Isolation of circulating tumor cells from seminal fluid of patients with prostate cancer using inertial microfluidics

doi:10.21203/rs.3.rs-1399684/v1

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Isolation of circulating tumor cells from seminal fluid of patients with prostate cancer using inertial microfluidics

https://doi.org/10.21203/rs.3.rs-1399684/v1

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Prostate cancer (PCa) diagnosis is primarily based on prostate-specific antigen (PSA) testing and prostate tissue biopsies. However, PSA testing has relatively low specificity, while tissue biopsies are highly invasive and have relatively low sensitivity at early stages of PCa. As an alternative, we developed a technique of liquid biopsy, based on isolation of circulating tumor cells (CTCs) from seminal fluid (SF). The recovery of PCa cells from SF was demonstrated using PCa cell lines, achieving the efficiency and throughput as high as > 82% and ~ 1.7 mL min^− 1, respectively, while > 99% of sperm cells was disposed of. The introduced approach was further tested in clinical setting by collecting and processing SF samples of PCa patients. The yield of isolated CTCs was measured as high as 613 cells per SF sample in comparison with that of 6 cells from SF of healthy donors, holding significant promise for PCa diagnosis. The correlation analysis of the isolated CTC amounts with the standard prognostic parameters such as Gleason score and PSA serum level showed correlation coefficient values at 0.40 and 0.73, respectively. Taken together, our results show promise of the developed liquid biopsy technique to augment the existing diagnosis and prognosis of PCa.

Microfluidics

Liquid biopsy

Circulating tumor cells

Prostate cancer

Seminal fluid

Prostate cancer (PCa) is one of the most common cancers worldwide and remains the third leading cause of cancer death in men ¹. Although PCa is common, there is still controversy regarding the utility of the existing screening and diagnostic methods. Prostate-specific antigen (PSA) blood tests in combination with invasive tissue biopsy are regarded the gold standard for early detection of PCa ² despite the following shortcomings. The specificity of PSA blood test is known to be relatively low, especially in case of the localized form of the disease. At the same time, while the sensitivity of the prostate tissue biopsy is moderate, it has to be accompanied by the needle-guiding radiological imaging and carries the risk of potential side-effects including local abscesses and bleeding ³. For the past decade, detection and assaying of circulating tumor cells (CTCs) has advanced to allow diagnosis and prognosis of diverse cancers, including PCa ⁴. However, in case of localized PCa, CTCs are relatively rare measured as few as several cells per milliliter of the blood. Besides, PCa CTC isolation remains cumbersome using the existing liquid biopsy techniques. As a result, the isolation and assaying of PCa CTCs from the peripheral blood is considered inadequate for early detection of PCa ⁵.

It has been reported that CTCs released from the primary tumor can escape to the blood stream and urine, while the escape pathway into the prostatic ducts has been largely overlooked. The detection of CTCs in urine reported to date ^6–8 speaks in favor of this possibility. An increase of the PCa biomarkers, including PCa antigen 3, in urine has been observed ⁹ during the digital rectal examination, which was known to squeeze some prostatic fluid into the urethra. One can conjecture that biomarkers detected in the urine originate from the prostatic gland ¹⁰. Therefore, the seminal fluid (SF) naturally abundant with prostatic fluid may represent an alternative to the blood and urine biological fluid rich of PCa CTCs.

In 1996, Gardiner et al. have described the presence of PCa CTCs in the ejaculate of PCa patients ¹¹. In this study, the SF was centrifuged, the resultant pellet was cast into smears, followed up by microscopic examination to detect CTCs. Atypical prostatic cells in the SF specimens were confirmed in 75% of the cohort and deemed inadequate. The CTC detection in the ejaculate smears was hindered by the overwhelming background of sperm cells measured in hundreds of millions per milliliter of the SF. Flow cytometry of the processed SF has allowed augmenting the procedure of the identification and counting PCa CTCs but at the expense of the cell viability compromise and wasting rare cells ¹². Therefore, an optimised automated method of isolating CTCs from SF is required to discriminate CTCs from unwanted background of sperm cells and seminal plasma debris and maintain the isolated cell viability. To this aim, we adopt the inertial size-based microfluidic technology.

