Figure 1.
EL-4 tumors grew in both SR/CR cancer resistant mice and C57BL/6 cancer susceptible mice.
No immune cell infiltration was observed in EL-4 tumor tissues from SR/CR cancer resistant mice (A) or C57BL/6 cancer susceptible mice (B) transplanted with EL-4 tumor pieces two weeks earlier (representative sections, n = 8). EL-4 tumor tissue contained mitotic figures and virtually no infiltration of immune cells (HE stain, bar 100 µm).
Figure 2.
Cancer growth susceptibility and resistance of SR/CR mice and nonresistant controls.
The SR/CR mice resisted the s.c. injection of S180 cells, whereas the C57BL/6 mice developed tumors 8–30 days after s.c. injection of S180 cells which was at a significantly higher frequency (p<0.0001). SR/CR and C57BL/6 mice both developed tumors 8–14 days after the transplantation or s.c. injection of EL4 cells. SR/CR mice were inoculated i.p. with 8×105 J774A.1 cells and ascites developed 34–40 days later, and this resistance was significantly lower (p<0.0081) compared to the resistance of SR/CR mice to S180 cells.
Figure 3.
Differences in tumor regression in S180-cancer susceptible C57BL/6 mice after the AT of leukocytes from either cancer resistant SR/CR mice or non-resistant controls.
(A) Survival of S180-cancer susceptible C57BL/6 mice after AT. Two mice that received SR/CR-leukocytes and four mice that received C57BL/6 leukocytes were euthanized due to excessive S180 tumor burdens. (B) Tumor volumes after AT. Progressive tumor growth was observed in untreated C57BL/6 mice after the bilateral s.c. injection of 106 S180 cells in the axillary region. The growth reached a plateau approximately 20 days post tumor cell inoculation. One mouse was euthanized when one of its tumors exceeded 12 mm in diameter. The vertical line at 40 days represents the time point when mice with completely regressed tumors were injected i.p. with S180 cells. A one-way ANOVA analysis revealed a significant difference (p = 0.0005) in tumor size between the two groups of mice with either efficient or inefficient AT. Each point represents the mean and SEM. (C) The numbers and the composition of the transferred SR/CR and C57BL/6 (B6) derived leukocytes. There were no significant differences in the numbers of total leukocytes (splenocytes + peritoneal leukocytes) transferred from the SR/CR and C57BL/6 mice (p = 0.969). The numbers of peritoneal immune cells derived from the SR/CR and C57BL/6 mice were not significantly different (p = 0.721). T = total leukocytes. Per = peritoneal leukocytes.
Figure 4.
Histology of tumor tissues from mice with or without AT.
(A) Fourteen days after inoculation, the tumor tissue was dense with mitotic figures, and no infiltration of immune cells was observed (HE stain, bar 20 µm). (B) A section of tumor tissue (TT) that reached the plateau phase of growth 22 days after inoculation. A large area of necrosis (N) was visible, but no infiltrating immune cells were present (HE stain, bar 20 µm). (C) Massive infiltration of PMNs in S180 tumors after AT of cancer-resistant SR/CR leukocytes to cancer susceptible C57BL/6 mice (representative sections, n = 8). Tumor tissue (TT) from a mouse with tumor regrowth showed large areas of infiltrating PMNs (PMN) and necrosis (N) (HE stain, bar 100 µm). (D) Tumor tissue (TT) from a mouse with tumor regrowth revealed the infiltration of PMNs (PMN) (HE stain, bar 20 µm). (E) The regression of tumor tissue (TT) in a cancer-susceptible C57BL/6 mouse after AT of SR/CR leukocytes. Large necrotic areas (N) in the tumor tissue were observed (HE stain, bar 100 µm). (F) An image of the same tumor as in C), showing a clear demarcation between the vital tumor tissue (TT) and the infiltrating PMNs (PMN), as well as large necrotic (N) areas (HE stain, bar 100 µm).
Figure 5.
Depletion of T- and B cells, demonstrated by FACS (representative plots, n = 6 in each group).
(A) Total leukocytes from a SR/CR mouse prior to depletion, stained for CD8+ (purple) and CD4+ (blue) cells. (B) After depletion, leukocytes were stained with the same antibodies as in A. No CD4+ or CD8+ cells were detected. (C) Total leukocytes from a SR/CR mouse prior to depletion, stained for CD19+ (y-axis) and CD45+ (x-axis) cells. (D) After depletion, leukocytes were stained with the same antibodies as in C. Two percent of the cells were double positive for CD45 and CD19.
Figure 6.
A significant decrease in the S180 tumor volume was observed after AT of SR/CR leukocytes compared to AT of leukocytes from nonresistant controls. Leucocytes were depleted of CD4+/CD8+ T cells and B cells (each point represents the mean and SEM, n = 6 in each group).
The tumors in the group that received an AT from nonresistant controls (n = 6) grew exponentially, whereas the tumors in the group that received an AT from SR/CR mice (n = 6) regressed 30–40 days after AT. The tumors in the nonresistant control group were significantly larger (p = 0.0001). The vertical line at 60 days represents the time point when the mice with regressed tumors were injected i.p with S180 cells.
Figure 7.
Massive infiltrations of PMNs and apoptotic areas in regressing tumor tissue 35 days after AT of SR/CR leukocytes depleted of CD4+/CD8+ T cells and B cells (representative sections, n = 6).
(A) Large necrotic areas with PMN infiltration (PMN) and clearly marked borders between vital and necrotic tumor tissues (TT) (HE stain, bar 100 µm). (B) The necrotic areas showed dense infiltration by PMNs (PMN) (myeloperoxidase staining, bar 100 µm). (C) CD3+ T cells were more sparsely distributed in the tumor tissue and were not found in areas with necrosis (N) (CD3 staining, 20x magnification). (D) PMNs (PMN) infiltrating the tumor tissue (TT) in trabecular formations (myeloperoxidase staining, bar 100 µm). (E) Many of the tumor cells were highly positive for caspase-3 (arrows) indicating apoptosis (caspase-3 staining, bar 20 µm). (F) The distribution pattern of the apoptotic cells had a trabecular formation, which resembled the distribution pattern of the PMNs in the tumor tissue in D (caspase-3 staining, bar 100 µm).
Figure 8.
Few infiltrating PMNs or apoptotic areas were observed in growing tumor tissue after AT of leukocytes from nonresistant controls depleted of CD4+/CD8+ T cells and B cells (representative sections, n = 6).
(A) Areas exhibiting the infiltration of PMNs (PMN) were observed within the vital tumor tissue (myeloperoxidase staining, bar 100 µm). (B) T cells were densely distributed in the vital tumor tissue (TT) (CD3+ staining, bar 100 µm). (C) Few apoptotic cells were present in the tumor tissue (TT) (caspase-3 staining, bar 100 µm). (D) Apoptotic tumor cells were distributed throughout the vital tumor tissue (TT) (caspase-3 staining, bar 20 µm).