A novel boron-conjugated SRC inhibitor for Proton Boron Capture Therapy in glioblastoma treatment

The ability of protons to deliver the maximum dose at the tumor region can work synergistically with boron atoms to emit alpha particles, enhancing therapy effects with less damage to healthy tissue. Protons and boron nuclear fusion reaction is the principle for the so-called Proton Boron Capture Therapy, that can contribute to high therapy efficiency by using smaller flux than conventional proton therapy, especially for radioresistant brain tumors such as glioblastoma. Glioblastoma is the most infiltrating and aggressive tumor of the brain with a very low life expectancy, ranging from 6 to 24 months. In this study we evaluated the protons combined effectwithanovelboron-conjugated compound, which is a pyrazolo[3,4-d]pyrimidine derivative, acting as SRC tyrosine kinase inhibitor. Indeed, this drug includes a boron cluster, that can be directed in tumor cells using the ATP-competitive mechanism of the pyrazolo[3,4 d]pyrimidine ring in activated SRC form of glioblastoma cells, achieving tumor uptake of boron for Proton Boron Capture Therapy reaction. This preliminary study showed interesting results that could offer important contributions for further experiments.


Introduction
The nuclear fusion reaction between protons and boron for alpha particles generation, represents a promising strategy for precise and powerful treatments of radioresistant tumors.Previously, the relative biological efficacy increase by applying a proton beam to tumor cells exposed with sodium borocaptate (BSH) has been investigated in vitro [1].The encouraging results stimulated further preclinical investigations to evaluate the benefits of Proton Boron Capture Therapy (PBCT) approach in a glioblastoma (GBM) mouse model [2].The results of the study demonstrated that the synergistic treatment with protons and BoronoPhenylAlanine (BPA) leads to a lower metabolic activity and increased tumor cell death [2].Nonetheless, one of the primary drawbacks of the PBCT strategy is an effective delivery of boron atoms into tumor cells, particularly regarding brain tumors.In fact, the presence of the blood-brain barrier, regulates and restricts the exchange of substances with the environment and extracellular fluids [3].Despite numerous boron compounds, only BPA and BSH have been effective in clinical trials so far, although there have been very few scientific updates on them.
In this preliminary study, we applied a boron-based compound, hereinafter named Si306+Boron, that was produced from a specific enzyme inhibitor belonging to SRC tyrosine kinase family, called Si306.This molecule was previously used in combination withboth X-ray and protons obtaining synergistic effectsfor GBMtreatments [4,5] (figure 1).
For the first time, our study shows the use of a Boron-enriched SRC inhibitor, to promote the nuclear fusion reaction between protons and boron.The pharmacological action of Si306 consists in blocking the enzymatic activity of SRC [6].In detail, Si306 acts with an ATP-competitive mechanism due to the isosterism of the pyrazolo [3,4-𝑑]pyrimidine ring with the adenine scaffold of ATP, binding the SRC activated form and blocking tyrosine kinase activity [7].Indeed, in pathological conditions such as cancers, SRC tyrosine kinase activity is involved in several upstream and downstream pathways.
-1 - Growth factor receptors such as epithelial (EGFR) or vascular (VEGFR) and other intracellular factors give rise to signal transduction mechanisms from SRC, resulting in the activation of transcriptional factors and promoting several GBM hallmarks [8,9].Therefore, the aim of the work was to use Si306+Boron for a double therapeutic action: the enzyme inhibition and the generation of high LET alpha particles able to increase the cellular damage (figure 2).The study was conducted on a GBM cell line that was treated with Si306+Boron in combination with protons and compared with Si306 [10].We showed thatin basal conditions, Si306+Boron has a lower cytotoxic effect than the non-borate molecule.However, Si306+Boron in combination with protons achieves higher lethality, as reported from the evaluation by clonogenic and LDH assay.
Figure 3. Experimental work-flow.Cells were plated in appropriate numbers and kept in incubation for 24 hours before receiving drug treatment.The drugs were maintained for 24 hours until irradiation.After irradiation, the medium was taken to perform the lactate dehydrogenase enzymatic assay and the supports were filled with fresh medium.Finally, the clonogenic assay was performed.Created with BioRender.com.

