Lowering of tumor interstitial fluid pressure reduces tumor cell proliferation in a xenograft tumor model

Abstract

High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.

Figure 1

Tumor volume and TIFP in A431 and A549 tumors in NMRI nude mice. (A) Increase of tumor volume after implantation of small fragments (2 mm³) of A431 or A549 tumors. The plots represent tumor volume versus time. (B) Effect of increasing tumor volume on the TIFP. (C) Effect of tumor puncture on the TIFP. Scatter plots show the TIFP of five individual animals 6 hours before, during, and 6 hours after tumor puncture. Plots in (A) and (B) show the results of n = 10 (A431/A549). Plots in (C) show results of n = 5 (A431) (mean ± SE).

Figure 2

Effect of puncture on tumor morphology. (A) (I) Untreated A431 control tumor features a tightly inflated tumor cortex. (II) After puncture of the central cystic region, the tumor collapsed, indicating loss of surface tension. (B) Sections from untreated A431 tumors stained with hematoxylin and eosin to highlight mechanically stretched tumor cells. (I) The central necrotic area (white arrow) is surrounded by proliferating tumor tissues (black arrow) and tumor capsule (blue arrow). Original magnification, x 100. (II) Black arrows indicate stretched cells in the proliferative and highly vascularized outer tumor cortex. Original magnification, x 320.

Figure 3

Reduction of TIFP decreased the phosphorylation of proliferation-associated p44/42 MAPK. (A) Time course of p44/42 phosphorylation in untreated A431 and A549 control tumors and in TIFP-lowered tumors. (B) Quantitative analysis of p44/42 MAPK phosphorylation in TIFP-reduced and control tumors. The values from TIFP-lowered tumors have been normalized to values of control tumors. Data are mean ± SE (*P < .001 vs untreated control tumors; **P < .01 vs untreated control tumors) for five independent experiments.

Figure 4

24-Hour TIFP lowering reduced Ki-67 expression. (A) Immunohistochemical staining of Ki-67 in A431 tumor slides in response to reduced TIFP. (I) Ki-67 expression in TIFP-reduced tumor tissue. (II) Ki-67 expression in untreated control tumors. (III/IV) control staining after withdrawal of the first or secondary antibody. (V) Overview of a tissue slide of a control tumor stained against Ki-67. Original magnification, x 240 (I–IV) and x 100 (V). (B) Immunohistochemical staining of Ki-67 in A549 tumor slides in response to lowered TIFP. (I) Ki-67 expression in TIFP-lowered tumor tissue. (II) Ki-67 expression in untreated control tumors. Original magnification, x 240. (C) Summarized graphical representation of Ki-67 expression. A significant reduction in Ki-67 expression is visible in TIFP-lowered A431 and A549 tumors. The results are the mean ± SE of seven tumors (*P < .001 and **P < .05).

Figure 5

Reduced TIFP shows no effect on vessel architecture. Immunohistochemical staining of vessels using CD31-PE antibodies in A431 tumor slides. (I) CD31 staining of drained tumors. (II) CD31 staining of control tumors. Original magnification, x 320.