Fibroblasts are an abundant cell type in tumor microenvironments. Activated fibroblasts, known as carcinoma‐associated fibroblasts (CAFs), interact with cancer cells through biochemical signaling and render cancer cells proliferative, invasive, and resistant to therapeutics. Targeting CAFs–cancer cells interactions offers a strategy to block cancer progression. 2D and 3D co‐cultures of human mammary fibroblasts and triple negative breast cancer (TNBC) cells are used to investigate the impact of heterotypic cellular interactions on the proliferation of matrix invasion of TNBC cells. The results show that fibroblasts secreting a chemokine, CXCL12, significantly enhance proliferation of TNBC cells expressing the chemokine receptor, CXCR4. Disrupting this interaction with a receptor antagonist normalizes cancer cell proliferation to that of a co‐culture model lacking this signaling. When co‐culture spheroids are embedded in collagen, fibroblasts producing CXCL12 promote collagen invasion of TNBC cells. Although co‐cultures containing normal fibroblasts also lead to TNBC cell spreading into the matrix, a morphological analysis of cells and inhibition of chemokine‐receptor signaling shows that this spreading is due to the incompatibility of fibroblasts and cancer cells leading to the segregation of the two cell types from the spheroid.
3D bioprinting improves orientation of
- Award ID(s):
- 1735968
- NSF-PAR ID:
- 10153976
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
Colorectal cancer (CRC) is the third-most leading cause of cancer-related deaths in the United States. To advance the understanding of CRC tumor progression, models which mimic the tumor microenvironment (TME) and have translatable study outcomes are urgently needed. CRC patient-derived xenografts (PDXs) are promising tools for their ability to recapitulate tumor heterogeneity and key patient tumor characteristics, such as molecular characteristics. However, as in vivo models, CRC PDXs are costly and low-throughput, which leads to a need for equivalent in vitro models. To address this need, we previously established an in vitro model using a tissue engineering toolset with CRC PDX cells. However, it is unclear whether tissue engineering has the capacity to maintain patient- and/or cancer stage-specific tumor heterogeneity. To address this gap, we employed three PDX tumor lines, originated from stage II, III-B, and IV CRC tumors, in the formation of 3D engineered CRC PDX (3D-eCRC-PDX) tissues and performed an in-depth comparison between the 3D-eCRC-PDX tissues and the original CRC-PDX tumors. To form the tissues, CRC-PDX tumors were expanded in vivo and dissociated. The isolated cells were encapsulated within poly(ethylene glycol)-fibrinogen hydrogels and remained viable and proliferative post encapsulation over the course of 29 days in culture. To gain molecular insight into the maintenance of PDX line stage heterogeneity, we performed a transcriptomic analysis using RNA seq to determine the extent to which there were similarities and differences between the CRC-PDX tumors and the 3D-eCRC-PDX tissues. We observed the greatest correspondence in overlapping differentially expressed human genes, gene ontology, and Hallmark gene set enrichment between the 3D-eCRC-PDX tissues and CRC-PDX tumors in the stage II PDX line, while the least correspondence was observed in the stage IV PDX line. The Hallmark gene set enrichment from murine mapped RNA seq transcripts was PDX line-specific which suggested that the stromal component of the 3D-eCRC-PDX tissues was maintained in a PDX line-dependent manner. Consistent with our transcriptomic analysis, we observed that tumor cell subpopulations, including human proliferative (B2M+Ki67+) and CK20+ cells, remained constant for up to 15 days in culture even though the number of cells in the 3D-eCRC-PDX tissues from all three CRC stages increased over time. Yet, tumor cell subpopulation differences in the stage IV 3D-eCRC-PDX tissues were observed starting at 22 days in culture. Overall, our results demonstrate a strong correlation between our in vitro 3D-eCRC-PDX models and the originating in vivo CRC-PDX tumors, providing evidence that these engineered tissues may be capable of mimicking patient- and/or cancer stage-specific heterogeneity.more » « less
-
Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bioprinting allows the deposition of cell-laden bioink, ensuring predefined scaffold architecture that may offer living tissue regeneration. With a combination of unique characteristics such as biocompatibility, less cell toxicity, and high water content, natural hydrogels are a great candidate for bioink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural integrity. To tackle this challenge, the rheological properties, specifically the shear thinning behavior (reduction of viscosity with increasing the applied load/shear rate on hydrogels) of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TO-NFC), carboxymethyl cellulose (CMC), and commonly used alginate, were explored. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TO-NFC, 1–4% of CMC, and 1–4% of alginate to analyze the shear thinning factors such as the values of n and K, which were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to tune shear thinning factors with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture.more » « less
-
more » « less
Abstract Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bio-printing allows the deposition of cell-laden bio-ink, ensuring predefined scaffold architecture that may offer living tissue regeneration. With a combination of unique characteristics such as biocompatibility, less cell toxicity, and high-water content, natural hydrogels are a great candidate for bio-ink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural ty. To tackle this challenge, we characterized the rheological properties of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TONFC), carboxymethyl cellulose (CMC) and commonly used alginate. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TONFC, 1%–4% of CMC, and 1%–4% of alginate to analyze the rheological properties. The shear thinning coefficients of n and K were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to control rheological properties with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture.
-
Abstract There is a need for new in vitro systems that enable pharmaceutical companies to collect more physiologically-relevant information on drug response in a low-cost and high-throughput manner. For this purpose, three-dimensional (3D) spheroidal models have been established as more effective than two-dimensional models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in shape, size, and aggregate forming tendencies. In this study, we overcome these challenges by coupling tissue engineering toolsets with microfluidics technologies to create engineered cancer microspheres. Specifically, we employ biosynthetic hydrogels composed of conjugated poly(ethylene glycol) (PEG) and fibrinogen protein (PEG-Fb) to create engineered breast and colorectal cancer tissue microspheres for 3D culture, tumorigenic characterization, and examination of potential for high-throughput screening (HTS). MCF7 and MDA-MB-231 cell lines were used to create breast cancer microspheres and the HT29 cell line and cells from a stage II patient-derived xenograft (PDX) were encapsulated to produce colorectal cancer (CRC) microspheres. Using our previously developed microfluidic system, highly uniform cancer microspheres (intra-batch coefficient of variation (CV) ≤ 5%, inter-batch CV < 2%) with high cell densities (>20×106 cells/ml) were produced rapidly, which is critical for use in drug testing. Encapsulated cells maintained high viability and displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. For PDX CRC microspheres, the percentage of human (70%) and CRC (30%) cells was maintained over time and similar to the original PDX tumor, and the mechanical stiffness also exhibited a similar order of magnitude (103 Pa) to the original tumor. The cancer microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with staurosporine (1 μM) and doxorubicin (10 μM). Expected responses were quantified using CellTiter-Glo® 3D, demonstrating initial applicability to HTS drug discovery. PDX CRC microspheres were treated with Fluorouracil (5FU) (10 to 500 μM) and displayed a decreasing trend in metabolic activity with increasing drug concentration. Providing a more physiologically relevant tumor microenvironment in a high-throughput and low-cost manner, the PF hydrogel-based cancer microspheres could potentially improve the translational success of drug candidates by providing more accurate in vitro prediction of in vivo drug efficacy. Citation Format: Elizabeth A. Lipke, Wen J. Seeto, Yuan Tian, Mohammadjafar Hashemi, Iman Hassani, Benjamin Anbiah, Nicole L. Habbit, Michael W. Greene, Dmitriy Minond, Shantanu Pradhan. Production of cancer tissue-engineered microspheres for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 175.more » « less
