Critical Reviews in Oncology/Hematology 95 (2015) 179–190

The relevance of ADCC for EGFR targeting: A review of the literature

and a clinically-applicable method of assessment in patients
Martino Monteverde a , Gerard Milano b , Giuliana Strola c , Monica Maffi a , Laura Lattanzio a ,
Daniela Vivenza a , Federica Tonissi a , Marco Merlano d , Cristiana Lo Nigro a,∗,1
a Laboratory of Cancer Genetics and Translational Oncology, Oncology Department, S. Croce General Hospital, Via Carle 25, 12100, Cuneo, Italy
b Oncopharmacology unit, Centre Antoine Lacassagne, Nice, France
c Laboratory Department, S. Croce General Hospital, Cuneo, Italy
d Medical Oncology, Oncology Department, S. Croce General Hospital, Cuneo, Italy

Accepted 26 February 2015

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
2.1. Compilation of ADCC data from the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
2.2. Tumor cell targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
2.3. Anti EGFR MoAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.4. Evaluation of cell viability and cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.5. PBMC isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.6. NK cell purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.7. Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.8. ADCC tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.9. Statistical analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.1. An overview of ADCC and EGFR targeting by MoAbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.2. Isolation and cryopreservation of PBMCs and enrichment of NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.3. Selection of the HSCCN cell line most suitable for ADCC tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.4. Selection of the effector cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
3.5. ADCC tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

∗ Corresponding author. Tel.: +39 0171 616338; fax: +39 0171 616331.
E-mail address: lonigro.c@ospedale.cuneo.it (C. Lo Nigro).
1 Cristiana Lo Nigro is research biologist in the Laboratory of Cancer Genetics and Translational Oncology, Oncology Dept at the S. Croce General Hospital,

Cuneo, Italy. She graduated in Biological Sciences in 1994 at the University of Genoa and completed her training in Medical Genetics in 2000 at the University
of Genoa, Italy. She has taught many Genetics courses in the Biological Science Degree at the University of Genoa and various courses in Laboratory Technician,
Radiology Technician and Nurse Medical Degree at University of Turin. She is co-author of more than 50 scientific papers published in peer-reviewed journals
with IF and has delivered more than 90 abstracts/communication in national/international meetings/congresses and reviewer for many peer-reviewed journals
with IF. She is member of the European Association for Cancer Research (EACR) and the Società Italiana di Genetica Umana (SIGU).

http://dx.doi.org/10.1016/j.critrevonc.2015.02.014
1040-8428/© 2015 Elsevier Ireland Ltd. All rights reserved.
180 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190

Abstract
Background: Advances in the understanding of tumor biology have led to the development of targeted therapies as monoclonal antibodies
(MoAbs) in clinical oncology. Among their suggested mechanisms of action monoclonal antibodies (IgG1) selectively directed against tumor
membrane receptors mediate of antibody–dependent cellular cytotoxicity (ADCC) by triggering Fc-␥RIII on natural killer (NK) cells.
Methods: This study reviews the clinical context of ADCC measurement with a particular focus on EGFR targeting and describes an ex vivo
ADCC method applied to MoAbs (cetuximab and panitumumab), against epidermal growth factor receptor (EGFR). The test performance
was evaluated on different target cells lines (CAL166, A431, HNO91, CAL27), with different effector cells (peripheral blood mononuclear
cells or natural killers -NK-) and in various experimental conditions, in order to establish a truly clinically applicable method.
Results: Using the experience available in the published literature, we optimized all variables involved in the experimental design: target
cells type, numbers and ratio target cells and NK cells (effector cells) per well, time of exposure and repeatability.
Conclusion: ADCC measurement may be of clinical relevance in the context of treatment with MoAbs. This study describes a non-radioactive
method which has proven satisfactory in terms of sensitivity, reproducibility, feasibility and cost effectiveness for the measurement of ADCC
activity mediated by NK with an orientation towards the EGFR target.
© 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: ADCC; Antitumor monoclonal antibody; Natural Killer; Cellular immunity; Cetuximab

