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Rath et al. J Cancer Metastasis Treat 2020;6:30 I http://dx.doi.org/10.20517/2394-4722.2020.51 Page 3 of 10

Cytotoxicity assays
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1 × 10 cells in the form of single cells or TOS in 100 µL medium were distributed to individual wells of 96-
well microtiter plates (TPP, Trasadingen Switzerland) and ten 2-fold dilutions of the test compounds were
added from stock dilutions as described previously. Assays were performed in triplicates. The plates were
incubated for four days under tissue culture conditions and viable cells then detected using a modified
MTT assay (EZ4U, Biomedica, Vienna, Austria). The respective dilutions of the compounds tested were
present for the entire incubation period. IC values were determined from dose-response curves using
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Origin 9.1 software (OriginLab, Northampton, MA, USA).

Scanning electron microscopy
For scanning electron microscopy (SEM), samples were washed twice with phosphate-buffered saline, fixed
in Karnovsky’s fixative (Morphisto®, Frankfurt am Main, Germany) and dehydrated in a graded ethanol
series. Ethanol dehydration was followed by hexamethyldisilazane drying (HMDS, Sigma-Aldrich) followed
by gold sputtering (Sputter Coater, SC502, Polaron, Fisons Instruments®, England) and examination
performed using a scanning electron microscope (JSM 6310, Jeol Ltd.®, Japan).

Western blot array
Biomarkers were analyzed using the ARY026 Proteome Profiler Array (R&D Systems, Minneapolis, MN,
USA), which detects 84 cancer-related proteins according to manufacturer’s instructions. Experiments were
performed in duplicate and the different tests were calibrated using the six reference spots included for
each individual membrane. Arrays were evaluated using ImageJ (NIH, Bethesda, MD, USA) and Origin 9.1
software (OriginLab, Northampton, MA, USA).

Statistical analysis
Statistical significance was tested by t-tests and P < 0.05 regarded as statistically significant difference using
Origin software (Originlab, Northampton, MA, USA).

RESULTS
Physical structure of SCLC CTC spheroids
In our laboratory, SCLC CTC cell lines were obtained from blood samples of patients diagnosed with ED-
SCLC prior to initiation of second-line chemotherapy. These cell lines show release of cellular fragments
by intact cells or covering of the SCLC CTC spheroids by layers of such fragments. The cellular fragments
were termed material (MAT). A CTC BHGc91 cluster encased in subcellular material is depicted in Figure 1A
in light microscopy. Physical removal of the cover by vigorous pipetting releases a core assembly of intact
tumor cells [Figure 1B].

Effects of MAT on the chemosensitivity of SCLC cell lines
MAT released into the cell supernatants were collected after centrifugation and added to chemosensitivity
assays which employ cisplatin and Topotecan, respectively. Two typical experiments and their data, using
BHGc10 CTCs are shown in Figure 2A and B. The dose-response curves reveal that addition of MAT91
decreases the chemosensitivity of CTC cells to cisplatin and topotecan to a large extent. In the presence
of MAT, a fraction of the SCLC CTC cells continue to survive at the highest concentrations of both
chemotherapeutics tested.

In a subsequent chemosensitivity test, the viable cell content of a MAT91 fraction was eliminated by
several freeze-thaw cycles. The absence of any contaminating living cells in this preparation resulted in a
resistance-enhancing effect on the topotecan sensitivity of BHGc10 CTCs [Figure 3]. In this experiment,
the protective effect was not exhibited for topotecan concentrations ranging from 2.5-10 µg/mL.