Download The genetics combined to the chemotherapy:cell survival after single

The genetics combined to the chemotherapy:cell survival
after single-dose irradiation
Poster No.:
C-1147
Congress:
ECR 2016
Type:
Scientific Exhibit
Authors:
A. Mazza , M. Ignatti , G. Spagnoletti , F. Sabatino , G. Bove ;
1
1
2
1
1
1
2
Foggia/IT, Bari, IT/IT
Keywords:
Radiotherapy techniques, Experimental investigations,
Experimental, Oncology
DOI:
10.1594/ecr2016/C-1147
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Page 1 of 16
Aims and objectives
From January 2014 to October 2014 at the Units of Radiation Oncology of the 'University
Hospital "Riuniti Hospitals" in Foggia, was conducted on a weekly basis an experimental
work on the effects of radiation on human tumor cells of colon and breast, radiated and
studied in vitro with clonogenicity test.
The purpose was to identify and optimize a technical and individual protocol for the
study of cell survival after irradiation with mono-fraction dose, combined with the use of
a chemotherapeutic drug.
Methods and materials
The cells used are represented by various tumor cell lines and colon cancer (HT-29,
HCT-116 and Colo-320) and breast (MCF7) stored in liquid nitrogen and plated in
culture in suitable soils for each cell line. They have been used plates of plexiglass with
dimensions of 12x8 cm, each with 6 wells. According to maximum opening field (40x40
cm), it was calculated that could be radiated up to 6 plates simultaneously [Figure 1].
From the information TC, were established the spatial coordinates to define the set-up,
the volumes to be irradiated and through TPS (treatment planning system) has been
identified the isocenter and field geometry [Figures 2]. In addition, the SSD (source to
skin distance) was set to 100 cm, the rotation of the gantry 180 ° and has highlighted the
need for the interposition of a thickness (bolus) of plexiglass [Figure 3].
Irradiation: the irradiation was carried out with 6 MX-rays pro V duced by a linear
accelerator Elekta at different doses : 4, 8 , 16, 24 Gy [Figure 4].
Test clonogenicity: completed the irradiation , the plates have been brought in a
incubator where they were kept in culture for 10 days , during which time the medium
was changed every other day. This allowed the surviving cells to organize into colonies
Subsequently, they were subjected to washing, fixing and staining with crystal violet.
The test of clonogenicity is based on the ability to produce colonies of more than 50 cells
in a suitable growth environment with subsequent counting of those surviving.
In practice it expresses the reproductive capacity of tumor cells and normal cells following
radiation , important and decisive for the outcome of therapy.
Page 2 of 16
Images for this section:
Page 3 of 16
Fig. 1: Stored cells in the culture medium and divided into the wells.
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Fig. 2: Shaping the dose.
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Page 4 of 16
Fig. 3: Placing the bolus plexiglass.
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Fig. 4: Irradiation with linear accelerator Elekta:gantry is rotated 180° and the SSD 100
cm.
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Page 5 of 16
Results
The irradiation of the plates have been numerous and realized investigating different
dosages, a different number of cells, different cell lines and use or less of chemotherapy.
This latter aspect of the work allows to count the cell colonies formed after combined
treatment of radiotherapy and chemotherapy: in general, the two treatments can exercise
its effect, independently of one another, the chemotherapy may increase the response of
radiation therapy or even inhibit it. Therefore, this association is not always a therapeutic
gain.
In the first experiments have been distributed 600 cells for well, but it was immediately
apparent that in this way, in the control sample, cell growth appeared so massive as to not
be able to count the different colonies. Therefore, in the subsequent experiments we were
distributed 200-300 cells for well. Moreover, the irradiation at 24 Gy has proved totally
effective in inhibit cell growth: the counting of the colonies was regardless of cell type,
equal to 0 (zero). In subsequent experiments it was, therefore, avoided the irradiation
at 24 Gy.
Irradiation of cells of the colon: Radiobiology show us that each tissue has a different
radiosensitivity and each cell line has a different degree of growth and a different way
of responding to the damage caused by radiation (intrinsic radiosensitivity ) and the
differences are caused by genetic constitution of each tumor . The reasons of this different
radiosensitivity in field of the same histological type to be found positioned in genetic of
each celto the radiation induced damage :specifically , the cell line Colo - 320 proved to
be the most radiosensitive and HT - 29 the most radioresistant.
