Ex-Vivo Skin Explant Culture Is a Model for TSLP-Mediated Skin Barrier Immunity

The skin is the outermost barrier protecting the body from pathogenic invasion and environmental insults. Its breakdown initiates the start of skin inflammation. The epidermal growth factor (EGFR) on keratinocytes protects this barrier, and its dysfunction leads to atopic dermatitis-like skin disease. One of the initial cytokines expressed upon skin barrier breach and during atopic dermatitis is TSLP. Here, we describe the expression and secretion of TSLP during EGFR inhibition and present an ex-vivo model, which mimics the early events after barrier insult. Skin explants floated on culture medium at 32 °C released TSLP in parallel to the activation of the resident Langerhans cell network. We could further show the up-regulation and activation of the AP-1 family of transcription factors during atopic-like skin inflammation and its involvement in TSLP production from the skin explant cultures. Inhibition of the c-Jun N-terminal kinase pathway led to a dose-dependent blunting of TSLP release. These data indicate the involvement of AP-1 during the early stages of atopic-like skin inflammation and highlight a novel therapeutic approach by targeting it. Therefore, skin explant cultures mimic the early events during skin barrier immunity and provide a suitable model to test therapeutic intervention.

Role of FBXW2 in explant cultures of bovine periosteum-derived cells

Objective Bone regeneration is a potential technique for treating osteoporosis. A previous study reported that F-box and WD-40 domain-containing protein 2 (FBXW2) localized with osteocalcin in bovine periosteum after 5 weeks of explant culture. However, the osteoblastic functions of FBXW2 remain unclear. In this study, double-fluorescent immunostaining was used to investigate the potential role of FBXW2 and its relationship with osteocalcin. Results At day 0, FBXW2 was expressed in the cambium layer between the bone and periosteum, while osteocalcin was expressed in bone. After explant culture, changes in the periosteum were observed from weeks 1 to 7. At week 1, partial FBXW2 expression was seen with a small amount of osteocalcin. At week 2, a layer of FBXW2 was observed. From weeks 3 to 7, tube-like structures of FBXW and osteocalcin were observed, and periosteum-derived cells were released from the periosteum in areas where no FBXW2 was observed. Bovine periosteum-derived cells can form a three-dimensional cell pellet, because multilayered cell sheets are formed inside of the periosteum in vitro. It is shown that in results FBXW2 is produced in periosteal explants near sites where initial osteogenic activity is observed, suggesting that it may be involved in periosteal osteogenesis.

EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES

The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.

DDRE-48. COMPARTMENT LOCKED IL-12 - INCREASED TISSUE RETENTION AND MINIMAL PERIPHERAL EXPOSURE ALLOW HIGHER TREATMENT EFFICACY AND TOLERABILITY IN LOCAL GLIOBLASTOMA THERAPY

Recent clinical studies in glioblastoma (GBM) highlight the potential of local IL-12 therapy, but they also bring back tolerability concerns due to leakage into the periphery. This leakage might thus hamper exploiting the full potential of local IL-12 therapy. Fusion with an IgG4 Fc portion increases the tissue retention of IL-12; but could also confer export into the blood and subsequent systemic recycling through the neonatal Fc receptor (FcRn), ultimately leading to potentially toxic IL-12 serum levels. We assessed the expression of FcRn in human and murine GBM and its role in IL-12Fc tissue retention and systemic exposure upon local delivery. Human or murine IL-12Fc was injected in GBM-bearing or naïve wt or FcRn-humanized mice continuously or as bolus via convection-enhanced delivery (CED). We screened combinations of amino-acid substitutions at the (IL-12)Fc:FcRn binding interface to abolish this interaction. Brain and blood concentrations were assessed via ELISA or cytokine bead arrays. FcRn affinity was measured by SPR/ELISA and bioactivity tested on PBMCs and human GBM explant cultures. Treatment efficacy and immunological correlates were assessed in GBM bearing mice. FcRn is upregulated in human and mouse GBM and contributes to brain export and subsequent peripheral recycling of IL-12Fc in the blood. IL-12Fc with abrogated FcRn binding due to a unique set of substitutions is fully functional and appears brain compartment locked (CL IL-12) as it exhibits enhanced tissue retention and reduced serum levels upon local injection, reaching up 100x higher brain to serum concentration ratios than regular IL-12. Compared to its non-modified counterpart, murine CL IL-12 shows significantly higher treatment efficacy at negligible systemic footprint in late stage murine GBM. In patient explant cultures, human CL IL-12 leads to successful inflammatory conditioning. Compartment locked IL-12 should thus allow a wide dosing window to fully harness its therapeutic potential for local GBM therapy.

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

Senescent Changes and Endoplasmic Reticulum Stress May Be Involved in the Pathogenesis of Missed Miscarriage

BackgroundSenescence is involved in many complications of pregnancy. However, whether senescent changes are also associated with missed miscarriage has not been fully investigated.MethodsThe levels of p16, p21, and γH2AX, markers of senescence, were measured in placentas collected from women with missed miscarriage by immunohistochemistry and Western blotting. Levels of misfolded proteins in missed miscarriage placentas or normal first-trimester placenta that had been treated with H2O2 (100 μM) or extracellular vesicles (EVs) collected from missed miscarriage placental explant culture were measured by fluorescent compound, thioflavin-T. The production of reactive oxygen species (ROS) by missed miscarriage placentas was measured by CellROX® Deep Red.ResultsIncreased levels of p16, p21, and γH2AX were presented in missed miscarriage placentas compared to controls. Increased levels of misfolded proteins were shown in missed miscarriage placentas, but not in EVs that were collected from missed miscarriage placentas. The ROS production was significantly increased in missed miscarriage placental explant cultures. Increased levels of misfolded proteins were seen in the normal first-trimester placenta that had been treated with H2O2 compared to untreated.ConclusionOur data demonstrate that there are increases in senescence and endoplasmic reticulum stress and ROS production in missed miscarriage placenta. Oxidative stress and an accumulation of misfolded proteins in missed miscarriage placentas may contribute to the changes of senescence and endoplasmic reticulum stress seen in missed miscarriage placentas.