Lukas Sommer

Although tumor innervation is known to promote cancer progression, underlying mechanisms remain poorly understood and have mostly been linked to axonal signaling. Here, we identify peripheral glia as critical drivers of cutaneous squamous cell carcinoma (cSCC), the second most common skin cancer in humans. Tumor-associated glial cells (TAGs) resemble embryonic Schwann cell lineage stages, are abundant in the stroma of premalignant lesions and established murine and human cSCC, and are essential for tumor formation. TAG activation in premalignant lesions is triggered by UV-induced anti-inflammatory macrophages, which in turn are polarized by TAGs into pro-tumorigenic FcεRIα⁺ macrophages, key components of the cSCC microenvironment. Consequently, TAG depletion reduces FcεRIα⁺ macrophages, cancer-associated fibroblasts, and epithelial-mesenchymal transition in keratinocytes, thereby impairing cSCC initiation by disrupting intercellular communication required for tumor development. As most solid tumors are innervated and harbor peripheral glia, our findings reveal TAGs as regulators of tumor initiation with potential relevance beyond skin cancer.

Engineered in vitro platforms are powerful systems to study information flow in the nervous system. While existing polydimethylsiloxane (PDMS)‐based microfluidic platforms offer precise architectures, the cultured neurons grow on two‐dimensional (2D) planar multielectrode arrays (MEA). To mimic the native microenvironment, where neurons grow in three‐dimensional (3D) extracellular matrices (ECM), 3D hydrogels can be designed to encapsulate cells and enable physiologically mimicked behaviors. Here, we describe ‘hydroMEA,’ a 3D platform fabricated by placing PDMS microstructures on a high‐density MEA and filled with a desired hydrogel, to offer controlled topologies, physiologically relevant microenvironments, and real‐time electrophysiological measurements. First, we developed a gelatin methacryloyl (GelMA) hydrogel with incorporated ECM components and tuned the mechanical properties to match those of nerve tissue. The hydrogel was able to support: (1) the growth of iPSC‐derived sensory neurons (hSNs) for >100 days; (2) co‐cultures of hSN with human embryonic stem cell‐derived Schwann cells (hSCs), to enable reliable 3D myelination. Next, hydroMEA were prepared for topologically defined 3D growth and myelination in designated compartments. Finally, electrophysiological evaluation of hSN‐hSCs co‐cultures revealed increased conduction speeds indicating functional myelin. This platform is a promising tool to study cell‐cell interactions and to functionally evaluate the effect of pharmacological compounds in a more translational manner.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with limited therapeutic options. Here, we present a novel sporadic murine model of Nf1 wild-type MPNST, driven by conditional expression of oncogenic BrafV600E and loss of Pten in the glial lineage using the Plp1::CreERT2 driver. This model allows for highly penetrant and rapid tumor induction through spontaneous formation, localized initiation, or cell transplantation. Comparative analysis with Tyr::CreERT2-driven melanoma revealed striking phenotypic divergence despite shared genetic alterations, underscoring the importance of the cell of origin in shaping tumor identity. In this system, MPNST cells show refractory capacities to induce melanocytic trans-differentiation upon melanoma-promoting signaling cues, such as canonical Wnt signaling gain of function or increased of levels of the epigenetic mark H3K27Me3 upon Ezh2 gain of function. Our findings emphasize the significance of lineage context in tumor initiation and provide a foundation for future mechanistic and therapeutic studies.

Skin injuries initiate a complex regenerative cascade that engages various cell types. Amongst those, peripheral glial cells play a critical role in ensuring the success of the repair process. However, our understanding of these processes remains incomplete due to limitations in current technologies. Therefore, the iterative-bleaching-extends-multiplexity (IBEX) method was employed to characterize changes to the cellular composition of the skin using antibodies directed against proteins expressed by key cell types and structures involved in tissue regeneration. Importantly, antibodies directed against cell proliferation markers often underestimate the number of dividing cells in the skin. To overcome this limitation, Click-iT EdU chemistry was combined with the IBEX method, allowing detailed characterization of proliferating cell types. Using a fully automated spinning disk confocal microscope, a high-throughput workflow was developed for iterative imaging of samples sectioned into 24-well plates. Beyond extending the IBEX method to evaluate cell states in situ with Click-iT EdU, a novel open-source image processing pipeline was also introduced to perform image correction, stitching, and registration, and can be used by researchers without extensive programming experience. Moreover, a detailed documentation on processing and visualizing highly multiplexed imaging data (using conventional image analysis software, including Fiji and QuPath) is also provided. In summary, this protocol highlights the versatility of the IBEX protocol and demonstrates its adaptation to an established conventional imaging platform. Notably, combining IBEX with Click-iT EdU labeling enables the detection and categorization of actively dividing cell types in intact tissues and during highly dynamic processes at spatial resolution.

