###### Abstract  
(1) Background: For advanced head and neck squamous cell carcinoma (HNSCC), boron neutron capture therapy (BNCT) is a potential therapeutic option, but high concentrations of boron within HNSCC are necessary. Therefore, this in vitro pilot study examined the uptake and concentration of boron in HNSCC cells using the trans-stimulation effect of L-tyrosine when compared to non-stimulated samples. (2) Methods: Two HNSCC cell lines were incubated with L-tyrosine for up to two hours, followed by incubation with three L-para-boronophenylalanine (BPA) concentrations (5, 20, 50 ppm) at eight incubation times (1-4.5 h in half-hour steps). Subsequently, cellular boron uptake was measured via inductively coupled plasma mass spectrometry. (3) Results: No differences between laryngeal and oral SCC cells were seen; therefore, data were pooled. In total, boron uptake was not significantly increased in trans-stimulated samples when compared to the control group (all \(p\) > 0.05). Nevertheless, with trans-stimulation, higher BPA concentrations resulted in higher intracellular boron concentrations (5 < 20 < 50 ppm; all: \(p\) < 0.05), whereas these differences were less distinct in the non-trans-stimulated group. (4) Conclusions: The effect of trans-stimulation for up to two hours seems to be less relevant for HNSCC, though trans-stimulated HNSCC cells seem to have a more distinct BPA-dose-dependent cellular boron uptake that might be addressed in further research. boron neutron capture therapy; trans-stimulation; head and neck cancer; squamous cell carcinoma; preloading +
Footnote †: journal: Applied Sciences  
tyrosine or phenylalanineine [6]. Because of the similarity of BPA and phenylalanine, this transport system was identified as important for BPA uptake [7]. In the oral mucosa, LAT-1 is located in the basal cell layer, and is both intracellular and membrane-bound. Whereas the expression of LAT-1 in normal mucosa is low [8], it increases in cases of HNSCC and decreases for the highest dedifferentiation levels [9]. HNSCC is stated as a suitable target for BPA-mediated BNCT with a tumour/tongue \({}^{10}\)B ratio of up to 4.1 [10] and a high rate of patients that respond to BNCT treatment, even with inoperable, locally recurrent and previously irradiated cancer [5]. Various approaches for enhancement of BPA uptake in carcinoma cells have been proposed, such as pre-treatments with low-dose gamma irradiation before or during BPA incubation [11] and pulsed focused ultrasound [12]. Different infusion methods for BPA such as intravenous, intra-arterial or direct infusion into the carotid artery in the case of brain tumours were studied as well [13]. Another way to increase boron uptake is based on the transport mechanism of amino acids that aims to reach an equilibrium between efflux and influx. In this regard, two relevant phenomena are described: cis-inhibition and trans-stimulation. Cis-inhibition, as seen in erythrocytes [14], inhibits productive signals, whereas trans-stimulation indicates an exchange of intracellular for extracellular amino acids via the L-system [15; 16]. Trans-stimulation effects are described for various cell lines. For example, gliosarcoma cells showed an increased BPA accumulation after incubation with L-tyrosine, whereas simultaneous incubation of BPA and L-tyrosine led to a decrease in boron uptake [17]. In a xenograft tumour model, preloading with L-phenylalanine reduced the boron uptake in brain tissues relative to the uptake in the tumour tissue, and therefore reduced the maximum irradiation dose in normal tissue [18]. Further in vitro and in vivo investigations showed that preloading with L-tyrosine as well was with L-DOPA before exposure to BPA increased the intracellular boron concentration by a factor of three, while normal tissue did not show an increased BPA uptake [17; 19; 20]. The aim of the present study was an evaluation of the potential increase in the uptake of BPA into HNSCC cell lines utilising trans-stimulation with L-tyrosine, which seems to be clinically feasible.