Page 6 of 16
Fig. 5
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
Irradiation of cells of the colon with addition of chemotherapeutic: In these
experiments, the cells of the colon was added the 5- fluorouracil chemotherapy at different
concentrations ( 5 mM , 15 mM , 25 mM ). From the graphs 2 , 3 and 4 and in Tables 4 ,
5 and 6 , we can calculate the growth of colonies of cells HT -29 , HCT - 116 and Colo320 with the addition of chemotherapy.
Page 7 of 16
Fig. 6
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
Page 8 of 16
Fig. 7
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
Page 9 of 16
Fig. 8
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
Irradiation of cells of the breast: between cells MCF7 and
MCF7 SCR SH - TRAP [ Chart 5 and Table 5 ), of breast cancer, there were significant
differences of radiosensitivity.
Page 10 of 16
Fig. 9
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
Conclusion
The protocol used is effective: during the trial were changed cell lines, the dosages used
and the number of cells well distribuited, but have never been necessary amendments
to the technical protocol.
Comparing the graphs and data of different cell lines, the cells of a breast tumor compared
to cells HT-29 and HCT-116 are more radiosensitive: 4 Gy colonies form are less in
number than formed with the same dose for colon tumors , while to 8 Gy there is no
growth of the colony. Between the two breast cancer cell lines analyzed ( SCRMCF7 and
MCF7SH - TRAP ) were most radiosensitive cells MCF7SCR, although the differences
are not significant [ Table 6 ] .
Page 11 of 16
Fig. 10
References: U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia
- Foggia/IT
The differences, however, are significant also between different cell lines of the same
tumor. Among the cells of the colon, for example, the HT-29 and HCT-116 continue to
form colonies even after irradiation by 8 Gy, while the Colo-320 after 8 Gy do not give
any colony. For all three cell types, 16 and 24 Gy there is no growth of colonies.
Therefore, the cells more radiosensitive are the Colo-320 and the more radioresistant
are the HT- 2 29 forming many colonies more than in all other cell types.
To the cells of the colon it was added the 5-fluorouracil chemotherapy at different
concentrations. The presence of the drug significantly reduces the growth of the colonies
and with a high concentration of 5-fluorouracil (25mM) in all samples, including the
control, there is no growth of colonies for only effect of chemotherapy. It highlights the
radiosensitivity of the cell line Colo-320 that, with the irradiation with 8 Gy and the addition
of the drug, even at a concentration of 5 mM, does not allow the growth of any colony.
The cells HT-29, without the addition of 5-fluorouracil are more radioresistant than the
cells HCT-116; with the addition of chemotherapy at a low concentration, however, the
cells HCT-116 continue to form colonies in the control sample and after the irradiation at
4 Gy, differently from how they behave the other cell lines for which the addition of the
drug, even without radiation therapy, does not allow the growth of the colonies.
So, if you consider only the effect of the drug, the HCT-116, proved to be the most
resistant to chemotherapy 5-fluorouracil. The drug, when administered together with
radiotherapy, has always improved the effect, even at low doses. All cell lines, finally,
even without the addition of chemotherapy, behave the same way with the irradiation at
8 Gy with the absence of colonies.
Page 12 of 16
Definitely, not all types of cancer can be effectively treated with radiotherapy for the
inherent radiosensitivity , for the patient's medical history , for the conformation , histology
and staging of the disease .
For colon and breast cancer, has been possible to make objective assessments after in
vitro testing, considering the different radiosensitivity of tumor cells in the arrangement
genetic basis the response of each cell line reagent differently to radiation induced
damage and the combination of the use in the treatment of chemotherapy drug .
Personal information
A. Mazza, Dipartimento di Radioterapia UOC, "Ospedali Riuniti" di Foggia, Foggia, Italia.
([email protected])
M. Ignatti, Dipartimento di Radiologia UOC, "San Paolo" di Bari, Bari, Italia.
([email protected])
G. Spagnoletti, Dipartimento di Radioterapia UOC, "Ospedali Riuniti" di Foggia, Foggia
Italia. ([email protected])
F. Sabatino, libero professionista, Foggia Italia. (francescasabatino @ @ tiscali.it
hptmail.it)
G. Bove, Direttore UOC di Radioterapia, Ospedali Riuniti "di Foggia, Foggia Italia.
([email protected])
Images for this section:
Page 13 of 16
Fig. 11: A. Mazza
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Page 14 of 16
Fig. 12: M. Ignatti
© U.O.C. di Radioterapia, U.O.C. di Radioterapia, Ospedali Riuniti di Foggia - Foggia/IT
Page 15 of 16
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