Cytotoxicity and metabolic turnover of Si306 and Si306+Boron
Firstly, we tested metabolic turnover on the U251-MG cell line exposed to Si306 and Si306+Boron at concentrations of 0.01, 0.1, 1, 10 and 50 μM for 24 hours, that was the same time point of drugs exposure before irradiation.We reported no significant differences in Si306 versus Si306+Boron groups for each concentration.Nevertheless, we observed a slight decrease of metabolic turnover for Si306 groups.Our results showed that neither Si306 nor Si306+Boron exerted significant changes on GBM cell viability as a single dose ranging from 0.01 to 10 μM as compared to vehicle-treated cells (DMSO only).However, we observed significant changes in cell viability at 50 μM of Si306, which reported a significant decrease as compared to the control group (figure 4).

Synergistic effects of proton irradiation with Si306 and Si306+Boron treatment
In order to compare the effects of increasing doses of protons (0, 1, 2, 4, 5 Gy) in combination with 10 μM of Si306 and Si306+Boron drugs, we performed a clonogenic assay on the U251-MG cell line.The surviving fraction (SF) values were plotted against the dose to obtain dose-response curves.The results showed a radiation dose-dependent decrease in clones number with a significant effect for all protons doses.Moreover, we observed that without irradiation (0 Gy), both drugs reported a significant reduction in survival compared to the vehicle-treated control.Furthermore, the pharmacological treatment without irradiation determined a lower SF in the samples treated with Si306 compared to Si306+Boron.Notably, the comparison between combined treatments and proton alone exposureshowed a significant reduction of Si306 cell-treated only at the dose of 2 Gy, whereas the dose of 5 Gy reported a synergistic effect in combination with Si306+Boron (figure 5A, B and C).
-3 - Given that cell morphology and growth can be affected by drugs and ionizing radiations, we compared the mean size of the clones between the experimental groups.A reduction in average size of clones was significantly observed in protons treatment with 4 Gy, 5 Gy alone and in combination with Si306 compared to treatment with Si306+Boron alone.As for SFs, a significant difference was observed between the pharmacological treatment without irradiation; indeed, clones from Si306 treatment were significantly reduced compared to Si06+Boron.Furthermore, Si306+Boron in combination with the maximum dose of 5 Gy reported a significant reduction in clones size compared to treatment with Si306+Boron and Si306 at 0 Gy.Si306 combined with the maximum dose of 5 Gy reported a greater reduction in clones size compared to treatment with 2 Gy (figure 6).
We analyzed the synergistic effect of drugs and protons by LDH assay on U251-MG cell line.Even if the basal cytotoxic effect of Si306 was confirmed, the overall cytotoxic effects of the treatments observed in this assay were less evident than those reported in the clonogenic assay.Indeed, there was no dose-response relationship within the vehicle and Si306 experimental groups; Si306+Boron did not show cytotoxicity without irradiation.However, the cytotoxicity of Si306 was greater not only compared to treatment with protons at 0 Gy and 2 Gy, but also compared to Si306+Boron at 0 Gy and 2 Gy.Si306 in combination with 5 Gy showed scattered data which determined an increasing trend in cell death which was not statistically significant.
Regarding the Si306+Boron combined with protons, it has been reported an increased cell death at both 2 Gy and 5 Gy compared tothe single treatment with Si306+Boron and vehicle.Si306+Boron showed a significant mortality in combination with 2 Gy compared to protons at the same dose (figure 7).
-4 -Figure 5. A-C) Surviving fraction and representative pictures of U251-MG clones treated with proton irradiation at 0, 1, 2, 4, 5 Gy alone and combined with Si306 and Si306 + Boron.Surviving fraction of U251-MG were reported with linear (A) and log10 scale (B); representative ROI were extracted from chambers slide, where cells were cultured and irradiated, reporting also the number of seeded cells for each experimental point (C); data are expressed as superimpose symbols as mean ± SEM and connected line of three independent experiments.*p-value < 0.05 and ** p-value < 0.01 in blue for vehicle versus 0 Gy; # p-value < 0.05 in green for Si306 versus only protons or versus Si306+Boron at the same dose.# p-value < 0.05 in red for Si306+Boron versus only protons at the same dose.Two-way ANOVA with Tukey post-hoc test were used.

