Meng Dong

Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.

High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts.

We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the “cytokine fingerprints” identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Abstract 2223: Membranous expression of programmed cell death-ligand 1 (PDL1) on cancer cells is induced by cisplatin in an ATR dependent manner

Cancer cells frequently develop strategies allowing to escape potential immune attacks by the host. One central component of this defense mechanism is the PDL1-PD1 axis with PDL1 expression on the surface of antigen presenting cells. DNA damaging chemotherapeutic are known to have suppressive effect on the immune system. Possible effects on immune checkpoints, however, are poorly understood. We investigated if Cisplatin, a widely used chemotherapeutic for treatment of ovarian and lung carcinomas, might influence expression of PDL1 on cancer cells of these pathologies. Treatment of cell lines as well as precision-cut tissue slices from patient tumors with clinically relevant dosages of cisplatin led to a significant induction of PDL1 protein in cancer cells as revealed by Western Blot and IHC analyses. This effect was independent on ATR or ATM activity since pre-treatment with ATR or ATM inhibitors had no significant effect on cisplatin induced total PDL1 protein levels. Interestingly, however, we observed an almost complete inhibition of cisplatin-induced membranous PDL1 upon ATR but not ATM inhibition as revealed by FACS analysis in unfixed cells. The role of the ATR axis was further corroborated by the finding that presentation of PDL1 at the cell surface was paralleled by phosphorylation of CHK1. Together our data indicate an important role of DNA damage induced ATR/CHK1 activity on the localization of PDL1 at the surface of cancer cells. These data implicate that combination of cisplatin with either anti-PD1 or anti-PDL1 antibodies or ATR/CHK1 inhibitors should reduce immunosuppressive mechanisms and enhance chemotherapy efficacy. Citation Format: Lea Schaaf, Meng Dong, Simon Heine, Heiko van der Kuip, Walter E. Aulitzky. Membranous expression of programmed cell death-ligand 1 (PDL1) on cancer cells is induced by cisplatin in an ATR dependent manner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2223.

Abstract 2869: p53 response of cancer associated fibroblasts (CAFs) to cisplatin is closely correlated with the p53 response of tumor cells in lung cancer tissue cultures

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL One of the major molecular players in cellular stress responses is the p53 tumor suppressor protein. The vast majority of the research of p53 activities upon DNA damage has focused on its cell autonomous functions, i.e. its ability to induce cell cycle arrest, DNA repair, apoptosis, autophagy, or senescence in a homogenous cell population. However, data on the impact of tumor cells on p53 activation in their neighboring cancer associated fibroblasts (CAFs) and also vice versa are still limited. We investigated p53 status and responses to the p53-activating drug cisplatin both in tumor cells and in their adjacent CAFs in intact tumor tissues derived from 28 individual lung cancer patients. For this, 200µm thick patient derived tissue slices were cultivated ex vivo in presence or absence of cisplatin for 72 hours. By comparing cultivated control slices with the corresponding tumor tissue material immediately fixed after surgery (pathological routine material) we could show that morphology and p53 staining pattern were highly preserved during cultivation. TP53 mutations were detected in 16 of the 28 cases (57.1%). Independent of their p53 mutation status, the tumor samples could be divided into 3 categories according to their p53 immunostaining characteristics in the tumor cell compartment: (I) tumor cells with constitutively low p53 protein which was accumulated upon cisplatin (12 cases, 7 with mutated p53); (II) tumor cells with constitutively high p53 and no further accumulation upon cisplatin (8 cases, 7 with mutated p53); (III) tumor cells with constitutively no detectable p53 and no accumulation upon cisplatin (8 cases, 2 with mutated p53). Importantly, regardless of the p53 mutation status of the tumor cell compartment we detected a CAF specific p53 accumulation and induction of the p53 target p21 selectively in category I tumors (i.e. tumor tissue samples characterized by p53 accumulation in the tumor cell compartment). In sharp contrast, tumor tissues with no detectable p53 accumulation in tumor cells (categories II and III) showed no p53 accumulation or p21 induction in their adjacent fibroblast compartment. Furthermore, a CAF specific induction of TUNEL positive cells in cisplatin treated samples (7 of 28 cases) was selectively observed in tumor tissues characterized by a parallel induction of tumor cell death. These data for the first time show a close correlation of p53 response in tumor cells and adjacent fibroblasts in intact lung tumor tissues from patient derived material indicating that tumor cells control molecular and functional responses of their neighborhood fibroblasts upon DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2869. doi:1538-7445.AM2012-2869