Tumors induce complex DNA damage in distant proliferative tissues in vivo

Abstract

That tumors cause changes in surrounding tissues is well documented, but whether they also affect distant tissues is uncertain. Such knowledge may be important in understanding the relationship between cancer and overall patient health. To address this question, we examined tissues distant to sites of implanted tumors for genomic damage using cohorts of C57BL/6 and BALB/c mice with early-stage subcutaneous syngeneic grafts, specifically, B16 melanoma, MO5076 sarcoma, and COLON26 carcinoma. Here we report that levels of two serious types of DNA damage, double-strand breaks (DSBs) measured by γ-H2AX focus formation and oxidatively induced non-DSB clustered DNA lesions (OCDLs), were elevated in tissues distant from the tumor site in tumor-bearing mice compared with their age- and sex-matched controls. Most affected were crypts in the gastrointestinal tract organs and skin, both highly proliferative tissues. Further investigation revealed that, compared with controls, tumor-bearing mice contained elevated amounts of activated macrophages in the distant gastrointestinal tissues, as well as elevated serum levels of several cytokines. One of these cytokines, CCL2/MCP-1, has been linked to several inflammation-related conditions and macrophage recruitment, and strikingly, CCL2-deficient mice lacked increased levels of DSBs and OCDLs in tissues distant from implanted tumors. Thus, this study is unique in being a direct demonstration that the presence of a tumor may induce a chronic inflammatory response in vivo, leading to increased systemic levels of DNA damage. Importantly, these findings suggest that tumors may have more profound effects on their hosts than heretofore expected.

Fig. 1.

γ-H2AX foci are elevated in the GIT tissues and skin of COLON26 carcinoma-bearing mice. (A) (Left) GIT tissues consist of crypt structures (H&E-stained paraffin sections of NC mice), which contain continually renewing cells. (Right) γ-H2AX immunostaining in touch-print preparations of the noted organs from NC, acute IC, and TST cohorts. Images are representative maximum projections of optical sections with average numbers of γ-H2AX foci per cell. Higher-magnification images (Insets) more clearly show nuclei (red, counterstained with propidium iodide) containing γ-H2AX foci (green). (B) γ-H2AX staining in frozen duodenum sections. γ-H2AX levels are higher in the duodenum of TST mice compared with NC mice. (Insets) Crypt cells at higher magnification. (C) γ-H2AX staining in frozen sections of skin from control (NC and IC) mice compared with skin sections from tumor-bearing mice taken proximal (TST1) to the tumor mass (TST, tumor mass), about 0.5 cm (TST2) and 2 cm (TST3) from the tumor, with average numbers of γ-H2AX foci per cell. (D) Representative images of perpendicular hair follicle sections of NC and TST mice showing increased γ-H2AX staining in tumor-bearing mice. Graphs show the incidences of γ-H2AX foci in the GIT (E) and skin (F). The numbers 1, 2, and 3 correspond to the skin-sample location. White bars, NC; black bars, IC; gray bars, TST cohorts. Error bars are SDs (n = 6 mice per group). *Statistically significant difference in TST mice separately compared with both NC and IC controls (P < 0.01 for GIT tissues and P < 0.05 for skin). Magnification, 400× for main panels; 200× for H&E panel.

Fig. 2.

OCDLs in the GIT and skin from NC, IC, and TST cohorts of COLON26 carcinoma-bearing mice. In TST mice, OCDL levels were measured in the tumor (diagonally marked), as well as in normal skin proximal to tumor (checkered), close to tumor (solid), and far from tumor (horizontally marked). Clusters measured: (A) hAPE1 (abasic), (B) EndoIII (oxidized pyrimidine), and (C) hOGG1 (oxidized purine). Error bars are SD (n = 6) and three to five independent measurements. *Statistically significant difference in TST mice compared with both NC and IC controls (P < 0.01).

Fig. 3.

(A) Elevated numbers of F4/80⁺ macrophages in colon, duodenum, and skin of M5076 sarcoma-bearing mice (TST) compared with NC mice as determined by immunoperoxidase staining of paraffin sections. (B) In tumor mass and mucosa-associated lymphoid tissue (MALT), F4/80 expression is often severe, and tumors are infiltrated with CD3⁺ and CD45⁺ cells. Nuclei are counterstained with propidium iodide. Magnification, 200×. (C) Mature/activated macrophage staining intensity determined by immunoperoxidase staining for F4/80 in colon, duodenum, and skin of M5076 sarcoma-bearing mice (TST, red) compared with control cohort (NC, black). F4/80 intensity grades were 1, minimal; 2, mild; 3, moderate. Each dot in each group represents data from one animal. F4/80⁺ cells in lymphoid aggregates were excluded from the analysis. Horizontal bars represent medians. (D) CCL2 levels are higher in serum of WT B6 tumor-bearing mice over corresponding controls, but not in CCL2 KO mice. CCL2 levels are shown relative to the NC cohort (white). TST1, M5076 sarcoma-bearing mice (black); TST2, B16 melanoma-bearing mice (light gray). Error bars are SDs (n = 3–6). (E) The incidences of γ-H2AX foci increase in colon and duodenum of WT B6J tumor-bearing mice (TST1, TST2) compared with control mice (NC, IC), but not in CCL2 KO mice. Error bars are SDs (n = 5). *Statistically significant difference in a TST mice compared with both NC and IC controls (P < 0.05). (F) OCDLs are increased in colon and duodenum of B6J WT but not of CCL2 KO mice. Error bars are SDs (n = 5). *Statistically significant difference in TST mice compared with both NC and IC controls (P < 0.05).