Abstract
Background/Aim: Conventional in vitro assays measure the effect of drugs on total cells, while separating the effect to those on tumor and non-tumor cells is important for assessing drug specificity. Our aim was to evaluate the feasibility of separating the efficacy of vemurafenib on tumor and non-tumor cells in a mixed culture. Materials and Methods: Melanoma A2058 cells and CCD18Co non-tumor cells were mixed and treated with vemurafenib. DNA was subjected to digital PCR to determine the ratio of the mutant 1799A to the wild-type 1799T alleles and viabilities of total cells were subsequently calculated as percentages of tumor and non-tumor cells. Results: The set-up proportion of tumor cells correlated well with the calculated one. The calculated viability of tumor cells decreased with increasing doses of vemurafenib while that of the non-tumor cells remained rather constant. Variability of digital PCR data was high. Conclusion: Using the BRAF mutation 1799T>A to separate the response of tumor and non-tumor cells to a drug, such as vemurafenib, is feasible, supporting a foundation for a genetic in vitro tool for testing drug efficacy and specificity.
The BRAF gene is a proto-oncogene encoding the BRAF protein, a serine/threonine kinase of the RAF family that acts downstream of RAS and upstream of MEK in the MAPK/ERK signaling pathway (1). Mutations in the BRAF gene are found in more than 50% of melanomas while the most frequent one is the coding DNA c.1799T>A (p.V600E). Vemurafenib is an inhibitor of BRAF which has demonstrated a highly potent antitumoral activity (2).
Antitumor drugs, such as vemurafenib, generally affect tumor as well as non-tumor cells and are, therefore, associated with undesired toxicities of various degrees (3). Mixed cell cultures of tumor and non-tumor cells provide a potential in vitro tool for assessing simultaneously the effect of a drug on the target cells and the undesired effect on non-target cells under identical cultural conditions (4). However, conventional in vitro assays, such as the viability assay, can only measure the efficacy of drugs on total cells, while separating the efficacy between tumor and non-tumor cells is not easy or in most cases not even possible. A strategy for determining the proportion of tumor cells in a mixed culture is therefore highly desirable. One possible strategy is to quantify a tumor-specific genetic alteration such as a mutation or an allele loss (5, 6). For melanomas with the BRAF mutation c.1799T>A, the ratio of the mutant 1799A allele to the wild-type 1799T allele should, at least in theory, enable the calculation of the proportion of the tumor cells in a mixed culture containing both tumor and non-tumor cells.
Conventionally, studies regarding genetic alterations in tumors focus on the presence or absence of mutations as well as on features of the mutations, while quantification usually has not been an issue, especially since this is not particularly easy and straightforward. However, with the rapid technological development in the field of molecular genetics, quantification of genetic alterations, such as mutations, is becoming increasingly feasible, precise and affordable. Digital PCR is one such state-of-the-art technology, based on partitioning the PCR solution into large numbers of separated oil droplets or nano-compartments on chips (7-9). Since only droplets or nano-compartments containing specific mutant target sequences will give fluorescence signal, genetic features or mutations can be precisely quantified.
Previously, we demonstrated the feasibility of using a tumor-specific allele loss for determining the proportion of tumor cells in a mixed culture (5, 6). The present study aims to further explore the feasibility of using the BRAF mutation c.1799T>A to determine the proportion of tumor over non-tumor cells in a mixed culture. We used a melanoma cell line A2058 known to have this BRAF mutation, and mixed it with non-tumor cells from the reference line CCD18Co. We further explored the feasibility of separating the response of mixed cells to the BRAF inhibitor vemurafenib between tumor and non-tumor cells.
Materials and Methods
Cell culture and drug treatment. A2058 and CCD18Co were purchased from the American Type Culture Collection (ATCC: www.atcc.org). A2058 is a melanoma cell line and is listed in the ATCC BRAF genetic alteration panel as having a coding DNA1799T>A mutation in a heterozygous manner which leads to a change of p.V600E at the protein level. CCD18Co is one of the recommended control cell lines which does not have the c.1799T>A BRAF mutation. The cells were cultured and harvested as described by the provider ATCC (https://www.lgcstandards-atcc.org/products/all/CRL-11147.aspx#culturemethod).
For testing the feasibility of calculating the portion of tumor cells in a mixed culture using the allele ratio 1799A/1799T, cells of the melanoma line, A2058, and cells of the reference non-tumor line, CCD18Co, were mixed in various ratios (0:100, 20:80, 40:60, 60:40, 80:20 and 100:0) and were cultured overnight for the cells to attach to the surface of the dish. On the next day, the cells were harvested using 0.25% trypsin (Thermofisher) and were used for DNA preparation using a conventional method (see DNA preparation below).
For the drug treatment, 400 cells of A2058 and 1,600 cells of CCD18Co were mixed in each well of a 96-well culture plate, giving a total density of 2,000 cells/well and an initial tumor to non-tumor ratio of 1:4. On the next day, the medium was changed to one containing vemurafenib at concentrations of 0, 0.3, 3, 10, 30, 100 and 300 μM. The treatment was continued for 5 days. For each drug and concentration 4 independent replicates were set up.