1. Introduction with FOLFIRI [11] in patients with mCRC tumors expressing

The mechanisms mediating the therapeutic effect of these
MoAbs are still unclear. For instance it is thought that
anti-EGFR MoAbs prevent the ligand-mediated activation
of the EGFR-dependent pathway. However, cetuximab and
other anti EGFR MoAbs do not induce significant tumor
cell apoptosis [1,2]. Moreover intracellular EGFR kinase
inhibitors are ineffective in colon cancer patients [3]. These
observations have shifted attention towards the mechanisms
triggered by the Fc region of these antibodies with in partic-
ular to the role of antibody-dependent cellular cytotoxicity
(ADCC) [4].
ADCC is triggered by the binding of the Fc region of the
MoAb, already bound via Fv regions to the target cell, to any
of the Fc-␥ receptors, i.e. CD16, CD32 and CD64, which
are expressed with different patterns by cells of the innate
immune system, namely monocytes, macrophages, granulo-
cytes and natural killer cells (NK). The contribution of the
different cell types to the anti-tumor ADCC exerted in vivo
by anti EGFR MoAbs is still debated, albeit the most likely
candidate are NK cells. In general, these cells are thought
to play a relevant role in controlling tumor growth and in
preventing metastatic dissemination in humans [5,6].
Monoclonal antibodies (MoAbs) binding to the extracel-
lular domain of epidermal growth factor receptor (EGFR,
HER1, c-ErbB-1) have proved effective for the treatment of
some EGFR-positive human tumors.
Cetuximab (C225, ErbituxTM ) is a recombinant chimeric
MoAb composed of the variable regions (Fv) of a murine
anti-EGFR antibody and of the constant regions (Fc) of a
human IgG1 kappa immunoglobulin. It is indicated for the
treatment of squamous cell carcinoma of the head and neck
(SCCHN), and of KRAS wild type metastatic colorectal can-
cer (mCRC) [7–9]. Panitumumab (ABX-EGF, VectibixTM )
is a humanized IgG2 kappa MoAb which has been shown
to significantly improve progression-free survival as first-
line therapy with FOLFOX4 [10] and as second-line therapy
wild-type KRAS.
Cetuximab is able to mediate NK-dependent ADCC
in vitro, and the magnitude of the response is related to
CD16 polymorphism [12,13]. Most importantly, it has been
suggested that NK cells are the main mediators of the ADCC-
dependent therapeutic effect of cetuximab [14]. Instead,
panitumumab is less effective in inducing NK-dependent
ADCC [1]. This is probably related to the reduced avidity of
IgG2 immunoglobulins for CD16, as compared to IgG1. Not
surprisingly, anti EGFR MoAbs of the IgG2 subclass induce
ADCC predominantly in cells of myeloid lineage [15].
The level of ADCC activity exerted by NK cells from
tumor patients in the presence of cetuximab might thus be
a useful parameter for predicting response to treatment with
this MoAb.
In this study, we first performed a review of ADCC and
EGFR targeting MoAbs. We then designed a standardized
and validated protocol for the ongoing evaluation over time
of ex vivo NK-dependent ADCC activity. The assay appears
to be reproducible and robust, and is simple enough to be
carried out in standard laboratories.
2. Materials and methods
2.1. Compilation of ADCC data from the literature
We compiled data on ADCC paying particular atten-
tion to EGFR targeting and ADCC analysis. The research
was done by key words (ADCC, cetuximab, method,
NK) on the pubmed database: (http://www.ncbi.nlm.nih.gov/
pubmed/)(Table 1).
2.2. Tumor cell targets
We used four cell lines that we had previously found to
express EGFR: CAL166, A431, HNO91 and CAL27 [24,25].
Table 1
ADCC and EGFR targeting by therapeutic antibodies.
Author, year Tumoral cells Concentration Cetuximab PBMC or purified Effector cells from E/T ratio Technique Comments
types NK
Kawaguchi Y, Nine lines 0,5 micrograms/ml PBMC Healthy volunteers 20/1-40/1 Cr51 assay Cetuximab was able to induce ADCC against
2006 [16] oesophageal and patients EGFR-expressing oesophageal SCC and
cell cancer activities correlated with EGFR expression on
the oesophageal SCC.
The activities of Cetuximab-mediated ADCC by
patients’ PBMC were impaired in comparison
with those by healthy donors’ PBMC.