The inertial microfluidics has demonstrated excellent performance for CTC isolation and assaying from the blood of cancer patients ^13–19 as well as CTC isolation from the urine of PCa patients ⁸. However, this is the first time to the best of our knowledge, CTC isolation from the SF of patients with early localized form of PCa by means of inertial microfluidics is reported and demonstrated superb diagnostic and prognostic performance. This technique is believed to complement the existing gold standard PCa diagnostics suite. Purification of sperm cells for cryopreservation is another worthwhile possibility afforded by our technology.

2.1. Microfluidic device and its fabrication

The deployed microfluidic device architecture has been reported by Warkiani et al. ^13,17−19, and was adopted for the isolation of CTCs from the SF. A microfluidic chip was structured as two polydimethylsiloxane (PDMS) layers (Sylgard 184 from Dow Corning, MI, USA) made by mixing liquid PDMS and curing agent in a 10:1 ratio (w/w). The top layer of the device fabricated by employing the previously reported micromolding process was bonded to a flat layer of PDMS following oxygen plasma treatment for 2 min (Harrick Plasma, USA); 1.5 mm holes were punched at the beginning and ends of the microfluidic channel and 1.5-mm tygon tubes were inserted into each inlet/outlet.

2.2. Cell culture

The proof-of-concept experiments were performed using DU145, PC3, and LNCaP PCa cell lines. These cell lines were cultured under standard conditions in a humidified incubator (Panasonic, South Korea) at 37°C, in 5% CO2. DU-145 and PC-3 cell lines were cultured in RPMI-1640 medium (Thermofisher Scientific, USA) with Glutamax supplement (Gibco, USA), 10% heat-inactivated, 0.22 µm filtered fetal bovine serum (Life Technologies, Inc), and 1% Penicillin-Streptomycin (Life Technologies, Inc). For LNCaP cell line, an RPMI-1640 medium (Thermofisher Scientific, USA) supplemented with 10% heat-inactivated, 0.22 µm filtered fetal bovine serum (Life Technologies, Inc), 1% insulin-transferrin (Paneco, Russia), 1% HEPES pH 7.2–7.5 (Paneco, Russia), 1% nonessential amino acid solution (Gibco, USA), 1% gentamycin (Paneco, Russia), was used. RAW264.7 cell line, used as a negative control, was cultivated by using DMEM-F12 (PanEco, Russia) media with 1% penicillin (Invitrogen, USA), 100% streptomycin (Invitrogen, USA) and 10% bovine serum albumin (BSA) (Invitrogen, USA). T-25 and T-75 filtered flasks (Corning, USA) were used for subculturing of the cell lines. Cell dissociation was performed using 0.25% (w/v) Trypsin- 0.53 mM EDTA solution (Thermofisher Scientific, USA). Replating was carried out at a 1 to 6 ratio. Cell culture medium was renewed every 2 or 3 days. Cell lines were STR profiled for authenticity.

2.3. Proof-of-Concept Experiments

The ability of the microfluidic chip to isolate PCa CTCs in the SF was initially evaluated using human SF samples spiked with a predetermined number of DU-145 cells at approximately 1000 cells per standard volume (2–3 mL) of healthy male’s SF. For proper characterization, DU-145 cells were first stained with 4',6-diamidino-2-phenylindole (DAPI) prior to PCa SF spiking. Separation was then performed at flow rates of 1.1, 1.3, 1.5, 1.7, 1.9, and 2.1 mL/min to briefly characterize an ideal flow rate for further testing. Following this, DU-145 (or LNCaP, or PC3) cells were spiked at approximately 1000 cells into a standard volume (2–3 mL) of healthy male’s SF. SF samples were liquified for 30 min in a humidified incubator (Panasonic, South Korea) at 37°C prior to the addition of PCa cells. The sample was then transferred to a 15 mL falcon tube, centrifuged at 1000 rpm for 7 min and the supernatant was replaced with 5 mL of Dulbecco’s phosphate buffered saline (DPBS). Then the solution was processed using a microfluidic chip with a syringe pump mounted at the device input pumping tested fluid (Baoding Shenchen Precision Pump Co., Ltd, Baoding, China) connected with Tygon tubing (0.02-inch inner diameter, 0.06-inch outer diameter). The flow rate was optimised as 1.7 mL/min. The microfluidic chip outlet was split into ‘target’ and ‘waste’ fractions, where the target fraction contained most of the PCa cells (DU-145, LNCaP, or PC3), while the waste fraction contained most of the sperm cells. The mixture of cells in the target fraction was fixed with 4% paraformaldehyde (PFA) (ThermoFisher Scientific, USA) solution in DPBS for 10 min, and therefore prepared for antibody labelling. After fixation, the solution was centrifuged at 1000 rpm and the supernatant was replaced with 5 mL of DPBS. Further, a potential of additional processing of the target fraction through the microfluidic chip was assessed in terms of the separation efficiency of the residual sperm cells. All the experiments were repeated 5 times.

