Abstract 1000P
Background
Dissecting the interaction of tumor cells with immune cells is essential to decipher the mechanism of immunotherapies. With our immune-competent 3D InSightTM tumor models we propose optimized in vivo-like conditions over standard cell cultures to assess how immune cells elicit effective responses against cancer. Moreover, we present a live-cell imaging approach to assess the efficacy of immune-targeting therapies with high-content imaging and analysis (HCA) techniques.
Methods
The lung carcinoma 3D in vitro model was generated using GFP-A549 cells in coculture with dermal fibroblasts and PBMCs in Akura™ plates. PBMCs were previously stimulated either with cytokines or anti-CD3/CD28 antibodies. Using the Yokogawa CQ1 HCA instrument, we monitored the immune cell attack on tumors in real-time and in 3 dimensions without any discernible phototoxicity. Tumor killing activity and T-cell effector function were evaluated by measuring tumor size by automatic stage fluorescence microscopy (Leica Dmi8) or confocal HCA (Yokogawa CQ1). Release of inflammatory cytokines IL-6, TNF, IFNγ and GM-CSF was measured by a multiplex assay (MAGPIX™ Luminex system). Finally, we calculated tumor and immune cell volumes from individual image stacks to evaluate tumor killing and proliferation/infiltration of immune cells. Furthermore, beside modulating the adaptive immune system through T-cell activation, we also integrated our immune-competent 3D tumors with myeloid cells to recapitulate the role of the innate immune system.
Results
Our real time confocal analysis of tumor-immune cell cocultures shows that activation of PBMCs with cytokines or CD3/CD28 antibodies drives T-cell-mediated tumor cell killing.
Conclusions
Our results demonstrate how this assay platform may be used for I-O studies with engineered T-cells (e.g., CAR-T) or immunomodulatory agents, e.g. bispecific antibodies and immune checkpoint inhibitors. Thanks to the integration of innate immune cells into our immune-competent 3D tumor models, we can also employ this system to reprogram immunosuppressive myeloid cells and unravel mechanisms of innate resistance to cancer immunotherapies.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
F. Chiovaro.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.