Boonyakorn Boonsri and Kiren Yacqub-Usman contributed equally to this work.
The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells.
ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles
CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib.
Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.
Cholangiocarcinoma (CCA) or bile duct cancer is a malignant transformation of the epithelial cells lining the biliary tract. While in North America and Europe, it is relatively rare (2–4/100,000 deaths), but rapidly increasing in incidence [
Gemcitabine and cisplatin combination have widely been used in advanced CCA and the combination is currently accepted as the standard treatment of care for first-line therapy in the United States and Europe. However, the outcome of using these drugs is still discouraging [
Epidermal growth factor receptor (EGFR) or ErbB are effective therapeutic targets in many cancer types [
However, there is still a poor current understanding of the differential expression of ErbB receptor family either in tissues from CCA patients or in CCA cell lines [
RNA was extracted and purified from surgically resected tumor, four primary CCA cell lines (CCA-UK5, CCA-UK6, CCA-UK7, and CCA-UK9) and patient-matched, histologically tumor-free resected margin as a control. One microgram of total RNA was reverse transcribed using 500 ng Oligo-d(T) and 250 ng random primers and M-MLV reverse transcriptase (Takara Shuzo Co., Tokyo, Japan). One microliter of the total 20 μL cDNA was added to 1 μL Taqman Probe (EGFR, Hs01076092; HER2, Hs01001580; HER3, Hs00176538; and HER4, Hs00955522), and 8 μL ddH2O with 10 μL droplet digital polymerase chain reaction (ddPCR) super mix for probes (Bio-Rad, Hemel Hempstead, UK). Droplets were generated by adding 20 to 70 μL Droplet generation oil (Bio-Rad, Hemel Hempstead, UK) using a QX100 droplet generator (Bio-Rad, Hemel Hempstead, UK). Amplification was carried out using standard Taqman protocols (10-minute activation step, 95°C, 40 cycles of 30 seconds denaturation at 95°C, 1 minute annealing and elongation 60°C and final extension 98°C 10 minutes using a PCR thermal cycler (Bio-Rad, Hemel Hempstead, UK). Samples were analyzed using QX100 droplet reader (Bio-Rad, Hemel Hempstead, UK).
Immunohistochemistry was used to examine expression of EGFR family receptors in CCA patient tissues from tumours with different etiologies (OV related in the Thai group and nonOV in the UK group). Formalin-fixed-paraffin-embedded (FFPE) tumor samples from thirty patients were retrieved from the Department of Pathology, Ramathibodi Hospital, Mahidol University and Department of Pathology, Rajavithi Hospital, Bangkok, Thailand, and thirty patients from the Department of Pathology, Nottingham Universities Hospital Trust, for immunohistochemistry assessment. The tissues were histologically confirmed as mass forming CCA by the two pathologists of each institute (NL, CS and AZ, AM). OV-related CCA in Thai patients was diagnosed by the patient fulfilling one of these five criteria: (1) a history of previous positive stool examination for OV or its eggs; (2) identification of OV or its eggs in stool or bile; (3) demonstration of a typical bead-like cholangiogram; (4) demonstration of dilated peripheral small intrahepatic bile ducts, compatible with biliary parasitic disease on sonography, computed tomography or magnetic resonance image; and (5) histopathological evidence of OV in the hepatic resected specimen. Each case was tested using the following primary antibodies: anti-EGFR, anti-HER2, anti-HER3, and anti-HER4 antibodies. Serial 5-μm-thick sections of FFPE tissue were cut for a standard method for immunohistochemical analysis. The immunoreactivity was scored based on membranous and/or cytoplasmic staining [
