Impact of TTFields and chemotherapy on mesothelioma cell line proliferation maligno
Monica Lupi, Federica Mirimao*, Nicolò Panini, Paolo Ubezio, Massimo Zucchetti, Cristina Matteo, Tommaso Ceruti
Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy
Background: Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis. The chemotherapy represents the primary form of treatment, but the 5-year survival rate is less than 10%.
The good efficacy demonstrated by TTFields in preclinical experiments and in clinical trials in glioblastoma and MPM patients makes the system a promising therapeutic tool to be further investigated.
Rationale: Treatment with TTFields alone can determine some alterations in cell cycle distribution and cell growth and increase cell membrane permeability. Both these effects might contribute to an enhanced sensitivity of MPM cells to the treatment with different anticancer drugs.
Aim: Antiproliferative and cell cycle effects of TTFields on MPM cell lines growing in vitro will be studied testing different schedules of treatment and combining TTFields with anticancer drugs such as cisplatin, carboplatin, pemetrexed, trabectedin, gemcitabine, paclitaxel and doxorubicin at different concentrations. The combined treatments that will be found synergistic will be further investigated in order to understand the mechanisms behind the synergism.
Study design: The impact of TTFields on cell proliferation and cell cycle at several intervals of time during and after treatment at different intensities will be investigated by combining the results coming from absolute cell counts (by coulter counter) and the distribution of cells in G1, S and G2M cell cycle phases (by flow cytometry) to discriminate cytotoxic from cytostatic effects.
The interference of the TTFields with different anticancer agents at several concentrations will be tested by sulphorodamine B and/or colony assay. Simultaneous and sequential schemes of treatments will be explored to provide indications of the best schedule of exposure to TTFields able to potentiate anticancer activity. The most promising combined treatment (i.e. those presenting a synergistic effect) will be considered for flow cytometric analysis of DNA distribution, cell membrane potential modification or ROS production and time-lapse microscopy for cell motility changes. Drug uptake and efflux will be studied dosing the intracellular drug concentrations by atomic absorption for platinum drugs and specific HPLC/MS methods for the other drugs. The measures will be performed at different times from treatment start and after drug washout.
Relevance: The investigation of the interaction of TTFields and anticancer drugs can be useful to plan more rational and effective clinical trials for MPM patients. It should be noted that any improvement of MPM therapy is of great interest, as part of the recently developed drugs failed to show a significant effect in this neoplasm.
* Fellow by Fondazione Buzzi Unicem
Introductory Statement: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a 5-year survival rate of less than 10% . For the majority of patients surgery is not an option due to the diffuse spreading of this tumor , thus chemotherapy represents the primary form of treatment. Combined pemetrexed and cisplatin constitute the backbone of most chemotherapy, while gemcitabine is used to treat patients unable to take pemetrexed. Despite the increasing knowledge of the biology of this tumor, patients with MPM have a poor prognosis, with estimated median survival times of only 4–12 months .
Background: Tumor-treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields delivered through transducer arrays placed around the anatomic region of the tumor. TTFields disrupt cell division, and their antimitotic effect has been demonstrated in vitro in different tumor types .
Clinical trials in glioblastoma patients suggested that the addition of TTFields to postoperative radiation therapy and chemotherapy represents an important advance in the management of newly diagnosed tumors . Ongoing clinical trials are investigating the efficacy and safety of TTFields in other tumor types, including pancreatic cancer, mesothelioma, ovarian cancer, and non–small cell lung cancer. In particular, the efficacy of TTFields in combination with chemotherapy in MPM has been investigated in a phase II clinical trial (NCT02397928), whose interim results demonstrated a 12-month survival rate of 79.7% (95% CI, 57.2–91.2) and median PFS of 7.3 months (95% CI, 5.6–NA) . Expected TTFields-related dermatitis was reported in 55%, but only two patients had grade 3 dermatitis.
Other clinical and preclinical studies aimed to understand the impact of TTFields on MPM are in progress, but the cellular effects of the treatments, with TTFields alone or in combinations with other chemotherapeutic agents, have not been thoroughly investigated yet.
