“First Use of GFP for In Vivo Imaging”

Remembrances of Takashi Chishima at AntiCancer, Inc., 1995-1997

 

The Night of the Nobel Prize Announcement

It was October 8, 2008.  I had my outpatient clinic and had many surgical cases as well and a staff meeting too.  After I finally arrived home, my wife told me “Hi darling! I heard that GFP won the Nobel Prize.”  My memory flashed back to the US more than 12 years ago as if I had seen a revolving lantern.  Four Japanese scientists won the Nobel Prize in 2008.　The Japanese got excited over this good news which contrasted with gloomy stories such as abnormal weather, recession, stock market crash, etc.

 

To me, a Nobel Laureate is a great person.  Even though he was born in Japan, I did not know Dr. Shimomura but I was impressed when I found out some years ago that he was the discoverer of GFP.

 

It was more than 12 years ago when I was a student at the Graduate School of Medical Sciences of Yokohama City Medical University.  At that time, I went to the US as a post-doc to study with Professor Robert M. Hoffman at AntiCancer, Inc., and the Department of Surgery, UCSD.  I came to replace my senior colleague Shinji Togo, who would come back to our Medical School.  At AntiCancer, I studied the process how cancer cells spread in live mice using GFP-expressing cancer cells, the first use of GFP for in vivo imaging.  Experiments that start from a small idea can have a big influence on subsequent cancer research.  The Nobel Prize announcement noted the work I started as the prime use of GFP.  I still clearly remember what my boss, Prof. Hoffman, said when I showed him my results:  “Takashi!  This is a big discovery that happens once in 10 years.”  This was in the summer of 1996.

 

Encounter with GFP

In the spring of 1995, I was unexpectedly invited to replace Dr. Togo at AntiCancer and UCSD, which is a sister-University of Yokohama Medical University, as a post-doc without much prior notice.  These arrangements were due to the close friendship of my Chairman, Professor Hiroshi Shimada, and Professor A.R. Moossa, Chairman, Department of Surgery, UCSD.  In the beginning, I was not very interested to study abroad; however, I thought that studying abroad might become a good chance to break the deadlock in my career which I was facing.  After I accepted the offer, everything proceeded very fast in a very short time and all arrangements were completed within one month.

 

My new life in the US had a great impact on me.  In addition to language problems, culture and customs and the way of thinking differed from Japan.  For the first week after I arrived in the US, I spent most of my energy getting over jet-lag and getting used to customs there.  Actually, I did not have time to spare because immediately my boss, Professor Hoffman, told me “Let’s talk about your experimental schedule.”  As soon as I organized all my things from Japan, Professor Hoffman gave me my topic, “Study how to change mouse white hair to black through gene and protein therapy of the hair follicle.”  The project was part of a very hot topic at AntiCancer, since my colleague at AntiCancer, Dr. Lingna Li, and Professor Hoffman had just published the first paper on gene therapy of the hair follicle in the new and already high-impact journal, Nature Medicine.  Newspapers, and magazines picked up this story around the world and it even made headlines, and Professor Hoffman appeared on the ABC Evening News anchored by Peter Jennings.

 

The experimental method I initially tried was rather simple, using liposomes containing melanin and applying them on mouse skin after shaving its white hair.  Restoring normal color to white hair by this simple procedure would be a great discovery and create a sensation in the cosmetic industry.  However, all the new hair that grew from the mouse skin was all white despite application with the melanin liposomes.  I repeated these experiments for several months by changing the ratio of various lipids in the liposomes; however, I could not get new black hair.  I felt much pressure since I thought people in the US value results above everything else.  Moreover, I did not want to be known as a Japanese guy who was “good-for-nothing,” which would rapidly spread.  In addition, I thought my boss may have wanted me to go home.