Cell lines used in this study included human CCA cell lines established from CCA tissue of Thai patients, HuCCA-1, KKU-M213, KKU-100, and KKU-M055 (obtained from the Japanese Cell Research Bank (HuCCA-1 [JCRB1657], KKU-M213 [JCRB1557], KKU-100 [JCRB1568], and KKU-M055 [JCRB1551]), and a normal cholangiocyte cell line, MMNK-1 [
ErbB protein expression was examined in CCA cell lines. Protein extracted using NP-40 lysis buffer containing 1% (vol/vol) Triton X (Calbiochem, Darmstadt, Germany), 1× protease inhibitor (Roche, Mannheim, Germany), 50 mM NaF (Sigma-Aldrich, St. Louis, MO), 2 mM sodium orthovanadate (Sigma-Aldrich, Dorset, UK) and phenylmethylsulphonyl fluoride (Sigma-Aldrich) and quantified using Pierce BCA Assay Kit (Thermo Scientific, Basingstoke, UK). Twenty-five micrograms of protein was separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels followed by wet transfer of the proteins to a nitrocellulose membrane using Bio-Rad transfer equipment (Bio-Rad, Hercules, CA), and transfer efficiency was confirmed by Ponceau S staining. The membranes were blocked in 5% BSA for 1.5 hours. Blots were then incubated with primary antibodies: anti-EGFR (Abcam, Cambridge, UK), anti-HER2, anti-HER3 (Cell Signaling Technology Inc., Beverly, MA), anti-HER4 (Abcam), and anti–α-tubulin antibodies (Cell Signaling Technology Inc.) at 4°C, overnight. The blots were then incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology Inc.) for 2 hours at room temperature and imaged using an Alliance Q9 mini Chemiluminescent gel imager (UVITEC, Cambridge, UK). The relative intensity of the protein band was quantified by ImageJ (from NIH website by Scion Corporation, Frederick, MD) and Image Studio (LI-COR).
Chemotherapeutic drugs used in this study included gemcitabine, cisplatin, and 5-fluorouracil (5-FU). The specific ErbB inhibitors included afatinib, lapatinib, and erlotinib, all were purchased from Selleckchem (Houston, TX). The drugs were dissolved in cell culture grade dimethyl sulfoxide (DMSO) (AppliChem, Barcelona, Spain). Frozen DMSO stocks were prepared together with the drug to be used as vehicle. For cell lines, all drug treatments were performed at 60%–80% cell confluence.
Fresh surgical material from tumor resections at Nottingham University NHS Trust, was immediately placed into tissue transfer media (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 1% 0.2 M L-glutamate, 1% penicillin/streptomycin/amphotericin B, 0.1% 1 mg/mL hydrocortisone, 0.75% 1 mg/mL insulin) at 4°C, and processed within 4–6 hours. The majority of tissue was finely minced for immediate use and further portions were FFPE for immunohistochemistry, or stored in RNAlater (Ambion, Austin, TX) for subsequent analysis.
The CCA cells were harvested and employed for close-to-patient three-dimensional tumor growth assays. Briefly, cells were resuspended in 9 mg/mL ice-cold Cultrex basement membrane extract (Trevigen, Gaithersburg, MD), diluted in modified RPMI-1640 containing phenol red-free and 6 mmol/L D-Glucose at pH 6.8) (Life Technologies, Inc., Rockville, MD). CCA cells (confirmed by human-specific antibodies) were suspended in basal membrane extract in the presence or absence of the CAFs or human bone-marrow-derived mesenchymal stem cells (MSCs, Sciencell, UK). Cells were then plated into low adherent, black-walled, clear bottom 384-well plates at 6,250 tumor cells±CAFs/MSCs. Drugs at 0.1, 1, 10, and 100 μM were added in six replicates on day 3. Drugs used in combination were premixed and serially diluted together before adding to the assay. Drug exposure was for 96 hours before final endpoint readings. An AlamarBlue assay (Invitrogen); 10% (v/v) was added for 1 hour at 37°C to monitor cell growth daily using fluorescent plate reader (FLUOstar Omega, BMG Labtech). Drug sensitivity was calculated as a percentage of a matched untreated control and IC50 were determined using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA), nonlinear curve fit of y=100/(1+ 10(Log1C50-X)×HillSlope).
