CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

Neurons burdened by DNA double strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

DNA double strand breaks (DSBs) are linked to aging, neurodegeneration, and senescence. However, the role played by neurons burdened with DSBs in disease-associated neuroinflammation is not well understood. Here, we isolate neurons harboring DSBs from the CK-p25 mouse model of neurodegeneration through fluorescence-activated nuclei sorting (FANS), and characterize their transcriptomes using single-nucleus, bulk, and spatial sequencing techniques. We find that neurons harboring DSBs enter a late-stage DNA damage response marked by the activation of senescent and antiviral-like immune pathways. We identify the NFkB transcription factor as a master regulator of immune gene expression in DSB-bearing neurons, and find that the expression of cytokines like Cxcl10 and Ccl2 develop in DSB-bearing neurons before glial cell types. Alzheimers Disease pathology is significantly associated with immune activation in excitatory neurons, and direct purification of DSB-bearing neurons from Alzheimers Disease brain tissue further validates immune gene upregulation. Spatial transcriptomics reveal that regions of brain tissue dense with DSB-bearing neurons also harbor signatures of inflammatory microglia, which is ameliorated by NFkB knock down in neurons. Inhibition of NFkB or depletion of Ccl2 and Cxcl10 in DSB-bearing neurons also reduces microglial activation in organotypic brain slice culture. In conclusion, we find that in the context of age-associated neurodegenerative disease, DSBs activate immune pathways in neurons, which in turn adopt a senescence associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of age-associated neuroinflammation.

Comparison of Long-term Human Precision-cut Lung Slice Culture Methodology and Response to Challenge: An Argument for Standardisation

As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air–liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared. The hPCLS were assessed in terms of their viability and responsiveness to challenge. The endpoints selected to compare the medium–method (M–M) combinations, which included histological features and viability, were evaluated at day 14 (D14) and day 28 (D28); protein and adenylate kinase (AK) content, and cytokine response to immunostimulants (lipopolysaccharide (LPS) at 5 μg/ml; polyinosinic:polycytidylic acid (Poly I:C) at 15 μg/ml) were evaluated at D28 only. Based on the set of endpoints assessed at D28, it was clear that certain culture conditions significantly affected the hPCLS, with the tissue retaining more of its native features and functionality (in terms of cytokine response) in some of the M–M combinations tested more than others. This pilot study indicates that the use of appropriate M–M combinations can help maintain the health and functional responses of hPCLS, and highlights the need for the standardisation of culture conditions in order to facilitate effective inter-laboratory comparisons and encourage greater acceptance by the regulatory community.

Bioorthogonal labeling of live dissociated and organotypic slice culture neurons

Over the past couple decades, the explosion in the development of high-resolution and super-resolution microscopy techniques has led to the need for the development of new protein labeling techniques. Click-labeling via genetic code expansion (GCE) has received particular attention given its potential has the ultimately small labelling probe for proteins. Click-labeling via GCE offers a reliable and sterically minimally demanding capacity to label proteins, but its application in non dividing cells such as neurons remains poorly exploited due to its low efficiency. Here, we describe a simple, efficient and reproducible protocol that allows to fluorescently label transmembrane proteins in live neurons using click-labeling via GCE, both in dissociated culture and organotypic brain slices.

Building on a Solid Foundation: Adding Relevance and Reproducibility to Neurological Modeling Using Human Pluripotent Stem Cells

The brain is our most complex and least understood organ. Animal models have long been the most versatile tools available to dissect brain form and function; however, the human brain is highly distinct from that of standard model organisms. In addition to existing models, access to human brain cells and tissues is essential to reach new frontiers in our understanding of the human brain and how to intervene therapeutically in the face of disease or injury. In this review, we discuss current and developing culture models of human neural tissue, outlining advantages over animal models and key challenges that remain to be overcome. Our principal focus is on advances in engineering neural cells and tissue constructs from human pluripotent stem cells (PSCs), though primary human cell and slice culture are also discussed. By highlighting studies that combine animal models and human neural cell culture techniques, we endeavor to demonstrate that clever use of these orthogonal model systems produces more reproducible, physiological, and clinically relevant data than either approach alone. We provide examples across a range of topics in neuroscience research including brain development, injury, and cancer, neurodegenerative diseases, and psychiatric conditions. Finally, as testing of PSC-derived neurons for cell replacement therapy progresses, we touch on the advancements that are needed to make this a clinical mainstay.

