Macrophage-recognized molecules of apoptotic cells are expressed at higher levels in AKR lymphoma of aged as compared to young mice.

While a direct relation between aging and tumorigenesis is well established, a slower tumor progression rate was reported in old as compared to young cancer patients. The mechanisms responsible for the less aggressive behavior of tumors in the aged, are largely unknown. We have recently shown an increase in apoptotic cell death in tumors derived from aged as compared to young animals in the AKR lymphoma. This was shown by DNA flow cytometry and by the ladder type DNA fragmentation in agarose gel electrophoresis. Analysis of the expression of genes involved in apoptosis in tumors derived from young and old animals showed a lower bcl-2 expression in those from the aged. The Fas antigen, on the contrary, displayed higher expression levels on lymphoma cells derived from old than on those from young mice. Apoptotic cells are recognized and phagocytosed mainly by macrophages. One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues. While comparing the "eat me status" phenotype of the tumor cells derived from young and aged animals, by the use of lectins recognizing sialic acid and galactose residues, FACS analysis showed a decrease in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis showed that a 130 Kda sialylated membrane glycoprotein was expressed at a lower level in tumors from the old as compared to young mice. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested, one of the mechanisms of the age-related decrease in tumor progression rate.

Sensitivity to macrophages decreases with tumor progression in the AKR lymphoma.

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-1), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified 3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, inMT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found moreover that WGA-treated tumor cells were more sensitive than nontreated cells to macrophages in vitro.