The proliferative effect of specific ErbB tyrosine kinase inhibitors (afatinib, lapatinib, and erlotinib) (Selleckchem) on human CCA cells was determined by MTT assay. In brief, CCA cells (HuCCA-1, KKU-M213, KKU-100, and KKU-M055) were grown overnight in 96-well plates at density of 5×103 cells/well, then incubated with different concentrations of specific ErbB tyrosine kinase inhibitors (0.1, 1, and 10 μM) for 48 hours. Cells (HuCCA-1 and KKU-M213) were also incubated with the chemotherapeutic drugs: gemcitabine, cisplatin, 5-FU (Selleckchem) at 0.1, 1, and 10 μM for 48 hours. Cells without drug treatment were used as a control group and 0.1% DMSO treated cells were used as a vehicle control group. After incubation, 100 μL of MTT solution (0.5 mg/mL, Sigma-Aldrich, St. Louis, MO) was added to each well and incubated for a further 4 hours at 37°C in the dark. Subsequently, 100 μL of DMSO (Merck, Darmstadt, Germany) was added to each well and absorbance of the sample was measured at OD490 nm by a Versamax microplate reader using SoftMax Pro 4.8 analysis software (Molecular Devices, San Jose, CA). The IC50 value was calculated based on the nonlinear regression curve fit method by GraphPad Prism 6.0 software (GraphPad Software, Inc.). The greatest best-fit curve was used for calculation and R2 value was more than 0.8.
The drug combination study was performed in HuCCA-1 and KKU-M213 cells by using a fixed concentration of gemcitabine (0.1 μM) combined with cisplatin, 5-FU, or with specific ErbB inhibitors (afatinib, lapatinib, and erlotinib) at 0.1, 1, and 10 μM. Interactions between the different drugs were evaluated using the combination index (CI) as described by Chou [
Cell cycle distribution was analyzed using flow cytometry. HuCCA-1 and KKU-M213 cells were cultured and starved with serum-free media overnight. Cells were treated with a single treatment of gemcitabine (0.1 μM) or afatinib (0.1 μM) for 24 hours. Concurrent treatment with gemcitabine and afatinib (G+A) and sequential treatments of gemcitabine (6 hours) followed with afatinib (18 hours) (G→A) and an inverted order with the same interval (A→G) were also performed. Cells were harvested and prepared for flow cytometry by incubated with propidium iodide (Sigma-Aldrich, St. Louis, MO). DNA content of cells was determined using a flow cytometer (BD FACSCanto model, BD Biosciences). The protein samples were applied to western blotting and incubated with the following primary antibodies: anti-cyclin D1, anti-cyclin E (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti α-tubulin antibodies (Cell Signaling Technology Inc.).
Experiments were performed in triplicate. Data are presented as means±standard error of mean and statistically analyzed using GraphPad Prism program ver. 6 (GraphPad Software, Inc.) by one-way ANOVA followed by Tukey’s multiple comparison tests and within-treatment group comparisons were performed using paired t test (2-tailed). Difference with p-values less than 0.05 were considered statistically significant.
