It has been accepted that the immune system undergoes changes in its ability to respond to immune challenge both with in the presence of tumor. The clinical observations of increased infection rates and wound healing complications in cancer-bearing patients are often attributed to this immune system defect. Some reports have documented T-cell functional defects in both tumor-bearing mice, and in cancer patients. However, the exact nature of and reasons for such defects and alterations of the immune system have not been fully elucidated. The purpose of this study was to quantify specifically the effect of tumor burden upon stem cell mobilization and immune system response. Female BALB/c mice were treated with 4T1 breast cancer cells (1 x 10 to the 5th) injected to the mammary fat pad. Control mice received no such injections. Animals were matched for age and tumor-bearing interval. At the time of sacrifice peripheral blood and spleens were collected and from which low density mononuclear cells (LD MNCs) were separated via density gradient centrifugation and utilized immediately. Assays performed included colony forming unit count, INF-g output, mitogenic response and cytotoxicity. Serum from animals was further analyzed for levels of several growth factors and cytokines including TGF-b (both bound and unbound), G-CSF and Flt-3 ligand using standard sandwich ELISA techniques. Finally, blood and splenic LD MNCs were tagged with fluorescent markers and percentage distributions determined via FACS analysis. All studies were performed in triplicate. Breast cancer bearing mice showed progressive increase in their splenic to body weight ratios with lengthening tumor-bearing interval (0.022 vs 0.0048). The corresponding increase in splenic weight and size paralleled the increased numbers of LD MNCs recovered form each organ (34 x 106 cells/spleen vs 580 x 106 cells/spleen). Output of INF-g, a general measure of cellular inflammatory activity, was markedly increased in LD MNCs from spleens of tumor-bearing animals in comparison to controls as measured via the ElispotŪ assay (78 plus/minus 16 vs 13 plus/minus 3). Mitogenic response was markedly increased in cells from tumor-bearing animals as compared to controls (0.88 vs 1.78, normal vs tumor-bearing). This effect was found in cells both from the blood and spleen. Cell killing activity (cytotoxicity) against 4T1 cancer cells was likewise increased as a function of tumor-bearing interval (1.83% vs 36.4% for normal and 3 week tumor-bearing interval at a 40:1 effector to target cell ratio). As a measure of hematopoietic stem cell mobilization, clonogenic assays demonstrated significant increases in CFU-C per cells plated and total CFU-C in splenic LD MNCs from tumor-bearing animals. FACS analysis of these same cells found a decrease in all cell lines except for Gr-1+ cells. Concerning output of growth factors and other regulatory cytokines, levels of biologically active TBF-b increased with lengthening of tumor bearing interval (2 vs 15 ng/ml). This was in contrast to bound levels of TBF-b, which decreased with increasing tumor-bearing interval (360 vs 75 ng/ml). Finally, G-CSF was elevated as a function of tumor-bearing interval (721 vs 35.8 pg/ml) as was Ftl-3 ligand (364 vs 294 pg/ml). Analysis of immune response and stem cell mobilization of splenic LD MNCs in breast cancer bearing mice of various ages demonstrated consistent increase in immune response by several standard measures. Concurrent with this general increase in immune system response was increased mobilization of stem cells to the spleen. These changes were mirrored by elevated output from splenic LD MNCs of several cytokines including TBF-b, G-CSF and Flt-3 ligand. These changes increased with lengthening of the tumor-bearing interval. These results suggest that in terms of in-vitro immune activity and capacitance splenic LD MNCs of tumor-bearing mice are competent, a situation that does not decrease with increasing tumor-bearing interval. These immune responses occurred conurrent with progressive stem cell mobilization to the spleen in tumor-bearing animals. Nonetheless, the results of these experiments suggest that immune system deficiency is not the reason for increased wound healing and infectious complications in post-surgical cancer patients.

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