Depletion of cutaneous macrophages and dendritic cells promotes growth of basal cell carcinoma in mice

Abstract

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.

Figure 1. MHCII expression in BCC-bearing skin of Ptch^flox/floxERT2^+/− mice.

H&E staining and immunohistochemical analysis of MHCII expression on paraffin sections derived from BCC-bearing skin of Ptch^flox/floxERT2^+/− mice. The secondary antibody alone did not result in any staining, hence the MHCII signals were specific (data not shown). double arrows: intraepidermal MHCII⁺ LC.

Figure 2. Clodrolip treatment results in depletion of cutaneous phagocytic cells in BCC-bearing skin of Ptch^flox/floxERT2^+/− mice.

(A) Immunohistochemical analysis of F4/80 expression on paraffin-sections of BCC-bearing skin derived from liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. Left panel shows micrographs; right panel shows the absolute number of F4/80⁺ cells per square millimeter of BCC-bearing tissue. **** P<0.0001 (mean +SEM). (B) Absolute numbers of MHCII⁺ cells per square millimeter of BCC-bearing tissue as counted on stained paraffin-sections from liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. MHCII⁺ cells were counted separately both in the epidermis (representing LC) and the stroma (representing DC). ** P<0.01.

Figure 3. Clodrolip enhances BCC growth in Ptch^flox/floxERT2^+/− mice.

(A) H&E stainings of paraffin-sections derived from BCC-bearing skin of liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. (B) Relative tumor areas of H&E-stained BCC-bearing skin samples of liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. The mean value of the liposome-treated controls was normalized to 1. Left panel shows the mean value (+SEM) of all liposome- and clodrolip-treated animals **P<0.01; right panel shows the mean value (+SEM) of all liposome-treated animals in comparison with 2 clodrolip-treated mice (mouse 2 and mouse 3 in Table 1), in which the phagocytic cells have been very efficiently depleted (due to the small sample size, statistical analysis was omitted). (C) Viability/metabolic activity of the BCC cell line ASZ001 after treatment with PBS, liposomes or clodrolip. Mean value of the PBS-treated controls was set to 100%. **P<0.01.

Figure 4. Clodrolip decreases the number of collagen-producing cells and the mRNA expression of the fibroblast markers vimentin and P4hb in BCC-bearing skin of Ptch^flox/floxERT2^+/− mice.

(A) Percentage of Ki67⁺ BCC cells in liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. (B) TUNEL⁺ BCC cells per mm BCC-bearing skin in liposome- or clodrolip-treated Ptch^flox/floxERT2^+/− mice. (C) Collagen-producing cells (mainly fibroblast) as estimated by Goldner staining (left panel; see main manuscript for detailed informations) and qRT-PCR analysis of vimentin (middle panel) and of P4hb (right panel) in BCC-bearing skin of Ptch^flox/floxERT2^+/− mice treated with liposomes or clodrolip. Data shows the mean value (+SEM). * P<0.05, ** P<0.01, *** P<0.001.