Tumors Alter Inflammation and Impair Dermal Wound Healing in Female Mice

Abstract

Tissue repair is an integral component of cancer treatment (e.g., due to surgery, chemotherapy, radiation). Previous work has emphasized the immunosuppressive effects of tumors on adaptive immunity and has shown that surgery incites cancer metastases. However, the extent to which and how tumors may alter the clinically-relevant innate immune process of wound healing remains an untapped potential area of improvement for treatment, quality of life, and ultimately, mortality of cancer patients. In this study, 3.5 mm full-thickness dermal excisional wounds were placed on the dorsum of immunocompetent female mice with and without non-malignant flank AT-84 murine oral squamous cell carcinomas. Wound closure rate, inflammatory cell number and inflammatory signaling in wounds, and circulating myeloid cell concentrations were compared between tumor-bearing and tumor-free mice. Tumors delayed wound closure, suppressed inflammatory signaling, and altered myeloid cell trafficking in wounds. An in vitro scratch "wounding" assay of adult dermal fibroblasts treated with tumor cell-conditioned media supported the in vivo findings. This study demonstrates that tumors are sufficient to disrupt fundamental and clinically-relevant innate immune functions. The understanding of these underlying mechanisms provides potential for therapeutic interventions capable of improving the treatment of cancer while reducing morbidities and mortality.

Fig 1. Tumors delay dermal wound closure.

(A) Average percent (±SEM) of original 3.5 mm dermal excisional wound size analyzed over 5 days using digital photography. (B) Representative photographs of wounds over time. Unhealed skin is circled. n = 10/group; *p<0.05.

Fig 2. Tumors alter absolute myeloid cell numbers in wounds examined 1 or 5 days after wounding.

Representative flow cytometry bivariate dot plots of Ly6C and Ly6G labeling of CD11b+ wound cells from mice (A) without tumors and with tumors 1 or 5 days post-wounding. Gates: R1 = Ly6C^high Mo/MΦ, R2 = Ly6C^lowMo/MΦ, and R3 = Neutrophils. Average (±SEM) (B) neutrophil (Cd11b/Ly6G⁺), (C) Ly6C^high Mo/ MΦ, and (D) Ly6C^low Mo /MΦ isolated from wound tissues and quantified using flow cytometry. (E) Average (±SEM) percent of these Ly6C^high or Ly6C^low Mo/ MΦ expressing IL-4Rα protein. n = 2-3/group; *p<0.05 within the same cancer treatment over time; ^#p<0.05 between treatments.

Fig 3. Tumors reduce relative gene expression of inflammatory markers in wounds.

Average (±SEM) quantitative expression of (A) Tlr4, (B) IL-1β, (C) Ccl2, (D) IL-10, (E) Ccl3, and (F) Cxcl1 mRNA extracted from wound tissue 1 or 5 days post-wounding. n = 8-10/group; *p<0.05 between cancer treatments.

Fig 4. Tumors alter gene expression pattern of select inflammatory/growth factors in wounds over time.

Average (±SEM) quantitative expression of (A) Tnfα, (B) Cxcl2, (C) Cxcl10, (D) Tgf-β, and (E) Igf-1mRNA extracted from wound tissue 1 or 5 days post-wounding. n = 8-10/group; *p<0.05 between cancer treatments.

Fig 5

Absolute circulating myeloid cell concentrations (CBC) with and without wounding. Average (±SEM) circulating (A) total white blood cells, (B) neutrophils, and (C) monocytes in tumor-bearing and -free mice with and without dermal wounding. n = 8-10/group; *p<0.05 within the same cancer treatment between wounding treatments; #p<0.05 between cancer treatments with same wound treatment; &p<0.05 within the tumor groups between Days 1 & 5.

Fig 6. Tumor cell-conditioned media reduces murine fibroblast migration and proliferation.

(A) Schematic of conditioned media paradigm and in vitro “wounding” scratch assay. (B) Average (±SEM) percent decrease in scratch “wound” width of adult murine fibroblasts 18 and 26 h post-scratching cultured with fibroblast-conditioned control media or tumor cell-conditioned media. n = 6 wells/group; *p≤0.05 between treatments (C) Representative photographs of scratch assay. Black bars represent margins of scratch.