Background
Head and neck squamous cell carcinomas (HNSCC) are tumours characterized by great phenotypic, aetiological and biological heterogeneity among individuals [
1]. Standard options for locally advanced HNSCC are surgery and adjuvant radiotherapy or cisplatin-based chemoradiotherapy, depending on associated risk factors [
2]. Chemoradiotherapy is used alone for non-resectable tumours [
2]. However, a large number of patients display locoregional and/or metastatic recurrence despite an adequate local treatment. Cetuximab, a monoclonal antibody directed against the Epidermal Growth Factor Receptor (EGFR), is the only targeted therapy approved for advanced HSNCC. It is used in combination with radiotherapy for locally advanced disease [
3] or with platinum-based chemotherapy for palliative purposes [
4,
5]. Epidermal Growth Factor (EGFR) is almost systematically overexpressed in HNSCC, but no clear correlation has been established between EGFR expression levels and individual response to cetuximab [
6]. Primary resistance to cetuximab is estimated to occur with a frequency of 13–35 % [
4,
5]. Acquired resistance also appears more or less rapidly during the treatment, through mechanisms that are complex and to date only partially understood [
7].
While every individual HNSCC bears a unique load of genomic alterations, therapeutic targeting is limited by the fact that the current level of genomic analysis does not translate into clear information regarding tumour sensitivity to most drugs [
8]. New companion assays that would help to predict individual tumour sensitivity to cetuximab and other treatments of HNSCC would be of medical and economic interest. Such companion assays would help to personalize the medical treatment of patients with HNSCC, and would also be helpful for the optimization of phase II and III clinical trials of new targeted therapies. We and others have examined the possibility of cultivating fragments of human solid tumours for a short-period of time, and exposing them to targeted therapies in vitro [
9‐
13]. Two preliminary studies in particular provide a proof of principle that short-term culture is applicable for exploring the heterogeneity of individual responses of HNSCC to cetuximab [
12,
13]. In the present study, we prepared tumour slices from HNSCC samples and exposed them to a panel of targeted therapies.
Methods
Tumour samples
This study was conducted in compliance with the French legislation and the declaration of Helsinki regarding ethical principles for medical research involving human subjects. The use of surgically-resected solid tumours for research purposes in the laboratory of Biochemistry of the University Hospital of Amiens was approved by the Comité de Protection des Personnes Nord-Ouest (CPP NO ref. 2009/14). A written consent was obtained from the patients. No samples were obtained from any patients that were minor or physically or mentally unable to understand and give their consent to the use of surgical samples.
Head and neck tumour specimens were obtained from 18 adult patients who had been referred to the Head and Neck department of Amiens University Hospital (France) for a surgical procedure between September 2013 and September 2014. Surgery was the first line of treatment, without previous radiotherapy or chemotherapy. Different localisations and stages of squamous cell carcinoma from the upper aero-digestive tract are represented (Additional file
1: Table S1) [
14].
Preparation of tumour slices and culture
About 1 cubic cm of non-necrotic tumour was selected by the pathologist from each fresh surgical specimen. Tumour samples were prepared as 300 μm thick slices with a vibrating blade microtome (VT1200S Vibratome, Leica). Slices were maintained for 48 h in 24-well culture plates in Dulbecco’s Modified Eagle Medium (DMEM) culture medium, supplemented with 10 % fetal calf serum (PAN-Biotech), penicillin / streptomycin, and 1 % glutamine at 37 °C in a 5 % CO2 atmosphere.
Drugs
Eight targeted therapies (cetuximab, sorafenib, erlotinib, tivaninib, masitinib, ponatinib, afatinib and rapamycin) were selected on the basis of their distinct reactivities toward oncogenic kinases (Table
1). Drug concentrations applied in the culture medium were chosen from previous pharmacological studies [
7,
15‐
18]. Cetuximab was purchased from Merck-Serono. Sorafenib, erlotinib, tivaninib, masi tinib, ponatinib, afatinib were purchased by Euromedex (Souffelweyersheim, France). Rapamycin was purchased from Sigma (Sigma-Aldrich, France). Except for cetuximab, which was kept in saline, all other drugs were dissolved in DMSO and kept at – 20 °C before use.
