Background The mechanisms underlying the spatiotemporal development of the tumor microenvironment (TME) present a challenge in evaluating drug efficacy and the underlying molecular mechanisms of tumor biology. 3D culture systems are an indispensable tool to delineate the dynamic interplay between the tumor and the host microenvironment.

Methods Four human PDX (patient derived xenograft) models, LXFA923, LXFA289, LXFL1121 and LXFL1674, and two murine, KP1 and KP4, non-small cell lung cancer (NSCLC) models were cultivated under different conditions in 3D: The co-cultivation with matching fibroblasts as well as immune cells was evaluated in a 96-well format. Tumor spheroid growth and immune cell infiltration (monocytes, NK & T cells) was followed over time via live cell imaging, light & confocal microscopy. The cross-talk between the different cell types was evaluated by analyzing the different cytokine profiles across the settings. At the end of the study spheroids underwent histological and immunohistochemical examination.

Results The four human NSCLC lines depicted very specific growth characteristics and displayed distinct infiltration patterns for monocytes, NK- or T cells. In general, the addition of fibroblasts in the human setting increased the infiltration rate of the immune cells and altered the invasiveness of the tumor cells into the surrounding matrix. Tumor lines with a high EMT score (based on RNAseq data) grew more invasive and displayed a cytokine pattern that supports the infiltration of monocytes as well as NK cells. This increased infiltration rate of immune cells was confirmed longitudinal by live cell imaging and at endpoint by confocal imaging. Analyzing the cytokine patterns across different tumor lines and co-culture settings by principal component analysis it became evident that the most prominent discriminator is the tumor line itself.

The murine cell lines KP1 and KP4 were derived from a genetic engineered mouse model KP but displayed differences in the expression of EPCAM, being KP1 EPCAM+ and KP4 EPCAM- and in the tumor mutational burden is higher in KP1 as compared KP4. These two characteristics aligned with the fact that the infiltration rate of monocytes in the KP1 was augmented, which was due to the increased levels of GM-CSF and IL-6 in the triple culture of KP1, BM-derived monocytes and murine lung fibroblasts.

Conclusions Taken together, the molecular and phenotypic profile of the tumor shapes the interaction with the TME essentially. However, the non-tumorigenic cells do also influence the biology of the tumor cells regarding invasiveness and tumor growth rate.