Promotion of tumor invasion by cooperation of granulocytes and macrophages activated by anti-tumor antibodies

Abstract

We investigated the potential role of anti-tumor antibodies and tumor antigens in the formation of immune complexes which promote matrix degradation and angiogenesis. B-cell deficient or B-cell depleted mice showed a reduction in tumor invasion and metastasis. In vitro invasion assays and in vivo models of metastasis showed that anti-sTn antibodies and sTn tumor antigens form complexes which induce granulocytes and macrophages together to mediate tumor invasion and metastasis by processes including extracellular matrix degradation and angiogenesis. These results suggest the existence of a tumor promoting role of a B-cell immune response induced by shed tumor associated antigens of solid, nonlymphoid tumors.

Figure 1

Reduction of metastasis in conditions of B-cell deficiency or depletion. (A) Reduced lung metastasis in µMT/µMT B-cell deficient mice. (B) Reduction of liver metastasis in C3H mice receiving B-cell depletion treatment immediately after tumor-cell injection. Unpaired t test has been used to compare the mean of each column. The two-sided P value is shown on the graph. The error bars represent the SEM.

Figure 2

Promotion of metastasis of tumor by tumor infiltrating B cells. (A) Increased liver metastasis of splenic implanted tumors in C3H mice after four injections (alternate days) of 10⁴ B cells (B-TIL) obtained from the lymphocyte infiltration of the same tumor type, compared with non treated mice, and mice receiving the same number of B cells obtained from the spleen of normal mice (SBL). (B) Increased number of lung metastases obtained by tail vein injection of 10⁵ tumor cells with or without B-TIL in immune competent C3H mice. (C) The same experiment repeated with nu/nu C3H mice also shows increased metastasis when the tumor cells are injected together with B-TIL. Unpaired t test has been used to compare the mean of each column. The two-side P value is shown on the graph.

Figure 3

In vitro promotion of tumor invasion by stromal cells, TAA and anti-TAA Ab. Each individual migration rate is expressed as the number of migrated tumor cells in each field (data) divided by the average number of migrated tumor cells of the control combination (CTRL) (data/CTRL). Therefore, the CTRL mean appears as 1.0±SEM. (A) Stromal cells alone inhibit tumor-cell migration. However, a MAb binding sTn TAA in the presence of stromal cells increases tumor-cell migration. An excess of Ig of the same isotype, but nonspecific for tumor antigens (neutral Ab) inhibits the increase of tumor-cell migration induced by anti-sTn TAA MAb. Finally, MAb binding sTn TAA secreted by tumor cells, without stromal cells, does not change the natural ability of tumor cells to migrate. The mean of each column has been compared by unpaired t-test. (B) Comparative effects on tumor-cell migration of increasing amounts of the specific anti-tumor antigen MAb, the Fab segment of the same MAb and a neutral IgG of the same isotype. (C) Comparative effects on tumor-cell migration of the presence of anti-TAA Ab (anti-sTn-MAb). Each of azide, PMN, or MO, alone did not enhance tumor-cell migration in the Matrigel assay. The simultaneous addition of both PMN and MO increases tumor-cell migration. Peroxidase inhibition (azide), mannose receptor blocking (mannan) or elastase inhibition (MeOSAAPV-CMK) constrains the increase of tumor-cell migration induced by the combination of PMN, MO and anti-sTn TAA MAb.

Figure 4

In vivo promotion of tumor growth by tumor antigens and anti-tumor antibodies. (A) One million SW620 human colon carcinoma cells shedding sTn TAA were injected: alone into eight mice (■); together with 5x 10⁴ P3X63Ag8 hybridoma cells producing B72.3 anti-sTn MAb into eight mice (▲); or together with 5x10⁴ P3X63Ag8 hybridoma cells producing the neutral 9D9 MAb into eight mice (▼). The survival curves represent the remaining fractions of mice with tumor smaller than 10 mm, following the method of Kaplan and Meier. Error bars show the 95% confidence interval (CI) for fractions at any particular time. (B) Representation of the individual tumor weight reached in 21 days of tumor progression in the same experimental groups. The horizontal bar represents the average. The two-tailed P value showed statistically significant differences between the media (control vs. B72.3 P<0.05; control vs. 9D9 P<0.01). (C) Average number of vessels per mm² of tumor tissue section, identified by immune histochemistry using fluorescein conjugated anti-CD31 MAb. The t-test showed significant differences between the mean of each group.