M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190

Inhibition of TGF-b could enhance
Cetuximab-mediated ADCC against
TGF-b-producing SCC.
Kimura H, 10 lines of 0.001–10 micrograms/ml PBMC Healthy volunteers 10/1 LDH assay ADCC-dependent antitumor activity results
2007 [17] NSCLC from the degree of affinity of cetuximab for the
extracellular domain of EGFR, independent of
EGFR mutation status.
Kurai J, 2007 Eight lines 0.0000025 to Purified NK and Healthy volunteers 40/1 Cr51 assay Low EGFR expression levels on lung cancer
[18] lung cancer 1000 micrograms/ml PBMC and patients cells are sufficient for maximum ADCC activity
mediated by cetuximab.
IL-2 ex vivo treatment of PBMCs can enhance
the cetuximab-mediated ADCC activity against
lung cancer cell lines mainly through activation
of CD3 CD56+ NK cells.
Cetuximab-mediated ADCC activity in lung
cancer patients is less susceptible to
chemotherapy-induced immunosuppression than
NK activity and is retained responsiveness to
IL-2 after chemotherapy.
Roda JM, Two human 100 micrograms/ml Purified NK Healthy volunteers 12.5–100/1 Cr51 assay NK cell IFN-g production and ADCC in
2007 [19] breast adeno- response to cetuximab was markedly enhanced
carcinoma in the presence of immune modulatory
lines and two cytokines, such as IL-2, IL-12 or IL-21
human
NSCLC lines,
three human
pancreatic cell
lines
Kawaguchi Y, Four lines 0,5–5 micrograms/ml PBMC Healthy volunteers 40/1 Cr51 assay the combination of cetuximab and trastuzumab
2007 [20]19 oesophageal and patients could induce synergistic antiproliferative effects
cell cancer and additional ADCC activities against several
oesophageal SCC cells
Loı́pez- PCI-15B 0,001-10 micrograms/ml PBMC Healthy volunteers 10/1 LDH assay Cetuximab mediated ADCC with doses much
Albaitero A, SCCHN cell lower than those required to block pEGFR,
2007 [1] line panitumumab-mediated ADCC with doses
similar to those required for full pharmacologic
blockade of pEGFR (1-10 ␮g/mL). It is of
interest that variability of ADCC mediated by

181
these mAbs is seen between individuals.
 182
Table 1 (Continued )
Author, year Tumoral cells Concentration Cetuximab PBMC or purified Effector cells from E/T ratio Technique Comments
types NK
Correale P, Five colon 1, 10 and PBMC Healthy volunteers 1, 12.5, 25, 50/1 LDH assay Some cytotoxic drug can up-regulate EGFR
2010 [21] carcinoma cell 100 micrograms/ml expression in colon cancer cells and that this