Table 1
A summary of the drugs used in this study and their inhibitory spectrum against the cellular kinome
Cetuximab | 30 μM | EGFR (Epidermal Growth Factor Receptor) | |
Erlotinib | 1 μM | EGFR | |
Afatinib | 10 μM | EGFR / ErbB2/ ErbB4 | |
Sorafenib | 10 μM | B-RAF, C-RAF, PDGFR-A, PDGFR-B (Platelet-Derived Growth Factor Receptor) | |
VEGFR-2 (Vascular Endothelial Growth Factor-2) |
Masitinib | 10 μM | KIT, PDGFR-A, PDGFR-B | |
Tivantinib | 10 μM | HGFR (Hepatocyte Growth Factor Receptor) | |
Ponatinib | 1 μM | FGFR1-4 (Fibroblast-Growth Factor Receptor), PDGFR-A, VEGFR-2 | |
Rapamycin | 1 μM | mTOR (mammalian Target Of Rapamycin) | |
Immunohistochemistry
Tumour slices were fixed in formalin and then paraffin-embedded; 3 μm sections were cut and stained with hematoxylin phloxin saffron (HPS) to select non-necrotic and non-fibrotic areas with a high density of tumour cells. The monoclonal antibody MIB1 (Immunotech, Marseille, France) was used for the immunostaining of Ki67. Two tumour slides were analyzed for each experimental condition. Ten microscopic pictures were taken from each slide focusing on non-necrotic and non-fibrotic tumour areas, as defined by a senior pathologist (J.-F.I.). The cell proliferation index (CPI) was calculated from tumour cells only, excluding cells from the matrix and vessels. The ratio of Ki67-positive cells (brown stained nuclei / total number of nuclei × 100) was automatically determined by using ImmunoRatio, an Image J plugin adapted to automated image analysis (
http://jvsmicroscope.uta.fi/sites/default/files/software/immunoratio-plugin/index.html). The total number of pictures analyzed was 20 per experimental condition, representing in total a minimum of 1000 tumour cells. Mean CPI was determined by calculating each time the average, using the results from all slides.
Immunoblots
Tumour slices treated as indicated were kept at −20 °C for immunoblotting. Total extracts were prepared as described previously and loaded on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes [
11]. Antibodies against Extracellular Regulated Kinase 1/2 (ERK) and phosphorylated ERK1/2 (pERK) were from Cell Signaling Technology (Danver, MA, USA). Antibodies against β actin were from Sigma (Saint-Quentin Fallavier France). Secondary antibodies coupled to peroxydase were from GE Healthcare (Aulnay-sous-Bois, France). Enhanced chemiluminescence reaction was used for revelation. Immunoblots were scanned and quantified using the software Image J (National Institute of Health, USA).
Statistical analysis
The Student’s
t-test was used for individual biological analysis and a value of
p < 0.05 was considered as threshold for significance. Pearson’s r test was used for correlation analysis. Hierarchical cluster analysis was performed with the software R3.02 (
http://www.r-project.org/). The hclust function in R was used, using the agglomeration method “complete”. The algorithm proceeds iteratively. At each stage distances between clusters are recomputed by the Lance–Williams dissimilarity update formula.
Discussion
Here, we explored the individual sensitivity of HNSCC tumours that were exposed in culture to various drugs, using a procedure that has been described by us and others and applied to a variety of human solid tumours in the past [
9‐
13]. The tumour samples were sliced and maintained in culture for 48 h. This time point was chosen as a compromise, in order to let the tumour cells undergo in theory two proliferation cycles without inducing any massive cell death due to the cell culture conditions [
13]. Following the identification of areas with high density of tumour cells, Ki67 labeling combined with automated image analysis was applied in order to examine the impact of a panel of targeted therapies on tumour cell proliferation. We show that short-term culture is a simple and robust method for the evaluation of the effects of targeted therapies on fresh human tumours, i.e., in conditions that resemble as closely as possible the clinical setting.
