A total of 5 human cell lines representing different histological subtypes of MPM were used: Epithelioid MPM: ACC-MESO-1, ACC-MESO-4, and NCI-H226; biphasic MPM: MSTO-211H; and sarcomatoid MPM: NCI-H28. The ACC-MESO-1 and ACC-MESO-4 cell lines were purchased from the Riken BioResource Research Center, while the NCI-H226, MSTO-211H, and NCI-H28 cell lines were purchased from the American Type Culture Collection. All the cell lines were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO₂.

To analyze EGFR expression, the cell lines were incubated with an anti-EGFR antibody (1:100 dilution; clone 528; cat. no. sc-120; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The cells were subsequently incubated with goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate for 30 min at room temperature (1:20 dilution; cat. no. 349031; BD Biosciences). Flow cytometry analysis was performed using an EC800 Cell Analyzer (Sony Biotechnology, Inc.), and the data was analyzed using FlowJo software version 10 (Becton-Dickinson and Company). The mean fluorescence intensity was calculated as the ratio of the positive control to that of the negative control (PBS with 1% bovine serum albumin; Nacalai Tesque, Inc.).

The CTC-chip was first coated with antibodies in a two-step process as previously described (14), then used for the experiments. Briefly, the CTC-chip was incubated with the base antibody overnight at 4°C followed by incubation with the capture antibody at room temperature for 1 h.

In the present study, the CTC-chip was built based on the following three combinations: i) previously established NZ-1.2-chip (16) with goat anti-rat IgG (200 µg/ml; cat. no. 3052-01; SouthernBiotech) as the base antibody and rat anti-human podoplanin antibody (5,000 µg/ml; clone NZ-1.2) (27) as the capture antibody; i) Cetuximab-chip with goat anti-human IgG (200 µg/ml; cat. no. 2043-01; SouthernBiotech) as the base antibody and cetuximab (5,000 µg/ml; Bristol-Myers Squib Company) as the capture antibody and iii) Cocktail-chip with goat anti-rat IgG (200 µg/ml) + goat anti-human IgG (200 µg/ml) as base antibodies and rat anti-human podoplanin antibody (5,000 µg/ml) + cetuximab (2,500 µg/ml) as capture antibodies.

Sample preparation and evaluation of cell-capture efficiency was performed as previously described (14). Tumor cells labeled using the CellTrace CSFE cell proliferation kit (Thermo Fisher Scientific, Inc.) were suspended in 1 ml blood collected from a healthy volunteer (obtained from author MK; single venous blood collection from the elbow; 100 cells/ml). This sample was added to each CTC-chip system at a constant flow rate (1.0 ml/h) and monitored using a fluorescence microscope (CKX41; Olympus Corporation). The total number of cells added to the CTC-chip (N-total) was determined by counting the number of cells that passed through the inlet of the CTC-chip, whereas the number of captured cells (N-captured) was determined by counting the number of cells that remained on the CTC-chip. Cell-capture efficiency was calculated as the N-captured/N-total ×100 (%), and the mean ± SE was calculated for each sample. Each experiment was performed in triplicate.