M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190

lines effect correlates with an enhanced sensitivity to
cetuximab-mediated ADCC by LAK cells. This
mechanism is independent of either
pre-treatment EGFR expression or k-ras
mutational status in the tumor cells.
Expression of an activating k-ras mutation in the
tumor cells is associated with a higher
susceptibility to either LAK-mediated
cytotoxicity or cetuximab-mediated ADCC.
Stephenson UM-22B and 10 micrograms/ml Purified NK and Healthy volunteers 50/1 Cr51 assay Cetuximab-mediated ADCC can be enhanced in
RM, 2013[22] PCI-15B HNC PBMC head and neck cancer (HNC) patient PBMCs
cell lines upon TLR8 stimulation.
In HNC patient PBMC demonstrated lower
levels of specific lysis than healthy donor
PBMC, this was attributed to the
immunosuppressed state of the HNC patient
PBMC.
The presence of a strong CD8+ TA-specific
cytotoxic T-cell response was associated with
improved clinical outcomes
Broussas M, A431, 10 micrograms/ml Purified NK Healthy volunteers 6.25– 100/1; best LDH assay The optimal quantity of target cells has to be
2013[23] epidermoid ratio 50/1 lower than 10,000 cells/well to limit the NK cell
human cells quantity to be added.
OUR method Cal166 10 micrograms/ml Purified NK Healthy volunteers 5, 10, 20, 30, 40/1 LDH assay The optimal conditions for LDH assay are
SCCHN cell obtained using target cells pre-seeded 48 hrs
line before experiments. The use of fresh purified
NK cells is preferable. Effector and target cells
have to be co-incubated for 4 hrs in serum-free
medium.
Abbreviations: PBMCs peripheral blood mononucleated cell; EGFR epidermal growth factor receptor; SCC Squamous cell carcinoma; TGF-b transforming growth factor-beta; LDH Lactate Dehydrogenase;
pEGFR phosphorylated epidermal growth factor receptor; mAbs monoclonal antibodies; SCCHN squamous cell carcinoma of the head and neck.
 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190
The CAL166 cell line derived from metastases of parotid
adenocarcinoma; A431 was a human epidermoid carcinoma
cell line; the HNO91 cell line derived from an oral cavity
carcinoma and CAL27 from an adenosquamous carcinoma
of the oral cavity. Two EGFR-negative cell lines were used as
controls [26,27]: SK-MEL-30, from a melanoma metastasis,
and OCM1, from a choroidal melanoma.
In particular, CAL166 cells showed epithelial markers
(CALAM, 3D7 and SM27) and the absence of the mesoder-
mic marker GC12, when analyzed by immunohistochemical
staining (IHC). They were also characterized for the p53
mutations and codon 72 polymorphism and selected for
the high EGFR expression (3253 fmol/mg protein) [24,25].
Moreover, they were HPV16 and HPV18 negative.
In our lab cell lines are routinely tested for mycoplasma
using PCR based techniques and STR profiled to
avoid misidentification (http://www.lgcstandards-atcc.org/
Services/Testing Services/∼/media/8B24D7A948D3400C9
D12FF20BF97BB30.ashx)”.
Cell lines were cultured in DMEM supplemented
with 5 mM glutamine, 1 mM sodium pyruvate, and 10%
fetal calf serum (all from Life Technologies, Carlsbad,
CA) in a humidified 37 ◦ C incubator with a 5% CO2
atmosphere.
2.3. Anti EGFR MoAbs
Cetuximab (Merck, Whitehouse Station, NJ) and panitu-
mumab (Amgen, Thousand Oaks, CA) were kindly provided
by the Pharmacy of S. Croce General Hospital, Cuneo, Italy,
and by Centre Antoine Lacassagne, Nice, France, respec-
tively.
2.4. Evaluation of cell viability and cell death
The viability of peripheral blood mononucleated cells
(PBMCs) and of cultured cell lines was routinely checked
by the trypan blue exclusion assay. The rate of cell deaths
in the preliminary experiments was measured using a
standard MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl
tetrazolium bromide) test [28]. Absorbance at 570 nm was
recorded using a microplate reader (Multiskan Ascent,
Thermo Electron Corp., Vantaa, Finland).
2.5. PBMC isolation
After informed consent, anticoagulated peripheral blood
was collected from healthy donors in our hospital and imme-
diately processed.
PBMCs were isolated by density-gradient centrifugation
following the four different procedures listed below. In pro-
cedures 2, 3 and 4, blood samples were diluted with PBS
supplemented with 5% bovine serum albumin (BSA) and
EDTA 2 mM (herein referred to as ADCC buffer).
183
(1) 5 ml of whole blood was layered on an equal volume
of Lymphoprep (Axis, Rodeløkka, Norway), and cen-
trifuged at 400g for 30 min at RT;
(2) 5 ml of blood was diluted 1:1 with ADCC buffer, lay-
ered on 5 ml of Lymphoprep and centrifuged at 300g for
25 min at RT;
(3) 5 ml of blood was diluted 1:1 with ADCC buffer, lay-
ered on 5 ml of Lymphoprep and centrifuged at 650g for
30 min at RT;
(4) 5 ml of blood was diluted 1:1 with ADCC buffer, lay-
ered on 5 ml of Lymphoprep and centrifuged at 800g for
30 min at RT.
The PBMCs were collected, diluted in ADCC buffer,
washed twice, counted and either used in experiments or
resuspended in DMEM supplemented with 50% fetal calf
serum (from Life Technologies) plus 10% DMSO (Sigma
Aldrich, St. Louis, MO) and cryopreserved using a pro-
grammable rate-controlled freezer Kryo 560-16 (Planer PLC,
Sunbury-on-Thames, UK). After thawing, cell viability was
measured.
2.6. NK cell purification
Enriched NK cells were obtained from PBMC pellets
using the human NK Cell Isolation Kit (Miltenyi Biotec,
Cologne, Germany) and exactly following manufacturer’s
instructions. NK cell purity was monitored using flow cytom-
etry, as described below.
2.7. Flow cytometry
Flow cytometric analyses were performed on FC500
(Beckman Coulter, Brea, CA). Lymphocytes were gated on
the basis of physical parameters (SSC vs FSC), and the
