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Page 2 of 10 Rath et al. J Cancer Metastasis Treat 2020;6:30 I http://dx.doi.org/10.20517/2394-4722.2020.51

Keywords: Small cell lung cancer, circulating tumor cells, shedding, topotecan

INTRODUCTION
Small cell lung cancer (SCLC) constitutes approximately 15% of all lung cancers and is characterized by
rapid metastasis, universal drug resistance upon recurrence which subsequently leads to a poor prognosis
[1]
in patients . The initial choice of chemotherapy for patients with extended disease (ED)-SCLC includes
using platinum-based regimens in combination with etoposide and immune checkpoint inhibitors. This
yields high response rates but tumors may invariably relapse and exhibit increased chemoresistanceresulting
[2]
in a 2-year survival less than 10% . A host of chemically unique therapeutics failed in clinical trials of
SCLC and the mechanisms of drug resistance have yet to be conclusively resolved thus far . The recent use
[3]
of novel drug combinations yields a minor prolongation of the overall survival but at a cost of increasing
side effects in light of frequent COPD exacerbations and sequelae of smoking. In contrast to NSCLC, SCLC
does not have clear dependence on driver mutations and shows inactivation of the tumor suppressor genes
[4]
p53 and RB1 . Recurrent tumors grow rapidly and may show universal necrotic regions due to the lack of
supply of blood vessels. Dissemination of SCLC appears to be correlated with a high count of circulating
tumor cells (CTCs), exceeding the CTC numbers found in carcinomas of breast, colon and prostate by
[5]
several magnitudes . Nevertheless, only a small fraction of the highly heterogenous CTCs is competent to
generate metastases. Several studies have reported a higher metastatic potential of CTC aggregates, defined
[6]
as clusters of different cells .

The extreme CTC count that is present in SCLC patients allowed us to expand eight permanent CTC lines
from blood samples in vitro. Our data show these CTCs include typical SCLC markers such as CD56/
[7]
NCAM, enolase-2 and chromogranin, which are EpCAM-positive . Furthermore, all of these CTC lines
demonstrated spontaneous formation of larger spheroids under regular cell culture conditions, exhibiting
[8]
high chemoresistance when compared with the corresponding single cell suspensions . The global drug
resistance to structurally unrelated chemotherapeutics may be appropriately explained by a physical factor
represented by the barrier of spheroids, limiting drug access to tumor cells in sufficient concentrations. A
unique feature of SCLC CTCs is the release or shedding of cellular particles, which appear as free-floating
debris or as coat containing cores of intact cell aggregates. Release of such cell fragments is not detectable
in established cell lines from SCLC tumor tissues. The present work investigates the properties of these cell
fragments and their possible role in protection of cells against chemotherapeutics.

METHODS
Cell lines and reagents
SCLC26A cell line was established in our laboratory from a pleural effusion fluid sample of an SCLC
patient prior to treatment. NCI-H417, DMS153 and NCI-H69 cell lines were established from primary
SCLCs before treatment and GLC16 from a recurrent tumor. These cell lines were obtained from the Finsen
Center, Copenhagen, Denmark. The SCLC CTC cell lines BHGc7, 10, 16, 26 and 91 were established from
[7,9]
blood samples of ED-SCLC patients at our institution . In brief, leucocytes and CTCs were isolated by
gradient centrifugation (Ficoll-Hypaque; Sigma-Aldrich, St. Louis, MO, USA) and the cell preparation was
cultivated in serum-free RPMI-1640 medium supplemented with insulin, IGF-1, transferrin, and selenite
until appearance of clonal outgrowth. Following expansion of the CTC clones, cells were transferred to
regular medium containing fetal bovine serum (FBS). Tissue culture medium consisted of RPMI-1640
medium (Sigma-Aldrich) supplemented with 10% FBS (Seromed, Berlin, Germany) andPenicillin-
StreptomycinSigma-Aldrich). Single cell suspension of the CTC lines spontaneously form tumorospheres
(TOS) in regular tissue culture. Blood collection and generation of cell lines was performed according to
the Ethics Approval protocol #366/2003 by the Ethics Committee of the Medical University of Vienna,
Vienna, Austria. All other chemicals were obtained from Sigma-Aldrich.