Regional control of tumor growth

Abstract

Tumors implanted near the scapulae have been shown to grow four times faster than the same tumors implanted at the iliac crest. Although there were marked differences in the vascularization of tumors from these two different sites, the mechanism controlling regional angiogenesis was not identified. Here, we show site-specific growth of intraperitoneal tumor implants in the mouse abdomen. Our data indicate that the angiogenic response of the host differs significantly between the upper and lower sites in the mouse abdomen and reveal that the expansion of tumor mass is restricted to sites with low angiogenic responses, such as the bowel mesentery in the lower abdomen. We show that, in this model, this suppression of angiogenesis is due to an expression gradient of thrombospondin-1 (TSP-1), a potent endogenous angiogenesis inhibitor. Mice with a targeted deletion of TSP-1 no longer show regional restriction of tumor growth. The physiologic relevance of these findings may be seen in patients with peritoneal carcinomatosis, whereby tumors spread within the peritoneal cavity and show differential growth in the upper and lower abdomen. We hypothesize that the difference in tumor growth in these patients may be due to a gradient of TSP-1 expression in stroma. Finally, our studies suggest that upregulation of TSP-1 in tumor cells is one method to suppress the growth of tumors in the upper abdomen.

Figure 1

Inoculation of CT26 tumor cells into the peritoneal cavity leads to differential tumor growth in the upper versus lower abdomen. A. Upper panel: CT26 carcinoma cells form large, angiogenic tumors in the upper region of the peritoneal cavity and small, non-angiogenic tumors on the mesentery in the lower abdomen. Arrows point to large angiogenic tumors located on or below the stomach in upper panel, and to small non-angiogenic tumors in the lower abdomen (n=10 mice per group). B. The size and weight of CT26 tumors isolated from the upper abdomen are significantly larger than tumors from the lower abdomen. Data are represented as mean +/− SEM. C. Schematic depicting the sites of tumor cell inoculation (quadrants I and IV) and the direction of the needle.

Figure 2

Microvessel density is significantly increased in tumors isolated from the upper abdomen. A. Sections from CT26 colon carcinomas isolated from the upper and lower abdomen were immunostained with anti-CD31antibody to detect endothelial cells and Hoechst dye to detect nuclei. Scale bar, 40μm. B. Quantification of microvessel density (MVD) in CT26 tumors isolated from the upper and lower abdomen per high powered field (hpf) (n=20 tumors per group). Data are represented as mean +/− SEM.

Figure 3

Differential expression of thrombopsondin-1 (TSP-1) in the stroma of the upper and lower abdomen. A. Tsp-1 mRNA expression in organs from the upper and the lower abdomen. The fold change is relative to liver and normalized to GAPDH. B. TSP-1 immunofluorescence of the serosal surface of organs in the upper (duodenum) versus lower (colon) abdomen. White arrows identify the serosal surfaces. Upper panel: H&E. Lower panel: Anti-TSP-1 (red) immunofluorescence and Hoechst dye (blue). Pictures are taken at 63×. Scale bar = 50 μm. C. The differential growth of Lewis lung tumors in the upper versus lower abdomen is lost in Tsp-1^−/− mice. The weight of tumors isolated from the upper (small intestine) and lower (large colon) abdomen was measured (n= 10 mice per group). Data are represented as mean +/− SEM.

Figure 4

Tumor growth is suppressed in the upper abdomen upon over-expression of TSP-1 in CT26 colon carcinoma cells. A. CT26 vector transfected control (CT26 NC-5) tumor cells demonstrated differential tumor growth in the upper and lower abdomen. However, CT-26 over-expressing TSP-1 tumor cells (CT26 TSP-1#6) show limited tumor growth in the upper abdomen. Arrows point to large and small tumors in the upper abdomen and small non-angiogenic tumors in the lower abdomen. B. The weight of tumors from the upper abdomen derived from CT26 TSP#6 tumor cells was significantly decreased in comparison to tumors derived from CT26 NC-5 control tumor cells. Inset graph represents the weights of the CT26 TSP-1#6 and CT26 NC-5 tumors from the lower abdomen demonstrating no significant difference in the weights of these tumors. (n= 5 mice per group). Data are represented as mean +/− SEM.

Figure 5

Decreased microvessel density in tumors derived from CT26 colon carcinoma cells with TSP-1 over-expression. A. CD31 and TSP-1 immunostaining of CT26 control tumors transfected with vector alone (CT26 NC-5) isolated from the upper abdomen. B. CD31 and TSP-1 immunostaining of CT26 NC5 tumors from the lower abdomen. C. CD31 and TSP-1 immunostaining of tumors derived from CT-26 over-expressing TSP-1 tumor cells (CT26 TSP-1#6) and isolated from the upper abdomen. D. CD31 and TSP-1immunostaining of CT26 TSP-1#6 tumors from the lower abdomen. Scale bar = 20 μm. E. Microvessel density (MVD) of CT26 NC-5 and CT26 TSP-1#6 tumors from the upper and lower abdomen was determined by counting the number of CD31-positive blood vessels per high powered field (hpf).