Discussion and conclusion
Scientific progression in molecular diagnostic areas has led to the definition of GBM tumor subgroups for treatment personalization and stratification.However, despite the identification of molecular and genetic tumor subgroups with slightly different prognostic characteristics, personalized treatments are not broadly applied in GBM clinical practice yet.In fact, aggressive treatments are still needed to eradicate tumor cells as much as possible sparing the surrounding healthy tissues, independently by GBM subgroup [11,12].To date, the standard treatment with the alkylating agent temozolomide in addition to photon beam therapy is only palliative [13].However, the recommendation for GBM treatment with radiotherapy after surgery resection is classified as "high quality evidence" by clinical guidelines [14].Despite the infiltrative pattern of GBM, clinical data support the treatment with partial irradiation rather than whole brain in order to spare healthy tissues.Indeed, partial irradiation has been demonstrated to not affect the overall survival and to improve the patient's conditions [15].Proton therapy may match this aim thanks to the physical characteristics of protons that show a depth-dose curve, with a dose peak (Bragg peak) at a well-defined depth in tissue [16].However, a weak point of protons is represented by the similar Relative Biological Effectiveness (RBE) of conventional cancer radiotherapy [17].In this context, the idea theoretically proposed by Do-Kun Y et al. (p + 11 B → 3α nuclear fusion reaction) generating a short-range high-LET α particles can be applied using novel molecules and radiosensitizing agents [18,19].Indeed, small molecules for the so-called targeted therapies can be used to follow specific molecular mechanisms of tumor cells, in order to find preferential pathways to selectively introduce boron in tumor cells.Pyrazolo [3,4-d]pyrimidines are potent ATP-competitive SRC kinase inhibitors that have been explored for their promising antitumor activity.Lead Discovery Siena (Siena, Italy), starting from several prodrugs, selected the most suitable SRC inhibitor in terms of permeability, aqueous solubility, metabolic stability and enzymatic inhibitory abilities.The candidate drug Si306, was tested alone and in combination with radiotherapy reporting good radiosensitive activity and anticancer properties, increasing GBM cell death, also demonstrating a synergistic effect with X-rays and protons [4,5].A boron-containing moiety was added to Si306 molecule, generating a compound that could then deliver boron into tumor cells.
In this preliminary study it has been observed that the novel boron-conjugated SRC inhibitor, achieves radiosensitizing effects in association with protons, although it reported a basal level of cytotoxicity lower than Si306.However, synergistic effect with protons and Si306+Boron was not significantly higher than protons and Si306.The encouraging data is represented by the mortality increase of Si306+Boron at the highest proton dose.It remains unknown whether the combinatorial effect of protons + Si306Boron is additive or synergistic.
It is worth noticing that from clonogenic assay, as compared to only 5 Gy of protons, a significant decrease of SF was achieved by combining 5 Gy and Si306+Boron, whereas the combination of 5 Gy and Si306 did not report a significant reduction.In the LDH assay, the combination of Si306 and Si306+Boron with 2 Gy determined a significant increase in cell death as compared to irradiation with protons alone.This result could encourage the application of low pharmacological doses to obtain higher therapeutic effects thanks to the synergy between boron cluster of SRC inhibitor and protons.
To deepen our understanding and to confirm our results, it is necessary to increase the number of tests and gather more evidence.To better elucidate and confirm the underlying mechanisms for alpha particles generation by 11 B capture it will be necessary to increase experimental data, especially about cells boron internalization and boron amount in the medium during irradiation.Another important

JINST 19 C04051
point to investigate is represented by the basal toxicity of the Si306 and Si306+Boron molecules.The first MTT assay showed an increasing toxicity trend for Si306, higher than Si306+Boron, but comparable with 10 μM, that was used in the experiments.However, in the subsequent clonogenic assay, the basal cytotoxic effects of both molecules were higher.In the LDH assay, the cytotoxicity of Si306+Boron was absent, whereas Si306 citotoxicity was confirmed.For these data, it is worth noticing that MTT assay is not reflecting necessarily cell proliferation and growth, but viable cell metabolism.Clonogenic assay chronically evaluates the loss or maintenance of cellular reproductive capacity on a few numbers of cells after treatments (over 10 days), whereas a cell lysate of approximately 40,000 cells is evaluated in early time point after treatment.(24 hours in our case).For these reasons, further investigation is needed in future studies to confirm the cytotoxic effects of drug at 0 Gy by comparing different incubation times and evaluating other markers of cell death.
In particular, it would be necessary to investigate cell death processes that are triggered by evaluating some specific biomarkers (i.e.γH2AX for double strand breaks) as well as to explore the molecular pathways of SRC involved in response to the treatment.Finally, it would be noteworthy to examine pharmacological changes that have occurred for the boron cluster integration in Si306, by testing the molecules in different cancer and healthy cell lines.

