Acute wounds accelerate tumorigenesis by a T cell-dependent mechanism

Abstract

We investigated the influence of acute wounding on tumor growth in a syngeneic mouse breast cancer model. Metastatic mouse breast cancer cells (4T1) were orthotopically injected into the mammary fat pads of BALB/c mice, and animals were wounded locally by full thickness dermal incisions above the mammary fat pads or remotely above the scapula 9 days later. Local, but not remote, wounding increased tumor size when compared with sham treatment. Injection of wound fluid close to the tumor site increased tumor growth, whereas in vitro wound fluid compared with serum increased the proliferation rate of 4T1 cells. Our results show that wound stroma can unfavorably influence growth of nearby tumors. This effect is T cell-dependent, as local wounding had no effect on tumor growth in nu/nu mice. The effect of wounding on tumor growth can be mimicked by acellular wound fluid, suggesting that T cells secrete or mediate secretion of cytokines or growth factors that then accelerate tumor growth. Here, we define an experimental model of wound-promoted tumor growth that will enable us to identify mechanisms and therapeutic targets to reduce the negative effect of tissue repair on residual tumors.

Figure 1

Wounding promotes tumor growth in a mouse model of breast cancer. A. Experimental design. 4T1 cells (10⁴ in 50ul DPBS) were orthotopically injected into the mammary fat pads IV/V and IX/X of syngeneic BALB/c mice, followed by local or remote dermal incision 9 days later. Tumor growth was assessed using calipers. Animals were euthanized and autopsied 23 days after inoculation with tumor cells. B. Local wounding increased tumor volume and tumor incidence in the syngeneic model described in A. Data points present the mean±SEM of n=18 tumors. C. Wounding significantly increased the cumulative tumor mass as measured by AUC (Mann-Whitney Test). D. Histology of tumors dissected from wounded and unwounded animals 23 days after tumor cell inoculation.

Figure 2

Local but not remote wounding promotes tumor growth in a T-cell dependent fashion. A. In contrast to local wounding, remote wounding by suprascapular dermal incision does not significantly alter the cumulative tumor mass as measured by AUC in an orthotopic syngeneic mouse model of breast cancer (Kruskal-Wallis Test / Dunn's Multiple Comparison Analysis). B. Local wounding but not induction of a systemic acute phase response by injection of turpentine (30μl) into the suprascapular pocket 9 days after tumor cell inoculation into the mammary fat pads IV/V and IX/X significantly increases the cumulative tumor mass (AUC, Kruskal-Wallis Test / Dunn's Multiple Comparison Analysis). C. In nude mice (BALB/c nu/nu) local wounding does not promote tumor growth (Mann-Whitney Test). D. Daily injection of wound fluid (40μl) into the proximity of tumor cell inoculation sites starting 9 days after tumor cell inoculation significantly increased the cumulative tumor mass (AUC) as compared to injection of DPBS (Mann-Whitney Test). Bars indicate medians.

Figure 3

Influence of wound fluid and wounding on tumor cell proliferation. A. Wound fluid increases tumor cell proliferation in vitro. Cells were stimulated with 3% wound fluid or serum or plasma, or were serum starved (0%) for 2 days. Cell numbers were counted and normalized to the average cell number of serum treated cells. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. B. Wound fluid generated from BALB/c mice but not wound fluid generated from nude mice (BALB/c nu/nu) promotes proliferation of 4T1 cells in vitro. The effect of wound fluid not only depended on the mouse strain, but also on the age of the wound fluid; wound fluid harvested 6 days to 9 days after wounding was most effective in stimulating tumor cell proliferation. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. C. Wounding increases tumor cell proliferation in-vivo. Mitotic tumor cells were counted in H&E stained sections of tumors obtained from wounded and unwounded animals 23 days after tumor cell inoculation. Student's t-test.

Figure 3

Influence of wound fluid and wounding on tumor cell proliferation. A. Wound fluid increases tumor cell proliferation in vitro. Cells were stimulated with 3% wound fluid or serum or plasma, or were serum starved (0%) for 2 days. Cell numbers were counted and normalized to the average cell number of serum treated cells. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. B. Wound fluid generated from BALB/c mice but not wound fluid generated from nude mice (BALB/c nu/nu) promotes proliferation of 4T1 cells in vitro. The effect of wound fluid not only depended on the mouse strain, but also on the age of the wound fluid; wound fluid harvested 6 days to 9 days after wounding was most effective in stimulating tumor cell proliferation. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. C. Wounding increases tumor cell proliferation in-vivo. Mitotic tumor cells were counted in H&E stained sections of tumors obtained from wounded and unwounded animals 23 days after tumor cell inoculation. Student's t-test.

Figure 3

Influence of wound fluid and wounding on tumor cell proliferation. A. Wound fluid increases tumor cell proliferation in vitro. Cells were stimulated with 3% wound fluid or serum or plasma, or were serum starved (0%) for 2 days. Cell numbers were counted and normalized to the average cell number of serum treated cells. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. B. Wound fluid generated from BALB/c mice but not wound fluid generated from nude mice (BALB/c nu/nu) promotes proliferation of 4T1 cells in vitro. The effect of wound fluid not only depended on the mouse strain, but also on the age of the wound fluid; wound fluid harvested 6 days to 9 days after wounding was most effective in stimulating tumor cell proliferation. ANOVA / Dunnet's Multiple Comparison Test. Data are presented as mean±SEM. C. Wounding increases tumor cell proliferation in-vivo. Mitotic tumor cells were counted in H&E stained sections of tumors obtained from wounded and unwounded animals 23 days after tumor cell inoculation. Student's t-test.