Stefan Koeck

Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells

Introduction We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Methods Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. Results Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. Conclusion We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

Development of a 3D angiogenesis model to study tumour – endothelial cell interactions and the effects of anti-angiogenic drugs

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling “signet ring cells” was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers

INTRODUCTION The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. METHODS 3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. RESULTS Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. CONCLUSION In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations

ABSTRACT The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Abstract 326: Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures

Introduction: Increasing efforts to integrate tumour-stroma interaction and 3-dimensional structures in innovative in-vitro models were made within the last years. We established a 3D co-culture system in non-small-cell lung cancer applicable for drug testing. In this work, we present data on the differences in RNA expression and drug sensitivity in the 2D and 3D culture system. Methods: A549 and Colo699 NSCLC cell lines were both cultivated in a 2D standard cell culture (flasks, PAA, Germany) and a 3D 96 well plate hanging drop system (GravityPlusTM, Insphero, Switzerland) for 5 and 10 days, respectively. 3D microtissues were cultured either in mono- or in co-cultures with the human lung fibroblast cell line SV80. RNA expression profiling via Affymetrix chip analyses was performed with 3 biological replicates per cell line (A549, Colo699 and SV80) in mono- and co-cultures. For this purpose, RNA was isolated by a TriReagent standard protocol. Cytotoxicity was determined for cisplatin and vinorelbine after 24 hours of incubation by ATP measurements of lysed microtissues/cells and modified AnnexinV/Propidium iodide staining for flow cytometry on Colo699 cells. Furthermore, standard flow cytometry cell cycle analyses and microscopic volume measurements were performed to analyse drug induced inhibition of proliferation. Results: RNA expression profiles revealed within the A549 monocultures 892 and the Colo699 monocultures 1,042 regulated genes (≥ two-fold change in expression) for 2D versus 3D cultures, respectively. Between the cell lines A549 and Colo699 only a limited overlap (131 genes) in differentially expressed genes was detected. However, significant differences in gene expression were experienced in co-cultures with SV80. The most frequently altered genes are involved in the regulation of the cell cycle, organelle fission, RNA polymerases, histone methylation, DNA repair, oxidative stress and WNT signalling pathways. To prove the potential in-vivo relevance of these findings, we assessed the chemosensitivity in the Colo699 for 2D and 3D cell cultures. A significant difference in LD50 could be observed for cisplatin between 2D (100μM) and 3D (250μM) cultures in both ATP release measurements and AnnexinV/PI flow cytometry analyses. In cell cycle analyses the maximum M arrest for vinorelbine was achieved with 25μM in 2D, whereas in 3D 100μM of vinorelbine were needed. Cytotoxicity assays did not reveal a significant difference for vinorelbine in 2D versus 3D models. Conclusion: Herein, we demonstrate that cultivation of cell lines either in a 2D or 3D environment lead not only to a difference in RNA expression but also in chemosensitivity. Thereby, these data support the urgent need of more in vivo like cell culture models for analysing drug related modes of action. Citation Format: Gabriele Gamerith, Johannes Rainer, Arno Amann, Stefan Koeck, Edith Lorenz, Heinz Zwierzina, Julia M. Huber. Differences in RNA expression and chemosensitivity in 2D versus 3D non-small-cell lung cancer cultures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2015-326

3D-cultivation of NSCLC cell lines induce gene expression alterations of key cancer-associated pathways and mimic in-vivo conditions

This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.

Abstract 4252: Establishment of a multicellular 3D cell culture model for tumor - endothelial cell interaction

Introduction: Due to the increasing understanding of the mechanisms relevant to the genesis of cancer, we are experiencing a transition from disease to target-oriented therapy. One major hurdle for the development of these targeted therapeutic regimens, however, is the limited availability of predictive in vitro models. The critical challenge is to develop culture models better reflecting in vivo conditions. We present data that highlights the differences of RNA expression of in vivo like 3D microtissues consisting of tumour cells, fibroblasts and two different endothelial cell lines compared to normal 2D cell culture conditions. Methods: 96-well hanging drop microtiter plates (InSphero AG, Zurich, Switzerland) were applied for the production of 3D mono-, co- and tri-cultures including the human lung cancer cell lines A549 or Colo699 alone or in combination with a human lung fibroblast cell line (SV-80) and either a human umbilical vein endothelial cell line (HUVEC) or the primary human lung microvascular endothelial cell line (HMVEC-L). In addition, to conventional histology (HE 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4252.

Infiltration of immune cells into cancer cell/stroma cell 3D microtissues

Meeting abstracts The pharmacological manipulation of the interaction between the immune system, fibroblasts and cancer cells has been proven to be a key target for cancer therapy. Based on our previously developed co-culture system, this work aims to develop an innovative 3D cell culture system

Abstract 316: Development of a novel 3D cancer model by cultivation of malignant effusions in a 3D cell culture system

Introduction: Metastatic spread and the occurrence of tumour cell resistance to therapeutic interventions can be induced by different molecular processes. Among those, tumour stroma interactions play a crucial role. So far only few cell culture models exist to study these cell processes. Therefore, we describe the development of a hanging drop technology for 3D culture of primary cells isolated from malignant effusion obtained from cancer patients. Methods: A 96-well hanging drop microtiter plate (InSphero AG, Zurich, Switzerland) was applied for the 3D cultivation of cells. Primary cells were isolated either from malignant ascites or pleura effusion of cancer patients. Cells were seeded either into 2D cell culture plates or into hanging drops for an incubation period of ten days. Apart from conventional histology (HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 316. doi:10.1158/1538-7445.AM2015-316

Abstract 3840: Development of an innovative 3D cell culture system to study tumor-stroma interactions in cancer cells.

Introduction: We describe a novel 3D co-culture method using non-small cell lung cancer (NSCLC) cell lines in combination with a lung fibroblast cell line in the hanging drop technology. This method allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Compared to other 3D models, it better reflects tumour growth in its microenvironment since no addition of growth factors or artificial extracellular matrix (ECM) substitutes are required. Methods: 96-well hanging drop microtiter plates (InSphero AG, Zurich, Switzerland) were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid aggregation was displayed either by scanning electron microscope, semi-thin sections, immunofluorescence or by immunohistochemistry (IHC). In addition to conventional histology (HE 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2013-3840