absolute cell numbers were calculated using white blood
cells count. We incubated whole blood cells 20 minutes at
room temperature with an APC-conjugated anti human CD56
(clone AF12-7H3, Miltenyi Biotec, Cologne, Germany) and
a FITC-conjugated anti human CD3 (clone bw264/56, Mil-
tenyi Biotec). NK cells were defined as CD56+/CD3−; T
cells as CD3+/CD56−. Three hundred thousand total events
were acquired and analyzed using CXPsoftware (Beckman
Coulter).
2.8. ADCC tests
Cell lines were washed, seeded in flat, round or conical
bottom 96-well tissue culture plates, and cultured for 4 h,
24 h or 48 h, as indicated in the Results section. After this
time, culture medium was replaced with DMEM plus 5 mM
glutamine and, effector cells at the indicated E/T ratios or
MoAbs at the indicated concentrations were added.
ADCC activity was measured with a standard lactate
dehydrogenase (LDH) assay (Cytotox 96® non radioac-
tive cytotoxicity assay, Promega, Madison, WI), following
184 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190
manufacturer’s instructions. Spontaneous lysis (untreated
cells), maximal lysis (cells incubated with 10% lysis solution)
and MoAb-dependent lysis (cells incubated with cetuximab
or panitumumab) were measured. Each test was performed
in triplicate. After 4 h incubation at 37 ◦ C in 5% CO2 ,
supernatants were collected and LDH release was evaluated
measuring absorbance at 492 nm on a Multiskan Ascent 96-
well plate reader (Thermo Fisher Scientific, Waltham, MA).
ADCC activity was calculated following the formula:
Experiment LDH − Effector spontaneous LDH − Target spontaneous LDH
%Cytotoxicity = 100 ×
Target maximum LDH − Target spontaneous LDH
2.9. Statistical analysis
Statistical analyses were performed using Prism soft-
ware (version 5.0, GraphPad Software, La Jolla, CA).
Differences between groups were evaluated using either
two-tailed Student’s t-test or one-way ANOVA with
Student–Newman–Keuls test, when appropriate. Results
were considered statistically significant with a P value < 0.05.
3. Results
3.1. An overview of ADCC and EGFR targeting by
MoAbs
Table 1 is a compilation of the main publications related
to ADCC and EGFR targeting with therapeutic antibodies
and especially cetuximab. It is clear that cetuximab is able to
induce ADCC against EGFR expressing tumor cells. Inter-
estingly cofactor like Il-2 treatment was shown to positively
modulate ADCC activity, thus demonstrating its potential
clinical value.
Concerning colorectal cancer cells, the presence of KRAS
mutations was examined as a parameter potential interfering
with ADCC activity and no clear consensus emerged from
the data compiled.
Of note, on the clinical side it was observed that cancer
patients may exhibit a lower ability to perform ADCC as
compared to healthy subjects; this observation has to be con-
sidered in the global context of the immunosuppress of cancer
patients.
Finally, while ADCC is specifically developed by IgG1
antibodies, it was interesting to note that panitumumab
(IgG2) was able to develop some ADCC activity. This point
merits further attention.
3.2. Isolation and cryopreservation of PBMCs and
enrichment of NK cells
To set up the protocol we initiated the first step, i.e. isola-
tion of the effector cells. Five different procedures for PBMC
isolation were carried out and cell recovery was calculated
(Table 2).
The best recovery results were obtained with procedures
3 and 4.
In order to evaluate the feasibility of using cryopre-
served effector cells for measuring ADCC activity, we also
checked which of the different separation procedures gave
the best viability after cryopreservation. PBMCs isolated
with the different procedures were frozen and cryopreserved.
After thawing, cell viability was measured for each sample
(Table 2). Best viability after cryopreservation was achieved
with procedure 1, but the best combination of recovery after
isolation and viability after cryopreservation was obtained
with procedure 4. This procedure was used in subsequent
tests.
Enriched NK cells were obtained by immunomagnetic
selection from fresh PBMCs. Cell samples were stained for
CD56 and CD3 before and after selection, in order to check
the quality of the cell enrichment (Fig. 1). The percentages
of CD56+/CD3− cells in PBMCs, in the enriched fraction
and in the leftover are shown in Table 3.
NK cell preparations were used for further experiments
only when purity was greater than 75% of total cell popula-
tion.
3.3. Selection of the HSCCN cell line most suitable for
ADCC tests
Cells suitable for use as targets in ADCC tests need to
have low levels of spontaneous cell death. Since our cell
lines require trypsinization in order to be collected, we per-
formed preliminary tests to check the time required by the
cells to fully recover. Cells from the four EGFR-positive
cell lines (CAL166, A431, HNO91 and CAL27), and two
EGFR-negative melanoma cell lines (OCM1 and SK-MEL-
30) were trypsinized and viability was evaluated by trypan
Table 2
Evaluation of recovery and viability after cryopreservation, using the differ-
ent procedures described in Section 2.
Procedure 1 Procedure 2 Procedure 3 Procedure 4
% Recovery 25 ± 9 27 ± 5 53 ± 5 53 ± 6
% Viability 60 ± 5 53 ± 6 38 ± 3 40 ± 5
Table 3
Mean percentage with standard deviation (St. Dev.) of CD3− and CD56+
NK cells in pre-depletion, in NK+ and in NK− fractions in five different
donors.
Pre-depletion NK+ fraction NK− fraction
% Total % Gated % Total % Gated % Total % Gated
Mean ± St. 10 ± 9 16 ± 15 78 ± 16 90 ± 7 0.9 ± 0.7 1.9 ± 1.8
Dev.
 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190 185

