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RESEARCH ARTICLE
Culture and Drug Profiling of Patient Derived
Malignant Pleural Effusions for Personalized
Cancer Medicine
Christian Ruiz
1☯
*, Stefan Kustermann
2☯
, Elina Pietilae
2
, Tatjana Vlajnic
1
,
Betty Baschiera
1
, Leila Arabi
1¤
, Thomas Lorber
1
, Martin Oeggerli
1
, Spasenija Savic
1
,
Ellen Obermann
1
, Thomas Singer
2
, Sacha I. Rothschild
3
, Alfred Zippelius
3
, Adrian
B. Roth
2‡
, Lukas Bubendorf
1‡
1Institute for Pathology, University Hospital Basel, Basel, Switzerland, 2Pharmaceutical Sciences, Roche
Innovation Centre Basel, Basel, Switzerland, 3Department of Medical Oncology, University Hospital Basel,
Basel, Switzerland
☯These authors contributed equally to this work.
¤Current address: Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
‡These authors share senior authorship.
*christian.ruiz@unibas.ch
Abstract
Introduction
The use of patients’own cancer cells for in vitro selection of the most promising treatment is
an attractive concept in personalized medicine. Human carcinoma cells from malignant
pleural effusions (MPEs) are suited for this purpose since they have already adapted to the
liquid environment in the patient and do not depend on a stromal cell compartment. Aim of
this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze
the effect of chemotherapeutic as well as targeted drugs.
Methods
MPEs from patients with solid tumors were selected for this study. After morphological and
molecular characterization, they were cultured in medium supplemented with patient-
derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-
time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erloti-
nib) according to the mutational status or chemotherapeutics based on the recommendation
of the oncologists.
Results
We have established a robust system for the ex-vivo culture of MPEs and the application of
drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithe-
lial cells before culture allowed treatment of effusions with only moderate tumor cell propor-
tion. Experiments using drugs and drug-combinations revealed dose-dependent and
specific growth inhibitory effects of targeted drugs.
PLOS ONE | DOI:10.1371/journal.pone.0160807 August 22, 2016 1/13
a11111
OPEN ACCESS
Citation: Ruiz C, Kustermann S, Pietilae E, Vlajnic T,
Baschiera B, Arabi L, et al. (2016) Culture and Drug
Profiling of Patient Derived Malignant Pleural
Effusions for Personalized Cancer Medicine. PLoS
ONE 11(8): e0160807. doi:10.1371/journal.
pone.0160807
Editor: Giancarlo Troncone, Universita degli Studi di
Napoli Federico II, ITALY
Received: February 18, 2016
Accepted: July 24, 2016
Published: August 22, 2016
Copyright: © 2016 Ruiz et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was supported by the Basel
Translational Medicine Hub and the Swiss National
Science Foundation (SNF 310030_138513, www.snf.
ch). The funders provided support in the form of
salaries for authors [LA, TL and MO], but did not have
any additional role in the study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. The specific roles of these authors
are articulated in the ‘author contributions’section.
Conclusions
We developed a new approach for the ex-vivo culture of MPEs and the application of drug
tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect
of clinically established treatments by standard chemotherapy and targeted drugs. This
sets the stage for future studies testing agents against specific targets from genomic profil-
ing of metastatic tumor cells and multiple drug-combinations in a personalized manner.
Introduction
The main goal of personalized cancer medicine is to provide “the right treatment for the right
person at the right time”, which increasingly relies on results from molecular analyses, such as
genomic profiling [1,2]. In the past, there have been multiple studies testing in-vitro assays
with patients’cancer cells to select the optimal drug treatment for patients with various tumor
types including lung cancer and ovarian cancer [3–5]. Despite some promising results, these
chemotherapy sensitivity and resistance assays (CSRAs) have not proved to be sufficiently reli-
able for clinical routine. Most of these assays were based on culture of tissue specimens from
solid tumors which poses several technical challenges such as contamination with normal non-
tumor cells or difficulties in disaggregation prior to culture which amongst other factors may
have led to the observed lack of predictivity.
