3D Bioprinted perfusable and vascularized breast tumor model for dynamic screening of chemotherapeutics and CAR-T cells

Abstract

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the rapid and early screening of anti-cancer agents. A major barrier in this endeavor, is the complex interplay between the tumor microenvironment and host immune response and lack of predictive biomarkers for clinical benefit. To tackle this challenge, we have developed a dynamic-flow based three-dimensionally (3D) bioprinted vascularized breast tumor model, responding to chemo and immunotherapeutic treatments. Heterotypic tumor spheroids, comprising metastatic breast cancer cells (MDA-MB-231), human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), precisely bioprinted at pre-defined distances from a perfused vasculature, exhibited tumor angiogenesis and cancer invasion. Proximally bioprinted tumors (~100 m) exhibited enhanced capillary sprouting, anastomosis to perfused vasculature and increased cancer cell migration as compared to distally bioprinted spheroids (~500 m). Proximally bioprinted tumors treated with varying dosages of doxorubicin for 72 h enabled functional analysis of drug response, wherein, tumors portrayed a dose-dependent drug response behavior with ~70% decrease in tumor volume for 1 M dose. Additionally, a cell based immune therapy approach was explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8+ T cells for 24 or 72 h through the central vasculature. Extensive CAR-T cell recruitment to the endothelium and substantial T cell activation and infiltration in the tumor site, resulted in ~70% reduction in tumor growth for high CAR treatment densities, after 72 h of treatment. The presented 3D model paves the way for a robust, precisely fabricated and physiologically-relevant 3D tumor microenvironment platform for future translation of anti-cancer therapies to personalized medicine for cancer patients.

Vascularization Strategies in 3D Cell Culture Models: From Scaffold-Free Models to 3D Bioprinting

“…However, even with the fabrication of a biomimetic 3D architecture, the lack of fluid flow perfusion limits 3D cell culturing models in the accurate simulation of the dynamic native tissue microenvironment. Dey et al addressed this limitation by devising a novel dynamically flow-based 3D vascularized breast tumor model with drug screening capabilities [ 90 ]. The model involved the aspiration-assisted bioprinting of heterotypic tumor spheroids, a composite collagen/fibrin-based matrix (C2F3) as the bioprinting substrate, central vasculature, and a perfusion chamber with connection ports.…”

Engineered Microphysiological Systems for Testing Effectiveness of Cell-Based Cancer Immunotherapies

“…For example, both lymphocytes and tumor spheroids can be combined inside hanging drops, such as a study which used this technique to assess the cytotoxicity of CAR T cells recognizing the human epidermal growth factor receptor 2 (HER2) in breast cancer [ 35 , 39 ]. Oil and water immersion microfluidic techniques can also be used to combine cancer cells with lymphocytes within droplets in order to image cytotoxicity or perform small-volume cytokine sampling [ 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ].…”

“…Bioprinting can also be used to generate 3D cancer models, with more controllable architecture than organoid models, but generally lacking the dense stromal structure and heterogeneity of a patient tumor [ 45 ]. Many bioprinting approaches exist, such as inkjet, extrusion, acoustic, and laser photopolymerization methods [ 48 ]. For cell therapy, bioprinting has been used to improve T cell expansion and function using alginate and alginate–gelatin scaffolds to mimic lymph vessels, resulting in the differentiation of CD4+ cells into the central memory type and differentiation of CD8+ cells into effector memory type [ 45 ].…”

“…Bioprinted tumor spheroids have been used to test infiltration and efficacy of novel CAR T cells, such as a study in neuroblastoma testing CAR T cells recognizing the L1 cell adhesion molecule [ 47 ]. The controllable geometry made possible by bioprinting also allows for the creation of multiple structures, such as models that print both large-scale vascular channels and tumor spheroids [ 48 ].…”

“…These models typically place tumor spheroids in microwells to image the infiltration and cytotoxicity of lymphocytes [ 41 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ]. Other studies encapsulate tumor spheroids in hydrogels such as collagen or Matrigel, either embedding the lymphocytes or applying them to the surface of the hydrogel [ 29 , 48 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ]. The advantage of performing these studies in 3D is that lymphocytes must actively migrate through the ECM toward and into the tumor spheroids in order to exert cytotoxic effects, rather than the passive interactions that occur in traditional cytotoxicity assays in which lymphocytes settle onto a 2D cancer cell monolayer.…”