Peripheral blood samples were collected from 19 patients with MPM between January 2018 and August 2020, to assess CTCs at diagnosis or prior to treatment. A total of 6 ml blood was collected in a collection tube (BD Vacutainer EDTA-2K; BD Biosciences), and after sufficient suspension, 1 ml blood was added to both the NZ-1.2- and Cocktail-chips. The characteristics of the patients with MPM are summarized in Table I. Clinical stage was determined according to the guidelines of the International Mesothelioma Interest Group, version 8 (28). All patients provided written informed consent to participate in the study. The cells captured on 2 of the CTC-chips were incubated with a primary antibody, a rabbit anti-cytokeratin (CK) antibody (1:100 dilution; cat. no. ab9377; Abcam) and a mouse anti-CD45 antibody (1:100 dilution; cat. no. 304002; clone HI30; BioLegend, Inc.) for 1 h at room temperature, followed by incubation with 30 min of incubation at room temperature with a secondary antibody, an Alexa Fluor 594 anti-rabbit IgG antibody (1:100 dilution; cat. no. A-11037; Thermo Fisher Scientific, Inc.) and an Alexa Fluor 488 anti-mouse IgG antibody (1:100 dilution; cat. no. A-11029; Thermo Fisher Scientific, Inc.) containing 1 µg/ml Hoechst 33342 (cat. no. 4082; Cell Signaling Technology, Inc.) Cells with round-to-oval morphology, Hoechst 33342-positive nuclei, CK-positive staining in the cytoplasm, and CD45-negative staining were identified as CTCs using a fluorescence microscope at ×10 magnification (DMi8-S2G; Leica Microsystems), and CTCs were independently identified by two investigators who were blinded to the clinical data. Survival analysis according to the CTC-counts in NZ-1.2-chip and Cocktail-chip was also performed. The median follow-up was 175 (range, 28–1,067) days.

In addition, to evaluate non-specific detection, peripheral blood samples were collected from five healthy individuals (6 ml; single venous blood collection from the elbow) and used to detect CTCs in the same manner as aforementioned. The healthy volunteers were recruited from our laboratory staff and provided formal written informed consent to participate. Data collection from the healthy subjects was also included in the Ethics Committee approval (approval no. H26-15).

Serial 4-µm-thick sections were cut from each 10% formalin-fixed and paraffin-embedded primary tumor specimen collected by pleural biopsy or radical surgery, then evaluated using immunohistochemistry staining according to standard protocols. Sections were heated in 0.01 M citrate buffer (pH 6.0; cat. no. RM102-C; LSI Medience Corporation) at 98°C for 15 min for antigen retrieval and incubated in 3% hydrogen peroxide (cat. no. 081-04215; FUJIFILM Wako Pure Chemical Corporation) for 10 min to inactivate endogenous peroxidase. After blocking with Protein Block Serum-Free (cat. no. X090930-2; Agilent Technologies, Inc.) for 15 min, sections were incubated with mouse anti-podoplanin monoclonal antibody (clone D2-40; cat. no. 413451; pre-diluted antibody; Nichirei Biosciences, Inc.) and rabbit anti-EGFR monoclonal antibody (1:50 dilution; clone D38B1; cat. no. 4267S; Cell Signaling Technology, Inc.) for 1 h. Sections were then washed and incubated with Histofine Simple Stain MAX PO (MULTI) (cat. no. 424152; Nichirei Biosciences, Inc.) for 30 min. Thereafter, sections were visualized with DAB+ Liquid (cat. no. K346811; Agilent Technologies, Inc.) for 10 min and counterstained with hematoxylin for 1 min (cat. no. 30002; Muto Pure Chemicals Co., Ltd.). All steps after the antigen retrieval step were performed at room temperature.

Differences between continuous variables were evaluated using a Wilcoxon signed rank test for paired data, that was not normally distributed. The correlation between two variables was analyzed using Spearman's rank correlation analysis. The Kaplan-Meier method was used to estimate the probability of survival, with survival differences being analyzed using the log-rank test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software (version 27.0; IBM Corp.).

The flow cytometry results of EGFR expression are shown in Fig. 1. EGFR was detected in all the cell lines, including biphasic and sarcomatoid MPM subtypes, which have low podoplanin expression.