Today, the focus in personalized cancer medicine has moved towards the detection of novel
predictive targets and the investigation of specific inhibitors of these targets [2,6]. Targeted
therapies based on a better molecular understanding of the disease have clearly improved the
armamentarium of oncological therapies in various solid tumors. For example, in non-small
cell lung cancer (NSCLC), administration of EGFR tyrosine kinase inhibitors (EGFR TKI) have
shown high efficiency in the EGFR mutated subpopulation and have led to improved progres-
sion-free and overall survival (reviewed in [7]). Similarly, most patients whose tumors harbor
an ALK gene rearrangement significantly respond to the ALK TKI inhibitors [8–10]. However,
resistance to these targeted treatments is inevitable in most patients due to different mecha-
nisms such as additional resistance mutations and/or activation of alternate signaling path-
ways, as it is well known for TKIs against EGFR and ALK [11–13]. The number of potential
predictive markers will continue to increase due to powerful genome screening methods, such
as next-generation sequencing, that are being applied in a number of ongoing large-scale can-
cer sequencing studies. However, translating potential driver mutations to an efficient drug
against the related protein still remains a major challenge requiring preclinical testing of poten-
tial inhibitors in cancer samples in-vitro and in mouse models.
Whereas genomic profiles of carcinomas have mainly been acquired using solid tumor spec-
imens, malignant pleural effusions (MPEs) have rarely been considered for these analyses.
MPEs in the serosal cavities frequently occur in patients with metastatic lung, breast and ovar-
ian cancer. The appearance of MPEs reflects the advanced or metastatic disease state beyond
the organ of origin leading to significant therapeutic implications and worse prognosis [14].
The MPE samples are routinely processed in cytological laboratories and are subjected to the
same diagnostic tests as for solid tumor specimens, such as morphological evaluation, immu-
nocytochemical analysis (ICC) of protein markers, fluorescence-in situ hybridization (FISH)
for detecting genomic rearrangements or amplifications, and sequencing specific mutations
[15–17]. In contrast to most solid tumor biopsies MPEs have already adapted to the liquid
environment and do not depend on stromal cells or vascularization. Therefore they may repre-
sent a promising cellular model representing the patient’s tumor. Of note, MPEs have been the
MPEs in Culture
PLOS ONE | DOI:10.1371/journal.pone.0160807 August 22, 2016 2/13
Competing Interests: SK, EP, TS and ABR are
employees of the Pharmaceutical Sciences, Roche
Innovation Centre Basel (Switzerland). This does not
alter the authors' adherence to PLOS ONE policies
on sharing data and materials.
source for many commercially available tumor cell lines. Previously, Basak et al used MPEs
from lung cancer patients for the validation of putative cancer stem cells [18]. More recently,
ascites cells from patients with high-grade serous carcinoma were used for exome sequencing
in order to determine the clonal composition of their origin [19].
In this study, we aimed at establishing a robust system for the efficient testing of targeted
therapeutics in MPEs from patients with solid tumors. For this purpose we used the xCELLi-
gence culture system, which enables impedance based live-cell monitoring of proliferation,
cytostasis or cytotoxicity [20]. Here, we show for the first time the utility of this approach in a
proof-of-concept study. In the future, this functional system may support oncologists in tailor-
ing therapy to individual patients based on the molecular profile of cancer cells in their MPEs.
Materials and Methods
Collection and processing of cells
All effusions processed in this study were acquired at the University Hospital Basel for routine
diagnostics and were subjected to diagnostic analyses in the department of cytopathology of
the Institute for Pathology. Effusions for culturing were processed as described [18]. Briefly,
cell suspension was applied onto the Ficoll density gradient medium (Histopaque-1077,
Sigma-Aldrich) and processed as recommended by the manufacturer. The cell layer (mononu-
clear cells) and the upper layer were subjected to a second centrifugation step and the resulting
cell pellet was resuspended in Opti-MEM medium (Thermo-Fisher Scientific). For EpCAM
based enrichment, the MACS (Miltenyi Biotec) system was used as recommended by the man-
ufacturer. Briefly, 50ul of FcR Blocking Reagent and 50ul of CD326 (EpCAM) MicroBeads
(Miltenyi Biotec) were added per 5x10
7
cells and incubated for 30 minutes at 4°C. After wash-
ing, resuspended cells were passed through the MACS separator (Miltenyi Biotec). Release was
performed by removing the column from the separator and flushing 5ml of buffer.
