Review Series to Celebrate Our 100th Volume
Studies on “Ascites sarcoma”
2024 年 100 巻 6 号 p. 309-319
詳細
2024 年 100 巻 6 号 p. 309-319
This review seeks to highlight and celebrate Professor Tomizo Yoshida’s famous work on “Establishment and characterization of a rat ascites sarcoma, later named “Yoshida ascites sarcoma”. Considering the tremendous contribution of this ascites tumor system to the subsequent promotion of research on cancer biology and cancer chemotherapy, his paper should be regarded as a monumental one in the cancer field. The research was carried out during 1943 and the results were submitted to this Journal in October 1944, when Japan was approaching a debilitating defeat in World War II in August 1945. In 1947, when “Research on Ascites sarcoma” was first comprehensively introduced to researchers in a special lecture at the Annual Meeting of the Japanese Society of Pathology, the whole audience was deeply impressed and was encouraged to resume scientific activity in Japan.
It was on a summer day in 1943, in the pathology laboratory of Professor Tomizo Yoshida (Fig. 1) at Nagasaki Medical Collage when a young researcher, Yoshio Muta, noticed a tumor in the surrounding tissue of the right testis of an albino rat that had been kept for a hepatocarcinogenesis experiment for 6 months (initial 3 months of feeding of o-aminoazotoluol followed by 3 months of skin painting with arsenic acid). After a month, the rat was sacrificed before the impending rupture of the tumor. Autopsy revealed an elastic, soft tumor occupying the tissue surrounding the bilateral testis, the soft part of the small pelvic cavity, and the mesentery. There was about 2 ml of milky ascetic fluid containing numerous tumor cells in the peritoneal cavity. The fluid was then transferred to the peritoneal cavity of two rats. One of the rats died of infection the next day but the other lived and developed ascites with tumor cells in 20 days. The second inoculation was performed in 8 rats resulting in 5 of them developing ascites. The third inoculation was done 18 days later resulting in complete success.
Professor Tomizo Yoshida.
Yoshida was convinced that a long-awaited ascetic tumor system had now been established. When he returned home that day, he told his family excitedly, “This is a very important discovery. This will be extremely useful for various kinds of research.”
By the time of submitting the paper 1 year after the discovery of the ascites tumor, 33 generations of transplantation(in 359 animals) had been performed, with an average success rate of 98%.
Inoculation of cell-free ascites fluid never produced ascites tumors, thus excluding the possibility of any virus participation.
At the end of World War II, Japan was suffering from a serious shortage of food. The government distributed wheat flour, potato, and pumpkin but no rice. Rice was available only on the black market. Since Yoshida had a law-abiding spirit, he did not buy rice and he and his family endured hunger. However, he dared to buy rice on the black market to feed his rats. In June 1945, Yoshida moved to Tohoku Imperial University in Sendai with his family and his rats. On August 9, 1945, Nagasaki was destroyed by the atomic bomb attack, and Yoshida’s former laboratory was completely destroyed and his successor as professor at the university and his family were killed at the house where the Yoshida family had lived.
When Yoshida visited Nagasaki in 1946, he found human bones piled up in the garden of his former house. He knew that there would have been no doubt that he would have died had he been there on the day of the attack, and he decided to devote his remaining life for the benefit of people.
By the day of his success, the Yoshida’s laboratory had been making efforts for 3 years in attempts to produce ascites tumors by inoculating transplantable tumors and primary hepatocellular carcinomas into the peritoneal cavity of rats, but all had failed.
One of his motivations to obtain an ascites form of cancer came from his philosophy or idea that cancer cells, in contrast to normal cells, may be able to live without stroma, ultimately in fluid, thus revealing their essential character.
His philosophy is described in the first paragraph of the original paper: ”From our past research on the stroma of malignant tumors, especially transplantable tumors, it is proven that in transplanted tumor tissue, no closed circulation system, histologically defined as blood vessels for nutrition supply, is observed. In sarcoma tissue, for example, the blood flows through irregular net-forming tissue spaces of tumors in which the blood directly contacts with tumor cells. Some of such spaces, by the effect of a continuous blood stream, show remarkable dilatation of the lumen together with thickening of the wall by increased ground substance and may look like small vessels. However, the cells lining the lumen are of course flattened tumor cells, not true endothelial cells. Furthermore, they have no muscle layer. In transplanted carcinomas, carcinoma cells, especially at the high stage of the growth, float in blood-lake (mainly plasma) so that through incompletely structured loose capillaries they flow out. From this finding, it is clear that in transplanted tumors the normal parenchyma-stroma setting is not absolutely necessary for the life of the tumor cells. Far from it, they live and thrive without a solid tissue arrangement. It can therefore be easily stated that it is an essential character of tumor cells; they are floating in nutritional fluid as their essential life style, i.e. structure-less fluid tumor.” He further considered that usual tumors grown in nodular form may artificially be changed into fluid form tumor under special condition.”
