3D bioprinting and the revolution in experimental cancer model systems-A review of developing new models and experiences with in vitro 3D bioprinted breast cancer tissue-mimetic structures

Abstract

Growing evidence propagates those alternative technologies (relevant human cell-based-e.g., organ-on-chips or biofabricated models-or artificial intelligence-combined technologies) that could help in vitro test and predict human response and toxicity in medical research more accurately. In vitro disease model developments have great efforts to create and serve the need of reducing and replacing animal experiments and establishing human cell-based in vitro test systems for research use, innovations, and drug tests. We need human cell-based test systems for disease models and experimental cancer research; therefore, in vitro three-dimensional (3D) models have a renaissance, and the rediscovery and development of these technologies are growing ever faster. This recent paper summarises the early history of cell biology/cellular pathology, cell-, tissue culturing, and cancer research models. In addition, we highlight the results of the increasing use of 3D model systems and the 3D bioprinted/biofabricated model developments. Moreover, we present our newly established 3D bioprinted luminal B type breast cancer model system, and the advantages of in vitro 3D models, especially the bioprinted ones. Based on our results and the reviewed developments of in vitro breast cancer models, the heterogeneity and the real in vivo situation of cancer tissues can be represented better by using 3D bioprinted, biofabricated models. However, standardising the 3D bioprinting methods is necessary for future applications in different high-throughput drug tests and patient-derived tumour models. Applying these standardised new models can lead to the point that cancer drug developments will be more successful, efficient, and consequently cost-effective in the near future.

Keywords: 3D bioprinting; biofabrication; breast cancer; cancer; disease models.

FIGURE 1

Timeline of cell biology and experimental cancer models—From complexity…to simplicity…and complexity again. The three research areas—in vitro cell- and tissue-culturing, organoid technology and 3D bioprinting—are developing, and their co-evolution with cancer research supports the establishment of new cancer models. A detailed explanation can be found in the text.

FIGURE 2

“All models are wrong but some are useful”—Advantages and disadvantages of different cancer models.

FIGURE 3

The number and distribution of cancer research papers regarding 3D bioprinting. (A) The number of research papers between 2011 and 2021 mentioning cancer models combined with 2D cell culture/organoid/3D bioprinting. The data show that the number of publications using 2D cell culture models is stagnating. Additionally, the organoid research area has increased faster for the last years and 3D bioprinted model systems have just been developing, but the application of 3D bioprinting technologies would also be increased rapidly in the future. (B) Original research papers which mention 3D bioprinting in relation with different scientific disciplines (>2260) mainly focused on 1. creating bone/cartilage and scaffold, 2. regeneration, 3. applications in the creation of vascular/cardio/skin/liver tissues/mini-organs and 4. cancer research. The distribution and the areas of the frontiers are also shown by Venn-diagram, the size of different circles represents the percentages of publications excluding review papers. (C) The distribution of cancer types among cancer research-related non-review and experimental papers (we excluded the papers where cancer research was only mentioned as a potential other research area or where 3D bioprinting was highlighted among technologies that are spreading or could help develop). Tumour types where the percentages of publications could not be higher than 1% merged into other studied malignancies groups.

FIGURE 4

Proliferation/tumour growth and morphological characteristics of different in vitro/in vivo models of ZR75.1. (A) The growth of in vivo xenotransplanted and 3D bioprinted scaffold of ZR75.1 tumour. Tumour growth was indicated by the calculated tumour volume of the xenograft (right scale), while the increase in cell amount in 3D scaffolds was estimated by both Alamar Blue (AB) and Sulforhodamine B (SRB) proliferation tests (left scale). (B,C) Microscope images of haematoxylin-eosin-stained slides from the human xenograft mouse model, 3D bioprinted scaffold (1-week maintaining), spheroid cell culture (1-week maintaining), 2D cell culture (prepared with cytospin) of ZR75.1 cells (B), human luminal B type breast cancer tissue section (C). (scale bar: 50 μm).

FIGURE 5

Metabolic alterations of different in vitro/in vivo models of ZR75.1. (A) Immunostainings of 2D cell cultures (prepared with cytospin technique), spheroid cell culture (maintained for 1 week), in vivo xenotransplanted ZR75.1 tumour, and 3D bioprinted scaffold (maintained for 1 week). The expression of ALDH1 (aldehyde dehydrogenase 1), Cleaved-caspase-3 (apoptosis marker), COXIV (Cytochrome c oxidase complex IV), LDHA (lactate dehydrogenase A), Phoshpo-Histone-H3 (mitotic marker). Immunohistochemistry was accomplished with brown (DAB, diaminobenzidine) substrate and haematoxylin counterstaining (scale bar: 50 μm). (B) Different maintaining condition (2D; scaffold; xenograft) affects protein expression pattern in ZR75.1 cells and xenograft tumour. WES^TM Simple was used to detecting metabolic enzymes (LDHA—lactate dehydrogenase A, COXIV—cytochrome c oxidase subunit 4) and Rictor expressions (left panel). Densitometric analysis was performed to present the normalised protein expression differences and was used β-actin as a loading control (right panel). *p < 0.05.