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Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics., The mechanics of the migration of T cells into tumours is an important aspect of tumour immunity. Here the authors engineer complex 3D environments to explore functions of microtubules and cell contractility as strategies to enhance T cell migration in tumour microenvironments.

While chemical signals play a strong role in drawing T cells into solid tumors, the physical features of the stroma, such as architecture and mechanics, also strongly influence T cell infiltration as well as their ability to effectively distribute throughout, and sample, the entire tumor volume. Indeed, the mechanically and chemically complex stromal reaction in solid tumors can limit access and effective distribution of T cells creating antitumor immunity-free sanctuaries. Furthermore, many solid tumors are rich with aligned extracellular matrix (ECM) networks, which provide contact guidance for carcinoma cells, but can also direct migration of infiltrated T cells within solid tumors. Yet, our understanding of how native and engineered T cells migrate through mechanically complex tumor microenvironments (TMEs) is quite incomplete. As such, defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding sanctuaries from antitumor immunity and for optimizing T cell-related therapeutic strategies. In order to decipher and enhance migration of T cells through complex microenvironments, we initially engineered nanotextured elastic platforms that mimic ECM architectures observed in breast and pancreatic carcinomas. These platforms allowed us to define how the balance between contractility localization-dependent T cell phenotypes (i.e. high cortical contractility distributed along the curved cell membrane versus forces imparted onto substrates) influences migration in response to tumor-mimetic mechanical and structural and cues. From this information we characterized a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and contractile force generation. In three-dimensional (3D) environments and live pancreatic tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration while clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. Indeed, we show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering with CRISPR results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. This suggests that T cell engineering to generate “mechanically optimized” therapeutic cells that better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics. Citation Format: Erdem Tabdanov, Nelson Rodriguez-Merced, Alexander Cartagena-Rivera, Vikram Puram, Mackenzie Callaway, Ethan Ensminger, Emily Pomeroy, Kenta Yamamoto, Walker Lahr, Beau Webber, Branden Moriarity, Alexander Zhovmer, Paolo Provenzano. Engineering T cells to enhance 3D migration through mechanically and structurally complex tumor microenvironments [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO010.

The paracrine interaction between tumor cells and adjacent stroma has been associated with the oncogenic activity of the Hedgehog (Hh) pathway in triple-negative breast tumors. The present study developed a model of paracrine Hh signaling and examined the impact of mesenchymal cell sources and culture modalities in the oncogenicity of the Hh pathway in breast tumor cells. Studies consisted of tumor cell monocultures and co-cultures with cancer-associated and normal fibroblasts, tumor cells that undergo epithelial&ndash, mesenchymal transition (EMT), or adipose-derived mesenchymal stem cells (ADMSCs). Hh ligand and pathway inhibitors, GANT61 and NVP-LDE225 (NVP), were evaluated in both cell cultures and a mouse xenograft model. Results in monocultures show that tumor cell viability and Hh transcriptional activity were not affected by Hh inhibitors. In co-cultures, down-regulation of GLI1, SMO, and PTCH1 in the stroma correlated with reduced tumor growth rates in xenografted tumors and cell cultures, confirming a paracrine interaction. Fibroblasts and EMT cells supported Hh transcriptional activity and enhanced tumor cell growth. Mixed and adjacent culture modalities indicate that tumor growth is supported via fibroblast-secreted soluble factors, whereas enriched tumor stemness requires close proximity between tumor and fibroblasts. Overall this study provides a tumor&ndash, mesenchymal model of Hh signaling and highlights the therapeutic value of mesenchymal cells in the oncogenic activity of the Hh pathway.