Resuspended effusion cells (from both Ficoll as well as MACS approach) were cultured in
BD Primaria (BD Biosciences) flasks at 37°C and 5% CO2 with FBS (PAN-Biotech) or sterile
filtered primary culture medium [18] and with the supplement of 1% Penicillin Streptomycin
(Amimed, BioConcept, Allschwil Switzerland). Cell growth was monitored by usage of light
microscopy and medium was replaced after every 5–7 days or when cells were splitted. For
drug experiments, 1000x concentrated stock solutions of test compounds were prepared by dis-
solving powder in 100%DMSO (Sigma-Aldrich, St. Louis, MO) under sterile conditions. For
cell treatment, stocks were diluted with culture medium and added to the cell culture wells at a
final concentration as indicated at the graphs in the figures. Final solvent (DMSO) concentra-
tion was adjusted to 0.1% DMSO in the culture medium. Concentrations noted at the graphs
are reflecting final compound concentration in solution of the cell culture medium. Com-
pounds were obtained from Roche’s local sample repository. H522 and H3122 cell lines were
maintained in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum (FBS,
PAN-Biotech GmbH, Germany). All cells were cultured at 5% CO2 and 37°C.
This study was approved by the local ethical committee in Basel, Ethikkommission Nord-
west- und Zentralschweiz (EKNZ), with the approval number EKBB 284/11.
Cytospins and immunocytochemistry (TTF-1, Ber-EP4, Calretinin)
Before culture and during each splitting, cytospins were performed on a Cytospin 4 Cytocentri-
fuge (Thermo Scientific Limited) at 800rpm for 2 minutes. Cytospins were fixed in Delaunay
solution and standard immunocytochemistry procedures were applied using the automated
BOND-MAX system (Leica Biosystems, Muttenz, Switzerland). Antibodies used: TTF-1
(Clone 8G7G3/1, 1:100 dilution, pretreatment: ER1, 5 min 80°C, Neomarkers/Thermo
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Scientific), Ber-EP4 (clone M804, 1:400 dilution, DAKO) and Calretinin (Clone Cal6, 1:100
dilution, pretreatment: ER1 10 min, 80°C, Leica). All cytospins were evaluated by board certi-
fied pathologists (LB, TV).
Monitoring of cell growth using real-time cell analysis
Cell growth experiments were performed by usage of the real-time cell analysis system xCELLi-
gence (ACEA) as previously described [20]. Briefly, 10’000 effusion cells were plated per well
(96-well format) and their growth was continuously monitored for at least 90 h in 15 min inter-
vals. All xCELLigence experiments were performed in triplicates. One day after seeding, test
compounds were added to the culture in the specified concentrations.
Results
Usage of a real-time monitoring system for drug target experiments with
commercially available carcinoma cell lines
As shown previously, the xCELLigence system is well-suited to analyze drug effects on cell pro-
liferation, cytostasis and cytotoxicity in real-time [20]. In contrast to single end-point assays,
the availability of drug effect profiles over the whole experimental period allows building of
dynamic growth curves. In order to assess the utility of this system for analysis of targeted ther-
apies, we subjected two established non-small cell lung cancer cell lines H522 and H3122 to cri-
zotinib treatment, a tyrosine kinase inhibitor (TKI) with activity against ALK, ROS1 and
CMET [21–23]. Whereas H3122 cells harbor a rearrangement in the ALK gene, H522 cells are
wild-type for ALK,ROS1 and CMET [21,24]. We treated the ALK-rearranged cell line H3122
and the control cell line H522 with different concentrations of crizotinib. As expected, treat-
ment with crizotinib led to a significant growth reduction in H3122 cells (Fig 1A), but not in
the control H522 cells (Fig 1B). Whereas lower concentrations of crizotinib (0.3nM/3nM) were
not effective, concentration of 150nM or above significantly reduced growth of H3122 cells,
but not of H522 cells. Thus, our data suggest that effects of targeted drugs such as crizotinib
can be reliably assessed using this real-time monitoring system.
