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Spontaneous Regression of Cancer Phenomena is Linked to Specific Mechanisms Decreasing with Age
Posted on: September 5, 2003

Regression of human cancers without treatment (spontaneous regression, SR) is well documented for many types of cancer, but occurs infrequently. The most intriguing implication of SR is that there might be a rare, but extremely effective, mechanism engaged to eradicate cancer cells after the development of advanced malignancy. Despite efforts over many decades, the mechanism(s) of SR in humans and animals has remained elusive.

Because of the absence of MHC, mouse S180 cells form highly aggressive cancers in all strains of laboratory mice and rats. When injected into the peritoneal cavity, S180 cells grow exponentially with a generation time of 12-18 hours. Growing primarily in suspension in the peritoneal cavity, S180 cells gradually plug peritoneal lymphatic drainage, leading to accumulation of ascites fluid within 2 weeks. S180 cells can also metastasize into major organs near the peritoneal cavity, such as liver, kidney, pancreas, lung, stomach, and intestine. Mice that develop ascites normally die in 3-4 weeks. S180-induced ascites represents one of the most aggressive transplantable cancers in experimental mouse models. Resistance to S180-induced ascites has never been reported to our knowledge. Because of their consistent response to transplanted S180 cells, BALB/c mice have become a standard strain for ascites production.

An S180-resistant founder mouse was initially identified within a group of BALB/c mice as a result of its failure to develop ascites upon an injection of 5·105 S180 cells. To verify that this failure was true resistance, the founder mouse was given two more injections of 2·106 S180 cells, as were control BALB/c mice, followed by two further injections of 2·107 S180 cells. No ascites developed in the founder mouse. This unique mouse remained healthy and cancer free and eventually died of old age at 26 months of age. This resistance was independent of tumor burden in the range tested (up to 10% of total body weight) and independent of whether the S180 cells had been passaged in vivo or through tissue culture.

In contrast to complete resistance CR to S180-induced ascites, a portion of the S180-resistant mice displayed SR, dependent on the age at the first injection of S180 cells. After injection of S180 cells, SR mice developed ascites for the first 2 weeks, which rapidly disappeared in <24 hours. The mice then became healthy and immediately resumed normal activities, including mating. S180 cells in the regressed ascites were equivalent to 3 g of solid tumor mass or 3·109 of cells.

The mice that underwent regression remained ascites-free thereafter. The mice that had once undergone regression became completely protected from S180 cells and never developed ascites again in response to subsequent injections of S180 cells. The initial development of ascites suggested that the anticancer mechanism might not be engaged immediately in response to the implantation of cancer cells in older animals. After an initial period of latency, an anticancer mechanism was rapidly engaged in these mice, leading to destruction of S180 cells, clearance of peritoneal lymphatic drainage, and regression of ascites. The lasting protection against S180-induced ascites after initial regression suggests that the anticancer mechanism, after being engaged once, is primed for later engagement in response to subsequent exposures to S180 cells.

The manifestation of the CR or SR phenotypes was related to the age of mice at the time of the first injection with S180 cells. When the first injection was given at the age of 6 weeks, essentially all of the resistant mice display the CR phenotype. When the first injection of S180 cells was given at the age of 12 weeks, about 50% of the S180-resistant mice showed the SR phenotype and the other 50% showed the CR phenotype. When the first injection of S180 cells was given at the age of 22 weeks, the majority of the resistant mice showed SR. In a small number of mice tested at 56 weeks, however, the first injection of S180 cells resulted in ascites and death even in mice whose offspring were cancer resistant.

Host immune cells are infiltrated in rapid destruction of cancer cells. SR/CR mouse was capable of destroying up to 20 million S180 cells in the first 12 h. After the majority of S180 cells were destroyed, residual S180 cells could be occasionally detected in the first 48 h, but were completely absent thereafter. At day 7, S180 cells became the dominant cell population in the peritoneal cavity of control mice, but were not detected in the SR/CR mice. A day 4 cancer cells were completely eliminated in the SR/CR mice. In contrast, some leukocytes in the control mice showed apoptosis. Interestingly, 6-12 h after injection, as many as 1.6·108 leukocytes migrated into the peritoneal cavity in SR/CR mice in response to the presence of S180 cells, yet disappeared after cancer cells were destroyed. S180 cells from the SR/CR mice were surrounded by immune cells forming rosettes and larger cellular aggregates (Fig. 1). Additionally, many S180 cells in rosettes were ruptured, suggesting a primary cytolytic event. Apoptotic morphology was not observed in the injected S180 cells.