Preliminary Data: A panel of primary MPM cells were isolated a few years ago by our group with the collaboration of Alessandria and Casale hospitals from pleural effusion and/or lavage of patients’ thoracic cavity, before administration of therapeutic treatments. The aim was to develop new clinically relevant MPM models, reproducing the biological and pharmacological features of the human tumor . Five cell lines derived from the three types of MPM have been cultured and characterized by our laboratory: CD473 and CD484 epithelioid, CD60 and CD432 sarcomatoid and CD487 biphasic. In vitro and in vivo activity of anticancer drugs such as cisplatin, pemetrexed, OTX015 and gemcitabine have been tested on these models .
Besides the use of human-derived MPM cells lines, it is also important to consider murine mesothelioma cell lines in view of future preclinical studies in mice to investigate the interactions of TTFields with the immune system and immunotherapy. For this purpose we plan to use AB1, AB12 and AB22 cell lines that have been obtained exposing mice to asbestos .
We do not have yet any preliminary data about the effects of TTFields on these cells. We present some data obtained treating AB22 cells with doxorubicin, as an example of studies that we will perform exposing this and all the other considered cell lines to TTFields alone or with chemotherapeutic agents.
Some of the results obtained by preliminary experiments aimed to characterize the unperturbed growth of the epithelioid cell line AB22 and its response to 24-treatment with doxorubicin are shown (Figure 1). From these data we can observe that doxorubicin induce a dose-dependent effect on AB22 proliferation (panel A) which is mainly due to the G2M block that the cells experienced at the end of the treatment (panel B: DNA distributions at 24h), but we can also derive the doubling time of control cells, mean phase durations and cell cycle percentages %G1, S and G2M (Table 1). The knowledge of these parameters represents the first step for a deep comprehension of the effects induced by TTFields or by the treatments with anticancer agents on cell cycle and proliferation [9,10].
Table 1: Parameters characterizing exponential growth of control AB22 cells.
Doubling time Td
16.7 ± 0.8 h
Mean duration G1
5.9 ± 1.0 h
42.4 ± 4.3
Mean duration S
7.8 ± 0.7 h
44.5 ± 4.4
Mean duration G2M
3.1 ± 0.2 h
13.1 ± 3.3
Figure 1: Results of cell count (panel A) and flow cytometric analysis of DNA (panel B) at different time during or after exposure of AB22 cells to 62, 125 and 250 nM doxorubicin. In panel A means and standard deviations of three replicates are shown, while representative DNA are reported in panel B.
Many papers published in the years by our laboratory demonstrate the long experience in the evaluation of the effects induced by combined therapies [9,11] and cellular drug uptake by HPLC/MS methods [12–15].
Rationale: Starting from the results already published about the effects induced by TTFields on cell proliferation [4,16] and those obtained by clinical trials [5,6,17], it is reasonable to suppose that the treatment with TTFields alone can determine some alterations in cell cycle distribution and cell growth of MPM cells. Possible cell cycle effects induced by TTFields might also determine a cell cycle mediated synergism with other chemotherapeutic drug that can be administered together or in sequence with TTFields.
Moreover, as demonstrated by Chang et al. , TTFields increase cell membrane permeability in glioblastoma cells and this alteration may lead the cells to a different drug uptake. The possibility to enhance intracellular concentration of different chemotherapeutic drugs might determine an increased sensitivity of MPM cells to the treatment.
Specific Aims: We plan to study in vitro antiproliferative and cell cycle effects of TTFields in MPM cell lines, starting from the patient-derived ones. Different schedules of treatment combining TTFields and anticancer drugs such as cisplatin, carboplatin, pemetrexed, trabectedin, gemcitabine, paclitaxel and doxorubicin at different concentrations will be tested in order to select those combinations and those schedules that are demonstrated to be synergistic.
The possible mechanisms that can be at the basis of the synergism will be further investigated with the experimental platforms available in our laboratory. In particular we will study cell cycle distribution and proliferation, cellular drug uptake, modification of cell membrane, oxidative stress and cell motility.
The most promising schemes of treatment will be investigated also in mouse-derived MPM cells with the hope that these results can be translated also in in vivo experiments once that the TTField device for small animals will be available.
