The orange carotenoid protein (OCP) plays an important role in photoprotection in cyanobacteria, which is achieved by the photoconversion from the orange dark state (OCP) to the red active state (OCP). Using Raman optical activity (ROA), we studied the conformations of the carotenoid chromophore in the active sites of OCP and OCP. This ROA measurement directly observed the chromophore conformation of native OCP in solution, and the measurement of OCP first demonstrated the ROA spectroscopy for the transient species. For OCP, the spectral features of ROA were mostly reproduced by the quantum chemical calculation based on the crystal structure of the OCP. Within the spatial resolution (∼2 Å), a slight modification of the polyene-chain distortion improved the agreement between the observed and calculated ROA spectra. While the crystal structure of OCP is not available, the ROA spectrum of OCP was reproduced by using the crystal structure of red carotenoid protein (RCP), an OCP proxy. The present results showed that the chromophore conformations in the crystal structures of OCP and RCP hold true for OCP and OCP in solution. Particularly, ROA spectroscopy of the native OCP provides a direct support for the 12 Å translocation of chromophore in the photoactivation, which was proposed by X-ray crystallography using RCP [R. L. Leverenz, M. Sutter, et al. Science 2015, 348, 1463-1466].

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune–cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune–cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune–cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic-flow based 3D bioprinted multi-scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre-defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose-dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8 + T cells for 24 or 72 h. Extensive CAR-T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically-relevant tumor model for future translation of anti-cancer therapies to personalized medicine.

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.