Abstract 2027: Novel 3D tumor-immune cell spheroid models for assessment of cancer immunotherapy agents
Authors: Morra Laura , Waldhauer Inja , Klein Christian , Umana Pablo , Bacac Marina 
Published: May 2015

Abstract
The pre-clinical development of anticancer immunotherapeutics is based on 2D co-culture systems that rely on use of allogeneic immune effector cells and established tumor cell lines. These are, however, suboptimal models unable to resemble the complex histopathological, morphological and immunosuppressive features present within tumor microenvironment and devoid of nutritional, metabolic and oxygen gradients established in primary tumors. In addition, when maintained in 2D, tumor cells undergo exponential growth and lack formation of proliferating, quiescent and necrotic cores established in tumors. Due to absence of cell polarity, the level and pattern of tumor antigen expression is different between 2D and 3D cultures and interactions between tumor cells and immune cells in 2D sub optimally represent those occurring in vivo.
To overcome these limitations, we established novel types of 3D models that more closely mimic the histological complexity of tumors and better resemble cell-cell, cell-matrix and tumor-stroma interactions, i.e. 1) the heterotypic 3D spheroids composed of tumor cells, fibroblasts, endothelial cells, matrix components and immune effector cells, 2) 3D ex vivo thick tumor sections offering an intact tumor microenvironment with autologous tumor-infiltrating lymphocytes (TILs). In the first model, lymphocytes were either admixed with tumor cells, fibroblasts, endothelial cells and matrix components (collagen, Matrigel or both) giving rise to heterotypic 3D co-cultures containing low levels of immune effector cells (resembling poorly-infiltrated tumors), or were stimulated with low dose of antibody-conjugated immunocytokine (DP47-IL2v, 10 nM, overnight), which promoted their infiltration into pre-formed tumor/fibroblast spheroids (resembling highly-infiltrated tumors). In the second model, primary tumors collected from naïve or DP47-IL2v-treated mice were sliced ex vivo to obtain 400 μm-thick tumor sections that were maintained in culture for up to 7 days. 3D models were characterized by quantification of size and IHC staining of tumor, stromal and endothelial cell antigens and immune effector cells.
We further validated our models by testing the activity of tumor- or stroma-targeted immunotherapies. Incubation of spheroids with 50 or 100 nM of tumor-targeted T Cell Bispecific (TCB) antibody led to crosslinking of T cells to tumor cells resulting in elimination of tumor areas (but not of the fibroblast core) and overall reduction in spheroid size. On the contrary, spheroids incubated with untargeted TCB remained intact.
Taken together, the novel models presented here provide the first example of heterotypic 3D microtissues containing the immune effector cells and offer a versatile system to characterize the activity of tumor- and stroma-targeted immunotherapies and explore their combinations.
Citation Format: Laura Morra, Inja Waldhauer, Christian Klein, Pablo Umana, Marina Bacac. Novel 3D tumor-immune cell spheroid models for assessment of cancer immunotherapy agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2014-2027