1. Comparative study of the radiobiological effects induced on adherent vs suspended cells by BNCT, neutrons and γ rays treatments L.Cansolino, A.M.Clerici, C.Zonta, C.M.Bianchi, P.Dionigi, G.Mazzini, R.Di Liberto, S.Altieri, F.Ballarini, S.Bortolussi, M.P.Carante, M.Ferrari, I.Postuma, N.Protti, C.Ferrari* *corresponding author: cinzia.ferrari@unipv.it

2. Introduction & experimental goal Extensive preclinical in vitro studies are mandatory in order to assess the applicability and efficay of new treatments against any neoplasia of interest. Inhibition of the continued reproductive ability of cells is an important consequence of radiation exposure -> The Plating Assay (PA) is one of the most common reference methods to verify the cell proliferative capacity after irradiation and radiotherapy. Looking to future comparisons of in vitro preclinical data of cell survival after radiation treatments performed using indifferently suspended or adherent cells, the aim of the present study is to investigate if the two modalities of cell exposure to radiations influence their radiosensitivity. Tannock et al., Basic Science of Oncology, second edition, 1999: “… cells grown in colture are irradiated either before or after preparation of a suspension of single cells …”

3. Materials and methods The present study is part of the experimental preclinical validations aimed to verify the applicability of BNCT to liver and lung coloncarcinoma metastases and limb osteosarcoma. Cell lines rat osteosarcoma UMR-106 cell lines; cells grow as confluent monolayers in medium composed by HAM'S F10 and DMEM high glucose (1:1 v/v), 10% Foetal Bovine Serum (FBS) and 40 μg/ml gentamicine. rat coloncarcinoma DHDK12TRb (DHD); cells grow as confluent monolayers in medium composed by HAM’S F10 and DMEM low glucose (1:1 v/v), 10% Foetal Bovine Serum (FBS) and 40 μg/ml gentamicine

4. Materials and methods Plating Efficiency test (PA) to assess cell survival CTR Treated 100 cells 100 cells Treated samples Incubation at 37ºC for ~ 10 days Fixation and counterstaining Counting of derived clones Cell suspensions Cell seeding in Petri disches at 3 different dilutions x5 Plating Efficiency (PE) (%) = n° of colonies n° of seeded cells Cell survival (%)= PE treated sample PE ctr cells

5. Materials and methods Radiation treatments:  Co60 γ rays: CGR ALCYON II; 3.5, 5, 7 and 10 Gy; electronic equilibrium condition  Neutrons: TRIGA Mark II reactor thermal column of Pavia University; 10 minutes irradiation; 1, 7.5, 30, 60 and 100 kW  BNCT: TRIGA Mark II reactor thermal column of Pavia University; 10 minutes irradiation; 1, 7.5, 30, 60 and 100 kW; after intracellular boron enrichement (80 μg/ml 10 BPA, 4 hours exposition)

6. Irradiation condition Field size: 32 cm x 32 cm SSD: 78,5 cm Dose rate: 0,815 Gy/min Co60 γ rays irradiation facility

7. Boral wall 40 cm 100 cm 20 cm Thermal neutron irradiation facility energyin air neutron flux (cm-2s-1)1 σ (%) 0.414 eV1.17 · 10 10 2.5 10.7 eV7.51 · 10 7 3.1 1.58 keV1.20 · 10 8 5.4 17.3 MeV4.39 · 10 7 9.0 - Open-pool thermal reactor; light water as moderator and cooler - First criticality: 15th of November 1965 - Nominal steady-state power: 250 kW in air γ dose rate: 1.8 mGy/s (≈ 80% from background) “Determination of gamma component in thermal column of Pavia Triga reactor by using alanine ESR detectors”, S.Altieri, Pa P6, Thursday 19th, 9:30-10:30