mRNA expression of ErbB receptors by ddPCR revealed high expression of EGFR, HER2 HER3, and HER4 (
We then investigated the effects of ErbB inhibitors on the growth of CCA cell lines. All three inhibitors (afatinib, lapatinib, and erlotinib) were cytotoxic in a dose-dependent manner (
To determine whether ErbB inhibitors could in addition to existing therapies we examined the effect of ErbB2 inhibitors in the presence of effective cytotoxic agents. We first confirmed the sensitivity of HuCCA-1 and KKU-M213 cells to chemotherapeutic drugs (gemcitabine, cisplatin, and 5-FU). This confirmed that HuCCA-1 cells were more sensitive to gemcitabine than (IC50=0.76 μM) 5-FU (IC50=4.64 μM), or cisplatin (18 μM) (
As gemcitabine seemed the most likely to provide additional benefit, we therefore determined the effect of gemcitabine combination with tyrosine kinase ErbB inhibitors (afatinib, lapatinib, and erlotinib) at a fixed ratio (0.1: 0.1, 1, 10 μM) and the efficacy of combination treatment was evaluated in the least and most sensitive cell lines (KKU-M213 and HuCCA-1). In HuCCA-1 cells, a synergistic effect was observed when afatinib (0.1, 1, and 10 μM), lapatinib (10 μM), or erlotinib (10 μM) were combined with 0.1 μM gemcitabine compared to gemcitabine alone (
We chose afatinib to further investigate a synergistic effect of the combined treatments using three different approaches: (1) concurrent treatment of gemcitabine (0.1 μM) with afatinib for 24 hours (G+A), (2) sequential treatment of gemcitabine (6 hours) followed with afatinib for 18 hours (G→A) and (3) inverted treatment with afatinib then gemcitabine (A→G) with the same interval (
We performed cell cycle analysis to determine whether the result of the synergistic effect of gemcitabine combined with afatinib resulted from cell cycle arrest or apoptosis. The cell cycle distribution of HuCCA-1 and KKU-M213 cells after single drug treatment, concurrent or sequential treatments of gemcitabine (0.1 μM) and afatinib (0.1 μM) for 24 hours are shown in
For KKU-M213 cells, either gemcitabine or afatinib single treatment showed no significant effect on cell cycle distribution compared to untreated control cells. Concurrent treatment of gemcitabine and afatinib significantly increased cells in subG1 population associated with a decrease in G0/G1 and S phases. Sequential treatments of either G→A or A→G significantly increased cells accumulation in S phase (
We next determined the expression of cyclin D1 and cyclin E cell cycle regulators. Western blot analysis showed that treatment with gemcitabine had no effect on cyclin D1 but upregulated cyclin E in HuCCA-1 cells (
We next assessed the response to ErbB inhibitors in primary CCA cells isolated from patients, in a 3D setting, in the presence of either CAFs or MSCs (
In this study, we show that CCA cells, a very aggressive malignant biliary epithelial cell transformation, resistant to chemotherapy both
We investigated the growth response of a panel of CCA cell lines to treatment with ErbB inhibitors: afatinib, lapatinib, and erlotinib. There was no correlation of the ErbB expression with IC50 of drugs that target ErbB. Others have also reported a lack of a close correlation between varying degrees of EGFR expression in tumor cells and their sensitivity to EGFR tyrosine kinase inhibitor (TKI) inhibitors [
Of the four CCA cell lines, two high ErbB proteins expressing CCA cell lines showed different sensitivity to all the drugs tested, HuCCA-1 cells showed the greatest growth-inhibiting activity (low IC50) while KKU-M213 cells showed the least sensitivity (high IC50). We found no apparent correlation between the response to the treatments of ErbB inhibitors and the expression of ErbB proteins. These suggest that in CCA cells level of ErbB receptor expression per se is not a relevant marker of drug response. Other factors such as the mutation status of ErbB receptors or their activation status, using molecules such as KRAS and TP53 may instead be important. Difference in sensitivity to ErbB targeted drugs may be due to difference in mutation status of the two cell lines. KKU-M213 cells harbor
As expected, the sensitivity of these two cell lines to chemotherapeutic drugs was different; HuCCA-1 cells were more sensitive to gemcitabine while KKU-M213 cells were less sensitive to all tested chemotherapeutic drugs. Afatinib is an oral, irreversible ErbB family blocker that covalently binds to the kinase domains of EGFR, HER2, and HER4, resulting in irreversible inhibition of tyrosine kinase autophosphorylation. Afatinib was found to show a superior progression-free survival over gefitinib or erlotinib in non-small cell lung cancer [
Cell response to a drug comprises a complex sequence of events; some die, some do not proliferate. Here, we examined if the additive effect resulted from alteration of the event in cell cycle. As observed in other cancer cells, the sensitive cells HuCCA-1 treated with gemcitabine (100 nM) alone induced increased subG1 and S and decreased G0/G1 cell population. This event was associated with upregulating of cyclin E, a regulator of cyclin-dependent protein kinases, which mediates the cell cycle transition from G1 to S phase [
In sequential treatment (G→A), more cells died (increased SubG1) and cells arrested in S phase. The low concentration of gemcitabine could allow viable cells in S phase to be responsive to afatinib. Thus, potentiation of the effects on cells traversing S phase may lead to the synergism. Similar results were found in a sequential administration of pemetrexed followed by erlotinib for non-small cell lung cancer [
We also show that the combined treatment of gemcitabine with 5-FU had a growth inhibition effect over either combination gemcitabine/cisplatin or each drug monotherapy in both HuCCA-1 and KKU-M213 cells. This finding provides a rationale for further clinical studies of chemotherapy treatment of the gemcitabine and 5-FU combination in Thai CCA patients.
