c Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 ؋ 10 5 and 2.5 ؋ 10 5 conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.

Squamous cell carcinoma (SCC) is one of the most common skin cancers and causes significant morbidity. Although the expression of the epithelial adhesion molecule collagen XVII (ColXVII) has been linked to SCC invasion, only little is known about its mechanistic contribution. Here, we demonstrate that ColXVII expression is essential for SCC cell proliferation and motility. Moreover, it revealed that particularly the post-translational modification of ColXVII by ectodomain shedding is the major driver of SCC progression, because ectodomain-selective immunostaining was mainly localized at the invasive front of human cutaneous SCCs, and exclusive expression of a non-sheddable ColXVII mutant in SCC-25 cells inhibits their matrix-independent growth and invasiveness. This cell surface proteolysis, which is strongly elevated during SCC invasion and metastasis, releases soluble ectodomains and membrane-anchored endodomains. Both released ColXVII domains play distinct roles in tumor progression: the endodomain induces proliferation and survival, whereas the ectodomain accelerates invasiveness. Furthermore, specific blockage of shedding by monoclonal ColXVII antibodies repressed matrix-independent growth and invasion of SCC cells in organotypic co-cultures. Thus, selective inhibition of ColXVII shedding may offer a promising therapeutic strategy to prevent SCC progression.

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The incidence of cutaneous squamous cell carcinoma (cSCC) and its precancerous forms is increasing worldwide. Here, we analyzed the role of complement classical pathway components C1q, C1r and C1s in the progression of cSCC. Analysis of cSCC cell lines (n¼8) and normal human epidermal keratinocytes (n¼11) with oligonucleotide arrays, RNA-seq and quantitative RT-PCR showed significantly elevated C1r and C1s mRNA levels in cSCC cell lines, whereas the expression of C1q was low. Western blotting analysis showed increased production of C1r (n¼8) and C1s (n¼ 7) in cSCC cells compared to NHEKs. The mRNA levels for C1r and C1s were markedly elevated in cSCC tumors (n¼6) compared to normal skin (n¼11) in vivo. Immunohistochemical analysis revealed strong tumor cell specific expression of C1r and C1s in invasive sporadic cSCCs (n¼164) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n¼16), whereas the expression of both C1r and C1s was clearly lower in cSCC in situ (n¼63), in premalignant epidermal lesions (actinic keratoses, n¼61), and in normal skin (n¼9). The expression of C1r was up-regulated by IFN-g and the expression of C1s was up-regulated by IFN-g and TNF-a in cSCC cells. Knockdown of C1r and C1s expression with two specific siRNAs inhibited proliferation of cSCC cells. Moreover, growth of human cSCC xenograft tumors in vivo was significantly inhibited by knockdown of C1r and C1s. These results provide evidence for the role of tumor cell-derived C1r and C1s in the progression of cSCC and identify them as putative therapeutic targets in cSCC.