Preparation and culturing of pleural effusions over time
To determine optimal conditions for culturing pleural effusions and real-time monitoring of
their growth, we tested different growth medium supplements, coatings of cell culture dishes
and tumor enrichment approaches. Patient-derived pleural effusions are adapted to the liquid
environment in vivo, and in order to best mimic their original environment, the use of com-
mercial fetal bovine serum might be considered to be sub-optimal. We thus followed the proce-
dure recommended by Basak et al and separated non-nucleated cells from pleural effusions via
Ficoll-gradient and used sterile-filtered effusion supernatant instead of commercially available
serum [18]. We confirmed that medium supplemented with supernatant from the same patient
led to significantly increased growth and viability of pleural effusions compared to effusions
grown in medium supplemented with 10% FCS (S1 Fig). Furthermore, we used commercially
available surface-modified polysterene plates to increase initial adherence of patient-derived
effusion cells (BD Primaria plates from BD Biosciences, S1 Fig). In order to monitor tumor cell
content over several passages, we performed Papanicolaou-staining of cytospin preparations
before culture (as part of diagnostic routine) and after each passage. We estimated tumor cell
content by cytology and by immunocytochemistry (ICC) using markers previously confirmed
to be positive when measured as part of routine clinical diagnostic procedure. In general,
EpCAM (epithelial cell adhesion molecule, BerEP4) (Fig 2B) and Calretinin (Fig 2C) were used
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PLOS ONE | DOI:10.1371/journal.pone.0160807 August 22, 2016 4/13
as pan-epithelial and mesothelial markers, respectively, and if necessary, additional tumor spe-
cific markers were analyzed, such as TTF1 (thyroid transcription factor 1) (Fig 2E) in effusions
originating from lung cancer patients. We observed a dramatic decrease of relative tumor cell
content after few passages in almost all of the malignant pleural effusions analyzed (Fig 2F).
This included effusions from lung, breast, ovarian, stomach and colon cancer. This decrease
was accompanied by a relative increase in mesothelial cells, which with increasing culture time
outgrew epithelial cells (Fig 2G). We therefore used magnetic beads coated with EpCAM anti-
bodies as an optional additional step to enrich for cells of epithelial origin. As depicted in Fig
2H, enrichment with magnetic beads led to a reduced number of mesothelial cells in the culture
in comparison to cultures treated with Ficoll-gradient alone. Thus, this protocol enabled the
use of pleural effusions in vitro, even when derived from patient samples with low tumor cell
Fig 1. Measurement of the growth inhibitory effect of the TKI crizotinib in real-time. A. H522 cells, which are wild-type for ALK do not
respond to crizotinib treatment. B. The ALK rearranged H3122 lung cancer cells show high sensitivity for even low doses of crizotinib. Arrow
head points towards time point used for normalization.
doi:10.1371/journal.pone.0160807.g001
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content. Of note, a very high tumor cell proportion was considered ideal when using the system
for tests with targeted therapeutics to minimize the impact of non-responding normal meso-
thelial cells in the culture.
Treatment of malignant cells in pleural effusions with cytotoxic agents
and targeted therapeutics
Similar to the cell line experiments described above (Fig 1), we treated patient-derived MPEs
with distinct cancer origins with cytotoxic agents based on the standard of care in the clinic set-
ting and specific recommendation from medical oncologists. As expected, treatment of pleural
effusions with the cytotoxic agents cisplatin or pemetrexed or a combination thereof led to a
dose-dependent reduction of cell growth in malignant pleural effusions from pulmonary ade-
nocarcinomas (Fig 3A–3C). The same effect was observed for MPEs from other tumor types
(data not shown), as well as an effusion from a malignant mesothelioma (S2 Fig) and in a
benign effusion (normal control, non-malignant mesothelial cells) (S3 Fig). The real-time
monitoring system allowed us to determine the latency time of the drug effect on cell growth.
The growth-reducing effect of cisplatin started before 40h (Fig 3A: 10ug/ml), whereas for
pemetrexed first effects on cell growth were detected after 50h only (Fig 3B: 10ug/ml). This dif-
ference was expected since cisplatin directly targets the DNA and thereby induces apoptosis,
whereas pemetrexed acts as an inhibitor of enzymes involved in the purine and pyrimidine syn-
thesis process.
