Prostate cancer cell lines PC3 and LNCaP were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS) in a humidified incubator supplemented with 5% CO₂. Upon confluency, cells were digested with 0.5% trypsin and passaged at a 1:4 ratio. Some cells were resuspended in culture medium, counted and used for experiments. Blood samples were obtained from healthy donors. Clinical blood samples from patients with metastatic prostate cancer were obtained at Henry Ford Health System (Detroit, MI, USA). The present study was approved by the Institutional Review Board (IRB) of the Henry Ford Health System and written consent was obtained from all patients. Following informed consent, blood samples from patients were acquired in 10 ml BCT Cell-Free DNA tubes (Streck, La Vista, NE, USA) or EDTA-coated Vacutainer^® tubes (BD Biosciences, Franklin Lakes, NJ, USA).

To test CTC enrichment efficiency using the Celsee PREP100 instrument, 250 PC3 and 50 LNCaP cells were spiked into 4 ml of blood from healthy donors. CD45-positive cells were removed using the RosetteSep Human CD45 Depletion Cocktail (Stemcell Technologies, Inc., Cambridge, MA, USA) following the manufacturer's protocol. The upper layer of plasma cells was collected and added into the inlet funnel of the Celsee PREP100. Cells were enriched in the microfluidic chip and stained for cytokeratins, CD45 and nuclei using anti-Pan cytokeratin antibody (1:100 dilution; cat. no. 914204), anti-CD45 antibody (1:100 dilution; cat. no. 368515; both antibodies were from BioLegend, Inc., San Diego, CA, USA) and DAPI, using Celsee PREP100 CTC Immunochemistry kit (Celsee Diagnostics, Plymouth, MI, USA). Cells enriched on the slides were counted using the Celsee Analyzer (14). Cytokeratins and DAPI-positive and CD45-negative cells were counted as CTC cells. The enrichment efficiency was calculated as the percentage of the enriched cells of the total spiked-in cells.

A different amount of PC3 and LNCaP cells were spiked into the priming buffer and retrieved in 2 ml phosphate-buffered saline (PBS) using the Celsee PREP100 instrument (Celsee Diagnostics) following the protocol provided by the manufacturer. The cells were then concentrated in 10–50 µl by centrifugation at 500 × g for 10 min, and counted using a hemocytometer. The retrieval efficiency was calculated as the percentage of the retrieved cells of the total spiked-in cells.

A different amount of PC3 and LNCaP cells were spiked into 4 ml of blood from healthy donors. After removal of the CD45-positive cells and enrichment of the spiked-in CTCs as aforementioned, cells enriched in the microfluidic chip were retrieved in 2 ml PBS using the Celsee PREP100 instrument (Celsee Diagnostics) following the manufacturer's protocol. These cells were then collected by centrifugation at 500 × g for 10 min and stored at −20°C for future analysis by PCR amplicon sequencing.

An aliquot of 4 ml of blood sample was used to capture or retrieve CTCs using the Celsee PREP100 instrument (Celsee Diagnostics) following the protocol provided by the manufacturer. CTCs were monitored using an inverted fluorescence microscope. CTC enumeration following antibody labeling was performed manually. PanCK⁺/CD45⁻ nucleated cells were identified as CTCs. Positive and negative controls for antibody performance and staining were included in each experiment. After removal of CD45-positive cells and enrichment of the spiked-in CTCs as aforementioned, cells enriched in the microfluidic chip were retrieved in 2 ml PBS using the Celsee PREP100 instrument (Celsee Diagnostics) following the manufacturer's protocol. These cells were then collected by centrifugation at 500 × g for 10 min and stored at −20°C for future analysis by PCR amplicon sequencing.