The optimal concentration of cetuximab to be used on the newly designed CTC-chip was determined using the NCI-H226 cell line. The cell-capture efficiencies were 89.2, 89.1 and 102.5% at cetuximab concentrations of 500, 1,000 and 5,000 µg/ml, respectively (data not shown). Therefore, 5,000 µg/ml was used as the optimal cetuximab concentration for the further experiments. The cell-capture efficiencies of the three types of CTC-chips are shown in Fig. 2. The epithelioid MPM cell lines, with high podoplanin expression, were effectively captured by the NZ-1,2-chip containing podoplanin, but the cell-capture efficiency for the non-epithelioid cell lines was low. The Cetuximab-chip, containing the anti-EGFR antibody, had high cell-capture efficiency in the majority of the cell lines, except for the ACC-MESO-4 cell line, in which there was a 40% cell-capture efficiency. The Cocktail-chip, containing antibodies targeting both podoplanin and cetuximab, reached 100% of a cell-capture efficiency in all the MPM cell lines; therefore, it was used for clinical evaluation.

Representative images of immunofluorescent staining of CTCs captured using the Cocktail-chip in patients with malignant pleural mesothelioma are shown in Fig. 3. Fig. 4 shows the distribution of CTC-counts using the NZ-1.2- and Cocktail-chips for each sample. No cells were detected in the five healthy subjects, and no non-specific detection was observed (Fig. 4D). CTCs were detected in 73.7% of the samples (14/19 patients) with both the NZ-1.2- and Cocktail-chips. Furthermore, the NZ-1.2- and Cocktail-chips detected CTCs in 90 (9/10 patients) and 70% (7/10 patients) of samples with epithelioid MPM, and in 55.6 (5/9 patients) and 77.7% (7/9 patients) of samples with non-epithelioid MPM, respectively. No clusters, such as clusters of tumor cells or clusters of tumor cells and white blood cells, were observed. The median CTC-counts using the NZ-1.2- and Cocktail-chips were 1 (range, 0–9) and 3 (range, 0–12) in the overall population (P=0.311), 1.5 (range, 0–9) and 2 (range, 0–12) in epithelioid MPM (P=0.332), and 1 (range, 0–4) and 3 (range, 0–9) in non-epithelioid MPM (P=0.106), respectively. There was no significant difference between the two chips; however, the Cocktail-chip detected more CTCs in non-epithelioid MPM, suggesting it was more effective at detecting CTCs. In addition, the Cocktail-chip achieved a CTC-detection efficiency equivalent to that of the conventional NZ-1.2-chip in epithelioid MPM, that is there was no change in the number of epithelial cells (Fig. 4B).

The correlation coefficient between CTC-counts and clinical stage in all patients was 0.194 (P=0.425) with the NZ-1.2-Chip and 0.413 (P=0.079) with the Cocktail-chip. Based on the histology data, the correlation coefficients in epithelioid and non-epithelioid MPM were 0.522 (P=0.121) and 0.199 (P=0.582) for CTC-counts with the NZ-1.2-Chip and 0.108 (P=0.766) and 0.763 (P=0.017) with the Cocktail-chip, respectively (Fig. 5). These data indicated that the CTC-counts using the Cocktail-chip were significantly correlated with the clinical stage of non-epithelioid MPM.

The survival analysis is shown in Fig. 6. There was no statistically significant difference between the presence of CTCs and prognosis in both the NZ-1.2- and Cocktail-chips; however, patients with CTCs detected had a poorer prognosis (P=0.274 and P=0.114, respectively). Furthermore, in patients with stage I MPM, one patient without CTCs detected survived without progression for >3 years following surgery, whereas 2 patients with CTCs detected showed early progression and died following treatment. More specifically, case 1 (NZ-1.2-chip:2, Cocktail-chip:12) died 4 months following chemotherapy due to tumor progression, including distant metastasis, and case 2 (NZ-1.2-chip:2, Cocktail chip:4) died 4 months following surgery due to locally advanced tumor progression (data not shown).

Podoplanin staining was strongly positive in all cases of epithelioid MPM, whereas it was negative or weakly positive in 3 out of 5 cases of sarcomatoid MPM. In biphasic MPM, the epithelial component was strongly positive in all four cases, whereas the sarcoma component was negative or weakly positive in some cases. In contrast, there were no negative cases of EGFR, and all were strongly positive except for 2 epithelioid and 1 biphasic MPM, which were weakly positive (Fig. 7).