(I-322) A Tissue-Engineered Model of Anisotropy at The Post-Surgical Tumor Site

Patients with early-stage lung cancer often undergo surgical resection to remove the primary tumor, causing a scarring response by the fibroblasts as a part of the normal wound-healing process. This process alters the extracellular matrix (ECM) greatly, introducing higher concentrations of collagen and thereby increasing tissue stiffness. The linear nature of the tumor removal causes this stiffened scar tissue to occur along an aligned axis. Aligned tissue affects cell shape, migration, and directionality. The activation of fibroblasts and the severe alteration of the ECM makeup is similar to the process by which cancer-associated fibroblasts (CAF) cultivate the tumor microenvironment (TME) for carcinoma cells. Due to this TME-like environment, cancer cells disseminated from the primary tumor can cause local recurrence. Utilizing highly active patient derived CAF, we modeled a local recurrence at the resection site to test the effect of aligned tissue on the migration of circulating cancer cells. Here we tested the hypothesis that the anisotropic tissues promote increased migration of cancer cells.

The characterization of HLF and CAF in 2D showed upregulation of TGFb, IL6, IL8 inflammatory genes and upregulation in CAF specific genes DKK3 and COL11a1 (Figure 1A) confirming CAF as the more aggressive, activated cell type. The characterization was similar in 3D (Figure 1B) showing the CAF upregulating TGFb, IL6, and IL8 as compared to HLF as well as upregulation in COL1a1 and VEGFA, integral ECM and angiogenic transcription factors, respectively. A549 co-cultures in 3D caused upregulation in inflammatory and angiogenic genes and downregulation of ECM transcription factors. The profibrotic nature displayed by these cells in 2D and 3D configurations confirm their validity as inputs to form an in vitro TME that mirrors native tissue.  These results establish the foundation for translating the baseline isotropic TME configuration to a post-surgical recurrence model that captures scarring anisotropy. A proof-of-principle test was performed by placing carcinoma cell spheroids within sculpted anisotropic tissues to test the hypothesis that embedded spheroids would alter growth and migration patterns due to an anisotropic TME. Immunochemistry analysis (Figure 1D) confirmed distinct morphological and migratory patterns of the spheroids (denoted by white dashed lines), which are not commonly observed in isotropic TMEs of numerous other models in our lab. Future investigations will systematically explore various parameters of carcinoma cell aggressiveness in anisotropic versus isotropic tissues. These parameters include the proliferation index, gene expression patterns using previously established panels, morphological analysis(elongation, alignment with ECM, cytoskeleton), and metrics of single cell and collective cell migration (invasion). Furthermore, the study will assess collective migration of the original spheroids and their adherence to patterns of anisotropy as a function of input tumor cell phenotype.

 The significance of this work lies in future screening approaches to locally deliver combination therapies for prophylactic treatment of recurrent metastasis at post-surgical sites that target mechanical activation of residual cells. Anti-metastasis drugs such as Rho kinase inhibitors have a high failure rate in translation from cell culture models to human subjects. Therefore, a model mimicking anisotropic, stiff tissues with aggressive cellular inputs is necessary for further mechanistic investigation and efficacy screening.