We observed that proliferation was inhibited by at least one drug in 10/14 HNSCC tumours in our experimental conditions. At this stage however, the aim of the present study was not to raise any definitive conclusions regarding the sensitivity of HNSCC to each of the drugs that was tested. Indeed, there are three major limitations to this study: i) the first limitation is the fact that a single pharmacological concentration of each drug was tested. Future studies should ideally assess a range of concentrations to determine an optimal inhibitory concentration for each drug. ii) The second major limitation of the use of tumour slices maintained ex vivo is that they reflect the consequences of the direct inhibition of signal transduction pathways inside tumour cells. Short-term culture of tumour slices cannot be used to evaluate anti-angiogenic or immunotherapeutic procedures, whose effects on tumour cells are indirect and require mid- to long-term exposure. ii) The third limitation of the present study is the limited number of tumours analysed (n = 14), which does not allow us to draw any definitive conclusions regarding the frequency with which tumours are resistant or sensitive to different drugs. Having in mind these limitations, it is nevertheless interesting to note that cetuximab, the only approved drug for the treatment of HNSCC that was tested in the panel, significantly inhibited the proliferation of only two tumour samples. An anti-proliferative effect was more often observed with sorafenib (n = 5), masitinib (n = 3), and afatinib (n = 3), thus opening the possibility that some HNSCC tumours might be individually sensitive to these drugs. At this stage, we conclude that short-term culture of tumour samples is a technique that could be useful for exploring the tumour-intrinsic determinants of drug sensitivity at the individual level.
Beyond this exploration of individual tumour sensitivity, cluster analysis that was built from the results of all drugs on tumour cell proliferation resulted in a pattern that was concordant with the known mechanisms of some of the drugs. Cetuximab, erlotinib and sorafenib, which are drugs that target the EGFR-RAF-MEK-ERK transduction pathway, presented related patterns of anti-proliferative efficacy. The observation validated the results of the assay, while providing another practical demonstration of the interest of short-term culture of tumour samples. Afatinib, whose mode of action is thought to be the inhibition of EGFR, was found to exhibit an activity spectrum distinct from those of the others EGFR-RAF inhibitors. In agreement with these observations, a correlation was established between the extent of the inhibition of ERK phosphorylation and the control of HNSCC proliferation with erlotinib, but not with afatinib. Given the poor specificity of most inhibitors of tyrosine kinase currently in clinical use, additional mechanisms of action beyond EGFR inhibition and the control of the EGFR-RAF axis can therefore be hypothesized for a drug like afatinib [
15]. On the basis of these data, we suggest that short-term culture of tumour samples can also be useful for addressing the mode of action of anticancer drugs on HNSCC.
Compared to other techniques aimed at exploring the sensitivity of individual solid tumours in general and HNSCC in particular, the preparation of slices offers the advantage of being relatively easily performed and rapidly implemented. It might be therefore suited for rapid exploration of the response of a relatively-large number of tumours exposed to anticancer drugs. In theory, we propose that short-term culture of slices might serve as a basis for new companion assays that might be useful for the design of clinical trials testing the efficacy of anti-cancer drugs [
19]. Ultimately, it is also tempting to propose that it could be used to select the most active targeted therapies for individual patients with advanced HNSCC. More studies are however required in order to establish the optimal culture conditions, as well as the biochemical and histological readouts that best reflect the efficacy of anticancer drugs against HNSCC. Validation of the short-term culture assay will require a comparison with the results obtained with other, more established models currently used for the determination of tumour drug-sensitivity, such as patient-derived xenografts [
18]. Finally, in the present study, we did not carry out a correlation of the in vitro behaviour of tumour cells with the clinical response, since most patients enrolled here were considered cured after surgery and thus did not receive any targeted therapy.
Acknowledgements
We thank Pr Henri Sevestre and Dr Patrick Votte for their support and Aline Houessinon for helpful comments on the manuscript. We thank Amiens University Hospital and Ligue contre le Cancer, Comité de la Somme, for financial support.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JD designed and performed the study, analysed the data and contributed to the writing of the manuscript; EL designed and performed the study and analysed the data; MP designed and performed the study and analysed the data; ZS performed statistical analyses and contributed to the writing of the manuscript; SD provided tissues and contributed to data collection; JFI contributed to data collection and analysis; BC designed the study, analysed the data and contributed to the writing of the manuscript; CP contributed to data analysis; AG designed the study, analysed the data and contributed to the writing of the manuscript. All authors read and approved the final version of the manuscript.