Finally, we showed that in 3D culture, the presence of fibroblasts has a significant impact on the sensitivity to standard chemotherapies, but insufficient effect on erlotinib to suggest likely efficacy
In conclusion, we demonstrated the potential therapeutic benefits of afatinib in combination with gemcitabine which exerted anti-tumor activity superior to each drug monotherapy particularly in the
Supplementary materials are available at Cancer Research and Treatment website (
Fresh surgical material from tumor resections at Nottingham University NHS Trust, were collected with informed patient consent and National Research Ethics Service approval (NRES REC 10/H0405/6). Samples were used in accordance with NRES approval (NRES REC 08/H0403/37). The study protocol in Thailand was approved by the ethical clearance committee on human rights related to research involving human subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (protocol no. 12-58-41) and Rajavithi Hospital (protocol no. 61042).
Conceived and designed the analysis: Wongprasert K, Bates DO, Tohtong R, Janvilisri T, Kumkate S, Grabowska AM, Larbcharoensub N.
Collected the data: Boonsri B, Yacqub-Usman K, Thintharua P, Myint KZ, Sae-Lao T, Collier P, Suriyonplengsaeng C, Egbuniwe IU.
Contributed data or analysis tools: Suriyonplengsaeng C, Kumkate S, Gomez D, Egbuniwe IU, Mukherjee A, Zaitoun AM, Kuakpaetoon T.
Performed the analysis: Boonsri B, Yacqub-Usman K, Balasubramanian B, Venkatraman S, Myint KZ, Thintharua P, Bates DO.
Wrote the paper: Boonsri B, Wongprasert K, Bates DO, Tohtong R, Yacqub-Usman K, Myint KZ, Venkatraman S, Balasubramanian B, Grabowska AM.
Conflicts of interest relevant to this article was not reported.
This work is supported by the Thailand Research Fund and the Medical Research Council (UK), Newton Fund MR/N01247X/1, Project Nos. DBG 5980006, Rajavithi Hospital Research Fund, “Young Researcher Development Program 2018” from National Research Council of Thailand (NRCT), and Research Assistantship, Faculty of Graduate Studies, Mahidol University. We would like to thank John Gordan for sharing cell lines.
Expression of ErbB receptors in surgically resected patient cholangiocarcinoma (CCA) tissue. (A) Absolute RNA expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER) 2, HER3, and HER4 transcript levels in paired CCA patient resected tissue and adjacent margin, detected by droplet digital polymerase chain reaction (ddPCR). Significant difference was determined by paired t test. (B) Absolute expression of EGFR, HER2, HER3, and HER4 transcript levels in primary CCA cells isolated from the tumor tissue and primary cells from normal tissue margins, detected by ddPCR. Significant difference was determined by Welch’s t test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with margin. (C) Immunohistochemistry for ErbB proteins in CCA tissues. EGFR: placenta (positive control EGFR); breast cancer (positive control HER2 and HER3) and normal liver (positive control HER4). Immunopositive cells stained with 3,3′-diaminobenzidine (brown) and counterstained with hematoxylin (blue). Asterisks indicate tumor margin. Scale bars=50 μm.
The percentage of cell survival of different four cholangiocarcinoma cell lines treated with various concentrations of ErbB inhibitors at 48 hours by MTT assay. Afatinib (A), lapatinib (B), and erlotinib (C). The percentage cell survival of the combined treatments of fixed concentration of gemcitabine (0.1 μM) with afatinib, lapatinib, and erlotinib (0.1, 1, and 10 μM) in HuCCA-1 cells (D) and KKU-M213 (E). *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group The combination effect is described as synergism (blue), or antagonism (red) according to the combination index value.
