Modular micro-physiological human tumor/tissue models based on decellularized tissue for improved preclinical testing

High attrition rates associated with drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies, our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates, and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model, finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia or toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model, we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments.

Drug induced micellization into ultra-high capacity and stable curcumin nanoformulations: Physico-chemical characterization and evaluation in 2D and 3D in vitro models.

Curcumin (CUR) is a natural extract from the plant Curcuma longa and part of turmeric, a spice and herbal remedy in traditional medicine. Thousands of papers claim a plethora of health benefits by CUR, but a growing number of reports and contributions caution that many experimental data may be artifacts or outright deny any suitability of CUR due to its problematic physicochemical properties. Two major issues often encountered with CUR are its extraordinarily low solubility in water and its limited chemical stability. Here, we report on a novel nanoformulation of CUR that enables CUR concentrations in water of at least 50 g/L with relative drug loadings of >50 wt% and high dose efficacy testing in 3D tumor models. Despite this high loading and concentration, the CUR nanoformulation comprises polymer-drug aggregates with a size <50 nm. Most interestingly, this is achieved using an amphiphilic block copolymer, that by itself does not form micelles due to its limited hydrophilic/lipophilic contrast. The ultra-high loaded nanoformulations exhibit a very good stability, reproducibility and redispersibility. In order to test effects of CUR in conditions closer to an in vivo situation, we utilized a 3D tumor test system based on a biological decellularized tissue matrix that better correlates to clinical results concerning drug testing. We found that in comparison to 2D culture, the invasively growing breast cancer cell line MDA-MB-231 requires high concentrations of CUR for tumor cell eradication in 3D. In addition, we supplemented a 3D colorectal cancer model of the malignant cell line SW480 with fibroblasts and observed also in this invasive tumor model with stroma components a decreased tumor cell growth after CUR application accompanied by a loss of cell-cell contacts within tumor cell clusters. In a flow bioreactor simulating cancer cell dissemination, nanoformulated CUR prevented SW480 cells from adhering to a collagen scaffold, suggesting an anti-metastatic potential of CUR. This offers a rationale that the presented ultra-high CUR-loaded nanoformulation may be considered a tool to harness the full therapeutic potential of CUR.

ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models.

Solid tumors impose immunologic and physical barriers to the efficacy of chimeric antigen receptor (CAR) T cell therapy that are not reflected in conventional preclinical testing against singularized tumor cells in 2-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors and evaluated the antitumor function of receptor tyrosine kinase-like orphan receptor 1-specific (ROR1-specific) CAR T cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic > static; MDA-MB-231 > A549). Treatment with ROR1-CAR T cells conferred potent antitumor effects. In dynamic culture, CAR T cells actively entered arterial medium flow and adhered to and infiltrated the tumor mass. ROR1-CAR T cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized, scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the antitumor function of CAR T cells and to obtain proof of concept for their safety and efficacy before clinical application.