3D Cell Printing of Stomach Cancer Model with Vasculature Using Stomach Decellularized ECM Bioink for Preclinical Assays

Abstract

Introduction: Stomach cancer is the second leading cause of cancer-based death worldwide. Chemotherapy is widely used for end staged cancer; however, there is a lack of clinically approved anticancer drug for stomach cancer treatment. Most of the anticancer drug candidates have failed during the preclinical trial due to the drug test models that did not successfully recapitulate native microenvironments. Therefore, development of in vivo-like cancer model is gaining increasing interest in drug discovery. Here, we developed a stomach decellularized extracellular matrix (st-dECM) bioink capable of providing optimized microenvironments conducive to growth of stomach cancer. Moreover, stomach cancer model with vasculature was fabricated using 3D cell-printing system with st-dECM bioink for drug tests.

Materials and Methods: Porcine stomach was obtained and decellularized following physical, chemical and enzymatic process. To verify the extent of decellularization, the residual DNA and ECM components were measured. To prepare the st-dECM bioink, lyophilized st-dECM was digested in a solution of 0.5M acetic acid and pepsin. Stomach cancer cell-lines were used for assessment of the effectiveness of st-dECM. Cells were laden in the st-dECM bioink for 14 days, and it was evaluated by analysis of gene expression and histological study. In addition, stomach cancer model with vasculature was fabricated using our cell-printing system. Immunohistology was conducted to verity the stomach cancer model with vasculature.

Results and Discussion: The method for printing of pre-vascularized stomach cancer model using st-dECM bioink consists of a few steps (Fig. 1A). st-dECM bioink showed appropriate viability and higher proliferation rate of stomach cancer cell-line. To verify the effectiveness of st-dECM bioink, conventional hydrogel, collagen and matrigel were designated as a control group. Interestingly, cells were aggregated in the st-dECM bioink (Fig 1B) and showed overexpression of MMP2, beta-catenin and integrin beta 1 (Fig1C). In drug resistance test, we determined the half maximal inhibitory concentration (IC50) against 5-fluorouracil (5-FU), and cells showed higher drug resistance in the st-dECM than the other hydrogel (Fig1D). To mimic the 3D stomach tissue environment, stomach cancer model with vasculature was fabricated using 3D cell printing system. Fabricated vasculature was perfusable, and HUVECs encapsulated in the printed vasculature formed an endothelium within 5 days (Fig1E). Cells showed higher drug resistance with the vasculature. Through the results of histology, gene expression, drug resistance, which are characteristics of cancer cell in vivo, the developed structure could recapitulate stomach cancer mimicking 3D environment that can provide a more relevant pre-clinical model.

Figure 1. (A) Schematic illustration of stomach cancer model with vasculature (B-D) Comparison of bioink 3D culturing GC cell-line: B. Immunohistology C. Gene expression D. IC50 value (E) Immunohistology image of cell-printed vasculature. Conclusions: We developed a st-dECM bioink, which provides an appropriate tissue-specific microenvironment to stomach cancer cells. Furthermore, the implementation of the 3D cell printing system allowed the fabrication of stomach cancer tissue and vasculature, which enabled the mimicry of in vivo environment. Our findings demonstrate the developed model can be applied to pre-clinical model for drug discovery.

Acknowledgements: This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2019R1A3A3005437)