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

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 …”

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

Materials and methods

• Plating Efficiency test (PA) to assess cell survival

CTR Treated100cells

100cells

Treatedsamples

Incubation at 37ºC

for ~ 10 days

Fixationand

counterstaining

Counting of derived

clones

Cellsuspensions

Cell seedingin Petri disches at 3 different dilutions

x5

x5

x5

Plating Efficiency (PE) (%) =n° of colonies

n° of seeded cells

Cell survival (%)=PE treated sample

PE ctr cells

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 10BPA, 4 hours exposition)

Irradiation conditionField size: 32 cm x 32 cmSSD: 78,5 cmDose rate: 0,815 Gy/min

Co60 γ rays irradiation facility

Boral wall

40 cm100 cm

20 cmThermal neutron irradiation facility

energy in air neutron flux (cm-2s-1) 1 σ (%)

0.414 eV 1.17 · 1010 2.5

10.7 eV 7.51 · 107 3.1

1.58 keV 1.20 · 108 5.4

17.3 MeV 4.39 · 107 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

Irradiation set-ups Co60 irradiation of suspended cells

Adherent cells: Co60 and thermal neutron irradiations

Thermal neutron irradiation of suspended cells

T-75 flask12.3 x 7.3 x 3.1 cm3 box (inner size), 1 mm thickpolystyrene (1.068 g/cm3)

Cell colture and medium75 cm2 x 10 m thick monolayer, average soft tissue ICRU-44 (1.05 g/cm3);20 mL, 75 cm2 x 0.27 cm, 99% H2O

Teflon (C2F4) container8.4 cm high x 10 cm ; 2.1 g/cm3

12 cylindrical holes 2.3 cm high x 0.9 cm

Cell colture and mediummicrovials; cells as pellets (0.05 cm thick layer); 700 L, 1.1 cm thick layer, 99% H2O

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

suspensionadherent

Photon flux in the flask monolayer and microvial pellet

background

Cross section of neutron elastic scattering on 1H

Protocols for cells exposition and post-irradiation assays

ADHERENT, γ or n

T-75 flask20 mL medium≈ 5 · 106 cells/mLsubconfluent

DNA profile (24h, 48h, 5d, 7d)

Plating Assay

Trispin and counted700 μL medium

≈ 5 · 106 cells/mL

SUSPENSION, γ or n ADHERENT, BNCT

10BPA (4h, 80µg/mL)

medium exchange

SUSPENSION, BNCT

10BPA (4h, 80µg/mL)

PBS

Trispin and counted

Trispin and counted Trispin and counted

I II III I II III I II III I II III

Results (I) Comparison between calculated dose rates delivered to adherent vs

suspended cells (“reference irradiation”: reactor power = 250 kW; 1 ppm 10B)

ADHERENT SUSPENDED

Dose component Dose rate Gy s-1

error Dose rate Gy s-1

error

10B(n,α)7Li 1.420 · 10-3 0.001 1.536 · 10-3 0.00314N(n,p)14C 2.994 · 10-3 0.001 3.210 · 10-3 0.0031H(n,n’)1H 7.534 · 10-4 0.01 9.072 · 10-4 0.051H(n,γ)2H 1.507 · 10-3 0.003 8.307 · 10-4 0.01

photon bg 1.044 · 10-3 0.003 1.465 · 10-3 0.01

total, n-irradiation 6.298 · 10-3 0.01 6.413 · 10-3 0.005

total, BNCT 7.718 · 10-3 0.01 7.949 · 10-3 0.008

Results (II)N

um

ber

of

cells G0/1

S

G2M

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 (50g/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

d= days after neutron irradiation

1d 2d 5d 7d 9d 12d 15d 21d

B+R+

B -R+

B+R -

B -R -

2n= 27.41 %

4n= 55.13 %

4,8x105 7,5x105 1,4x106 4,8x106 8x106

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 %

5x105 9x105 4,5x106 10x106

2n= 40.10 %

4n= 32,91 %

2n= 44.84 %

4n= 20.60 %

2n= 64.01 %

4n= 16.29 %

5x105 1,2x106 6x106

2n= % of cells in G0/1 4n= % of cells in G2M

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 %

4,7x105 4x105 9,5x105 6,5x105 7x105 1,4x106 1,4x106 2x106

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)

60Co -ray irradiation of DHD suspended cells

60Co -ray irradiation of DHD adherent cells

Results (II)

Results (III) Cell survival by PA

Results (III) Cell survival by PA

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-106 DHD

G0/1 S G2M G0/1 S G2M

CTR 39.79 23.45 36.76 ---- ---- ----

CTR B+ 43 31.9 25 ---- ---- ----

Adh. B- 49.29 24.79 25.93 ---- ---- ----

Adh. B+ 50.79 30 19.21 ---- ---- ----

Susp.B- 50.71 23.10 26.19 ---- ---- ----

Susp.B+ 42.21 34.98 22.81 ---- ---- ----

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 cm2

accessible air volume = 212.175 cm3

SUSPENDED CELLS

pellet size = 0.64 cm2

accessible air volume = 0.75 cm3

(adapted from Little 1973)

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

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.

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! …