The discovery of angiogenesis inhibitors: A new class of drugs: Transcript: Part 1
Judah Folkman:
Thank you Dr. Speck for that very elegant introduction. I greatly appreciate the invitation to participate in this prestigious Friday Evening Lecture Series. For any talk after dinner, I follow my wife's advice. She says, "No matter how short your talk is after you have it all written out, cut it by one-third." Her entreaty reminds me of the story of the great Russian author, Dostoyevsky, who received the following letter from his publisher: "Thank you for the draft of your new book. We think it will become a great classic, but it is much too long. We cannot publish it unless you cut it by one-third. Therefore we are returning your manuscript entitled, Crime, Punishment and Redemption" (laughter). So this will be a truncated talk on a very large field--angiogenesis research, now moving at 35-40 publications a week. I will try to address just one of the central questions which preoccupies our laboratory group and colleagues in other labs. How do tumors switch on the angiogenic phenotype?
What is Angiogenesis?:
But first I need to give you a brief background about what angiogenesis is and to tell you that capillary blood vessels, thinner than a hair, supply every cell in the body. Ramzi Cotran (former Chairman of Pathology at the Brigham and Women's Hospital, now deceased) told me that from confocal microscopy it had been determined that a pound of fat contains approximately one mile of capillary tubing. And it is most likely the reason that if you have high blood pressure and your physician says to please lose a few pounds, you lose a few miles of tubing, which require less eventual head of pressure.
These tubes are lined by cells called endothelial cells. However, they rarely divide, in contrast to bone marrow, for example. In the bone marrow, 6 billion cells divide every hour, per person. So that the whole bone marrow is replaced every 5 days, approximately. That is called the turnover time. The replacement or turnover time for endothelium is 3-5 years in most tissues, and 10 years in the eye vessels. However, in the neighborhood of a tumor, endothelial cells are so stimulated that they can divide rapidly with a turnover time similar to bone marrow. Once a tumor has recruited its own private blood supply, tumor cells can enter the circulation and can form metastases. Metastasis means "changing places." When tumor cells arrive at a remote site, lung or brain, the whole angiogenic process begins again.
The Angiogenic Process:
The process of angiogenesis is now very well recognized as a powerful control point in tumor growth. The hypothesis that tumors are angiogenic, which as Dr. Speck said, we first proposed in 1971, has now been confirmed by genetic methods and has stimulated research in many laboratories. As a result, angiogenesis inhibitors, which stop blood vessel growth, have now emerged as a new class of drugs. These drugs are either selective or specific inhibitors of proliferating or migrating microvascular endothelial cells. And this is the rationale for the proposal that treating both the cancer cell and the endothelial cell in a tumor may be more effective than treating the cancer cell alone, which has been the conventional strategy for the past 50 years, and still is. Furthermore, about 20 different angiogenesis inhibitors are in clinical trials in more than 100 medical centers in the U.S., plus others in U.K. and Europe.
Presentation of Slides:
1. Sheep's heart
2. Microvasculature of sheep's heart
2. Microvasculature of sheep's heart
4. Comparative microvessel packing densities
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What does this process look like? Here is the microvasculature of a sheep heart. The magnification bar is 200 microns (two-tenths of a millimeter). These microvessels are densely packed in the heart muscle. The microvessel density is similar in most other organs. In this slide, magnification is increased so that the bar is now 10 microns. Each cardiac muscle cell is contiguous to a capillary blood vessel. There is little room for additional cells to obtain nutrients and oxygen from the same capillary vessel. So if tumor cells arise as a primary tumor in a tissue or an organ, they may form a tiny colony of tumor cells of approximately 0.5 mm diameter or less. But until they can become angiogenic and recruit their own new capillary blood vessels, they will remain as an in situ cancer. In this slide, showing comparative packing densities of microvessels in the hearts of different species, the human heart contains 2500 mm of capillary blood vessel length per cubic millimeter of tissue. |
5. Schematic of human breast
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In the human breast there are 8,000 to 10,000 small ducts. They are less than a pencil tip in size and they drain to 7 to 9 major ducts, which open at the nipple. They are lined by two cell layers. The cell layer facing the lumen rarely divides. When the cells in this layer begin to divide together with the cells beneath--and they fill up this hollow lumen, that is called "in situ" cancer. The right side of this diagram shows a large vascularized tumor of approximately 1 cm drawn to scale. |
6. Histological section-breast tissue
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It is possible to actually see in situ carcinomas before they have become vascularized by examining biopsies of breast tissue. This histological section is from Noel Weidner in a paper that we wrote in 1991. You see the normal lining of one of those ducts. And here is an early in situ carcinoma. The new blood vessels converging toward the carcinoma are highlighted by an antibody, which specifically identifies blood vessels (antibody to von Willebrand factor--orange-brown stain in this slide). |
