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Tumour development


Isolating 500 cells
Renato Dulbecco Scientist
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The other important thing is that the people that carried out this type of work using Microarrays, generally used tumour samples, a piece of the tumour, thus, they take all the messengers. But this tumour is a mix of many types of cells, these are not tumour cells, they are normal cells, connective tissue, blood capillaries, molecules that infiltrate the tumour. In fact, the tumour is probably only half of what can be seen, thus already obscure, although it is possible to correct this a little, but it is something that obscures the results. And instead, therefore, we decided in collaboration with the pathologist from the National Institute of Tumours in Milan to isolate tumour cells, to isolate them one by one and the lady that carried out this work would look at them under the microscope and then isolate the cells that are characteristic of tumour cells using a small glass stick, therefore they are distinct tumour cells and there is no doubt about their origin and certainly there is no more contamination because this has been checked. However, it is necessary to collect a good number of cells, because if there are too few you can't do anything and the minimum you can use is 500 to isolate 500 cells one at a time...

[Q] How long does it take?

One at a time you need quite a while. But as these cells are... the tumour is above a fixed stratum on the glass. It is fixed, so it doesn't change, you can work on successive days, in order to obtain from the same sample. Then once you have these 500, you must extract the RNA, the messenger, but there are not sufficient quantities of this RNA to then use the SAGE technique that I told you about, because you need a lot more. So what you need to do is to amplify this RNA and there are...

[Q] How do you amplify it?

There are methods of amplification that work pretty well and starting with this sample of 500 cells, the necessary quantities to use this SAGE technique can be obtained and this is checked to see if there are alterations, etc. This is looked at for certain specific genes, to see if they are represented normally after amplification and they are. Therefore, there is no danger of change, too many artefacts during multiplication. This was... we spent a lot of time on this to be certain.

L'altra cosa che e'importante, che quelli che hanno fatto lavori di questo tipo, usando i 'microarray', generalmente adoperano campioni del tumore, un pezzo del tumore, perciò, prendono tutti i messaggeri. Ma questo tumore è una miscela di cellule di molti tipi, non ci sono le cellule del tumore, ci sono cellule normali, del connettivo, dei capillari sanguigni, delle molecole che infiltrano il tumore. Insomma, il tumore probabilmente è solo metà di quello che si vede, perciò questo già oscura, sebbene insomma si possa un po' correggere per questo, ma è una cosa che oscura i risultati. E invece, perciò, noi abbiamo deciso, in collaborazione lì con il patologo dell'Istituto dei Tumori di Milano, di isolare cellule tumorali, isolarle una per una e questa signora che fa questo lavoro, lei guarda al microscopio e poi ha la possibilità, con un bastoncino di vetro, di isolare le cellule che sono caratteristiche di cellule tumorali, perciò sono cellule tumorali distinte e non c'è dubbio sopra la loro origine e certamente non c'è più contaminazione, perché questo è stato verificato. Però bisogna raccogliere un numero buono di cellule, perché se son troppo poche, non si può fare niente e il minimo che si può usare sono 500 per isolare 500 cellule una per una...

[Q] Quanto tempo prendono?

Una per una ci vuole parecchio tempo, come vedi. Ma siccome queste cellule sono- il tumore è sopra uno strato fisso sul vetro è stato fissato, per cui non cambia, si può lavorare in giorni successivi, insomma, per ottenere. Dallo stesso campione. Poi una volta che hai queste 500, devi estrarre l'RNA, il messaggero, ma questo RNA non è in quantità sufficienti per fare poi quel S.A.G.E. che ti ho detto, perché ce ne vuole molto di più. Allora, quello che bisogna fare è amplificare questo RNA e ci sono...

[Q] Come si fa a amplificarlo?

Ci sono, appunto, dei metodi di amplificazione, che funzionano abbastanza bene e partendo da questo campione di 500 cellule, si può ottenere le quantità necessarie per fare questo S.A.G.E. e questo è stato verificato per vedere se ci sono delle alterazioni, ecc. Questo si guarda, appunto, a certi geni specifici, per vedere se vengono rappresentati normalmente dopo l'amplificazione e lo sono. Perciò non ci sono pericoli che ci siano dei cambiamenti, troppo, insomma, artefatti durante la moltiplicazione. Questo è stato appunto abbiamo passato un mucchio di tempo su questo per essere sicuri.

The Italian biologist Renato Dulbecco (1914-2012) had early success isolating a mutant of the polio virus which was used to create a life-saving vaccine. Later in his career, he initiated the Human Genome Project and was jointly awarded the Nobel Prize in Physiology or Medicine in 1975 for furthering our understanding of cancer caused by viruses.

Listeners: Paola De Paoli Marchetti

Paola De Paoli Marchetti is a science journalist who graduated with an honours degree in foreign languages and literature from the University Ca’Foscari, Venice. She has been a science journalist since the 1960s and has been on the staff of the newspaper Il Sole 24 Ore since 1970. She was elected president of UGIS (Italian Association of Science Journalists) in 1984. She has been a Member of the Board of EUSJA (European Union of Science Journalists’ Associations, Strasbourg), and was its president in 1987-1988 and 1998-2000. In May 2000 she was unanimously elected president emeritus. She was a member of the National Council of Italian Journalists (1992-1998). From 2002 to 2004 she was member of the working group for scientific communication of the National Committee for Biotechnology. She has also been a consultant at the Italian Ministry of Research and Technology and editor-in-chief of the publication MRST, policy of science and technology. She has co-authored many publications in the field of scientific information, including Le biotecnologie in Italia, Le piste della ricerca and Luna vent’anni dopo.

Tags: National Institute of Tumours in Milan

Duration: 3 minutes, 26 seconds

Date story recorded: May 2005

Date story went live: 24 January 2008