Fig. 1. NK staining by flow cytometry. Upper panels: the lymphocytes population is gated on a FSC/SSC dot plot in pre-depletion sample (a), NK+ fraction
(b) and NK− fraction (c). Lower panels: the dot plots show the staining of the gated lymphocyte populations with anti-CD3 and anti-CD56. Lymphocytes were
gated on the basis of physical parameters (SSC vs FSC), and the absolute cell numbers were calculated using white blood cell count. An APC-conjugated anti
human CD56 (clone AF12-7H3, Miltenyi Biotec, Cologne, Germany) and a FITC-conjugated anti human CD3 (clone bw264/56, Miltenyi Biotec). NK cells
were defined as CD56+/CD3−; T cells as CD3+/CD56−. 30,000 total events were acquired and analyzed using CXPsoftware (Beckman Coulter).

blue after 4, 24 and 48 h. The highest level of cell viability was
recorded with last time point (data not shown). Consequently,
we decided to measure ADCC activity after the target cells
had been left undisturbed for 48 h. This implied measuring
the proliferation rate of the cell lines, in order, after 48 h in
culture, to reach the required number of target cells per well.
We also had to identify the most suitable type of tissue cul-
ture plate to use for this preincubation. Thus, the six cell lines
were grown for 48 h in 96-well plates with either flat, round,
or conical bottoms, and duplication time (DT) was measured
for each of them. In round-bottom plates the cells grew too
slowly and showed a high percentage of dead cells, while in
conical-bottom plates they grew too rapidly (data not shown).
For cell lines grown in flat-bottom plates, the following DT
values were obtained: CAL166 15 h, A431 18 h, CAL27 21 h,
HNO91 21 h, SK-MEL-30 25 h, and OCM1 15 h. The DT
was used to calculate for each cell line the initial number of
cells needing to be seeded per well in 96 well flat-bottom
plates, in order to obtain 5 × 103 , 104 , and 2 × 104 cells per
well after a 48 h incubation. Cultures were started in these
conditions, and after 48 h LDH release was measured. Cell
lines CAL166, CAL27 and SK-MEL-30 showed the lowest
spontaneous LDH release. In these lines, the release from
5 × 103 and 104 cells per well, but not from 2 × 104 cells per
well, was considered acceptable (Fig. 2).
We also checked whether serum deprivation during the 4 h
incubation required for the ADCC test affected on target cell
viability. An MTT test showed no difference in terms of cell
viability between cell lines incubated 4 h in medium with or
without serum (Supplementary Fig. 1).
By comprehensive consideration of the above data, we
decided to use 104 CAL166 or SK-MEL-30 cells as targets
for the following tests.
3.4. Selection of the effector cells
Two remaining parameters required adjustment, i.e. the
cell type to use as effector (PBMCs or NK cells either fresh
or frozen) and the range of the effectors/target ratio (E/T).
Flow cytometry analysis showed that among our donors
NK cells accounted for roughly 5% of PBMCs (data not
shown). Hence, 106 PBMCs needed to be seeded per well
in order to reach an NK cell/target cell ratio of 5/1. The spon-
taneous LDH release we recorded with this concentration
186 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190

Fig. 2. Spontaneous release of LDH of different amounts of cells/well during 4 h of incubation. (a) Spontaneous release from CAL166 (circle), A431 (square),
CAL27 (triangle) and HNO91 (rhombus) cell lines. (b) Spontaneous release from LDH from SK-MEL-30 (circle) and OCM1 (triangle).

of PBMCs in the culture conditions described above was
unacceptably high (data not shown).
To check whether NK cells could be used as effector cells,
10 × 104 , 20 × 104 , 30 × 104 , and 40 × 104 NK cells, either
freshly isolated or freshly thawed were seeded per well, and
LDH spontaneous release was measured after a 4 h incuba-
tion. Low levels of spontaneous LDH release were recorded
with both fresh and frozen NK cells (Fig. 3A and B).
Lastly, we checked whether NK cells exerted any direct
cytotoxic activity on the tumor cell lines we had chosen as
targets. NK cells, either freshly isolated or thawed, were incu-
bated at E/T ratios ranging from 10/1 to 40/1 with 104 target
cells. No NK activity was found against SK-MEL-30 cells
(data not shown), while low levels of NK cytolitic activity
were found against CAL166 cells at a 40:1 E/T ratio (Fig. 3A
and B).
3.5. ADCC tests
In a set of preliminary ADCC experiments we tested two
different MoAb concentrations, i.e. 1 and 10 ␮g/ml of cetux-
imab and panitumumab (Fig. 4A). Note that, in this case, NK
cells were derived from five mCRC and five HNSCC patients
enrolled randomly. Even though the basal lysis rate was 5%,
significantly lower than the rate found in our set of healthy
donors (Fig. 3A), we confirmed that panitumumab is unable
to induce ADCC.
On the basis of these data we decided to use the high-
est dose of cetuximab (10 ␮g/ml) to determine ADCC in ex
vivo experiments. At this concentration neither cetuximab
nor panitumumab affect LDH release from targets and from
effectors cells, even at the highest concentration of the latter
(Fig. 4B).
Having set up the different parameters, we measured the
ADCC activity of fresh and frozen NK cells on CAL166 and
SK-MEL-30. In the presence of cetuximab, ADCC activity
mediated by fresh NK cells on CAL166 appeared to be ratio
dependent, with the highest releases obtained with 1/30 and
1/40 E/T ratios., even though the response was already sta-
tistically significant at E:T 5 (p < 0.05) (Fig. 5A). Instead, in
the same conditions, freshly thawed NK cells were unable to
mount an effective ADCC (Fig. 5B).
In a cohort of 10 donors, the ADCC displayed by fresh NK
cell against CAL166 in the presence of panitumumab was