We next interrogated the usability of our system for treatment of patient-derived malignant
effusions with targeted therapeutics. For this purpose, we selected MPEs with genomic markers
known to qualify for treatment with a specific targeted drug. The first malignant pleural effu-
sion was from a 73 year old male patient with metastatic adenocarcinoma of the lung, positive
for TTF1 and BerEP4. Routine diagnostic sequencing procedure revealed an activating muta-
tion in the EGFR gene (p.E476-T751 delinsA, exon 19) but no mutations in KRAS,BRAF and
HER2, as well as no ALK gene rearrangement as determined by FISH. Based on these molecular
characteristics, we assumed this lung cancer to be responsive to an EGFR-TKI. In addition, and
as a control, we selected a pleural effusion from a 62 year old patient with metastatic TTF1 pos-
itive adenocarcinoma of the lung. Genomic analysis revealed neither an ALK gene rearrange-
ment nor EGFR,BRAF or HER2 gene mutations, but an activating point mutation of the KRAS
gene (p.G12C, exon 2) and therefore this tumor could be considered a non-responder case to
EGFR-TKI therapy. We thus processed both pleural effusions as described above and treated
the cultured cells with the EGFR TKI erlotinib. The pleural effusion with the EGFR mutation
showed a dose-dependent growth reduction upon treatment with erlotinib (Fig 4A). Impor-
tantly, this was not the case for the MPE of the KRAS mutated but EGFR wild-type adenocarci-
noma (Fig 4B). Concordantly, reviewing of the clinical follow-up of the patient with the EGFR
Fig 2. Determining tumor cell content in cultured malignant effusions. A-C. Second passage (P2) of a malignant effusion
of a metastatic ovarian carcinoma. BerEP4 (B) and Calretinin (C) were used as epithelial and mesothelial markers, respectively.
In this case, the number of mesothelial cells in passage 2 was very low (1–10%) (This case corresponds to the light blue line in
the histogram of 2G, Ovarian Ca.). D-E. First passage (P1) of a malignant pleural effusion of an adenocarcinoma of the lung.
TTF1 (nuclear) was used as specific marker for epithelial cells. F. In most of the effusions, relative tumor cell content decreased
during the first passages in culture. Y-axis denotes relative tumor content as determined by the pathologist. G. Example of the
tumor cell decrease in a malignant effusion from a patient with lung adenocarcinoma. In P0 and P1 most of the cells were from a
pulmonary adenocarcinoma, as shown by TTF1 nuclear positivity (small box). In contrast, cells in P2 were mostly of non-
epithelial origin (no specific nuclear TTF1 staining). Of note, mesothelial cells can change their morphology in culture and show
pronounced atypia, and may be mistaken for tumor cells (green arrowhead). H. Substantial increase in tumor cells (EpCAM
positive) and decrease in mesothelial cells (Calretinin positive) after enrichment with EpCAM antibody coated magnetic beads
(MACS). Red arrowheads point towards epithelial tumor cells, green arrowheads towards mesothelial (non-neoplastic) effusion
cells.
doi:10.1371/journal.pone.0160807.g002
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mutant effusion (Fig 4A) showed a partial response (>50%) by computer tomography (CT)
scan after the first two months of treatment with erlotinib. These findings provide further evi-
dence that our system of in-vitro culturing of patient-derived malignant effusions can assess
the response to specific inhibitors (targeted therapeutics) based on their genomic
vulnerabilities.
Discussion
The use of genomic markers for therapy decisions has become a fundamental part of personal-
ized medicine in clinical oncology. Based on the presence of druggable somatic driver muta-
tions, patients are selected for specific therapeutic treatment. The number of such targets and
corresponding drugs is continuously increasing. However, a full translation of somatic driver
mutations into an effective, tailor-made therapy for an individual patient is still a challenge.
We therefore aimed to establish a robust real-time in vitro system that can be used for the
testing of targeted inhibitors on patients’cancer cells obtained from malignant effusions, which
are a common manifestation of metastatic disease. In line with previous publications monitor-
ing drug effects on cell growth and survival, we showed that the impedance based real-time
monitoring system can reproduce known effects of chemotherapeutic agents and targeted
inhibitors on commercially available cell lines [20]. Here, we could successfully develop a pro-
tocol for culturing and drug testing of primary cultures from malignant pleural effusions of
pulmonary adenocarcinomas and a malignant mesothelioma as demonstrated by experiments
with standard chemotherapy and targeted inhibitors selected based on the genomic profile of
tumor cells. Only the lung cancer effusion with the sensitive EGFR mutation, but not the EGFR
wild-type one, responded to erlotinib in a dose-dependent manner, which confirms the
Fig 3. Treatment of effusions with cytotoxic agents. Treatment of a malignant pleural effusion from pulmonary adenocarcinoma with different
concentrations of cisplatin (A), pemetrexed (B) and a combination thereof (C).