DNA preparation. For large numbers of cells harvested using 0.25% trypsin, DNA was prepared using a conventional method, yielding DNA of high quality and quantity (Gentra Puregene, Qiagen), according to manufacturer's instructions. For cells in 96 well plates, DNA was prepared using a directPCR lysis method (VWR International GmbH, Darmstadt. Catalog No.: 732-3259). The direct method was chosen because of the small number of cells in each well. Furthermore, large numbers of samples also require less expensive and simple methods for DNA preparation. For this method, cells in each well of a 96-plate were lysed in 50μl of the directPCR reagent plus 0.2 mg/ml proteinase K at 55°C by shaking overnight. On the next day, the lysis was heated at 85°C for 45 min to denature the proteinase K, and 2-10 μl were directly used for digital PCR.
Dual Color Digital PCR (ddPCR). A validated ddPCR Mutation Detection Assay for the BRAF mutation c.1799T>A was purchased from BioRad (www.bio-rad.com:10049550). The assay contains primers for amplifying the region of the mutation, a FAM (blue)-labelled TaqMan probe for the mutant allele 1799A and a HEX (green)-labelled TaqMan probe for the wild-type 1799T allele. All other components and consumables for droplet formation, PCR and droplet-reading, including droplet PCR supermix, cartridge, gaskets and foil seals were also purchased from BioRad. The set-up and reaction of digital PCR was as described in the protocol provided by BioRad. Droplet formation and reading were carried out using the BioRad QX100 digital PCR system.
The raw data of digital PCR were analyzed automatically by an integrated program of the QX100 system, which performed a Poisson correction accounting for positive droplets and calculated the ratio of mutant to wild-type alleles (1799A/1799T). However, for each reaction, the events of positive and negative droplets were inspected manually and thresholds were corrected accordingly. Data not meeting the criteria were excluded from further analyses. The resulting mutant/wild-type allele ratios of 1799A/1799T were exported to an excel data sheet and were used to calculate the portion of tumor cells in the mixed culture.
Results
Dual color digital PCR confirmed the BRAF mutation c.1799T>A in the melanoma cell line A2058 and lack of the mutation in the reference non-tumor cell line CCD18Co (Figure 1). These results ensured the sensitivity and specificity of the assays, however, the ratio of the mutant 1799A to the wild-type 1799T allele in A2058 was not 1:1, as expected, but rather 1:2 (Figure 1A). This result suggests that there are two wild-type alleles and one mutant allele in each tumor cell. Upon this unexpected finding, we contacted the ATCC and obtained the information of T=73.4% and A=26.4% from the data of next generation sequencing, which was consistent with our finding.
We, therefore, presumed the ratio of 1:2 for the mutant allele 1799A in tumor cells and used this presumption for calculating the proportion of tumor cells in a mixed culture as (A/T)/(1-A/T).
To check the feasibility of using the ratio A/T to calculate the proportion of tumor cells in a mixed culture, we carried out a titration experiment by mixing DNA from tumor cells and non-tumor cells at various proportions. In addition, we also mixed tumor and non-tumor cells at various proportions, and prepared DNA from the mixed cells. This DNA was subjected to digital PCR, which gave the A/T ratio for each sample. Using these ratio data, the proportion of tumor-DNA over the non-tumor DNA and the proportion of tumor cells over the non-tumor cells was calculated, and this correlated well with the set-up proportion (Figure 2). However, the variability was large, especially from samples of mixed cells (Figure 2B).
Next, the melanoma A2059 cells were mixed with the non-tumor CCD18Co cells at a ratio of 1:4, and were treated with 0, 0.3, 3, 10, 30, 100 and 300μM vemurafenib for 5 days. Following treatment, DNA from the mixed cells was subjected to digital PCR analysis to obtain the ratio of the mutant to wild-type alleles A/T (Figure 3). Droplet formation in digital PCR was successful, however, positive droplets for the wild-type T and the mutant A allele were generally small, less than 100 in most cases. Using a higher drug concentration (>30 μM) only very few cells survived. As a consequence, no adequate data could be obtained from digital PCR in this case.
Dual color digital PCR detecting the mutant 1799A allele in blue and the wild-type 1799T allele in green droplets. (A) Fluorescence intensity of all detected negative and positive blue (mutant 1799A) droplets. (B) Fluorescence intensity of all detected negative and positive green (wild-type 1799T) droplets. (C) Numbers (events) of positive blue droplets containing the 1799A allele and the green droplets containing the 1799T allele. Both blue and green droplets were detected in the melanoma cells A2058. By contrast, only green (wild-type 1799T) droplets were detected in the reference non-tumor cells CCD18Co.
Calculating the proportion of tumor cells based on the ratio of 1799A/1799T. (A) DNA purified from A2058 melanoma cells and from CCD18Co non-tumor cells were mixed at various proportions and were subjected to dual-color digital PCR. The obtained ratios of 1799A/1799T were used to calculate the proportion of tumor cells which was subsequently compared against the set up proportion. (B) A2058 tumor cells and CCD18Co non-tumor cells were mixed at various ratios and were seeded into wells of a 96-plate. Following overnight attachment, the cells were lysed using the directPCR reagent and they were subjected to digital PCR. The calculated proportion of tumor cells was compared to the set-up ones. Dotted lines are the trend lines of the real data which are close to the ideal lines of perfect correlation.
