Spontaneous regression͞complete resistance (SR͞CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR͞CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR͞CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites. cellular cancer immunity ͉ adoptive transfer ͉ cancer therapy ͉ macrophages ͉ leukocyte depletion

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We have established and studied a colony of mice with a unique trait of host resistance to both ascites and solid cancers induced by transplantable cells. One dramatic manifestation of this trait is age-dependent spontaneous regression of advanced cancers. This powerful resistance segregates as a single-locus dominant trait, is independent of tumor burden, and is effective against cell lines from multiple types of cancer. During spontaneous regression or immediately after exposure, cancer cells provoke a massive infiltration of host leukocytes, which form aggregates and rosettes with tumor cells. The cytolytic destruction of cancer cells by innate leukocytes is rapid and specific without apparent damage to normal cells. The mice are healthy and cancer-free and have a normal life span. These observations suggest a previously unrecognized mechanism of immune surveillance, which may have potential for therapy or prevention of cancer.

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d 9 -choline containing three deuterium atoms on each of the three methyl groups, d 3 -PC and d 6 -PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d 9 -choline. The synthesis of d 3 -PC and d 6 -PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.In hepatocytes, PC 1 is synthesized via two pathways. The CDP-choline pathway is catalyzed sequentially by three enzymatic activities present in all mammalian cells: choline kinase, CTP:phosphocholine cytidylyltransferase, and cholinephosphotransferase (1, 2). The PE methylation pathway is catalyzed by PEMT1 and PEMT2, two distinct hepatic PE methyltransferases that catalyze identical reactions but differ in structure and cellular localization (3). The CDP-choline pathway uses exogenous choline as the initial substrate and generates a pool of PC that is comprised primarily of short chain (16/18 carbons and 18/18 carbons), saturated and monounsaturated fatty acids (4). The PE methylation pathway on the other hand synthesizes a pool of PC predominantly with long chain (18/20 carbons and 18/22 carbons), polyunsaturated fatty acids (4). The two pathways also have opposite roles in liver growth and hepatocyte proliferation. Liver growth and liver carcinogenesis are associated with activation of the CDP-choline pathway (5, 6) and inactivation of the PE methylation pathway (5-7). Increased activity of PE methylation specifically and quantitatively inhibits cellular division of hepatoma cells (8). Inactivation of PEMT via gene knockout is not lethal when choline is present in the diet, but is lethal when choline is absent from the diet (9). During long-term choline deficiency, rat liver PEMT...

BackgroundSpontaneous regression/complete resistance (SR/CR) mice are a unique colony of mice that possess an inheritable, natural cancer resistance mediated primarily by innate cellular immunity. This resistance is effective against sarcoma 180 (S180) at exceptionally high doses and these mice remain healthy.MethodsIn this study, we challenged SR/CR mice with additional lethal transplantable mouse cancer cell lines to determine their resistance spectrum. The ability of these transplantable cancer cell lines to induce leukocyte infiltration was quantified and the percentage of different populations of responding immune cells was determined using flow cytometry.ResultsIn comparison to wild type (WT) mice, SR/CR mice showed significantly higher resistance to all cancer cell lines tested. However, SR/CR mice were more sensitive to MethA sarcoma (MethA), B16 melanoma (B16), LL/2 lung carcinoma (LL/2) and J774 lymphoma (J774) than to sarcoma 180 (S180) and EL-4 lymphoma (EL-4). Further mechanistic studies revealed that this lower resistance to MethA and LL/2 was due to the inability of these cancer cells to attract SR/CR leukocytes, leading to tumor cell escape from resistance mechanism. This escape mechanism was overcome by co-injection with S180, which could attract SR/CR leukocytes allowing the mice to resist higher doses of MethA and LL/2. S180-induced cell-free ascites fluid (CFAF) co-injection recapitulated the results obtained with live S180 cells, suggesting that this chemoattraction by cancer cells is mediated by diffusible molecules. We also tested for the first time whether SR/CR mice were able to resist additional cancer cell lines prior to S180 exposure. We found that SR/CR mice had an innate resistance against EL-4 and J774.ConclusionsOur results suggest that the cancer resistance in SR/CR mice is based on at least two separate processes: leukocyte migration/infiltration to the site of cancer cells and recognition of common surface properties on cancer cells. The infiltration of SR/CR leukocytes was based on both the innate ability of leukocytes to respond to chemotactic signals produced by cancer cells and on whether cancer cells produced these chemotactic signals. We found that some cancer cells could escape from SR/CR resistance because they did not induce infiltration of SR/CR leukocytes. However, if infiltration of leukocytes was induced by co-injection with chemotactic factors, these same cancer cells could be effectively recognized and killed by SR/CR leukocytes.