Fig. 3. LDH spontaneous release (sp. release) of CAL166 target cells (T) and fresh (a) and frozen (b) NK effector cells (E) at different concentrations of either
alone or in the presence of target cells. The NK effector cells were incubated at concentrations of 5 × 104 , 10 × 104 , 20 × 104 , 30 × 104 , and 40 × 104 per well
(or 1/5;1/10;1/20;1/30;1/40 E/T ratio respectively).
 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190 187

Fig. 4. (A) ADCC from CAL166 with fresh NK effector cells (T + E 1/40) obtained with cetuximab (CTX) or panitumumab (PNT) at 1 and 10 ␮g/ml
concentration. (B) LDH spontaneous release (sp. release) from CAL166 target (T) or from fresh NK effector (E) cells with 10 ␮g/ml of cetuximab or
panitumumab. Of note, in this case, NK cells are derived from five mCRC and five HNSCC patients enrolled randomly.

Fig. 5. ADCC activity mediated by cetuximab (CTX) on CAL166 at different E:T ratios with fresh (a) and frozen (b) NK effector cells.

consistently and remarkably lower than the ADCC mediated

by cetuximab (Fig. 6, cetuximab 81.0 ± 18.4, panitumumab
7.5 ± 5.8, p < 0.0001), confirming that the latter MoAb exerts
its therapeutic effect mainly via ADCC-independent mecha-
nisms.
In accordance with the lack of EGFR expression by SK-
MEL-30, no ADCC could be detected against this cell line
using either cetuximab or panitumumab, or fresh or freshly
thawed NK cells (Fig. 7).
In order to rule out that the mediation of the ADCC we
observed by contaminating non-NK cells, e.g., monocytes,
tests were performed using as effectors either enriched NK
cells or the NK-negative cell fraction from the same enrich-
ment. The ADCC activity displayed by the latter cells was
negligible, as compared to the ADCC exerted by the NK cells
(Table 4, p = 0.0003).
In order to evaluate the variability over time of ADCC
response, ADCC mediated by cetuximab was performed
weekly for three consecutive weeks in three different donors. Fig. 6. Comparison between ADCC induced by 10 ␮g/ml of cetuximab and
In these conditions we found a high inter-donor variability, by 10 ␮g/ml of panitunumab on CAL166 with fresh NK effector cells (T + E
but lower intra-donor variability (Fig. 8). 1/40).
188 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190

Fig. 7. ADCC activity mediated by cetuximab (CTX) on SK-MEL30 at different E:T ratios. The activity of fresh (a) and frozen (b) NK effector cells is shown.
The T + E total spontaneous release was compared with the corresponding release mediated by CTX (ANOVA with Student-Newman-Keuls test p < 0.05* ).

Table 4 response of tumor cells to attack from therapeutic MoAbs.