7. In situ breast carcinoma
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In this second patient, the in situ breast carcinoma has filled the duct. A cluster of new blood vessels has invaded into the duct. This is thought to be facilitated by enzymes (metalloproteinases) produced by proliferating or migrating endothelial cells. The tumor cells now begin to escape from the duct along the path of the invading blood vessels. |
8. Invasive breast cancer
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In this slide the tumor has now invaded tissue outside the duct and has grown rapidly. The vessels are highlighted, and around each one the tumor cells form microcylinders. |
9. Breast cancer cells; vessels
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In the next slide this is shown at higher power (the tissue section is 4 microns thick). The tumor cells are seen in sharp detail, but the capillary blood vessels would be difficult to see without this special stain (antibody to von Willebrand factor). Then 4 years ago, Usha Tedrow, a Harvard medical student, working with Rick Rodgers of the Harvard School of Public Health, took the sections to a thicker size--50 microns--did confocal microscopy, and immediately saw that the microvessels in each were clear cut, and each was surrounded by several layers of tumor cells. Recently, Donald McDonald, a professor of anatomy at the University of California, San Francisco, took breast cancer cells and implanted them under the skin of mice. After a tumor grew, the mice were euthanized and the vessels were perfused with fixative to keep them open.
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11. Breast cancer-higher magnification
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Here at higher magnification than in the previous slides you can see the perivascular cuffs of tumor cells around each capillary blood vessel. The thickness of each cuff (its radius) is approximately 100 to 120 microns, which is the oxygen diffusion limit for most tumors. This was shown by Rakesh Jain at the Massachusetts General Hospital by infrared spectroscopy in transparent skin windows in experimental animals. Other tumors, once they have become angiogenic, look similar at this magnification, with only small variations in the intercapillary distances. New vascular sprouts are growing from the vessels (right panel). Each endothelial cell supports approximately 50 to 100 tumor cells. |
12. electron micrographs of tumor cells surrounding capillary
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In a paper published in the December 2000 issue of Proceedings of the National Academy of Sciences, McDonald and Jain showed that the tumor cells are able to slowly cross into the vessel lumen. This trip averages about 24 hours for each tumor cell. Approximately 1 million tumor cells can enter the circulation per gram of tumor per day (they studied human colon cancer in mice). At any time, 4% of the vessel wall is made up of tumor cells traversing the vessel wall into the circulation. They are called mosaic vessels. Approximately 15% of vessels in a tumor are invaded by tumor cells at any given time. This slide is from an invited commentary on the McDonald/Jain paper that I wrote in the January 2001 issue of Proceedings of the National Academy of Sciences. I overlaid electron micrographs of tumor cells in this diagram so that they can be visualized surrounding the capillary blood vessel. |
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So this diagram summarizes that tumor cells recruit new vessels and grow around them, and thus can continually increase the mass of the tumor. These new vessels solve the oxygen diffusion problem for the growing tumor and also serve as conduits for delivery of nutrients and for the removal of waste metabolites. The endothelial cells also produce paracrine growth factors and survival factors, which can stimulate growth of tumor cells. Rak and Kerbel, as shown here, reported that at least 20 such factors including basic fibroblast growth factor, heparin binding epithelial growth factor, insulin-like growth factor, interleukin-6, and others generated by the endothelial cells in these vessels also contribute to tumor cell growth.
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14. Angiogenic vs non-angiogenic tumor cells
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In this second summary slide, tumor cells start out inherently non-angiogenic. Only a subpopulation of the tumor cells need to become angiogenic to supply the whole tumor with enough blood vessels for it to grow. We have learned that many human and animal tumors, once they are vascularized and growing, contain two populations of tumor cells, angiogenic and non-angiogenic. Both cell types may enter the circulation, but the non-angiogenic tumor cells appear to be responsible for dormant microscopic metastases while the angiogenic tumor cells can become rapidly growing metastases. This is based on recent experimental work in mice. |
15. Angiogenic vs non-angiogenic tumor cells-references |
If you teach medical students, you may have had the experience that in the front row sit the supercompulsive, straight A, type A summas, who often interrupt the lecture to ask, "What is the reference for that?" "How do you know that?" "Do we have to know that on the exam?" So I carry an occasional slide like this one, which has all of the references to support what I have told you so far. But, it is a crowded slide that can only be read by the first row, because beyond the first row no one seems to care about all of these references. |
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