Mutations p.K139fs*3 of TP53 in PC3 and p.T877A of AR in LNCaP cells have been previously reported (15,16). To detect these mutations, nested PCR was employed. Tables I and II lists outer and inner primer sets designed to amplify each of the mutations. The retrieved cells were resuspended in 5 µl ddH₂O and incubated at 4°C for 10 min, and used as the template for PCR amplification. The PCR assay was set up in a 20-µl reaction containing 10 µl KAPA HiFi HotStart Ready Mix (Kapa Biosystems, Inc., Wilmington, MA, USA), 300 nM of each outer primer and 1 µl dimethyl sulfoxide (DMSO) at the following cycling conditions: 2 min at 95°C, followed in turn by 3 cycles of 20 sec at 98°C, 20 sec at 64°C and 30 sec at 72°C, 3 cycles of 20 sec at 98°C, 20 sec at 61°C and 30 sec at 72°C, 3 cycles of 20 sec at 98°C, 20 sec at 58°C and 30 sec at 72°C, 35 cycles of 20 sec at 98°C, 20 sec at 57°C and 30 sec at 72°C and 10 min at 72°C. One microliter of amplified PCR products with the outer primer set was used as the template for the PCR reaction using the inner primer set under the same conditions. The final PCR products were examined on 1% agarose gel and subjected to Sanger sequencing using the inner primers after purification.

PC3 and LNCaP cell lines are established prostate cancer cells and have been widely used in studies on prostate cancer. It has been reported that PC3 cells possess a deletion in the TP53 gene, p.K139fs*3 and LNCaP possess a missense A to G mutation in the AR gene, p.T887A mutation (15,16). To detect these mutations, we designed primers (Table I) targeting the mutations, performed nested PCR and subsequently sequenced the amplicons by Sanger sequencing. To detect the p.T877A mutation of the AR gene in the blood samples of patients, a second set of inner primers were used to improve the sensitivity of the assay (Table II). Fig. 1 reveals the PCR products and Fig. 2 reveals the Sanger sequencing results. The p.K139fs*3 and p.T887A mutations were successfully detected from 28–1,200 PC3 cells, and 10–200 LNCaP cells, respectively.

We next tested CTC enrichment efficiency of the Celsee PREP100 by inputting a total of 110, 220, 330, 440, 550, 880 and 1,100 PC3 cells, and 60, 300, 600, 900, 1,200 and 1,500 LNCaP cells, respectively. The results are shown in Table III. Overall, the efficiency for cell retrieval was ~70% with a range from 40 to 121% for PC3 and LNCaP cells. The observation that some of the cell recovery rates were >100% was due to the variation of cell counts by hemocytometer at low cell numbers.

To test the enrichment and retrieval efficiencies of CTC in blood samples, we spiked PC3 and LNCaP into 4 ml of whole blood samples from healthy donors and stained the enriched cells for cytokeratins, CD45 and nuclei. Fig. 3 reveals typical images of the enriched cells in the microfluidic chip, where cytokeratins and DAPI-positive (green and blue) and CD45-negative cells were considered as CTC cells. The results (Table IV) reveals that ~40% of 250 spiked-in PC3 cells and 74% of 50 spiked-in LNCaP cells were retrieved after enrichment. Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

To test the enrichment and retrieval efficiencies of CTC the blood samples of patients, we stained the enriched cells for cytokeratins, CD45 and nuclei. Fig. 3 reveals typical images of the enriched cells in the microfluidic chip, where cytokeratins and DAPI-positive (green and blue) and CD45-negative cells were considered as CTC cells. For the patient samples that had CTCs, we processed another 4 ml of the blood samples and retrieved CTCs for subsequent mutation analysis.

In order to test whether or not the mutations p.K139fs*3 of TP53 in PC3 and p.T877A of AR in LNCaP cells could be detected in the enriched cells from blood samples, we spiked 50, 100, 300 and 1,000 PC3 cells, and 25, 50, 100 and 250 LNCaP cells into 4 ml of blood samples, depleted CD45-positive cells, enriched and retrieved the cancer cells using Celsee PREP100. The cells were then spun down, resuspended in water and used as templates for PCR amplification and subsequent Sanger sequencing. Fig. 4 reveals the PCR products. Fig. 5 shows the Sanger sequencing results of the PCR amplicons, indicating that both mutations could be successfully detected in the enriched cells. For the CTCs retrieved from 14 clinical blood samples, we successfully performed the mutation analysis of p.T877A. The AR mutation (heterozygous mutation) was identified in the 1 sample (CTC 37) tested (Fig. 6) and all other samples were negative for this mutation. Sixty-three CTCs were identified in sample CTC 37.