁵ In addition, the experimental results obtained by PAFC showed that damage or extrusion of tumor tissue resulted in an increase in the number of CTCs in the circulatory system, which promoted tumor metastasis.⁴⁰ The Wang group also proposed the photoacoustic imaging flow cytometry to trigger a high-power laser to kill CTCs once CTC signal was detected without damaging normal blood cells.⁴¹ For tumor cells (such as breast cancer cells) that have no obvious specific absorption, the nanoparticle labeling technology could increase the specific light absorption of CTCs and the amplitude of photoacoustic signals. Due to the extremely low quantity of CTCs in the body, the Zharov group enriched the magnetic nanoparticle-labeled CTCs in the blood circulation system by magnets, efficiently enhancing the photoacoustic signals of CTC.²⁸ In addition to PAFC, a number of advanced IVFC technologies, including diffuse fluorescence flow cytometry.^42,43,44 Reflectance confocal microscopy-based IVFC,^45,46 retinal flow cytometer,⁴⁷ Raman flow cytometry,⁴⁸ spectrally encoded flow cytometry,⁴⁹ multiphoton intravital flow cytometry,^50,51 have been proposed and demonstrated.

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Signal Processing Approaches for Diffuse in Vivo Flow Cytometry

Source: https://www.worldscientific.com/doi/10.1142/S1793545819300088

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