 

One day, Professor Hoffman brought me an issue of Science with a GFP-expressing C. elegans round worm on the cover.  Inside was an article by Martin Chalfie on using the GFP gene to make the round worm glow green by introducing the GFP gene.  He was also awarded the Nobel Prize for Chemistry for this work.  The article reported that GFP was extracted from Aequorea victoria and was identified by Dr. Shimomura in 1962.  Dr. Hoffman jokingly asked “Is Dr. Shimomura Japanese?  Is this protein made in Japan?”  Moreover, Professor Hoffman said “Let’s stop the experiment using melanin.  Let’s start an experiment that makes mouse hair fluoresce green using GFP.”  This was another difficult demand that seemed to me a joke again, but Professor Hoffman was serious.  This was the moment when I learned about GFP for the first time.  I thought, I could not deliver melanin into the hair follicle and it would not be possible for me to introduce the GFP gene into the hair follicle either.  I listened to Professor Hoffman with a feeling of resignation.  However, an idea then came to my mind, that GFP-labeled cancer cells could visualize the process of cancer metastasis. AntiCancer had the best mouse models of cancer metastasis in the world, and I thought my idea was a natural to do at AntiCancer.

 

 

I found a ray of hope in my study in the US which had come to a seemingly dead end.  Right away, I obtained the GFP gene and a cancer cell line from the cell bank at AntiCancer.  While I engaged in the project Professor Hoffman suggested in the daytime, I was engaged in my experiment “to introduce GFP-gene into cancer cells” far into the night, everyday.  This was in the autumn in 1995.　

 

Seize an Opportunity

However, in spite of my expectation and much support from my co-workers at AntiCancer, especially Dr. Yuying Tan, a very senior researcher, I could not succeed in introducing the GFP gene into the cancer cells.  Experiment after experiment failed.  It was not clear whether my technique was wrong or the experimental materials were insufficient.  I worried that I would be sent home.  One day, Professor Hoffman brought an issue of Science to me.  There was an article from a UCSD scientist who developed a brighter GFP molecule.  At this moment, I thought this was what I was looking for.  The scientist was Professor Roger Tsien, who also won the 2008 Nobel Prize in Chemistry for GFP.  I immediately wrote a letter to Professor Tsien to share his newly modified GFP gene with me.  Two weeks later, the GFP gene arrived.  This was an epoch-making event for me.  I had further good fortune.  Dr. Yohei Miyagi of the Kanagawa Cancer Center, who taught me Introduction to Bioscience in Yokohama came to San Diego and stayed one year at the Scripps Clinic for research.  I discussed my experiments with Dr. Miyagi and found that there were some problems with the vector I used.  He told me “I will help you before you leave for Japan.”  Immediately, I asked Dr. Miyagi to construct a new vector for me.  After a short time, he handed me the new vector and joked, “You should go home if you will not succeed with this vector.”  I brought the vector to AntiCancer and made up my mind to go home if I would fail.  I made a last-ditch effort in the next experiments.  I carried out my work with the greatest possible care.

 

Two weeks later, when I looked at a flask taken out from a CO2-incubator under a microscope, I shouted “My, my!” in spite of myself.  They were bright green cells!!  One year after I went to the US, I succeeded in making cancer cells express GFP.  In the next moment I thought this was the steepest part of a path near the mountaintop.  In order to observe cancer metastasis, I had to select cells expressing GFP and then inject the cells into mice.  In order to isolate a population of pure GFP-expressing cells, I cultured hundreds of Petri dishes of cells.  Day after day, I simply changed the culture medium and transferred cells from dish to dish far into the night.

 

One day, I found the temperature of the CO2-incubator was changed from 37°C to a far higher temperature.  All of my GFP-expressing cells resulting from my super-hard work were completely destroyed.  People around me saw me shouting “Who changed the temperature?”  At the time, I thought it was done on purpose.  Looking back, it was most probably an accident, due to a cleaning person, who unknowingly changed the temperature knob by accident.

 

“You should try your experiments again and reproduce the results,” Dr. Miyagi told me.  After much effort, I succeeded in isolating and establishing GFP-expressing cells about one month later.  I was ready to observe GFP-expressing cells in vivo.  I injected GFP-expressing cells into the back of a nude mouse given to me by my AntiCancer colleague.  When I checked the mouse one week after injection, a small tumor growing in the back of the mouse could be observed.  I wondered what it looked like under excitation light.  Immediately, I saw the mouse under excitation light.  Next moment, I shouted “Oh my!  Is that true?”  The tumor sparkled bright green.  Moreover, the green fluorescence from the cancer cells combined with the red color of the surrounding blood vessels to produce an exquisite contrast.  I continued my experiment further with excitement raised from the depths of my body.  In addition, when I observed a lung isolated from this mouse under the microscope, I saw an unbelievable scene there.  I could not find the appropriate words, I shouted “Heavens” in spite of myself.  I could see many tiny metastases giving out vivid green color like the stars in the night sky.  I lost control of myself until a colleague called out to me “Hi! Takashi.”  I was standing absentmindedly in the dark culture room.  This was the moment when GFP was imaged in vivo for the first time, in this case used to see cancer micrometastasis for the first time in live tissue.