2.4. Clinical samples

The second component of this study included the collection and processing of SF samples obtained from PCa patients and healthy volunteers, which was performed under the ethical approval provided by Sechenov University Local Ethics Committee (extraction from protocol № 17–19). The samples were collected at Sechenov University Clinical Hospital №2, Moscow, Russia. All donors provided written informed consent for collection of their SF samples and provision of their clinical data. The study was conducted in accordance with the Declaration of Helsinki (2013). In total, 15 patients with diagnosed localized PCa and 15 healthy volunteers provided SF samples, which were collected and analyzed in a non-blinded manner. SF samples were collected in sterile collection cups, and ranged between 0.5 mL and 3.5 mL in volume. Samples were transported from the hospital to the testing lab in a thermal container for microfluidic processing and further analysis. Each SF sample was incubated for 30 min in a humidified incubator (Panasonic, South Korea) at 37°C for liquefaction of the SF. Then, each sample centrifuged at 1000 rpm for 7 min and the pellets were resuspended in 5 mL of DPBS. After, the cells were fixed with 4% PFA solution in DPBS for 10 min, centrifuged again, and resuspended in 5 mL of DPBS. The solution was then processed using the microfluidic chip connected to the syringe pump (Baoding Shenchen Precision Pump Co., Ltd, Baoding, China) at the flow rate of 1.7 mL/min.

2.5. Immunofluorescence staining

For the proof-of-concept experiments using cultured PCa cells lines, collected fractions were centrifuged onto four adhesive glass slides (ThermoFisher Scientific, Australia) using a Thermo Scientific™ Cytospin™ 4 Cytocentrifuge (ThermoFisher Scientific, Australia) and air-dried. Further, three primary and three secondary antibodies were applied. The primary antibodies were as follows: rabbit monoclonal recombinant anti-prostate specific membrane antigen (PSMA) antibody (Abcam, catalog number ab76104, Cambridge, UK), mouse monoclonal anti-cytokeratin (CK) antibody (Abcam, catalog number ab86374, Cambridge, UK) and rabbit monoclonal recombinant anti-Glypican 1 (GPC-1) antibody (Abcam, catalog number ab199343, Cambridge, UK). The secondary antibodies were as follows: goat anti-rabbit IgG H&L Alexa Fluor 568 (Abcam, Cambridge, UK), goat anti-mouse IgG H&L Alexa Fluor 488 (Abcam, Cambridge, UK), goat anti-rabbit IgG H&L Alexa Fluor 647 (Abcam, Cambridge, UK). After incubations with the primary and secondary antibodies, the cells were mounted with a ProLong Gold Anti-Fade Mountant (Abcam, Cambridge, UK) containing DAPI, covered with a coverslip and counted under a fluorescence microscope (Zeiss Axio Imager Z2 Upright Microscope). The same number of DU-145 (or PC3, or LNCaP) cells, as the one added to the SF sample, was deposited onto the glass slide and labelled with anti-CK + AlexaFluor488 antibodies, then mounted with a ProLong Gold Anti-Fade Mountant (Abcam, Cambridge, UK) containing DAPI, covered with a coverslip, and counted under a fluorescence microscope (Zeiss Axio Imager Z2 Upright Microscope).