Fig. 1. Formation of rosettes between cancer cells and leukocytes in day 1 peritoneal samples of the SR/CR mice. Cytopreps of peritoneal samples fromWT or SR/CR mice injected with S180 cells on the previous day show prominent rosette formation around cancer cells (arrows) in the SR mouse, but not in the WT mice. Cancer cells from the WT mice (arrow) appeared intact and were often adjacent to apoptotic leukocytes (arrowheads), whereas the few remaining cancer cells in the SR/CR mice (arrow) often appear to have undergone cytolysis (arrowhead).

In the peritoneal samples of the SR/CR mice challenged with 2·107 S180 cells for 24 h, S180 cells displayed a variety of morphological changes, including swelling, flattening and simplification of microvilli, tight contact with leukocytes, and surface erosions consistent with membrane damage (Fig. 2).

Fig. 2. Scanning electron microscopic images of S180 cell-leukocyte interactions. Cells recovered from the peritoneum of a WT mouse show the normal surface morphology of S180 cells with extensive microvilli (A). Cells recovered from an SR/CR mouse after S180 injection show rosettes of leukocytes (arrows) surrounding a tumor cell (B) and ballooning, loss of microvilli, and membrane defects in tumor cells undergoing lysis (C and D).

The SR/CR mouse model represents a unique opportunity to examine cancer-host interactions. The killing of tumor cells primarily by cytolysis in SR/CR mice was extremely rapid and effective, yet was achieved with profound selectivity, with most normal cells being unharmed. The efficiency of this cell killing has a number of striking features. Once primed by the initial challenge of S180 cells, the SR/CR mice could withstand repeated daily challenge of >2·107 S180 cells and could also remain ascites free after a single challenge of up to 10% of body weight. Tumor cell killing was accompanied by a dramatic migration of leukocytes that form rosettes and aggregates with cancer cells. After cell contact, tumor cells undergo lysis. This cellular debris was then engulfed by peritoneal macrophages. The mice were then subsequently tumor free. Histological examination of tissues in SR/CR mice showed normal morphology. Although life span studies have not been completed, there was no sign of a shortened life span in SR/CR mice.

Several intriguing implications derive from the properties of the SR/CR mouse. First, this model demonstrates the existence of a host resistance gene that can prevent the growth of advanced, MHC-negative cancers. The existence of host cancer resistance genes has been postulated to be one explanation for the existence of individuals in the human population who fail to develop cancers, despite prolonged and intense exposure to carcinogens. The gene(s) responsible for the SR/CR phenotype may well be an example of such a resistance gene that might have a direct human ortholog. Second, the concept of immune surveillance has been debated for decades and has been difficult to prove, although recent studies have lent support to this concept. The SR/CR mouse may also provide a potential example of such a surveillance mechanism. Third, the alteration in the type of response seen with age in these mice suggests an intriguing possibility. The appearance of cancer in older individuals at a much higher frequency may not solely be caused by the accumulation of mutations in individual preneoplastic cells. This mouse model suggests that there may also be host resistance mechanisms that decline with age. Fourth, the rare phenomenon of SR of cancers has been documented in humans, but has been difficult to study because of a lack of an appropriate animal model. The SR/CR mouse may provide such a model and allow identification of the cellular and genetic machinery necessary to reject a fully developed malignancy. The ability of adoptively transferred infiltrating leukocytes from SR/CR mice to protect control mice from S180 cells may suggest a potentially feasible strategy for treatment of advanced cancers that could be translatable into human patients.

Source: Zheng Cui, Mark C. Willingham, Amy M. Hicks, Martha A. Alexander-Miller, Timothy D. Howard, Gregory A. Hawkins, Mark S. Miller, Holly M. Weir, Wei Du, and Cynthia J. DeLong; Spontaneous regression of advanced cancer: Identification of a unique genetically determined, age-dependent trait in mice. PNAS, May 27, 2003, vol.100, no.11, pp 6682-6687.
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