The percentage cell survival of HuCCA-1 (A) and KKU-M213 (B) cells treatment with gemcitabine (0.1 μM), afatinib (0.1 μM blue, 1 μM red, 10 μM green), concurrent treatment of gemcitabine (0.1 μM) and afatinib (0.1, 1, and 10 μM), sequential treatment of gemcitabine (0.1 μM) (6 hours) followed with afatinib (0.1, 1, and 10 μM) (18 hours) and sequential treatment of afatinib (0.1, 1, and 10 μM) (6 hours) followed with gemcitabine (0.1 μM) (18 hours) of survival. *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group, †p < 0.05 compared with afatinib treated group at respective dose of drug.
Effect of gemcitabine and afatinib combination on cell cycle distribution in HuCCA-1 (A) and KKU-M213 (B) cells. Representative Western blotting showing the expression of cyclin D1 and cyclin E proteins in HuCCA-1 (C) and KKU-M213 (F) cells treated with gemcitabine (0.1 μM), afatinib (0.1 μM), concurrent treatment of gemcitabine and afatinib (G+A), sequential treatment of gemcitabine (6 hours) followed with afatinib (18 hours) (G→A) and sequential treatment of afatinib (6 hours) followed with gemcitabine (18 hours) (A→G). Corresponded densitometric analysis of cyclin D1 (D, G) and cyclin E (E, H) proteins expression normalized with α-tubulin in HuCCA-1 and KKU-M213 cells, respectively. Expression value is expressed as fold of control (mean±standard error of mean) from three independent experiment. *p < 0.05 compared with untreated control group, #p < 0.05 compared with the gemcitabine-treated group.
Effect of ErbB inhibitors on primary cholangiocarcinoma (CCA) lines in 3D-tumor growth assay (TGA). (A) Four primary cells were derived from surgically resected CCA tissue samples; CCA-UK5, CCA-UK6, CCA-UK7, and CCA-UK9. Primary cells (dashed), matched cancer associated fibroblasts (CAFs) also derived from the same tissue samples (solid), a human mesenchymal cell line (mesenchymal stem cell [MSC] not shown) or a mixture (CAF+cancer cells shown as dotted line) were employed for the 3D-TGA. (B) Growth of cells, CAFs, and primary cells and CAF (red line) or MSC (green line) mixed with the primary cells was measured using AlamarBlue. Percent cell survival of CCA1 and CCA2 treated with gemcitabine/cisplatin (Gem/Cis) (C), erlotinib (D), or afatinib (E). The mean±20% peak serum concentration for patients is given as the dotted and shaded lines (grey).
Percentage of ErbB stained tumor from the UK and Thai patients
EGFR family | Origin | Immunohistochemistry | p-value | ||
---|---|---|---|---|---|
Weak | Moderate | Strong | |||
EGFR | UK | 13.3 | 53.3 | 33.3 | 0.430 |
Thai | 26.7 | 46.7 | 26.7 | ||
HER2 | UK | 60 | 40 | 0 | 0.027 |
Thai | 40 | 40 | 20 | ||
HER3 | UK | 80 | 20 | 0 | 0.206 |
Thai | 80 | 13.3 | 6.7 | ||
HER4 | UK | 0 | 20 | 80 | 0.061 |
Thai | 6.7 | 40 | 53.3 |
Values are presented as percentage. EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor.
IC50 value (μM) of the specific ErbB inhibitors at 48 hours
CCA cell line | IC50 at 48 hr (μM) | ||
---|---|---|---|
Afatinib | Erlotinib | Lapatinib | |
KKU-M213 (JCRB1557) | 4.305 | 8.058 | 11.140 |
HuCCA-1 (JCRB1657) | 0.729 | 4.519 | 0.3237 |
KKU-100 (JCRB1568) | 1.609 | 7.948 | 0.3074 |
KKU-M055 (JCRB1551) | 3.823 | 23.65 | 3.892 |
CCA, cholangiocarcinoma.