Chordomas grow relatively slowly in vitro and the doubling time of human chordoma U-CH1 cells in vitro is reported as 7 days . Because of its characteristic kinetics as slow-growing, radiobiological characterization of chordoma cells is still lacking, though many clinical trials have been conducted and their outcomes reported [6, 16]. Recently, however, an established cell line with a shortened doubling time of 3 days was developed with cell cycle kinetics thereby of particular usefulness towards acquiring invaluable biological information on chordoma cell growth and sensitivity to radiation or anti-tumor drugs in vitro and ultimately, in vivo.
In the present study we chose to focus on the cell survival of chordoma cells in vitro irradiated with two very different clinically relevant hadron beams in terms of quality, namely carbon ions and protons, currently used for charged particle radiation therapy in a limited number of highly specialized centers throughout the world [5, 22–24]. Qualitatively and quantitatively, our understanding of chordomas and data accrual for carbon treatments of this tumor lag far behind those for protons in light of the much smaller number of carbon treatment facilities worldwide. In this study, we show that exposure to carbon ions results in cell survival fractions lower than those for protons near the Bragg Peak. In the OptiCell™ system, RBE values against gamma-rays were 1.69 for higher LET (>30 keV/μm) carbon ions, 0.85 for low LET (13–20 keV/μm) carbon ions, 1.27 for low LET (20–30 keV/μm) carbon ions, and 0.89 for the entire range of protons (1-10 keV/μm) (Figure 3) and thereby demonstrate a marked superiority of carbon to proton charged particles in killing chordoma cells, explaining at least in part carbon’s superior ability to treat tumors in the clinic. Dose–response curves also show these differences between carbon ions and protons. While the latter in vitro cellular determinations reflect the clinical course of these actual chordoma tumors following carbon versus proton therapy in vivo, they do define a larger quantitative therapeutic differential between the two particle beams in question.
The cell killing effectiveness of carbon ions in our study reveals for the first time a dependency of the chordoma cell lethality of these hadrons on both the radiation dose and the particles’ LET values in a three-dimensional in vitro setting . The present study extends and refines the qualitatively similar effects of carbon ions in a standard tissue culture environment as we have previously shown . A marked novelty and advantage of our irradiating U-CH1-N cells in stacked OptiCell™ chambers with accelerated carbon beams is that we concurrently include both high LET particles with values near the Bragg peak and low LET particles with values outside of the Bragg peak area. In this study, we show that high LET carbon beams which are at present used in clinical treatment  do indeed kill more U-CH1-N cells than do low LET carbon particle beams. Moreover, we show that while proton particle beams which are considered to be of low LET also result in a cell-kill profile which consists of both a plateau and a Bragg peak areas, the dose response curve for chordoma cell-kill as a function of exposure to protons presents no significant differences when the survival data are segregated into outside- and near-Bragg peak areas. Thus, low LET protons and carbon are noticeably similar effective in killing chordoma cells (proton D10 = 0.89 and 13–20 keV/μm carbon ions D10 = 0.85). Cell kill data following proton beam exposure in our study, moreover, suggest that the effect of protons on cell kill would be far less therapeutically controllable or modifiable than that of a carbon beam as the former depends on the dose of alone. From the results above, the use of carbon ions in particle therapy of chordoma offers a distinct advantage with respect to both effectiveness and efficiency in that its therapeutic gain depends on LET and a demonstrated higher cell killing. These tendencies confirm chordoma cells patterns of superior cell kill by carbon ions recently shown in other cell lines, namely Chinese Hamster Ovary (CHO) cells  and human salivary gland cancer HSG cells .
OptiCell™ tissue culture chambers are cell culture systems optimized for cell growth, culture monitoring, and transportation. They are designed for maximizing tissue culture incubator space in light of their narrow cross-sectional profile and their highly specific enclosed-system which is readily applied to edifying three-dimensional stacks of monolayer culture systems as in vitro models for cell [20, 28]. In the present study, we devised stacked three-dimensional in vitro OptiCel™ arrays in configurations modeling in vivo clinically relevant anatomical constructs which we then used to determine cell survival fractions at each depth of beam energy deposition. Thus, development and alignment of these live-cell biological phantoms in three dimensions along a beam path more relevantly mimic a desired intravital geometry. To study relative in vitro survivals at varying depths, several other physical culture methods have been used, including Petri dishes fixed in a particular device , and a “cell stack chamber” . Relative to these other specialized cell culture methods, the most advantageous feature of the stacked OptiCell™ chamber system is the acquisition of many more - and greater detailed - data points, with an optimal resolution of 2 mm. Since the maximum dose of protons accelerated to 70 MeV was delivered at a depth of ~4 cm in this study, a detailed analysis was necessary to acquire data near the proton Bragg peak. One significant difference between our present study protocol and previously used methods is the relative timing of cell plating and that of irradiations. There are essentially two different approaches to performing studies involving colony formation assays. A first strategy involves the plating cells before exposure to drugs or radiation, while the other relies on treating cells prior to subsequent passaging them to assess their resulting clonogenic ability . Survival fractions are known to depend on whether plating of cells is performed before or after irradiation [32, 33]. In the present study, cells were diluted and plated prior to irradiation and maintained undisturbed for the subsequent three weeks, which contrasts with the methodology employed in our previously reported study . In this study, cell survival fractions following carbon or proton irradiation within OptiCell™ chambers demonstrate a marked radioresistance when compared with cells cultured in standard tissue culture dishes (Figure 3). The survival difference in both culturing conditions can also be explained in part by the respective sequences of plating and irradiation.
In summary, this is the first report of a direct and therapeutically relevant in vitro comparison between the cell killing effects of carbon ions and protons on human chordoma tumor derived cell lines. Our data describe an experimental setting in which we have begun to investigate the superior therapeutic index of high LET carbon charged particle beams over all other charged particle and photon radiation modalities. We contend that use of the OptiCell™ system provides an overall more reliable understanding of the biology of cells within a native in vivo chordoma tumor environment and should form the basis of future chordoma studies.