Sequential labelling was employed to identify isolated PCa CTCs in the clinical samples processed by using the microfluidic chip. The same antibodies as described above were applied. These antibodies were used at the concentrations recommended by the manufacturer. Staining was performed by first permeabilizing cells with a 0.2% Tween-20 in phosphate buffered saline (PBS) (ThermoFisher Scientific, USA) for 10 min at room temperature. After permeabilization, the glass slides were washed with DPBS 3 times for 5 min and air-dried. Glass slides were then incubated with a 3% solution of BSA in phosphate-buffered saline with Tween® detergent (PBST) (0.05% solution of Tween-20 in PBS) for 1 hour at room temperature, for minimization of unspecific binding of the antibodies. As the next step, the BSA in PBST solution was gently washed from the glass slides, and the solution of first primary antibody in PBST with 1% BSA was added onto the slides and incubated overnight at 4°C. Afterwards, the glass slides were gently washed with DPBS 3 times for 5 min, air-dried and incubated with 3% BSA in PBST solution of secondary antibody for 1 hour. Then, the glass slides were washed with DPBS 3 times for 5 min and air-dried. The incubations with other two primary and secondary antibodies were performed as described above. Finally, cells were mounted with a ProLong Gold Anti-Fade Mountant (Abcam, Cambridge, UK) containing DAPI, covered with a coverslip and observed under the confocal microscope.

2.6. Cell enumeration and data analysis

Fluorescent laser-scanning confocal microscope Zeiss LSM880 (ZeissAG, Germany) supported by Zen Black software (ver 3.1), was employed to image glass slides with tested specimens. To establish a benchmark for identification and enumeration of the isolated СTCs, healthy SF samples were spiked with approximately 1000 PC3 or DU-145 cells, processed by the chip, and immunocytostained as described above. Images of the immunocytostained cells were then obtained using laser scanning microscope Zeiss LSM880 and Zen Black software. The acquired fluorescence signal intensity of cells was quantified by using Fiji ImageJ software. Further, the mean (± SD) value of the signal intensity was measured and assigned as a threshold for fluorescence signal intensities from different fluorescent wavelengths. The isolated cells were assigned as PCa cells if the signal intensity was above the established thresholds for all three fluorescence channels (PSMA+, CK+, GPC-1+). In addition, to evaluate the potential diagnostic and prognostic value of the technique, a correlation between the amounts of the isolated CTCs and PSA serum levels, or Gleason score (GS) was estimated.

3.1. Recovery of CTCs from Spiked SF

The primary aim of this study was to characterize the performance of a microfluidic cell separation device in terms of its efficiency in isolating PCa CTCs (Fig. 1).

The inertial microfluidic device comprised of a microfluidic channel with a trapezoidal cross-section: one inlet and two outlets separated by a bifurcation of the channel (Fig. 2D). To determine the optimal flow rate for CTC isolation from SF cell suspensions, a range of flow rates were tested using healthy SF samples spiked with DU-145 cells (Fig. 2B). The resultant cell suspensions from the target (mostly tumor cells) and waste (mostly sperm cells) outlets can be seen in Fig. 2A (a video of the high-speed cell separation is available in Supplementary Video 1). The flow rates ranging from 1.1 to 2.1 mL/min were tested. The device performance, operated at the flow rates of more than 1.7 mL/min, exhibited a distinct drop in the isolation efficiency of tumor cells. At the same time, the device performance operated at the flow rates of less than 1.5 mL/min was similar to that of 1.7 mL/min but the operation rate was slower. The flow rate of 1.7 mL/min was chosen as the most optimal to produce the average recovery of tumor cells at 89.01%±3.8%. The sperm recovery rates were above 97% for all tested flow rates. As a result, the majority of cancer cells were isolated and presented into the target fraction (Fig. 2E), while the majority of the sperm cells were separated and presented into the waste fraction (Fig. 2F).