doi:10.1371/journal.pone.0160807.g003
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relevance of this new in vitro test system. The targeted nature of the treatment was emphasized
by the lack of an effect in control experiments using EGFR wild-type cell lines or primary cul-
tures. In contrast, the effect of standard chemotherapy did not differentiate between normal
proliferating mesothelial cells from benign effusions and carcinoma cells, both of which are
vulnerable to unspecific inhibition of proliferation and DNA synthesis. This study may be con-
sidered as proof-of-concept since we could reproduce the treatment effect known from clinical
practice.
Malignant effusions have rarely been considered as a substrate for basic research although
they represent a suitable model for studying the characteristics and behavior of metastatic car-
cinoma. Based on the previous work by Basak et al [18], we have optimized the (pre)culturing
conditions and adapted them for their use in drug testing experiments with the real-time
impedance-based growth monitoring system xCELLigence. Whereas culture and drug treat-
ment of commercially available cell lines was straight forward, culturing of primary effusion
cells required substantial modifications. Similar to Basak et al, we experienced higher growth
Fig 4. Treatment of lung cancer MEs with the TKI erlotinib based on their genomic profile. Only the effusion with the activating EGFR gene
mutation (A) responded to treatment with the tyrosine kinase inhibitor erlotinib, but not the effusion with wild-type EGFR (B).
doi:10.1371/journal.pone.0160807.g004
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rates when sterile-filtered MPE supernatant was used as growth serum [18]. The use of surface-
modified polystyrene cell culture dishes further increased the number of attached effusion
cells. One of the major issues of solid tissue culturing, besides tissue disaggregation, is the
prominent outgrowth of normal fibroblasts when cells from solid tissue specimens are placed
into culture. As expected, we did not detect any fibroblasts, but after few passages mesothelial
cells had outgrown the carcinoma cells to such an extent that a drug testing would not have
been representative of the tumor. Importantly, morphological analysis is often not reliable
enough to discern carcinoma and mesothelial cells, since the morphology of both carcinoma
cells and mesothelial cells often changes in culture [25]. The resulting morphological overlap
between benign and malignant cells in culture might be partly due to the high proliferative
activity of the mesothelial cells and epithelial-mesenchymal transition (EMT) of the carcinoma
cells. Thus, we used immunocytochemistry with cancer-specific markers in effusions such as
TTF-1 or the epithelial marker EpCAM combined with the mesothelial marker calretinin to
better quantify tumor cells across passages [25,26]. Enrichment of tumor cells can be achieved
by a positive selection strategy using EpCAM antibody-coated magnetic beads before placing
the effusion cells in culture. This optional step reduces the number of mesothelial cells to negli-
gible levels even after passaging. However, other methods, such as fluorescence activated cell
sorting (FACS) or a negative selection strategy by actively removing mesothelial cells with anti-
bodies may also be considered. Importantly, the aim of this study was not the optimization of
parameters for long-term culture of primary effusion cells, but their growth for a limited time
period (one to three weeks) in order to perform the analyses and the drug experiments. The
time needed for cytological and predictive biomarker analysis ranges from a few days (immu-
nocytochemistry or FISH) to up to two weeks (mutation analysis). Thus, freezing the tumor
cells to initiate culture at the time of completed biomarker testing might also be considered.
Otherwise, continuous growth could lead to a decreasing proportion of tumor cells while wait-
ing for the results of mutation profiling. In fact, short-term (up to 3 months) DMSO-based
freezing and re-culturing of primary effusion cells did not negatively affect vitality of the tumor
cells in our study (data not shown). The long-term storage of patients’malignant effusion
might also be interesting since it would allow for biomarker and drug sensitivity testing in case
novel targeted treatments should become available at a later point in time.