Cell culture and Si306+Boron synthesis
Experiments were carried out using the U251-MG human glioblastoma cell line.Cells were purchased from European Collection of Authenticated Cell Cultures (ECACC, Public Health England, Porton Down Salisbury, U.K.) and cultured as previously described [4].Cells were maintained in an exponentially growing culture condition in an incubator at 37 • C in a humidified atmosphere (95% O 2 and 5% CO 2 ) and were routinely sub-cultured in 75 cm 2 standard tissue culture flasks.
Both compounds, Si306 and Si306+Boron, were provided by Lead Discovery Siena (Siena, Italy) as a stock powder and were dissolved in dimethylsulfoxide (DMSO) (Merk, Milan, Italy).
For the synthesis of Si306 + Boron, anhydrous NaHCO3 (1 mmol; 6.0 equiv.) is added to a solution of Si306 (0.17 mmol; 1.0 equiv.) in dry dichloromethane (DCM, 15 mL).After 5 min. of stirring at room temperature, the solution is cooled to 0 • C and triphosgene (0.17 mmol; 1.0 eq.) is added to the solution.After 30 minutes, the reaction mixture is warmed to room temperature and left under magnetic stirring for 12 hours.After this time, a solution of boron reagent (0.17 mmol; 1.0 equiv.)prepared following a reported procedure in dry DCM is transferred by double needle to the reaction mixture.The reaction is stopped after 16 hours [20].The mixture is filtered over celite, the solvent is evaporated under reduced pressure, and the crude obtained is purified on a chromatographic column under isocratic conditions, using a mixture of DCM/methanol (MeOH) 98:2 as eluant.The product is obtained as a colorless oil with 54% yield.
Both compounds were stocked in DMSO anddiluted at the final concentration (10 μM) with fresh medium, where GBM cells were maintained for 24 hours.After irradiation, cells were replaced with fresh medium in order to remove the drugs.The control samples for all biological tests were supplemented with vehicle (no more than 0.5% DMSO).

Proton irradiation configuration for cells irradiation
Cell irradiation with protons was performed using the synchrotron-based clinical scanning beams (fixed horizontal beam line) at Centro Nazionale di Adroterapia Oncologica (CNAO, Pavia, Italy).The flasks were placed vertically inside a water phantom put in the isocenter on the treatment table, at the depth of 15 cm, corresponding to the mid spread-out Bragg peak (SOBP).The SOBP (6 cm width, from 12 to 18 cm depth in water) was achieved with active beam energy modulation, using 16 different energies (131.5-164.8MeV).The proton dose-averaged Linear Energy Transfer (LET) in the mid SOBP, evaluated with a Monte Carlo FLUKA simulation, was 3.6 keV/m.Cell samples were irradiated in the dose range 0-5 Gy.

MTT assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to compare viability effect on cells exposed to Si306 and Si306+Boron independently from proton treatment.Cells were distributed in 96-well plates (Costar, Milan, Italy) at a final density of 10,000 cells/well/100 μL and incubated for 24 hours.The day after, cells were exposed to drugs at specific concentrations (0.01, 0.1, 1, 10 and 50 μM), and incubated for 24 hours.Then, MTT at a final concentration of 1 mg/mL was added to each well and incubated for 3 hours under standard culture conditions.Media were then gently removed, 200 μL of MTT solvent (DMSO) was added, and cells were stirred on an orbital shaker for 10 min at room temperature.The absorbance was measured using a Multiskan SkyHigh Microplate spectrophotometer (Thermo Scientific, Milan, Italy) at 570 nm.Data were expressed as the percentage of MTT reduction versus control cells.Each experiment was performed three times with six replicates per condition during each experimental run [21].