 

Just after this historical moment, I knocked on the door of Professor Hoffman’s room.  He asked me “Takashi!  What are you so excited about?”  I told him, “Please come to the culture room, immediately.”  I guided my boss to the culture room with my heart leaping for joy.  He looked into the microscope with trembling hands.  He told me excitedly, “Takashi!  This is an experiment that happens once in 10 years.”  From that day, the possibility to visualize tumor metastasis using GFP-expressing cancer cells began.  It was in the summer of 1996.

 

Publishing Our Results

However, all things did not go well and many things remained to be solved because everything was being done for the first time.  For example, there were many apparatus needed for the studies using GFP in vivo.  We especially needed a fluorescence microscope with a camera.  In order to take pictures of faintly shining cells, I had to wait until night and take pictures of GFP-expressing cells using a conventional single-lens reflex camera.

 

Dr. Hoffman told me the fact that more scientists got the Nobel Prize in the US than in Japan may not depend on a language barrier but on the motivation of scientists.  I could not help changing my thoughts.  I started to think GFP work could get the Nobel Prize someday.  While I was collecting my results using GFP-expressing cancer cells in mice, I simultaneously started preparing manuscripts for scientific journals and for patent applications.  Professor Hoffman helped me submit a paper to Nature Biotechnology.  We did not receive an answer from the journal after two months.  Professor Hoffman then asked the editorial office how the review was going.  The editorial office only told him “It is under review,” and we could not obtain a formal answer.  After three months, the editorial office gave us the official answer in which one of two reviewers gave positive comments, but another reviewer gave very negative comments, that this article did not deserve publication in this journal because there was nothing worthwhile.

 

I remember that I was greatly disappointed with the answer, because I strongly expected the manuscript would be accepted.  Professor Hoffman felt bitter about the rejection, saying “We were cheated.”  According to Professor Hoffman, the reviewer who gave negative comments may have been a competitor.  We worried that the reviewer’s group had begun similar experiments.  Therefore, Professor Hoffman asked me to carry out new experiments as soon as possible.  I understood later that the date of receipt is very important in contributing an article to a scientific journal and in filing patent applications.  At that time, I felt that Professor Hoffman was aiming for the Nobel Prize for the use of GFP in vivo.  Now, I realize Professor Hoffman is a modest man and I was projecting my own fantasies on him.  When we began to write up our work, I told Professor Hoffman that I needed some papers in order to get my Ph.D.  Professor Hoffman then worked with me for many weeks often staying up all night writing together.  We submitted almost 10 papers during this time.  Our first results appeared in Cancer Research 57, 2042-2047 (1997).  It was in the spring of 1997.  This was the first paper describing GFP imaging in vivo.  This paper has now been cited more than 200 times.  It was the first of many OncoBrite papers published by the AntiCancer team on GFP imaging.  AntiCancer is world famous for this work.

 

Real World

In March of 1997, I returned to my country.  Professor Hoffman offered me a chance to stay on, but I thought my future was to become a famous doctor in Japan.  My life during two years at AntiCancer gave me more than what I could obtain from experiences over 10 years in Japan.  After returning home, many colleagues and mentors asked me about my life in the US.  They were equally surprised and impressed with my great accomplishments.  However, when I told them I thought Professor Hoffman was aiming for the Nobel Prize for GFP, they gave me a questioning look saying it must be tough.  Moreover, most of them said to me partly for fun, “But if you can win the Nobel Prize, we will treat you to dinner.”  Only one friend seriously asked me to tell the story in detail.  I reconfirmed the difference between the US and Japan with regard to motivation for scientific research.

 

At this point, I reached a turning-point in my life.  Unfortunately, the value of my results in the US was very low at my University.  I was given notice from the staff of the University at that time that they did not need my results in the US.  I was forced to choose between continuing research in the US or training myself as a surgeon at a general hospital in Japan.  Then, I reconsidered which dream was mine, “basic cancer research” or “clinical cancer treatment.”  When I remembered that I was originally challenged to become a medical doctor prompted by grandmother’s death due to cancer, to my surprise I could easily find a conclusion. I reconfirmed that my dream was not “the forefront of basic cancer research” but “the forefront of clinical cancer treatment.”  I decided to train myself as a clinical surgeon.  Fortunately, the GFP-expressing cancer cells that I left in the US started a new project called “OncoBrite,” as noted above.