Research Design and Methods: For each cell line we will measure proliferation and cell cycle perturbations in time during the exposure to TTFields at different intensities and after their discontinuation. The impact of TTFields will be studied by combining the results coming from absolute cell counts (by coulter counter) and percentages of cells in G1, S and G2M cell cycle phases (by flow cytometry) to discriminate cytotoxic from cytostatic effects [9,10,19,20].
The interference of the TTFields with different anticancer agents at several concentrations will be tested by sulphorodamine B and/or colony assay. Simultaneous and sequential schemes of treatments will be explored to provide indications of the best schedule of exposure to TTFields able to potentiate anticancer activity. In selected conditions where TTFields will be proved to modify the activity of the chemotherapeutic agent, we will perform mechanistic studies, to achieve a detailed picture of the antiproliferative effects. These will include data coming from different experimental platforms, such as flow cytometric analysis of cell membrane potential modification or ROS production and time-lapse microscopy for cell motility changes. In particular, time-lapse microscopy will allow us to perform the wound-healing assay, but also the most accurate single cell motility analysis. In this case each cell in the field of view will be monitored over a selected interval of time and once that the positions are registered the distance migrated and the persistence (ratio of the direct distance from start point to end point divided by the total track distance) can be quantified .
Drug uptake and efflux will be studied dosing the intracellular drug concentrations by atomic absorption for platinum drugs  and specific HPLC/MS methods already available in our laboratory for the other considered drugs. The measures will be performed at different times from treatment start and after drug washout [12,13,15]. For doxorubicin treated cells, drug uptake and efflux will be simultaneously monitored by HPLC/MS and flow cytometer exploiting drug fluorescence .
Significance and Statement of Relevance: Starting from the success obtained in clinical trials in different tumor types treated with TTFields, the comprehension of the mechanisms that underlie the interaction between cancer cells and TTFields and between TTFields and other anticancer drugs represents an important goal for preclinical research. The investigation of the interaction of TTFields and anticancer drugs can be useful to plan more rational and effective clinical trials for MPM patients.
It should be noted that any improvement of MPM therapy is of great interest, as part of the recently developed drugs failed to show a significant effect in this neoplasm.
All the facilities necessary to carry out this research project are available in our Institute and the people involved possess the necessary skills to perform the analysis planned in the present study.
Flow cytometry facility comprises two FACSCalibur (Becton Dickinson) flow cytometers equipped with 488 and 630-nm lasers, one Gallios flow cytometer (Beckman Coulter) equipped with 488, 561 and 640-nm lasers and one MoFlo Astrios cell sorter (Beckman Coulter) equipped with 355, 488 and 640-nm lasers.
Time-lapse microscopy facility comprises:
- X81 motorized inverted microscope (Olympus) with UPlanFLN 10x, 20x and 40x or UPlanSApo 60x and 100x objectives
- Incubator (OKOlab) to maintain optimal environmental conditions for cell growth – control of temperature, humidity and CO2
- Illumination system MT20, equipped with a 150W Xe arc burner with light output between 320 nm and 720 nm, a filter wheel with 8 positions and a fast attenuator with 14 grades of illumination intensity. The light is coupled via a single quartz light fiber into the microscope.
- ORCAER CCD camera (Hamamatsu)
- XY positioning stage (Marzhauser)
This equipment allows the repeated acquisition of transmission or fluorescence images over a long period of observation at determined intervals of time. The X-Y positioning stage allows the monitoring of different sample positions at any time. Image capture was controlled by cellSens software (Olympus).
HPLC and Mass Spectrometry facility is equipped with high performance liquid chromatography with UV and fluorescence detector (Waters) and API 4000 triple quadrupole mass spectrometer (AB SCIEX) used to conduct classical pharmacokinetic studies and with MALDI 4800 TOF-TOF (AB SCIEX) used for Imaging mass spectrometry analysis. For atomic absorption an Atomic Absorption (AA) with an Analyst 600 (Perkin Elmer) is available.
Tissue Culture facility comprises all the necessary instrument for tissue culture: laminar flow cabinets, incubators, Multisizer 3 Coulter Counter (Beckman Coulter) for absolute cell count and microscopes
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