The TGFβ signaling pathway is known for its pleiotropic functions in a plethora of biological processes. In melanoma, TGFβ signaling promotes invasiveness and metastasis formation. However, its involvement in the response to therapy is controversial. While several studies have linked TGFβ signaling to elevated resistance to targeted therapy in melanoma, separate findings have indicated a favorable treatment response through TGFβ-mediated increase of cell death. We now found that the outcome of TGFβ signaling in the context of targeted therapy is dose dependent. Unlike low doses, high levels of TGFβ signal activation induce apoptosis upon simultaneous MAPK pathway inhibition, even in targeted therapy resistant melanoma cell lines. Using transcriptomic analyses, combined with genomic target identification of the critical TGFβ signaling effector SMAD4, we demonstrate that parallel activation of TGFβ signaling and MAPK pathway inhibition causes a complete switch of TGFβ target genes from promoting pro-invasive processes to fueling pro-apoptotic pathways. Investigations of underlying mechanisms identified a novel apoptosis-inducing gene signature. Functional validation of signature members highlighted a central role of the pro-apoptotic BCL2 family member BCL2L11 (BIM) in mediating apoptosis in this condition. Using a modified, synthetic version of the TGFB1 mRNA for intra-tumoral injections, we additionally showcase a potential therapeutic application of this treatment combination.

A hallmark feature of pancreatic ductal adenocarcinoma (PDAC) is massive intratumoral fibrosis, designated as desmoplasia. Desmoplasia is characterized by the expansion of cancer-associated fibroblasts (CAFs) and a massive increase in extracellular matrix (ECM). During fibrogenesis, distinct genes become reactivated specifically in fibroblasts, e.g., the disintegrin metalloprotease, ADAM12 . Previous studies have shown that immunotherapeutic ablation of ADAM12 ⁺ cells reduces fibrosis in various organs. In preclinical mouse models of PDAC, we observe ADAM12 expression in CAFs as well as in tumor cells but not in healthy mouse pancreas. Therefore, we tested prophylactic and therapeutic vaccination against ADAM12 in murine PDAC and observed delayed tumor growth along with a reduction in CAFs and tumor desmoplasia. This is furthermore associated with vascular normalization and alleviated tumor hypoxia. The ADAM12 vaccine induces a redistribution of CD8 ⁺ T cells within the tumor and cytotoxic responses against ADAM12 ⁺ cells. In summary, vaccination against the endogenous fibroblast target ADAM12 effectively depletes CAFs, reduces desmoplasia and delays the growth of murine PDACs. These results provide proof-of-principle for the development of vaccination-based immunotherapies to treat tumor desmoplasia.

Fibroblast growth factor 23 (FGF23) levels are highly elevated in patients with chronic kidney disease (CKD); however, whether it serves merely as a biomarker or actively contributes to kidney inflammation remains unclear. Full-length FGF23 is cleaved into C-terminal FGF23 (cFGF23), which acts as a natural antagonist of FGF23 by inhibiting its binding to the FGF receptor (FGFR) and the co-receptor Klotho. Here, we show that chronically elevated FGF23 levels in a mouse model Hyp-Duk cause sustained-ERK signaling and an inflammatory and immune responses in the kidney. cFGF23, delivered via adeno-associated virus (AAV) gene therapy, successfully mitigated renal sustained-ERK signaling and inflammatory and immune responses in Hyp-Duk mice. On the other hand, acute physiological FGF23 levels in vitro elicited transient-ERK signaling with the expression of canonical early-ERK targets ( EGR1, JUNB, FOSB ). Consistent with in vivo findings, prolonged pathological FGF23 treatment in-vitro revealed sustained-ERK signaling with upregulation of late-ERK targets ( ETV4/5, SPRED1/2, SPRY2/4 ) and unique inflammatory and immune gene signatures. These effects were significantly mitigated by FGFR and ERK inhibitors, as well as by recombinant cFGF23 treatment. In summary, chronically high levels of FGF23 induce kidney inflammation through the FGFR-Klotho complex and sustained-ERK activation, which are successfully mitigated by cFGF23 gene therapy. TRANSLATIONAL STATEMENT: Chronically elevated circulating levels of Fibroblast Growth Factor 23 (FGF23) are strongly associated with inflammation and adverse outcomes in chronic kidney disease, but the underlying mechanisms remain poorly understood. Our study identifies sustained activation of the ERK signaling pathway as a key mechanism by which chronic FGF23 elevation induces inflammatory transcriptional programs in the kidney. Importantly, gene therapy using the C-terminal fragment of FGF23 (cFGF23) mitigates these effects, highlighting a potential therapeutic strategy to counteract the excessive FGF23 signaling.