8. Irradiation set-ups Co60 irradiation of suspended cells Adherent cells: Co60 and thermal neutron irradiations Thermal neutron irradiation of suspended cells T-75 flask 12.3 x 7.3 x 3.1 cm 3 box (inner size), 1 mm thick polystyrene (1.068 g/cm 3 ) Cell colture and medium 75 cm 2 x 10  m thick monolayer, average soft tissue ICRU-44 (1.05 g/cm 3 ); 20 mL, 75 cm 2 x 0.27 cm, 99% H 2 O Teflon (C 2 F 4 ) container 8.4 cm high x 10 cm  ; 2.1 g/cm 3 12 cylindrical holes 2.3 cm high x 0.9 cm  Cell colture and medium microvials; cells as pellets (0.05 cm thick layer); 700  L, 1.1 cm thick layer, 99% H 2 O

9. Dose estimation Doses delivered by neutron and BNCT treatments were calculated by Monte Carlo simulations: MCNP5 Main reactions induced by thermal neutrons in tissues and photon contribution taken into account to calculate the dose delivered to cells BNCT suspension adherent Photon flux in the flask monolayer and microvial pellet background Cross section of neutron elastic scattering on 1 H

10. Protocols for cells exposition and post-irradiation assays ADHERENT, γ or n T-75 flask 20 mL medium ≈ 5 · 10 6 cells/mL subconfluent DNA profile (24h, 48h, 5d, 7d) Plating Assay Trispin and counted 700 μL medium ≈ 5 · 10 6 cells/mL SUSPENSION, γ or nADHERENT, BNCT 10 BPA (4h, 80µg/mL) medium exchange SUSPENSION, BNCT 10 BPA (4h, 80µg/mL) PBS Trispin and counted IIIIIIIIIIIIIIIIIIIIIIII

11. Results (I) Comparison between calculated dose rates delivered to adherent vs suspended cells (“reference irradiation”: reactor power = 250 kW; 1 ppm 10 B) ADHERENTSUSPENDED Dose componentDose rate Gy s -1 errorDose rate Gy s -1 error 10 B(n,α) 7 Li1.420 · 10 -3 0.0011.536 · 10 -3 0.003 14 N(n,p) 14 C2.994 · 10 -3 0.0013.210 · 10 -3 0.003 1 H(n,n’) 1 H7.534 · 10 -4 0.019.072 · 10 -4 0.05 1 H(n,γ) 2 H1.507 · 10 -3 0.0038.307 · 10 -4 0.01 photon bg1.044 · 10 -3 0.0031.465 · 10 -3 0.01 total, n-irradiation6.298 · 10 -3 0.016.413 · 10 -3 0.005 total, BNCT7.718 · 10 -3 0.017.949 · 10 -3 0.008

12. Results (II) Number of cells G 0/1 S G2MG2M Fluorescence Intensity (DNA content) Radiation damage delays the progression through the cell cycle -> cytofluorimetric DNA analysis. At the prefixed observation times irradiated and control cells were harvested by trypsinization, washed with PBS and fixed in ethanol 70%. For cytofluorimetric analyses, cells were counterstained with propidium iodide (50  g/ml). DNA histograms, performed 24 h, 48 h, 5 days and 7 days post 60Co irradiation, of suspended cell samples, at 3.5, 5, 7 and 10 Gy. Measurements were performed by a PARTEC PAS II cytofluorimeter acquiring data in linear or logarithmic mode. DNA content spectrum of a normal ctrl cell colture