 

On the other hand, I could not completely give up basic research at heart.  Over a period of three years, whenever I had a holiday of more than 3 days, I went to the US to help the “OncoBrite” project.  However, as the reputation of GFP in biology became higher and higher, a lot of laboratories with GFP-expressing cells joined in, and it became hard for me to find my position.  Moreover, as GFP-expressing cancer cells went to higher levels at AntiCancer, like whole-body imaging, invented by my successor, Dr. Meng Yang, the “OncoBrite” project went far from me.  It was in the spring of 2000.

 

Nobel Fantasies

One day, my wife and I were standing at the Blue Hall located at the City Hall in Stockholm. I told my wife, “The celebration dinner after the Nobel Award Ceremony is held here every year.  Don’t you think this place is filled with a solemn atmosphere?  I talked about my dream that we would go to Stockholm some day with Professor Hoffman.”  I spoke to my wife while looking up the ceiling.  She asked me, “Who took over your study using GFP-expressing cancer cells?”  In that instant, the memory of my study abroad crossed my mind.  I answered, “The project is going well, I hear.”  I jestingly talked to my wife, “However, my dream to come to Stockholm for the Award Ceremony of Nobel Prize seems to be an impossible dream.”  Then, we entered the Golden Room.  It was in the autumn of 2004.

 

Next to the Nobel Prize

On October 8, 2008, GFP finally won the Nobel Prize in Chemistry 2008.  The morning after the day when the Nobel Prize in Chemistry was announced, I had a phone call from a TV station.  The TV news reporter said, “As you know Dr. Shimomura won the Nobel Prize in Chemistry for the discovery of GFP.  I heard that you succeeded in observing cancer metastases using GFP-expressing cancer cells for the first time.  I would like to hear this story from you.”  I was fully booked on that day; however, the reporter said, “Timing is the point for this kind of news story.  I would like to see you today.”  In response to his request, finally I agreed with his offer.

 

In announcing the Nobel Prize, the Nobel Assembly stated “With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread.”  The development of ways to watch processes of how cancer cells spread was started with my project and continued at AntiCancer by many great scientists.

 

I was really surprised that the project I started was mentioned in the Nobel Prize announcement.  I became increasingly excited.  It was a strange moment that I felt close to the Nobel Prize.

 

After surgery in the morning, TV and news reporters waited for me.  I explained my experiments using GFP-expressing cells in the US with a variety of slides.  When news reporters asked me to comment on the Nobel Prize: “The most impressive point for me is the high motivation of researchers in the US.”  What I said was a message for Japanese young researchers.

 

The following morning a patient spoke to me after their medical examination, “I saw the TV program where you appeared last night.  I was very surprised that my doctor is close to the Nobel Prize, because I thought that the Nobel Prize is unrelated to me.  The Nobel Prize is now not so far from me and I can boast about you to my friends.  It is too bad you missed the Nobel Prize.  However, I am still alive thanks to your good diagnosis and skillful technique; I cannot thank you enough.  I would like to give you our Nobel Prize.”  Then, a small child next to her gave me a “Thank you letter” and “Gold Medal” made of origami (Japanese colored paper work).  This was the happiest moment that I had experienced during the past 12 years as a clinical surgeon.  I felt that this was the Nobel Prize I have been looking for.  This took place on October 10, 2008, two days after the Nobel Prize announcement.  That day was my 44th birthday by a curious coincidence.

 

Message to Young Researchers

Dr. Shimomura’s comments on his Nobel Prize Award were the following: “Young scientists should be more confident about their own projects.  Once you start an interesting project, you should carry it out completely, overcoming every difficulty encountering you.”  What he said means “Always do your best.”  The Nobel Prize seems distant from every researcher; however, it may be close to everybody if he would do his best with confidence.  If you would do your best, even if you could not win the Nobel Prize, someone will give you “a special gold medal.”  If you have a chance, do your best.  Even if you could not reach the goal after doing your best, you can accept the situation.  But if you would fail without doing your best, you would regret the results.  We can only have a few chances in life.