Collection and processing of cells
Only MSE with unequivocal diagnostic results from the department of pathology were accepted including the tumour types of lung adenocarcinoma (LADC), breast carcinoma (Breast-Ca), ovarian carcinoma (Ovarian-Ca), gastro-intestinal carcinoma (GIT-Ca) and melanoma. After centrifugation the supernatant was removed and preserved. The cellular sediment was resuspended with the RPMI 1640 culture medium (Gibco, Waltham, MA, USA) followed by a cell-counting step using NucleoCounter (ChemoMetec, Denmark).
For 2D cell culture, 1 million cells were cultured in a T-25 culture flask (Corning, NY, USA) in an incubator at 37°C and 5% CO2 with 10ml complete culture medium RPMI 1640 with 10% fetal calf serum (FCS, Gibco) and Antibiotic-Antimycotic® (Gibco). The medium was replaced every 3 - 5 days. After reaching 70 - 80% confluence, the cells were harvested by incubation with TrypLE Express (Gibco) for up to 5 minutes at 37°C.
For 3D hanging drop culture, GravityPLUS™ plates (InSphero, Schlieren, Switzerland) were used as described previously . Briefly, MSE cells were seeded at a density of 1000 cells per drop and co-cultured with normal human dermal fibroblasts (nHDF) at a ratio of 1:1 in an InSphero Proprietary medium at 37°C and 5% CO2. After 2 - 4 days of spheroid formation, microtissues were transferred into GravityTrap™ ULA plates (InSphero) for further analysis.
For the low-attached 3D cell culture, the cell pellet was resuspended in 50% of complete culture medium and 50% MSE supernatant and seeded in a cell culture dish (100 mm). Since MSE samples were normally preserved at 4°C before processing, we put the dish at 37°C and 5% CO2 in the incubator for 24 hours on a rotator (30 rpm) to mimic the motion as the MSE cells would experience in the patient’s cavity. Afterwards, the cells were dispensed into a 96 well plate with ultra-low attachment surface (Corning) at a density of 7000 cells per well in complete culture medium overnight, for further in vitro drug screening assays.
Cell blocks and immunohistochemistry (IHC)
Cell pellets from harvested 2D cultured cells and original MSE effusions were prepared and processed to make cell blocks as previously described . Briefly, cell pellets were supplemented with thrombin and plasma for clot formation. After 4% para-formaldehyde fixation for 1 hour at 4°C, clots were paraffin-embedded and haematoxylin-eosin (H&E) stained. Microtissues were collected in 1.5ml tubes and washed once with PBS. Consequently, they were fixed in 4% para-formaldehyde for 1 hour at 4°C. Fixed microtissues were collected in the tip of a 1.5 ml microtube, embedded in 2% agarose (Amresco, Solon, OH) and covered with PBS. For paraffin-embedding, the agarose plugs were taken out of the microtubes and the tip containing the microtissues was cut and placed in formalin for 12-14 hours, followed by gradual dehydration. Finally, the plugs were embedded in paraffin (microtissues facing downwards) in order to facilitate sectioning.
For IHC analysis, sections (3 μm) were prepared and stained for antibodies against CD3 (T cells), CD45 (immune cells), Pan-CK (epithelial tumour cells), calretinin (mesothelial cells), MIB-1 (cell proliferation marker), and diagnostic markers for each MSE tumour entity: thyroid transcription factor 1 (TTF-1, LADC), CDX2 (GIT-Ca), estrogen receptor (ER, Breast-Ca and Ovarian-Ca), melan-A and S100 (melanoma) were performed on a Benchmark Ultra platform (Roche, Ventana Medical Systems, Oro Valley, AZ, USA) with protocols used for routine diagnostics. PD-L1 antibody clone E1L3N (Cell Signaling Technology, Danvers, MA, USA) was used. PD-L1 immunoreactivity was dichotomised into low (0 to 49%) and high (≥ 50%), taking into account only membranous staining of tumour cells. Diagnostic markers were scored 0 (negative -) or 1 (positive +). All primary antibodies used for IHC analysis were listed in Additional file 2: Table S1.
Digital Image analysis
Immunohistochemically stained sections were scanned with a high-resolution whole-slide scanner (Hamamatsu Nanozoomer Digital Pathology) using a x40 objective with spatial resolution of 0.23 µm / pixel. As for the colour-based segmentation, IHC results were quantified using ImageJ software (National Institutes of Health, USA, version 1.47t), which could separate positive stained areas (brown / red) from non-stained areas (blue / grey) by colour thresholding using the Lab colour space. Fixed thresholds were used for each set of images. The ratio of positive pixels to the total number of pixels per image was quantified (n = 3).
In vitro drug test
Drug efficacy test was applied only to the low attached cell culture systems. Drug sensitivities of carboplatin (Sigma-Aldrich, St. Louis, MO, USA), pemetrexed (Santa Cruz Biotechnology, Dallas, TX, USA) and pembrolizumab (KEYTRUDA®, Merck & Co., USA) were evaluated. There were six testing groups including 1. carboplatin (50 µM), 2. pemetrexed (5 µM), 3. pembrolizumab (2.5 nM), 4. carboplatin (50 µM) + pemetrexed (5 µM), 5. carboplatin (50 µM) + pemetrexed (5 µM) + pembrolizumab (2.5 nM) and 6. control group (complete culture medium only), (n = 4). After incubation for 48 hours, cell proliferation was assessed using CCK-8 kit (Dojindo, Japan). Briefly, 10µl of CCK-8 was added into each testing well (containing 100 µl medium), followed by incubation for one hour at 37°C and 5% CO2. The optical density was determined using a spectrophotometer (Infinite F Plex, Tecan, Switzerland) at 490 nm with background correction at 630 nm.
All data were expressed as mean ± SD. All statistical analyses were performed on SPSS software, version 23 (IBM, USA) or environment R, version 3.4.2 (R Core Team). Results were analysed with Student’s T-test and p-values < 0.05 were considered statistically significant.