Further, DU-145, PC3, and LNCaP cells lines were individually spiked into healthy SF samples and processed at the flow rate of 1.7 mL/min. The recovery rates were 85 (± 8) %, 82 (± 9) % and 83 (± 6%) for DU-145, PC3 and LNCaP cells, respectively [Figure2 (C)]. The recovery performance was comparable across the tested cell lines and demonstrated a similar level of recovery at more than 82% on average after a single processing with the microfluidic device, while more than 99% of the sperm cells could have been removed. The additional processing of the target fraction resulted in a loss of 15 (± 3) %, 12 (± 6) % and 17 (± 4) % of DU-145, PC3 and LNCaP cells, respectively, and additional removal of 13 (± 5) % of the sperm cells, calculated from the total amount of the cells presenting in the target fraction. Thus, the additional processing of the targeted fraction resulted in a loss of the cancer cells and removal of the sperm cells in comparable percentage amount of 15%. Therefore, additional purification of the target fraction from sperm cells by means of the second processing was deemed unreasonable. As a result, each SF sample was processed through the microfluidic chip just once.

3.2. Isolation of putative CTCs from the patients’ SF

SF samples from the group of 15 patients with diagnosed PCa at its localized stage, and from 15 healthy males aged from 18 to 30 years as a control group, were acquired. Cells collected from the target outlet of the device were considered CTCs if they were at more than 13 µm in mean diameter; featured the cytoplasm area at more than 232 µm² determined from measurements of DU-145 and PC3 cells of the mean area of (284 ± 52) µm² ; had the cell nucleus mean diameter at more than 92 µm², measured from DU-145 and PC3 cells; and were positive for 3 antigens: CK (CK+), PSMA (PSMA+), and GPC-1 (GPC-1+). CK+, PSMA+, and GPC-1+, defined as the cells exhibiting a fluorescence signal of more than 1572, 1589, and 1150 arbitrary units, respectively. The confirmation values were based on the mean fluorescent intensities of DU-145 and PC-3 cells measured as 1771 ± 199, 1867 ± 278, and 1423 ± 273 for the corresponding antigens, respectively.

As a result, CTCs were found in all processed SF samples collected from the PCa patients. The number of the isolated CTCs varied from 67 to 307 per mL (Table 1), with the median value of 104 and the mean value of 135 cells. However, cells overexpressing CK, PSMA and GPC-1 were identified in 2 of 15 samples in the healthy donor SF samples, although the number of identified abnormal cells was significantly lower ranging from 1.2 to 3 cells per mL for each of two samples, with the total amount of such cells not exceeding 6 units per sample. Figure 3 shows representative images of the antibody-based fluorescent labelling of CTCs isolated from the patients’ samples. It is worth noting that a significant level of the heterogeneity in the intensity of expression between the different antigens was observed [Figure 3 (B)].

Table 1

Number of isolated CTCs from SF samples and parameters of PCa in patients.
Patient number	n*	V**	n/V	TNM stage	Gleason score	PSA level
1	217	2.1	103.3	T1cN0M0	7	10.8
2	321	1.6	200.6	T2cN0M0	9	23
3	238	2.3	103.5	T2cN0M0	7	8.5
4	289	3.5	82.6	T2cN0M0	6	6.3
5	460	1.5	306.7	T1bN0M0	8	19
6	183	1.9	96.3	T1cN0M0	7	6.5
7	174	2.6	66.9	T2bN0M0	7	16
8	357	3	119.0	T2bN0M0	8	32.4
9	187	2.7	69.3	T1cN0M0	7	4.1
10	63	0.5	126	T1cN0M0	7	8.9
11	145	1.9	76.3	T1cN0M0	6	2.3
12	329	1.4	235	T1cN0M0	7	14.7
13	115	1.6	71.9	T1cN0M0	7	8.2
14	613	2.7	227	T2cN0M0	10	10.2
15	340	2.4	141.7	T2cN0M0	7	9.2
* - total amount of PSMA+, CK+, GPC-1 + putative tumor cells in the patients’ SF samples
** - total volume of a patients’ SF samples