TAMI-71. EFFECTS OF THE TREATED MICROENVIRONMENT ON GLIOMA CELL PROPERTIES IN AN ORGANOTYPIC BRAIN SLICE MODEL

Glioblastoma is the most aggressive primary brain tumor. Despite treatment all patients invariably recur. Treatment resistance is attributed to the presence of glioma stem-like cells. Initially thought to be a distinct and static cell population, it is becoming increasingly clear that the glioma stem-like cell phenotype represents one of many cellular states and that glioma cells show plasticity between stem-like and non-stem like states. These plastic cell states are affected by the tumor microenvironment. In our lab we have shown that irradiated and hypoxic astrocytes increase the stem-like cell properties of glioma cells. In this study, we aim to evaluate how the treated microenvironment alters glioma cell properties and use ex vivo organotypic brain slices generated from tumor bearing and tumor naïve mice to assess all aspects of the microenvironment. We first characterized organotypic brain slices cultured in different oxygen tensions. We saw that tumor-bearing slices survive for at least 14 days in culture at 21%, 5% or 1% oxygen tension (O2). Tumor cells were more viable in all culture conditions and timepoints compared to non-tumor cells. Moreover, we found that astrocytes seem to be attracted to tumor areas in both 5% and 1% O2 cultures. We then used the organotypic glioma slice culture system to address how preconditioning the microenvironment using radiation or temozolomide affects the properties of glioma cells that are seeded in these pretreated, tumor naïve slices. We saw that fluorescently labelled glioma cells seeded in treated slices can be isolated after two days of culture in the slices and can be used for downstream analyses, such as temozolomide or radiation treatment and colony formation. This study will elucidate the effect of the treated microenvironment on glioma cell properties by using the medium throughput method of organotypic slice cultures.

Using Organotypic Tissue Slices to Investigate the Microenvironment of Pancreatic Cancer: Pharmacotyping and Beyond

Organotypic tissue slices prepared from patient tumors are a semi-intact ex vivo preparation that recapitulates many aspects of the tumor microenvironment (TME). While connections to the vasculature and nervous system are severed, the integral functional elements of the tumor remain intact for many days during the slice culture. During this window of time, the slice platforms offer a suite of molecular, biomechanical and functional tools to investigate PDAC biology. In this review, we first briefly discuss the development of pancreatic tissue slices as a model system. Next, we touch upon using slices as an orthogonal approach to study the TME as compared to other established 3D models, such as organoids. Distinct from most other models, the pancreatic slices contain autologous immune and other stromal cells. Taking advantage of the existing immune cells within the slices, we will discuss the breakthrough studies which investigate the immune compartment in the pancreas slices. These studies will provide an important framework for future investigations seeking to exploit or reprogram the TME for cancer therapy.

Nivolumab Reduces PD1 Expression and Alters Density and Proliferation of Tumor Infiltrating Immune Cells in a Tissue Slice Culture Model of Renal Cell Carcinoma

Background: In the treatment of clear cell renal cell carcinoma (ccRCC), nivolumab is an established component of the first-line therapy with a favorable impact on progression free survival and overall survival. However, treatment-related adverse effects occur and, to date, there is no approved predictive biomarker for patient stratification. Thus, the aim of this study was to establish an ex vivo tissue slice culture model of ccRCC and to elucidate the impact of nivolumab on tumor infiltrating immune cells. Methods: Fresh tumor tissue of ccRCC was treated with the immune checkpoint inhibitor nivolumab using ex vivo tissue slice culture (TSC). After cultivation, tissue slices were formalin-fixed, immunohistochemically stained and analyzed via digital image analysis. Results: The TSC model was shown to be suitable for ex vivo pharmacological experiments on intratumoral immune cells in ccRCC. PD1 expression on tumor infiltrating immune cells was dose-dependently reduced after nivolumab treatment (p < 0.01), whereas density and proliferation of tumor infiltrating T-cells and cytotoxic T-cells were inter-individually altered with a remarkable variability. Tumor cell proliferation was not affected by nivolumab. Conclusions: This study could demonstrate nivolumab-dependent effects on PD1 expression and tumor infiltrating T-cells in TSC of ccRCC. This is in line with results from other scientific studies about changes in immune cell proliferation in peripheral blood in response to nivolumab. Thus, TSC of ccRCC could be a further step to personalized medicine in terms of testing the response of individual patients to nivolumab.