Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Tissue Engineering

Tissue Engineering - Poster Session B

Poster L1 - Docetaxel alters matrix-mediated transport in tissue-engineered lymph node stroma

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The lymphatic system is a network of approximately 450-600 lymph nodes (LNs) throughout the body which enable immune response by extending their lymphatic endothelial cells (LEC) for immunosurveillance of lymph extracted from the circulatory system. After antigens are captured, fibroblastic reticular cell (FRC) conduit networks in the LN help transport lymph to the necessary immune cells to target and destroy these foreign invaders. Like many cells in the human body, LN stroma are surrounded by a specialized extracellular matrix (ECM) responsible for defining structure and interstitial fluid flow. Various pathways are related to ECM function, with special emphasis on genes governing matrix metalloproteinase formation (MMP) and their tissue inhibitor counterparts (TIMP) that control matrix degradation and deposition respectively.

Due to the biofluidic nature of the LN, lymph transport is implicated in many instances of cancer metastasis. One such drug that has been utilized for the treatment of commonly metastasizing breast cancers is docetaxel (DTX), a taxane which inhibits proper microtubule formation during the G2/M phase of the cell cycle. Despite its efficacy, an off-target effect that has been observed in recent years is increased lymphatic-tumor crosstalk, exacerbating metastasis, however the exact mechanism remains unclear. This study hypothesized that DTX induces ECM remodeling via activation of MMP and TIMP pathways, altering flow on a microscale that compounds to global metastatic increase.

Materials and

Methods:  Tissue-engineered lymph node models were created by seeding LECs on the underside of a permeable cell culture insert, and FRCs were seeded in a hyaluronic acid-collagen hydrogel crosslinked within the insert. Treatment groups were incubated for 5-days with 1 µM DTX VascuLife® Complete Media. Models were then analyzed using three methods: dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) flow mapping, immunohistological staining (IHC), and reverse transcription-polymerase chain reaction (RT-PCR). DCE-MRI-designated models were injected with 300 µL of gadolinium and imaged in 3-minute intervals under DCE-MRI for 12 minutes. Images were stacked and analyzed to compute vector fields and streamlines using pixel brightness differentials. Whole-gel flow metrics were recorded. IHC-designated models were fixed with 4% paraformaldehyde and stained for DAPI, F-actin, and CD31. These models were imaged under spinning-disk confocal microscopy, and quantified using ImageJ to measure FRC networking coverage, LEC monolayer coverage, and junctional integrity. Lastly, gels from RT-PCR-designated models were placed in Trizol, and RNA was extracted and purified. Samples were converted to cDNA and run using RT-PCR to test for the presence of GADPH (housekeeping gene), TIMP1, TIMP2, MMP2, and MMP9. All RT-PCR runs were analyzed using QuantStudio™ Design & Analysis software.

Results, Conclusions, and Discussions: This study reported a significantly increased average gadolinium flowrate in the DTX-treated group as compared to the control (Figure B). This phenomenon is consistent with the original hypothesis and can be attributed to the results of RT-PCR, which measured increased gene expression for TIMP1, TIMP2, MMP2, and MMP9 across all DTX-treated models (Figure A), with this effect especially pronounced in the MMP genetic targets. As MMPs lead to enhanced matrix proteolysis, their increased expression would degrade ECM components and subsequently increase any interstitial space to allow faster fluid transport throughout the system. It is important to make the distinction that this phenomenon has only been measured within simplified tissue-engineered models and not within an in vivo system, so there are key-factors missing that represent the full scope of the lymphatic-metastasis pathway. However, this pipeline provides a niche outlook into the engineering-derived aspects of lymphatic function, giving a more complete understanding of the LN in conjunction with related studies.

Additionally, results from IHC staining indicated a drastic decrease in stromal viability, as FRC networking is significantly suppressed after DTX-treatment (Figure C). This decrease possibly contributes to a disruption in LEC-FRC cytokine signaling, implicated in the MMP-pathway mentioned above, although further study is required.

Continuing with future studies, investigating how breast cancer invades within the DTX-conditioned model is crucial to understanding the link between intramatrix flow and metastasis. Preliminary trials added 50K MCF-7 cells to 5-day pre-treated models and allowed for a 24-hour incubation period. Such models will undergo similar DCE-MRI, IHC, and RT-PCR analysis.

Acknowledgements (Optional):  This work was supported by NIH-NIBIB NCATS grant U01EB029127, as well as by the Fralin Biomedical Research Institute’s 2024 cardioSURF program. Further acknowledgement is for Dr. Maosen Wang of the Fralin Biomedical Research Institute, who aided in the MRI imaging process.