Sunlight causes skin cancer by directly damaging DNA as well as by suppressing antitumor immune responses. A major mechanism whereby sunlight exerts immunosuppressive effects is by modulating the migration of chemokine (C-X-C motif) receptor 4 (CXCR4)-expressing dermal mast cells into and away from the skin. We have demonstrated the importance of this by showing that the systemic administration of the CXCR4 antagonist AMD3100 prevents sunlight-induced immunosuppression as well as the consequent carcinogenic response. Our results highlight the therapeutic potential of antagonizing CXCR4 signaling, especially in individuals who are at high risk of developing skin cancer.
Mast cells are part of the immune system and characteristically contain histamine- and heparin-rich basophilic granules. While these cells are usually associated with allergy and anaphylaxis, they also promote wound healing and angiogenesis and confer protection against pathogens. The presence of these cells is sometimes indicative of a poor prognosis, especially in skin cancer, pancreatic cancer, and lymphoma. Toluidine blue staining of acid-fast granules is an established method for the identification and quantification of mast cells. Generating detailed information on the location of mast cells within tissues is problematic using this technique and often requires serial sections from adjacent tissue to be separately stained with hematoxylin and eosin (H&E). Staining serial sections is not always possible, particularly if the sample is very small or rare. In such cases, a method of simultaneously identifying and localizing mast cells in a tissue would be advantageous. Toluidine blue and H&E are not commonly combined because H&E includes repetitive washes in water, which may affect the efficacy of the aqueous-soluble toluidine blue. We have developed and tested a novel staining technique that integrates toluidine blue between hematoxylin and eosin in one simple procedure. This protocol works on both frozen and formalin-fixed, paraffin-embedded tissue and readily allows for the identification of purple-stained mast cells against a clean H&E background. This facilitates a more accurate localization and proper counting of mast cells in normal and affected tissue.
One way sunlight causes skin cancer is by suppressing anti-tumor immunity. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4-C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. We have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-only developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. Our results indicate that interfering with UV-induced CXCL12 by antagonizing CXCR4 significantly inhibits skin tumor development by blocking UV-induced effects on mast cells. Hence, the CXCR4-CXCL12 chemokine pathway is a novel therapeutic target in the prevention of UV-induced skin cancer.