Another of Yoshida’s motivations to develop an ascetic tumor system was to provide an effective model for cancer drug screening. Although nothing is mentioned about this in this paper, at the special lecture held at the Annual Meeting of the Japanese Society of Pathology in 1947, he clearly mentioned this possibility when presenting early data on screening 47 chemicals. When his lecture finished, the whole audience was deeply impressed and excited to know that such original and stimulating research had been carried out in the difficult days during the war. The proposal of the meeting president, Ryojun Kinoshita, to call this ascites tumor “Yoshida ascites sarcoma” was met with thunderous applause.
Yoshida’s description in the original paper was as follows: “The size of cells was 25-35 \(\mu\) in average, occasionally reaching over 45 \(\mu\) . Oval to kidney-shaped notched nucleus located eccentrically. By Giemsa-staining, abundant cytoplasm stained sky-blue with smoked nuance. As a specific feature of the tumor cells in Giemsa staining, so-called “Azur-Rosett”, which are comprised of centrally assembled red-violet azurophil granules, should be emphasized. The granules in general were arranged radially (refer to the arranged pictures). Janus-green-granules were scattered diffusely throughout the cytoplasm outside of neutral-red rosettes.The Oxydase-reaction was moderately positive. Phagocytic activity for Carmin or Indian ink was not clearly observed. The character of the tumor cells in the ascetic fluid reminded morphologically generally-named monocyte. However, the hemogram of the tumor-bearing animal was non-specific. Leukemic change was never observed. Even the infusion of ascetic fluid into the vessels did not induce abnormal hemogram, but did develop large tumors in the kidney or pericardium in association with ascites or simultaneous tumor-filled hydrothorax.
The histological feature of the infiltrated tumor tissue corresponded so-called round-cell sarcoma, in some aspects reminiscent of reticulum-cell sarcoma. However, as to the silver-fiber production, it fluctuated remarkably according to the site of invasion.”
In those days, Amano’s monocyte-origin theory of leukemia and lymphoma and Akazaki’s reticulum-cell origin theory of malignant lymphoma were popular in Japan. Yoshida may have simply paid respect to the then fashionable tumor terminology of his colleagues in his description of tumor histology.
Because the original tumor was found in the peritoneal cavity of a rat during experimental hepatocarcinogenesis, speculation remained that Yoshida ascites sarcoma may have originated from a hepatocellular carcinoma, although there was no description of primary hepatic tumors at autopsy. Later in 1960, a report on the production of AFP (a marker of hepatocellular carcinoma) by Yoshida ascites sarcoma and another stating that during transplantation generations, the sarcoma cells began to present epithelial features including multicellular nests came out, and the Yoshida ascites sarcoma began to be considered to be a strain of ascites tumor of hepatocellular carcinoma origin and put together with 60 other ascites hepatocellular carcinomas that were produced by Yoshida’s group during the 1950s.4)
From experience in experimental hepatocarcinogenesis and general pathology, it is unlikely that a macroscopically undetected small hepatocellular carcinoma (if there was one) induced by short-term feeding (or low total dose) of carcinogens caused widespread metastasis in the retroperitoneum. Hepatocellular carcinomas induced by low-dose carcinogens require more than 1 year to develop and are all of well-differentiated types that do not cause metastasis.7) On the other hand, if the tumor was a malignant lymphoma, the pattern of tumor distribution seen at the autopsy would be quite natural.
In 1994 Okio Hino’s group made a precise genetic and immunohistochemical analysis of Yoshida ascites sarcoma cells and clarified its T cell nature: cytotoxic T lymphocyte-associated gene transcript CTLA( \(+\) ), rearrangement of T-cell receptor \(\beta\) and \(\gamma\) chain genes( \(+\) ), CD2( \(+\) ), CD3( \(+\) ), AFP( \(-\) ).8)
The above-mentioned reports suggesting the hepatocellular origin of Yoshida ascites sarcoma can now be considered to be derived from experimental errors. The astonishing story of HeLa cell contamination in many culture lines around the world ( \(>18\%\) ) is well known.9)
Yoshida, his group, and many other researchers performed extensive observations and experiments to clarify the characteristics of over 60 ascites tumors including Yoshida ascites sarcoma. Initially, they were expecting to find specific feature(s) common to all the ascites tumors and essential markers of malignancy. However, what they found were quite unexpected, extensive differences among individual tumors, even ones derived from the same animal treated with a carcinogen. They were different in terms of:
The heterogeneity of tumors is well known by pathologists in terms of morphology and by physicians through clinical observations. However, this was the first example where the heterogeneity or individuality of tumors was clarified at the cellular level among broad biological aspects.
