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Page 8 of 10 Rath et al. J Cancer Metastasis Treat 2020;6:30 I http://dx.doi.org/10.20517/2394-4722.2020.51

largely independent of MAT containing viable material, since this effect is preserved during freeze-thaw
cycles, except for very high topotecan concentrations which are not found in patients. Changes in the
chemosensitivity of SCLC cells to cisplatin in the presence of MAT was tested but yielded less discernable
effects (data not shown). In all likelihood, MAT is still able to bind topotecan via DNA and topoisomerases
left, whereas the binding of cisplatin to DNA is impaired by the high sodium concentrations of the medium
by preventing the exit of chlorides [15,16] . SEM pictures of MAT depict cell fragments without membranes
and a sponge-like cytoskeleton. Further analysis of the MAT demonstrates a gradual decrease of the size
of the fragments down to approximately 2 µm. Nevertheless, this dimension remains too large for classical
[17]
extracellular vesicles which are typically less than 1 µm .

The role of cellular debris in tumors, either by spontaneous formation or induction by chemotherapy is
under investigation. Evidence from animal models indicates that chemotherapy may stimulate tumor
initiation, growth, and metastasis partially by tumor-derived cellular debris (e.g., apoptotic/necrotic cells,
and cell fragments) [18-21] . Similarly, radiation-induced apoptotic tumor cells may promote tumor growth
via the Révészphenomenon [22-24] . In colon cancer, chemotherapy triggers tumor cell death and the resulting
[24]
dead cells, or debris, may stimulate angiogenesis, inflammation, and growth . Debris induces the release
of osteopontin (OPN), which is a marker of poor prognosis. OPN plays important roles in angiogenesis, cell
proliferation and metastasis [25-28] . Apoptotic tumor cells further stimulate chemotaxis of macrophage and
[29]
the production of pro-inflammatory cytokines . In invasive breast cancers, patients with a high apoptotic
index resulted in a shorter overall survival . Persistent apoptotic cells may progress to become necrotic
[30]
cells that release macromolecules including that of degradative enzymes and inhibitory or protumor factors
[31]
into the local microenvironment . The cancer-related protein array showed expression of OPN by BHGc7
and derived MAT, which may possibly play a role in modulating the tumor microenvironment.

Taken together, the SCLC CTCs shed cellular fragments spontaneously without exposure to cytotoxic
drugs or environmental stress. In addition to the fragments described for the CTC BHGc91 cell line, other
CTC lines which have been established in our laboratory show a comparable and reproducible release of
such structures suggesting a general role in SCLC CTCs. The possible release of mediators by this MAT
was not tested in these experiements, but the role of these fragments to decrease the chemosensitivity of
SCLC cell lines has been established in other cytotoxicity assays. This novel characteristic of SCLC CTC
cells may participate in clinical refractoriness to chemotherapy. Furthermore, SCLC can be monitored by
the detection of routine neuroendocrine serum markers. In an extension of these assays, blood may be
used in the search for particle-associated SCLC markers, which could be more indicative of tumor cell
dissemination.

DECLARATIONS
Acknowledgments
We wish to thank Dr. T. Hohenheim for continuous endorsement.

Authors’ contributions
Performed experimental work: Rath B, Plangger A
Made the SEM preparations: Moser D
Planned and discussed the investigation: Hochmair M, Ulsperger E
Supervised the study and the writing of the manuscript: Hamilton G

Availability of data and materials
Not applicable.