ADCC answer from CAL166 target (T) with NK+ or NK- effector cells with ADCC offers a good example of what must not be neglected
cetuximab (CTX) at 10 ␮g/ml in five donors. Results were normalized with
the formula reported in Materials and Methods. Data are given in percentage
when considering the spectrum of factors which can modulate
for NK+ and NK- fraction separately and their ratio; mean and standard tumor response to attacks by targeted monoclonal antibodies.
deviation (St. Dev.) are also reported. Apart from the impact on the membrane receptor resulting
donors ADCC NK+ (%) ADCC NK- (%) NK-/NK+ in specific antitumoral activity, the role of tumor environ-
#1 84,25 2,50 3%
ment and particularly of immune cells involved in ADCC
#2 57,18 1,66 3% was proven to be of clinical importance and a global view of
#3 97,90 5,40 6% this situation is summarized in Table 1 for a fully clinically
#4 82,00 3,06 4% validated approach with MoAbs targeting EGFR.
#5 73,10 5,38 7% We dedicated much of our study to developing a stan-
mean 78,89 3.60 5%
S.D. 15,04 1,71 1,9%
dardized and optimized protocol to evaluate the ex vivo,
NK-dependent ADCC activity mediated by anti-EGFR
MoAbs.
We chose to measure ADCC activity using a method
based on LDH release, instead of using the 51 Cr release
assay, or flow cytometry-based techniques. The formed LDH
release method is easy to perform, does not involve radioac-
tive labelling and requires no specific expertise or expensive
instruments.
We first screened different procedures for isolating
PBMCs, to be used directly as effector cells or to isolate
NK cells. We recommend procedure 4 or 5 in order to obtain
the best recovery after gradient separation. We also sought to
determine the separation procedure which delivered the high-
est viability after cryopreservation. In this case the best results
were obtained with procedure 2. Had we been able to use
Fig. 8. Intradonor ADCC variability mediated by cetuximab is shown for
cryopreserved effector cells? If so, the cells could have been
three different donors. In all weekly experiments, each donor was tested in
triplicate. The figure showed the three different experiments (with standard collected by clinical laboratories and sent as frozen samples
deviation) and the relative means. ADCC was tested from CAL166 with to reference laboratories for ADCC testing. We would also
fresh NK effector cells (T + E 1/40) plus 10 ␮g/ml of cetuximab. have been able to perform retrospective ADCC tests. How-
ever, we had to rule out the use of cryopreserved effector cells
4. Discussion since, in our system, frozen NK cells showed an unaccept-
ably low level of ADCC activity. We had already excluded
In this era of therapies designed to specifically target the the use of PBMCs, whether fresh or frozen, as effector cells
immune system rather than cancer cell [29] it is still neces- since spontaneous LDH release in these conditions was too
sary to fully evaluate the parameters which can modulate the high.
 M. Monteverde et al. / Critical Reviews in Oncology/Hematology 95 (2015) 179–190
The other preliminary tests led us to design an ADCC
assay with the following characteristics: (i) flat-bottom 96-
well plates, (ii) CAL166 as target cells, (iii) pre-seeded target
cells in order to obtain 10,000 cells per well after 48 h, (iv)
cetuximab at a concentration of 10 ␮g/ml, (v) fresh NK cells
used as effector cells and (vi) effector and target cells co-
incubated for 4 h in serum-free medium.
We also found that at a 40:1 E/T ratio, NK cells displayed
direct, albeit low-level, cytotoxic activity against CAL166
cells. We therefore suggest that ADCC tests should be con-
ducted in the above-mentioned conditions at ratios ranging
from 5:1 to 30:1.
Our protocol proved robust and fast, and can be per-
formed in a standard laboratory. Using this protocol, we
confirmed that the NK+ fraction accounts for most of the
cetuximab-mediated ADCC activity displayed by PBMCs,
and that panitumumab is by far less effective than cetuximab
in inducing NK-dependent ADCC [1,22].
We are currently applying this protocol to assess whether
measuring the cetuximab-mediated ADCC activity exerted
by NK cells from mCRC and HNSCC patients might be a use-
ful tool for predicting and monitoring response to treatment
with this MoAb. We are confident that the reproducibility
and the repeatability of this protocol will allow this study to
be conducted in different laboratories, and will enable the
results to be compared with a high level of reliability. This is
the main objective of the present study.
Conflict of interest statement
We state explicitly that a potential conflict of interest do
not exist.
Reviewers
Ingeborg Tinhofer
Translational Radiooncology Research Laboratory
Dept. of Radiooncology and Radiotherapy
Charité University Hospital
Charitéplatz 1
Virchowweg 20
10117 Berlin
GERMANY
Dariusz M Kowalski
The Maria Sklodowska-Curie Memorial Cancer Centre
and Institute
Department of Lung Cancer and Chest Tumours
5 Roentgen St.
02-781 Warsaw
POLAND
Acknowledgments
We are grateful to Dr. G Frumento for reviewing
the manuscript. We also thank the personnel of the
189
Immunohematology and Transfusion Medicine Service, S.
Croce General Hospital, for providing buffy coats.
We are also grateful to the Fondazione Veronesi that
granted M. M. and D.V. in the 2014 with Post-Doctoral Fel-
lowship, supporting the clinical progression of this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.critrevonc.2015.02.014.
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