During drug screening experiments for chemotherapy described in the following session, Yoshida noted that in every positive case there remained a small fraction of viable cells, although most were dying, and he considered that a tumor might consist of heterogeneous cells from in the early phase of development. This idea was confirmed much later by the discovery of cancer stem-cells or the recent finding of cancer cell individuality by single cell analysis.
Yoshida considered that chemotherapy might be the final measure to win the fight against advanced cancer with metastasis, although there had not yet been any positive examples of successful chemotherapy. In fact, there had not been any extensive work to find effective drugs for chemotherapy. Yoshida invited and encouraged pharmaceutical researchers to collect as many synthetic compounds, antibiotics and plant extracts as possible. For screening, he proposed to utilize Yoshida ascites sarcoma, because ascites tumor systems may be more beneficial than using animals with transplanted tumors for screening. A large number of rats with ascites tumors can easily be provided, and the effects of drugs can be readily assessed by aspirating ascetic fluid and studying cytological changes in the tumor cells. It is possible to follow the effects repeatedly over a short interval, and such systems are neither expensive nor require complex instruments to carry out the research. It should be noted that cell-culture systems were not available then.
Many researchers collaborated with Yoshida and produced a wave of cancer chemotherapy research in Japan that influenced research around the world.
Among promising drugs found in 1950s were nitromin, sarcomycin, carcinophilin, mitomycin C, chromomycin A3, and colchicine.
Yoshida acted as the organizer of the 9th UICC World Cancer Congress held in Tokyo in 1966, which became the largest international congress in the world since the war, with 4,000 participants (3,000 from outside Japan). It was the year when the Nobel Prize for Medicine or Physiology went to Charles Brenton Huggins for his success in the hormonal treatment of prostate cancer, which was the first time this award was given to a researcher working in chemotherapy. The co-winner was Francis Peyton Rous for his discovery of Rous sarcoma virus.
In the 1950s and 1960s, the rates of cancer morbidity and mortality showed steep increases in Japan. The idea was born to create a major National Cancer Institute of Japan to promote cancer research. In a governmental advisory committee, however, Yoshida proposed to make an “Invisible National Cancer Institute” by creating a cancer research laboratory or unit in as many universities and research centers as possible in Japan. Yoshida was worried that a single national institute may gather the most prominent cancer researchers to it, and there would remain only a limited number of good cancer researchers at other sites in Japan, who were expected to attract and encourage young researchers to enter the cancer research field. Yoshida was also concerned that a “big national institute” might easily be spoiled by sectionalism and bureaucracy, which had often been the case in Japan.
Yoshida suggested to establish a comprehensive system for cancer research in Japan with a distinct large fund to be distributed without time limit. The proposal received the agreement of both scientific leaders and officials and became reality, although it was unusual and unprecedented for a governmental funding system in Japan. The Special Priority Grants for Cancer Research (Gann-Toku) was launched in 1941 and lasted for 30 years, receiving plaudits as a unique and effective system for cancer research both in Japan and from abroad.
The term “Tumor” indicates: 1) Mass in the body, including inflammatory swelling. 2) Neoplasm, both benign and malignant. Malignant neoplasms are classified into either carcinoma of epithelial cell origin or sarcoma of mesenchymal cell origin. Yoshida initially used the term “Tumor” in the meaning of malignant neoplasm, when the nature of the ascites tumor, carcinoma or sarcoma, was not clear. When he believed it was mesenchymal neoplasm, he called it ascites sarcoma.
Yoshida might be considering the term “Ascites tumor” is not appropriate because tumor cell-carrying ascites fluid is not a tumor, but he used it for convenience.
Edited by Tadao KAKIZOE, M.J.A
Correspondence should be addressed to: T. Kitagawa, The Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo 135-8550, Japan (e-mail: tkitagawa@jfcr.or.jp).
This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Yoshida, T., Muta, Y. and Sasaki, Z. (1944) Studien über das ``Ascites-Sarcom'' (I). Proc. Imp. Acad. 20 (8), 611-616 ( https://doi.org/10.2183/pjab1912.20.611).
[From Proc. Imp. Acad., Vol. 20 No. 8, pp. 611--616 (1944)]
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Tomoyuki Kitagawa was born in 1936 and graduated from the School of Medicine, the University of Tokyo in 1963. He obtained his Ph.D. in 1968 and became a Lecturer in the Department of Pathology, the University of Tokyo in 1968. He became a research associate of the Cancer Institute of Japanese Foundation for Cancer Research (JFCR) in 1970, Head of its Department of Pathology in 1978, Director of the Cancer Institute of JFCR in 1993, and its Director Emeritus from 2006 to present.
He received a UICC International Fellowship and worked in the McArdle Laboratory for Cancer Research in Wisconsin, USA, 1973-1975. He was awarded the Mataro Nagayo Prize by the Japanese Cancer Association in 2003.
He has been interested in experimental hepatocarcinogenesis, human pathology and cancer education, especially for school children.