The proportion of tumor cells over non-tumor cells at each drug concentration was calculated using the A/T ratios from the digital PCR data (Figure 4A). Despite the high variability, a trend of decreasing proportion of tumor cells concomitant with an increasing drug concentration was visible. Using this proportion of tumor cells, the viability of tumor cells and the viability of non-tumor cells could be separated from each other at each drug concentration. Consequently, tumor cells seemed to respond more to the drug while non-tumor cells seemed to be less affected.
In order to decrease the high variability in the digital PCR data, we increased the volume of lysed cells containing DNA from 2 μl to 10 μl for a single digital PCR reaction. However, this failed completely following failure in droplet formation, likely due to increased amount of salts, detergents and cell debris (data not shown).
Discussion
In this study, we demonstrated the feasibility of using a tumor-specific BRAF mutation c.1799T>A to determine the proportion of tumor over non-tumor cells in a mixed culture. We further demonstrated the feasibility of using the proportion of tumor cells to separate the response of tumor cells and the response of non-tumor cells of a mixed culture to a drug from each other. In this case of using vemurafenib as the test drug, the tumor cells responded more than the non-tumor cells, possibly providing an in vitro indication for good specificity of vemurafenib.
In the ATCC panel, the melanoma cell line A2058, is given as has the BRAF mutation c.1799T>A in a heterozygous manner, which means that the ratio of the mutant 1799A to the wild-type allele 1799T is 1:1. However, our digital PCR revealed a ratio of approximately 1:2. Upon this finding, we resolved that the while type T and the mutant “A” alleles are at 73.4% and 26.4%, respectively, based on the data of next generation sequencing provided by the ATCC. This finding also demonstrated the high resolution power of digital PCR.
Digital PCR of DNA from mixed tumor and non-tumor cells treated with vermurafenib at 0, 0.3, 1, 3, 10, 30, 100 and 300 μM, giving ratios of 1799A/1799T. At a high drug concentrations (>30 μM) only few cells remained viable and consequently, less than 10 or no positive droplets were detected.
Separating the response of tumor cells and the response of non-tumor cells to vermurafinib. (A) The proportion of A2058 tumor cells at each vermurafenib concentration was calculated using the ratio of 1799A/1799T from digital PCR. (B) The viability of total number of cells (tumor and non-tumor cells). (C) Calculated viability of tumor cells and viability of non-tumor cells at each concentration of vermurafenib. Gray-shaded area indicates the range of high drug concentrations where only few cells survived and lacked adequate data from digital PCR.
A major problem in the present study is the high variability of the digital PCR data. This is mainly because of the insufficient quality and quantity of DNA from the lysed cells (7). First, each well of a 96 plate contained only 2,000 cells at seeding. We presumed a final cell number of 4,000 after 5 days of the treatment period. However, with increasing drug concentrations, cell numbers decreased. Even for 4,000 cells, only 2 μl out of the total of 50 μl lysis could be used for one digital PCR reaction, corresponding to DNA from only 160 cells. It is therefore reasonable that the positive droplets were less than 100 in most cases. In addition, the lysis buffer contains salt, detergents and other contaminants, which may interfere with droplet formation and inhibit the PCR reaction to various extents, consequently intensifying data variability. Indeed, increasing the amount of lysis solution for digital PCR resulted in failure of droplet formation. Increasing the amount and improving the quality of DNA, therefore remains a key issue to be solved. Due to limited amounts of cells, conventional purification methods are not applicable in such cases and alternative strategies are needed, such as dialysis and filtering of the cell lysis containing DNA. As expected, this kind of treatment will lead to drastic increases of the total cost. An inexpensive alternative for improving DNA quality may be precipitation using ethanol, however this may lead to loss of DNA. To increase the yield of DNA, glycogen can be used as a carrier for the precipitation (10).
In summary, we demonstrated the feasibility of determining the proportion of tumor cells over non-tumor cells in a mixed culture by means of quantifying a tumor-specific mutation. Mixed cultures containing tumor and non-tumor cells provide a potential in vitro tool for assessing drug efficacy and specificity simultaneously under identical cultural conditions. Such a genetic-based in vitro tool may have a strong application potential in drug discovery and in personalized cancer care.
Acknowledgements
We thank Mrs. Sylvia Andrich who supported this study. We would like to thank Dr. Volz and the research group of Dr. Dandri for providing us with access to their digital PCR device.
Footnotes
Authors' Contributions
MK conceived and carried out the study, LK supervised the experiments and provided data evaluation, MB conceived the study, interpreted the results, and provided critical editing of the manuscript, including finalizing the manuscript. CM conceived the study, supervised the drug treatment and prepared the manuscript.
Conflicts of Interest
No conflicts of interest to declare.
- Received February 19, 2019.
- Revision received March 21, 2019.
- Accepted March 21, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.