3.3. Potential diagnostic and prognostic value of the method

The potential diagnostic and prognostic applicability of the developed method was evaluated taking into consideration the absolute amount of CTCs (n), the amount of CTCs per mL of the SF (n/V), and conventional diagnostic and prognostic parameters such as PSA serum level or GS (Table 1). The correlation coefficients (r) for n/V vs PSA and n/V vs GS were measured as 0.40 and 0.63, respectively. Further, r-values for n vs PSA, n vs GS, and GS vs PSA were measured as 0.42, 0.73, and 0.51, respectively. Thus, a weak positive correlation was found between the PSA serum level and total numbers of the isolated CTCs, as well as the number of CTCs per mL of the SF, with an insignificant difference between the r-values. At the same time, a moderate positive correlation was found between the GS and the total number of the isolated CTCs, as well as the number of CTCs per mL of the SF, with no significant difference between the r-values (Fig. 4). A moderate correlation was found between the GS and PSA values (See Supplementary Fig. 1). Further, a weak positive correlation was identified between the number of the isolated CTCs and the total volume of the SF sample (r estimated as 0.33).

During the past decades, in clinical practice, PSA blood test has been the most trusted tool for early detection of PCa and its prognostics ². Another tool, conventionally used in diagnosing PCa, has been the tissue biopsy. However, both diagnostic tools have significant disadvantages. Thus, PSA blood test has a relatively low specificity, while tissue biopsy has only moderate sensitivity if performed without being accompanied by radiological techniques and may also have serious complications ³. Recently, in PCa management, liquid biopsy by means of microfluidics-based isolation of CTCs presenting in biological fluids was proposed as the perspective alternative to the existing conventional diagnostic and prognostic techniques ²⁰. However, according to the published data, liquid biopsy of neither blood ^21,22 nor urine ⁸ demonstrated an outstanding performance in detecting PCa at its early stage. Therefore, investigation of other biological fluids as the potential sources of CTCs, particularly – semen, is of a great interest.

We tested this hypothesis by employing microfluidic label-free isolation of PCa CTCs from the SF of the patients diagnosed with PCa at its localized form. According to the obtained results, the technique based on inertial microfluidics demonstrated remarkably high efficiency in separating PCa CTCs from sperm cells presenting in the SF. Initially, flow rates ranging from 1.1 to 2.1 mL/min were tested for PCa cell isolation by using healthy SF spiked with DU-145 cell line. Due to the particular structure and cross-section of the microchannel, sperm and PCa cells experience two unique forces upon introduction into the microchannel under a continuous fluid-flow, which are inertial lift and Dean drag forces. These two forces are strongly dependent on the particle sizes, shape, and morphology. The microchannel of the microfluidic device was designed to focus sperm cells towards one side of the channel and PCa cells towards the opposite side of the channel ²³. In this pilot experiment with healthy SF samples spiked with DU-145 cells, it was possible to separate on average more than 89% of the spiked DU-145 cells from more than 99% of the sperm cells. As a result. an optimal flow rate of 1.7 mL/min was indicated and further used for spiking experiments with DU-145, PC3, and LNCaP cells. This approach offers the opportunity to isolate CTCs for early PCa diagnosis and prognostics, and also to purge SF from the tumor cells with the intent of its cryopreservation in men with cancer of reproductive organs. Conventional methods of cancer therapy, including chemotherapy and radiotherapy, often prove detrimental to the fertility potential and spermatogenesis in PCa patients ²⁴. Furthermore, patients with cancer of the reproductive organs typically present lower quality sperm with decreased concentration, motility, and DNA integrity ²⁵. Therefore, the isolation of sperm cells from cancer cells achieved in this study has great potential for bio-banking before treatments are applied to patients. The isolation of sperm cells from cancer cells serves to preserve sperm quality as cancer cells and other epithelial cells can release reactive oxygen species which can induce sperm DNA fragmentation, leading to potentially lower rates of sperm recovery post-cryopreservation and lower rates of fertilized embryo implantation ²⁶.

