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Page 2 of 6 Vander Borght et al. J Cancer Metastasis Treat 2019;5:57 I http://dx.doi.org/10.20517/2394-4722.2019.0010

Keywords: Small cell lung cancer, tumor biomarker, monoclonal antibodies, NCAM exon 18

INTRODUCTION
Small cell lung cancer (SCLC) is strongly associated with smoking. It is a very aggressive form of cancer
due to resistance to chemotherapy and rapidly dividing metastatic disseminations. SCLC represent about
15% of all lung cancer cases and annually kills about 250,000 people . At the initial diagnosis metastatic
[1]
lesions are already present in about two thirds of the patients . Patients die as a result of the disease at a
[2]
median of 10 to 12 months after diagnosis . Over many decades survival rates have not improved . SCLC is
[3]
[4]
a neuroendocrine tumor. There are three other types of neuroendocrine lung tumors [Table 1]. A hallmark
of these tumors is the presence of neuroendocrine peptides such as NCAM. NCAM is expressed as 120, 140
and 180 kDa isoforms, all derived through alternative splicing from a single gene. NCAM has been described
as a biomarker for SCLC, but is also present on other cell types .
[5]

Biomarkers for SCLC could lead to an important improvement in the treatment of SCLC. These markers
could be used to monitor the tumor load in patients, allowing early diagnosis and monitoring the effect of
therapeutic interventions. We recently described the expression of NCAM exon 18 as a potential biomarker
for SCLC . More recently we have further validated this biomarker. More SCLC biopsies were tested. These
[5]
were all positive for NCAM exon 18 expression. Expression was also found in large cell neuroendocrine
carcinoma (LCNEC) (unpublished data). In this paper we describe the generation and characterization of
monoclonal antibodies directed to E18, the exon 18 encoded moiety of NCAM. We intend to use these
antibodies for the quantification of E18 in extracellular vesicles present in the blood of SCLC patients.

METHODS
Production of monoclonal antibodies
Preparation of antigen
Recombinant his-tagged E18 was prepared in E. coli, using a plasmid with a codon usage optimized for E. coli
(GeneArt/Thermo Fisher Scientific), and purified on a Ni -NTA agarose column and E18-encoding DNA was
2+
cloned into the pCI mammalian expression vector as described .
[5]
Immunization of mice and preparation of hybridoma’s
Three Balb/c mice were immunized 4 times. At zero time intramuscularly with 2 times 50 µg in 50 µL of the
pCI vector containing E18-encoding DNA and at 3 and 8 weeks subcutaneously with 10 µg his-tagged E18
and 12 µg (in 25 µL) AbISCO-100 (Isconova, Sweden). The final immunization was at 10 weeks with 10 µg
his-tagged E18 without adjuvant subcutaneously and the same amount intraperitoneally. Three days after
the last immunization spleen cells were isolated and fused with SP2/0 myeloma cells according standard
procedures . Cells were plated in nine 96 well microtiter plates.
[9]
Screening and characterization
The supernatants of the microtiter wells were tested in an enzyme-linked immunosorbent assay (ELISA)
as described . Wells were coated with his-tagged E18 or an unrelated his-tagged protein. This resulted in 6
[5]
candidates positive for E18 and negative for unrelated his-tagged proteins. These hybridoma’s were subcloned
and characterized by four methods. First, an ELISA was carried out on full length E18 and a shortened
version of E18 (Supplementary Figure 1 by Vander Borght et al. ). Second, immunohistochemistry on SCLC
[5]
cell line NCI-H82 and control cell line HCT116. Third flow cytometry of intact and permeabilized NCI-H82
cells and last western blotting. Experimental details can be found in the study by Vander Borght et al. .
[5]
Isotyping was carried out with the MMT1 kit of AdD Serotec (presently Biorad).