13. d= days after neutron irradiation 1d2d5d7d9d12d15d21d B+ R+ B - R+ B+ R - B - R - 2n= 27.41 % 4n= 55.13 % 4,8x10 5 7,5x10 5 1,4x10 6 4,8x10 6 8x10 6 2n= 26.90 % 4n= 46.47 % 2n= 39.24% 4n= 36.26% 2n= 49.90 % 4n= 23.61 % 2n= 54.40 % 4n= 23.89 % 2n= 45.42 % 4n= 24.79 % 2n= 44.02 % 4n= 22.35 % 2n= 61.39 % 4n= 24.38 % 2n= 71.94 % 4n= 19.74 % 5x10 5 9x10 5 4,5x10 6 10x10 6 2n= 40.10 % 4n= 32,91 % 2n= 44.84 % 4n= 20.60 % 2n= 64.01 % 4n= 16.29 % 5x10 5 1,2x10 6 6x10 6 2n= % of cells in G 0/1 4n= % of cells in G 2 M Number of recovered cells 2n= 31.52 % 4n= 46.41 % 2n= 22.88 % 4n= 59.70 % 2n= 2 %2n= 7 %2n= 21 % 2n= 61.07 % 4n= 20.10 % 2n= 61.33 % 4n= 24.67 % 2n= 72.77 % 4n= 19.38 % 4,7x10 5 4x10 5 9,5x10 5 6,5x10 5 7x10 5 1,4x10 6 2x10 6 DNA histograms, performed starting from 24 hours up to 21 days after neutron irradiation, of suspended cell samples, with or without B10 enrichment. Results (II)

14. 60Co  -ray irradiation of DHD suspended cells 60Co  -ray irradiation of DHD adherent cells Results (II)

15. Results (III) Cell survival by PA

16. Results (III) Cell survival by PA

17. Discussion Factors influencing cell survival:  radiation quality (low vs high LET)  cell-cycle position → comparison of cell cycle position of suspended and adherent cells at the time of neutron exposure UMR-106DHD G0/1SG2MG0/1SG2M CTR39.7923.4536.76------ CTR B+4331.925------ Adh. B-49.2924.7925.93------ Adh. B+50.793019.21------ Susp.B-50.7123.1026.19------ Susp.B+42.2134.9822.81------

18. Factors influencing cell survival:  oxygen effect and hypoxic condition  suboptimal growth conditions of treated cells in the post-irradiation period (DHD and UMR cells use to grow in monolayers, that is as adherent cells → suspension can be considered a suboptimal condition for cell proliferation → increased radioresistance) Discussion ADHERENT CELLS monolayer size = 75 cm 2 accessible air volume = 212.175 cm 3 SUSPENDED CELLS pellet size = 0.64 cm 2 accessible air volume = 0.75 cm 3 (adapted from Little 1973)

19. Cell survival from suspended and adherent cells compared to survival of “suspended cells in flask”

20. Conclusions and future perspectives The present study evidences that adherent cells are much more radiosensitive to both low and high LET radiations than suspended cells. The enhanced block of the cell cycle progression in G2 phase, observed in suspended with respect to adherent cells, suggests the presence of a higher repair capability in the formers. Currently, the most convincing explanation of the observed differences in cell radiosensitivity depending on exposition modality seems to be suboptimal conditions for cell proliferation (either in the form of hypoxia or suspension cell phase) during the short time (around 1 hour) after irradiation of cells. Further experiments are planned to verify this hypothesis. The modality of cell exposure to irradiation must be considered as an additional factor influencing cell survival to radiation treatments. The modality of cell exposure must be taken into account in comparing intra and inter laboratory radiation survival results, also in case of BNCT treatment.

21. Conclusions and future perspectives The present study evidences that adherent cells are much more radiosensitive to both low and high LET radiations than suspended cells. The enhanced block of the cell cycle progression in G2 phase, observed in suspended with respect to adherent cells, suggests the presence of a higher repair capability in the formers. Currently, the most convincing explanation of the observed differences in cell radiosensitivity depending on exposition modality seems to be suboptimal conditions for cell proliferation (either in the form of hypoxia or suspension cell phase) during the short time (around 1 hour) after irradiation of cells. Further experiments are planned to verify this hypothesis. The modality of cell exposure to irradiation must be considered as an additional factor influencing cell survival to radiation treatments. The modality of cell exposure must be taken into account in comparing intra and inter laboratory radiation survival results, also in case of BNCT treatment. … kiitos! …