(131f) Extracellular Matrix-Inspired 3D Microprinted Tumor Microenvironment Models

Background: Physical barriers formed by extracellular matrix (ECM) fibers within the tumor microenvironment (TME) significantly limit the effectiveness of emerging cancer immunotherapies, such as chimeric antigen receptor (CAR) T cell therapy. These barriers shield tumor cells from the infiltration and actions of CAR T cells. To elucidate the influence of TME architecture on T cell migration, in vitro 3D cell culture models that capture microscale ECM fiber networks are essential. Yet, current 3D models, typically created via self-assembly, lack precise control over microscale network connectivity. Furthermore, a quantitative understanding of the 3D organization of ECM networks is notably lacking. Building upon our prior work on 3D microprinted cell scaffolds, we introduce a nature-inspired engineering approach that combines graph theory-driven design with high-resolution 3D printing to recapitulate intricate TME structures [1–3].

Methods: We employed second harmonic generation microscopy for label-free imaging of tumor-associated collagen structures from in vivo BxPC3 tumor models. Network topology and morphology were analyzed using a bespoke MATLAB application. By conceptualizing the networks as nodes connected by edges, we utilized graph theory to quantify their clustering coefficients and path lengths. These measures enabled us to assess the extent of “small-world” characteristics – high clustering and low path length – which are critical for efficient intercellular communication and prevalent in a variety of biological networks[4]. Additionally, we quantified the anisotropy, widths and lengths of collagen fibers. The extracted information was then used to algorithmically generate 3D models, which were 3D microprinted via two-photon polymerization (2PP).

Results: Tumor samples exhibited both intra- and inter-tumor heterogeneities in network organization. Some regions possessed a pronounced small-world topology, which may facilitate more efficient T cell navigation. However, others demonstrated considerably weaker “small-worldness”. Skeletalization of the networks for analysis initially produced artefacts, where nodes were connected solely to two adjacent nodes. We refined the network extraction process by removing these artefact nodes and reestablishing connections between neighbors. Using 2PP, ECM-inspired spatial networks were successfully physicalized using both acrylic and gelatin hydrogel-based resins.

Conclusions: Beyond the common focus on morphological analysis, our approach leverages graph theory to elucidate a more profound, mathematical understanding of the physical ECM wiring, thereby contributing to the design of complex cell scaffolds. Furthermore, our work facilitates comparison with other biological networks known to influence T cell migration, such as fibroblast networks within lymph nodes and tumors. We envision that this platform would enable the investigation of T cell migration in complex TMEs, which holds the potential to inspire new strategies for microenvironment engineering and cell delivery methods to improve cancer immunotherapies.

References:

[1] M.-O. Coppens, Nature-Inspired Chemical Engineering for Process Intensification, Annu Rev Chem Biomol Eng 12 (2021) 187–215. https://doi.org/10.1146/annurev-chembioeng-060718-030249.

[2] E.C. Goldfield, M.-O. Coppens, Developmental bioengineering: recapitulating development for repair, Mol Syst Des Eng 5 (2020) 1168–1180. https://doi.org/10.1039/D0ME00062K.

[3] M.H.W. Chin, B. Reid, V. Lachina, S.E. Acton, M.O. Coppens, Bioinspired 3D microprinted cell scaffolds: Integration of graph theory to recapitulate complex network wiring in lymph nodes, Biotechnol J 19 (2024) 2300359. https://doi.org/10.1002/BIOT.202300359.

[4] M. Gosak, M. Milojević, M. Duh, K. Skok, M. Perc, Networks behind the morphology and structural design of living systems, Phys Life Rev 41 (2022) 1–21. https://doi.org/10.1016/J.PLREV.2022.03.001.