Summary Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

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Adipose tissue-derived microvascular fragments (ad-MVF) represent effective vascularisation units for the seeding of dermal substitutes. However, particularly in case of extensive skin defects, the required amounts of donor fat tissue for the harvesting of ad-MVF may not always be available. Therefore, we herein determined the lowest ad-MVF density needed to induce a sufficient vascularisation and incorporation of seeded implants. Collagen-glycosaminoglycan matrices (Integra ® ; diameter: 4 mm) were seeded with 15,000 (HD), 10,000 (MD) and 5,000 (LD) ad-MVF and implanted into full-thickness skin defects within mouse dorsal skinfold chambers, to analyse their in vivo vascularisation and incorporation. Intravital fluorescence microscopy showed a comparable vascularisation of HD and MD ad-MVF-seeded Integra ® , which was significantly higher when compared to LD ad-MVF-seeded Integra ® . As assessed by photoacoustic imaging, this was associated with an increased oxygenation of the implants. Additional histological and immunohistochemical analyses revealed an enhanced cellular infiltration, collagen content, microvessel density and epithelialisation of HD and MD ad-MVF-seeded Integra ® , indicating a better incorporation compared to LD ad-MVF-seeded implants. These findings demonstrate that 80,000 ad-MVF/cm² is the least required density to guarantee an effective vascularisation of the dermal substitute.

Split-thickness skin grafts (STSG) are still the gold standard for the treatment of most skin defects. Hence, there is an ongoing need to improve this procedure. For this purpose, we herein analyzed dermal matrices seeded with adipose tissue-derived microvascular fragments (ad-MVF) in a bradythrophic wound model. In additional experiments, the matrices were covered with autologous STSG 10 days after implantation. Green fluorescence protein (GFP)+ ad-MVF were isolated from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and seeded onto collagen-glycosaminoglycan matrices. Non-seeded and prevascularized matrices were implanted into full-thickness skin defects on the skull of CD1 nu/nu mice for 21 days. Vascularization, lymphangiogenesis and incorporation of the matrices were analyzed using photo-acoustic imaging, trans-illumination stereomicroscopy, histology, and immunohistochemistry. The survival rate of STSG was assessed by planimetry. After 21 days, the density of microvascular and lymphatic networks was significantly higher in prevascularized matrices when compared to controls. This was associated with an improved implant integration. Moreover, prevascularization with ad-MVF allowed successful autologous skin grafting already at day 10, while coverage of non-seeded controls at day 10 resulted in STSG necrosis. In conclusion, ad-MVF represent powerful vascularization units. Seeded on dermal substitutes, they accelerate and enhance the healing of full-thickness skin defects and allow early coverage with STSG.

Reactive oxygen species ( ROS ) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum ( ER )–mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX 1 and TMX 3 oxidoreductases, which promote ER –mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial‐ and NOX 4‐derived ROS . The TMX ‐knockdown‐induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT 1. Furthermore, we identified NFAT 1‐positive and NFAT 1‐negative melanoma subgroups, wherein NFAT 1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial‐ and redox‐related proteins are under NFAT 1 control and indicated that TMX 1, TMX 3, and NFAT 1 are associated with poor disease outcome. Our study unravels a novel redox‐controlled ER –mitochondria– NFAT 1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

Taken together, these experimental findings suggest that xanthohumol inhibits the development of endometriotic lesions in mice without inducing serious side effects in the reproductive organs. Thus, xanthohumol represents a promising dietary phytochemical that, after further testing, may be considered for the use in the selective treatment of endometriotic lesions.

Secondary lymphedema is a common complication of cancer treatment characterized by chronic limb swelling with interstitial inflammation. The rodent hindlimb is a widely used model for the evaluation of novel lymphedema treatments. However, the assessment of limb volume in small animals is challenging. Recently, high-resolution three-dimensional (3D) imaging modalities have been introduced for rodent limb volumetry. In the present study we evaluated the validity of microcomputed tomography (μCT), magnetic resonance imaging (MRI) and ultrasound in comparison to conventional measuring techniques. For this purpose, acute lymphedema was induced in the mouse hindlimb by a modified popliteal lymphadenectomy. The 4-week course of this type of lymphedema was first assessed in 6 animals. In additional 12 animals, limb volumes were analyzed by μCT, 9.4 T MRI and 30 MHz ultrasound as well as by planimetry, circumferential length and paw thickness measurements. Interobserver correlation was high for all modalities, in particular for μCT analysis (r = 0.975, p < 0.001). Importantly, caliper-measured paw thickness correlated well with μCT (r = 0.861), MRI (r = 0.821) and ultrasound (r = 0.800). Because the assessment of paw thickness represents a time- and cost-effective approach, it may be ideally suited for the quantification of rodent hindlimb lymphedema.