Background: Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2′-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells. Methods: Human p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestin^pos/GFAP^neg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model. Results: SAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestin^pos/GFAP^neg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment. Conclusion: The Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 μm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco’s Modified Eagle’s Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

Despite novel immunotherapies being approved and established for the treatment of non-small cell lung cancer (NSCLC), ex vivo models predicting individual patients’ responses to immunotherapies are missing. Especially immune modulating therapies with moderate response rates urge for biomarkers and/or assays to determine individual prediction of treatment response and investigate resistance mechanisms. Here, we describe a standardized ex vivo tissue culture model to investigate individual tumor responses. NSCLC tissue cultures preserve morphological characteristics of the baseline tumor specimen for up to 12 days ex vivo and also maintain T-cell function for up to 10 days ex vivo. A semi-automated analysis of proliferating and apoptotic tumor cells was used to evaluate tissue responses to the PD-1 inhibitor nivolumab (n = 12), from which two cases could be successfully correlated to the clinical outcome. T-cell responses upon nivolumab treatment were investigated by flow cytometry and multispectral imaging. Alterations in the frequency of the Treg population and reorganization of tumor tissues could be correlated to nivolumab responsiveness ex vivo. Thus, our findings not only demonstrate the functionality of T cells in NSCLC slice cultures up to 10 days ex vivo, but also suggests this model for stratifying patients for treatment selection and to investigate in depth the tumor-associated T-cell regulation.

Diagnosis in an advanced state is a major hallmark of ovarian cancer and recurrence after first line treatment is common. With upcoming novel therapies, tumor markers that support patient stratification are urgently needed to prevent ineffective therapy. Therefore, the transcription factor FOXM1 is a promising target in ovarian cancer as it is frequently overexpressed and associated with poor prognosis. In this study, fresh tissue specimens of 10 ovarian cancers were collected to investigate tissue cultures in their ability to predict individual treatment susceptibility and to identify the benefit of FOXM1 inhibition. FOXM1 inhibition was induced by thiostrepton (3 µM). Carboplatin (0.2, 2 and 20 µM) and olaparib (10 µM) were applied and tumor susceptibility was analyzed by tumor cell proliferation and apoptosis in immunofluorescence microscopy. Resistance mechanisms were investigated by determining the gene expression of FOXM1 and its targets BRCA1/2 and RAD51. Ovarian cancer tissue was successfully maintained for up to 14 days ex vivo, preserving morphological characteristics of the native specimen. Thiostrepton downregulated FOXM1 expression in tissue culture. Individual responses were observed after combined treatment with carboplatin or olaparib. Thus, we successfully implemented a complex tissue culture model to ovarian cancer and showed potential benefit of combined FOXM1 inhibition.

Regulator of Chromatin Condensation 1 (RCC1) is the only known guanine nucleotide exchange factor that acts on the Ras-like G protein Ran and plays a key role in cell cycle regulation. Although there is growing evidence to support the relationship between RCC1 and cancer, detailed pancancer analyses have not yet been performed. In this genome database study, based on The Cancer Genome Atlas, Genotype-Tissue Expression and Gene Expression Omnibus databases, the potential role of RCC1 in 33 tumors’ entities was explored. The results show that RCC1 is highly expressed in most human malignant neoplasms in contrast to healthy tissues. RCC1 expression is closely related to the prognosis of a broad variety of tumor patients. Enrichment analysis showed that some tumor-related pathways such as “cell cycle” and “RNA transport” were involved in the functional mechanism of RCC1. In particular, the conducted analysis reveals the relation of RCC1 to multiple immune checkpoint genes and suggests that the regulation of RCC1 is closely related to tumor infiltration of cancer-associated fibroblasts and CD8⁺ T cells. Coherent data demonstrate the association of RCC1 with the tumor mutation burden and microsatellite instability in various tumors. These findings provide new insights into the role of RCC1 in oncogenesis and tumor immunology in various tumors and indicate its potential as marker for therapy prognosis and targeted treatment strategies.

Purpose: Presence of tumor-associated macrophages (TAM) and high levels of ferritin and lipocalin 2 (Lcn2) in the tumor microenvironment are associated with poor prognosis in many types of cancer. Here we investigate whether iron deprivation influences TAM phenotype and chemotherapy resistance in tumor slice cultures (TSC) of gastric cancer. Results: TAM remained morphologically and functionally stable for four DIV. DFO treatment for 72 h decreased ferritin expression in TAM and in the tumor stroma but did not alter Lcn2 expression. TAM phenotype was altered after 72 h of cisplatin or DFO treatment compared with control conditions. Single DFO treatment and combined treatment with cytotoxic drugs significantly increased tumor cell apoptosis in TSC of gastric cancer. Methods: TSC were manufactured by cutting tissue of gastric cancer resection specimens in 350 μm thick slices and cultivating them under standard conditions on a filter membrane, at an air-liquid interface. After 24 h ex vivo, TSC were treated with irinotecan (100 nM) or cisplatin (10 μM) alone and in combination with deferoxamine (DFO; 10 μM, 100 μM), respectively, for 72 h. After four days in vitro (DIV) the TSC were fixated with paraformaldehyde, paraffin embedded and analyzed by immunohistochemistry for apoptosis (cPARP), proliferation (Ki67), TAM (CD68, CD163), ferritin, and Lcn2 expression. Conclusions: TAM are well preserved and can be studied in TSC of gastric cancer. Iron deprivation significantly increased tumor cell apoptosis.