Following the successful isolation of PCa cell lines from the spiked SF samples with the microfluidic chip using the optimized fluid-flow rate at 1.7 mL/min, the same technique was applied to the SF samples from PCa patients and healthy donors as a control. To ensure high specificity of the technique, an immunocytochemistry panel detecting several PCa-specific antigens, such as anti-CK, anti-PSMA and anti-GPC-1, was applied for identifying isolated CTCs. All 15 out of 15 patients with localized PCa (100%) had CTCs (CK+, PSMA+, GPC-1 + cells) in their SF, varying from approximately 66.9 to 306.7 cells per mL and from 63 to 613 units per sample, representing a 20-30-fold improvement in CTC isolation when compared with previously reported studies where CTCs were isolated from blood or urine ^21,27. At the same time, among the SF samples collected from the healthy volunteers, 2 of 15 had 1.2 and 3 abnormal cells (CK+, PSMA+, GPC-1 + cells) per mL, with the maximal number of the isolated abnormal cells at 6 units per sample. Considering that the patient with the lowest CTC number had 63 cells in his SF sample, all 15 patients had far higher number of CTCs than both healthy volunteers whose SF samples were positive for the abnormal cells. Therefore, the developed technique demonstrated an outstanding specificity and sensitivity in detecting putative PCa CTCs. Furthermore, the described method of CTC isolation from SF is relatively straightforward and quick, with the sample processing time at less than 5 min, and without the need for complex equipment, requiring only the microfluidic chip, syringe and syringe pump.

The correlations between the number of isolated СTCs, PSA serum level or GS represent another significant result of this study. It is worth noting that, while the correlations between the absolute number of СTCs or the number of СTCs per mL and PSA serum level were modest, a moderate positive correlation between the numbers of СTCs and GS were observed. This indicated that the microfluidic СTC isolation from the SF may potentially have prognostic value in PCa risk stratification and treatment, particularly, as an alternative to the PSA test known for its notoriously poor sensitivity. Also, the proposed technique may potentially be applied for evaluating the expression of different antigens by the isolated СTCs, which may be valuable for decision making during such conventional approaches for treating localized PCa as watchful waiting or active surveillance.

Overall, the conducted technique demonstrated great potential for being applied as the diagnostic and prognostic tool in PCa management. Notably, the proposed technique allowed to isolate a significantly higher number of CTCs from the SF in comparison with the techniques, reported in the literature, previously developed for isolating CTCs from blood and urine. To better reveal diagnostic and prognostic potential of the developed technique, a study involving large cohorts of PCa patients and healthy volunteers is required. Also, longitudinal investigation based on the analysis of multiple semen samples, provided by each PCa patient at different time points, is highly relevant. Further, comparison of diagnostic and prognostic values between diverse antibody panels, used for immunocytochemical dentification of the isolated CTCs, is of a great interest. It is anticipated that the developed technique may be applicable for isolating CTCs originating from not only PCa but also adenocarcinoma and testicular cancer, as well as for purifying semen from epithelial cells and large debris with the purpose of its further cryopreservation.

Acknowledgements

We would like to thank the clinical and administrative staff of University Clinical Hospital №2 of Sechenov University for helping with collection of body fluid samples from patients, and for providing patients’ relevant clinical and demographic data. Additionally, we are grateful to all patients and healthy volunteers participated in this study.

Author contributions

Alexey S. Rzhevskiy – writing original draft, investigation, methodology; Alina Yu. Kapitannikova – writing original draft, methodology; Steven Vasilescu – methodology, writing, investigation; Maxim Y. Gavrilov – investigation; Sajad Razavi Bazaz – formal analysis, drawing figures, microchannel fabrication; Mark S. Taratkin – resources; Dmitry V. Enikeev – visualization; Vladimir Yu. Lekarev – validation; Evgeniy V. Shpot – resources; Denis V. Butnaru – project administration; Sergey M. Deyev – resources; Jean Paul Thiery – funding acquisition; Andrei V. Zvyagin – funding acquisition; Majid Ebrahimi Warkiani – supervision.

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests statement

The authors have no conflicts of interest to declare.

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Isolation of circulating tumor cells from seminal fluid of patients with prostate cancer using inertial microfluidics

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1. Microfluidic device and its fabrication

2.2. Cell culture

2.3. Proof-of-Concept Experiments

2.4. Clinical samples

2.5. Immunofluorescence staining

2.6. Cell enumeration and data analysis

3. Results

3.1. Recovery of CTCs from Spiked SF

3.2. Isolation of putative CTCs from the patients’ SF

3.3. Potential diagnostic and prognostic value of the method

4. Discussion

5. Conclusion

Declarations

Author contributions

Data availability statement

Competing interests statement

References

Additional Declarations

Supplementary Files

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