Associate Scientist Sanford Research / University of South Dakota School of Medicine Sioux Falls, South Dakota, United States
Introduction: Ovarian carcinoma (OC) is one of the deadliest forms of cancer in women. Patients with OC are treated with chemotherapy but over 80% of these patients develop resistance to treatment within 5 years. Chemoresistance in OC has been associated with the dysregulation of the tumor extracellular matrix (ECM) composition and structure which critically contributes to a cancer cell’s ability to resist treatment, ultimately leading to over 90% of deaths from cancer. Tumor ECM is a non-cellular network secreted by cells, made up of several diverse proteins, that are biochemically and biomechanically distinct in their composition compared to normal tissue ECM. Highlighting its clinical significance, ECM remodeling signatures in OC are associated with an increased risk of treatment failure and patient mortality. The effects of ECM composition and structure on therapeutic response have been well explored, however, the bidirectional effects of ECM changes associated with chemotherapy remain elusive. There is, therefore, a critical need for technologies to determine spatiotemporal dynamics of newly synthesized ECM in response to chemotherapy in OC.
Our overall objective is to accelerate the development and functional characterization of cancer biomimetic tissue-engineered technologies that are necessary for profiling spatiotemporal ECM dynamics in response to chemotherapy on a single-cell basis using metabolic labeling. In the absence of these models, the development of effective anticancer therapies that mitigate chemotherapy resistance in OC will likely remain difficult.
Materials and
Methods: A cancer biomimetic tissue-engineered technology known as the 3D extracellular matrix (3D-ECM) was developed through the chemical crosslinking of a hybrid hydrogel formed from ECM-based collagen I and fibrinogen ensuring viscoelastic and fibrillar microstructure which are critical for 3D mechanosignaling. OC cell lines (Kuramochi and SKOV3) were incubated with cancer-associated fibroblast (CAFs) and co-cultured for 7 days in the absence or presence of cisplatin treatment (5-10uM). Using our 3D-ECM model, the evolution of newly synthesized or nascent ECM over time in response to chemotherapy was visualized through bio-orthogonal chemistry where a non-canonical amino acid (azidohomoalanine, AHA) is incorporated into all deposited nascent ECM components and fluorescently labeled via copper-free cycloaddition with dibenzocyclooctyne-amine (DBCO) where it could be imaged, and results quantified (Fig A). Co-stains were performed to label known ECM proteins. Proteomic analysis of pre-enriched nascent ECM was performed by liquid chromatography-tandem mass spectrometry. Gene ontology analysis of exclusive AHA-proteins or differentially expressed proteins was performed. Additionally, cell-ECM interactions via mechanosignaling through ECM-dependent integrin activation were assessed by visualization and quantification of the phosphorylation of focal adhesion kinase (pFAK). Image-based spatiotemporal ECM dynamics quantification was performed at single cell resolution for nascent ECM and FAK expression using predefined and optimized pipelines with the Cell Profiler software, where at least 100 cells were profiled. High-throughput ECM targeting drug screening was also performed in order to identify whether a platinum resistant cell line could be re-sensitized to platinum therapy.
Results, Conclusions, and Discussions: The 3D-ECM model was engineered to recapitulate key OC parameters such as the cell-cell interactions between the cancer and stroma cell types and high collagen type I content with associated increased stiffness, which are essential for recapitulation of the tumor and its response to treatment. Bio-orthogonal metabolic labeling of the 3D-ECM model revealed that nascent ECM generation was significantly increased in response to cisplatin treatment compared to PBS control, this was quantified using higher intensity signal and major projection from cell, and also increases further over time (Fig. B). Acknowledging that metabolic labeling will mark any protein, we coupled metabolic labeling with immunofluorescence staining to confirm the presence of known ECM proteins involved in chemoresistance including collagen, laminin and fibronectin (Fig. C). Proteomics analysis of enriched nascent ECM utilized an approach where a cleavable biotin-alkyne linker and NeutrAvidin beads were used to selectively enrich for AHA -labeled nascent proteins (Fig D). These studies indicated significant upregulation of ECM biological process terms after cisplatin treatment (Fig E). Additionally, pFAK expression per cell was also shown to be significantly higher after cisplatin treatment compared to the PBS control in several OC cell lines within the 3D-ECM model (Fig F). Finally, we identified that ECM targeting in combination with cisplatin restores the efficacy of cisplatin, overcoming platinum resistance. While the platinum resistant cell line shows a 5-fold increase in the half-maximal inhibitory concentration (IC50) when treated with cisplatin alone, cisplatin in combination with an ECM targeting agent reveled total re-sensitization to the level of the parental platinum sensitive cell line (Figure G). Ultimately, these results suggest that chemotherapy itself induces changes in the ECM, which could further be linked to increased chemoresistance.
In conclusion, our results present a functionally characterized 3D-ECM model enabling a spatiotemporal profile of ECM dynamics in response to chemotherapy on a single-cell basis. These results will have a significant positive impact by improving chemotherapy effectiveness and subduing chemoresistance while providing strong-evidence for further development of therapeutics that are capably of targeting chemotherapy-induced ECM changes which could prove to be the best therapeutic window of opportunity in ovarian cancer.
Acknowledgements (Optional): This project was supported by 1P30GM145398-02