The concept of culturing tumor cells from patients in order to test their response to thera-
pies has been intensively discussed and assayed over the last decades [27]. Commercially avail-
able immortalized cell lines are not suited for this purpose given the heterogeneity of cancer in
general and the acquired genetic changes after months or years of growing in culture [28]. Fur-
ther, the majority of the cell lines used in research are matched based to the tumor subtype and
not to the molecular profile as recently shown for ovarian cancer [29]. Culturing of solid tumor
biopsies may be regarded as an optimal approach, but they have to be dissociated and often
depend on their microenvironment with normal stromal cells or vascularization. Furthermore,
the number of cells from a solid tumor specimen remaining after resection and histological
examination is very low and would restrict any potential in vitro experiments to a minimum–if
possible at all. These limitations make them less suitable for efficient drug testing analyses in
daily practice. On the other hand, major advances have been made in developing more sophis-
ticated cell culturing systems such as the hanging drop mechanism [30] or the bioreactor [31].
It remains to be seen how these systems can be used in a routine clinical setting. Another
approach currently under consideration is xenografting patient tumor tissue for drug testing
(reviewed in [32]). Whereas this approach is very interesting for research purposes, its routine
clinical applicability may be challenging due to the time required until cells start to grow and
thus testing can be initiated. More importantly, the very low tumor take rates for certain tumor
types may make drug testing using this approach impossible [33]. We believe that our
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PLOS ONE | DOI:10.1371/journal.pone.0160807 August 22, 2016 10 / 13
approach using patients’MPEs could be superior compared to approaches based on use of
solid tumor tissue, but further studies are needed in order to validate this in-vitro testing.
Importantly, regardless of the in-vitro system chosen, monitoring and quantifying the propor-
tion of tumor cells in culture is indispensable. For the here proposed system, ICC with cancer-
specific markers (such as TTF-1 or BerEP4) as well as a mesothelial marker (such as calretinin)
on cytospins at the tie of initial culture and after each splitting is recommended.
Taken together, in this proof-of-concept study, we have developed a new system for in vitro
sensitivity testing of primary cultures from malignant effusions that overcomes some of the key
limitations of previous approaches due to technical improvements allowing precise measurement
of cell growth in a real-time manner. While only a limited number of effusions with specific tar-
geted therapeutics have been tested so far, the data presented here are very encouraging and
underline the potential of this approach. Further studies using larger numbers of malignant effu-
sions with druggable mutations and a correlation with the clinical course of the affected patients
are therefore needed. It is anticipated that in the future this system could be used in cooperation
with clinical oncologists as part of a personalized medicine approach to support selection of tar-
geted therapies specific to an individual. Of note, the here proposed approach can only be applied
to patients with a MPE. Additionally, the system provides an easy tool with a reasonable through-
put to test a multitude of novel drug combinations in a personalized medicine manner where a
patient with a MPE could directly benefit from. This might not be a priority for some currently
approved treatments with EGFR or ALK inhibitors, given the high response rates. However, with
the upcoming large-panel NGS mutation testing in routine practice, oncologists are confronted
with an increasing number of mutations whose predictive effect in individual patients is not opti-
mal characterized. In-vitro testing of new inhibitors based on the profile of the patient’stumor
could be used for decision making for early access programs or clinical trials. In addition, not yet
approved targeted drugs and combinations thereof could be safely tested using these malignant
effusions showing respective mutations to estimate the potential clinical benefit of such drugs if
provided within compassionate use programs.
Supporting Information
S1 Fig. Optimization of growth conditions. A. Increased growth of pleural effusion cells
when patient-derived effusion supernatant was added to the medium. B. Increased adherence
of pleural effusion cells when surface modified polystyrene culture plates was used.
(TIF)
S2 Fig. Malignant mesothelioma. Treatment of a malignant mesothelioma with different con-
centrations of cisplatin (A), pemetrexed (B) and a combination thereof (C).
(TIF)
S3 Fig. Normal mesothelial cells. Treatment of normal mesothelial cells with pemetrexed (A)
and cisplatin (B).
(TIF)
Acknowledgments
We thank Sina Wyttenbach, Rosemarie Chaffard and the whole technical staff of the Division of
Cytology, Institute for Pathology, University Hospital Basel for their excellent technical support.
Author Contributions
Conceived and designed the experiments: CR SK LB ABR TS.
MPEs in Culture
PLOS ONE | DOI:10.1371/journal.pone.0160807 August 22, 2016 11 / 13
Performed the experiments: CR SK EP TV BB LA MO SS EO.
Analyzed the data: CR SK LA ABR TL.
Contributed reagents/materials/analysis tools: SIR AZ.
Wrote the paper: CR SK ABR LB SIR AZ.
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