Clonogenic survival assay
Cells were seeded in 6-well plates in triplicate at a number ranging from 50 to 1200 cells, according to the dose delivered and to the vehicle or drug concentration.Then, irradiation was performed using the dose values of 1, 2, 4 and 5 Gy.After irradiation, cells were incubated for 7-10 days until the colony formation.The colonies were stained with 0.05% crystal violet diluted in 50% methanol (Sigma-Aldrich, Milano, Italia) for 30 min at room temperature.SF was determined according to the plating efficiency (PE).Briefly, we calculated the PE, dividing the counted colony by the total plated cells.Then, we calculated the SF as a ratio of sample PE over control PE.For each experiment, the effect of each dose of radiation alone and combined with Si306+Boron or Si306 was evaluated on three individual wells of cell culture and each experiment was performed in triplicate [22].

Lactate dehydrogenase assay
For the LDH assay, 40.000 cells were plated in an 8-well slide chamber and kept in an incubator at 37 • C in a humidified atmosphere (95% O 2 and 5% CO 2 ).Then, 10 μM of Si306 and Si306+Boron was added for 24 hours.The samples were irradiated with 2 and 5 Gy of protons and after irradiation the medium was changed to remove the pharmacological effect.Media were collected after 24 hours of incubation.The LDH analysis started with 100uL of each group that was centrifuged in Eppendorf at 300 g for 5 min.The supernatant (40 μL) was collected in a 96-well and 10 μL of LDH reagent (Sial, Roma, Italia) was added to each well and incubated for 30 minutes in the dark at room temperature.Positive control (100% of cytotoxicity) was assumed as cells treated with 10% Triton X-100, a -9 -surfactant used to permeabilize cell membranes, for 10 min.The absorbance (OD) was measured using a Multiskan SkyHigh Microplate spectrophotometer (Thermo Scientific, Milan, Italy) at 450 nm [23].The percentage of relative cytotoxicity for samples was calculated as: [(OD sample − OD negative control )/(OD positive control − OD negative control )] × 100 Negative controls were cells exposed to fresh growth media (0 Gy and no drugs) and assumed as 0% cytotoxicity.

Statistical considerations
Data analysis was performed using GraphPad Prism software version 8.2.1.Data were tested for normality using a Shapiro-Wilk normality test and subsequently assessed for homogeneity of variance.For data that did not pass normality test, Kruskal-Wallis test was used for comparisons between groups.For comparison of n > 2 groups, two-way analysis of variance (ANOVA), followed by Tukey post-hoc test for multiple comparisons.Data are presented as the mean ± SEM.A value of p < 0.05 was considered statistically significant and symbols used to indicate statistical differences are described in figure legends.

Figure 1 .
Figure 1.Synthesis of derivative Si306 + Boron starting from Si306, via the triphosgene pathway.The new compound contains 10 boron atoms.

Figure 2 .
Figure 2. Synergistic action of Si306+Boron.The SRC protein is active in GBM to trigger several tumor hallmarks.The Si306+Boron molecule not only inhibits the tumorigenic action of SRC but also promotes the intake of boron for the reaction with protons and the generation of alpha particles which would determine cell death mechanisms.Created with BioRender.com.

Figure 4 .
Figure 4. Metabolic turnover profile of U251-MG after Si306 and Si306 + Boron compounds exposition.U251-MG cell line was exposed for 24 hours to increasing doses of Si306 and Si306 + Boron.Data are shown via standard box and whiskers of n ≥ 4 independent replicates for each experimental; *p-value < 0.05 versus vehicle.Statistical analysis was performed using the non-parametric Kruskal-Wallis test.

Figure 6 .
Figure 6.Average size of U251-MG clones treated with proton irradiation at 0, 1, 2, 4, 5 Gy alone and combined with Si306 and Si306 + Boron.Data of average size of clones are shown via separated scatter bar graphs with mean ± SEM of three independent experiments.*p-value < 0.05 and ** p-value < 0.01 for multiple comparisons using two-way ANOVA with Tukey post-hoc test.

Figure 7 .
Figure 7. U251-MG were treated with protons at doses of 0, 2 and 5 Gy, in combination with 10 μM Si306 or Si306 + Boron.LDH released by cells after treatments was evaluated to assess cell death.Data are shown via standard box and whiskers of n ≥ 4 independent replicates for each experimental group; *p-value < 0.05 and ** p-value < 0.01 for multiple comparisons using two-way ANOVA with Tukey post-hoc test.++ p-value < 0.01 is referred only for Tr-X-100 vs all groups.