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Tissue Engineering

Tissue Engineering - Poster Session C

Poster L19 - Investigating the Effect of IDO Inhibition as a Mechanistic Driver of Lymphatic/Blood Vessel Plasticity in the Tumor Microenvironment

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Angiogenesis and lymphangiogenesis, the creation of new blood and lymphatic vessels from a pre-existing branch, are key components to tumor growth and the spread of cancer. Under normal conditions, blood and lymphatic vessels rarely interact. However, in a tumor microenvironment, examples of plasticity such as mosaic vessels, lymphatic/blood vessel connections, gaps in lymphatic patterning, and vascular remodeling have been recorded. In prior studies, our lab has confirmed the presence of plasticity in the tumor microenvironment and determined that indoleamine 2,3-dioxygenase (IDO), an enzyme that contributes to tumor growth and the suppression of the immune system may also be involved in plasticity. However, the role IDO plays in tumor angiogenesis, lymphangiogenesis, and lymphatic/blood vessel plasticity remains unclear. Therefore, it is vital to be able to simultaneously investigate the effects of IDO on blood and lymphatic interactions to study microvascular remodeling in the tumor microenvironment. Our lab developed the ex vivo rat mesenteric model cultured with tumor spheroids, which allows the visualization of complex microvascular interactions and vessel dynamics. With the use of this model, the objective of this study was to quantify the effect of IDO inhibition via 1-methyltryptophan (1-MT) on angiogenesis, lymphangiogenesis, and vessel plasticity.

Materials and

Methods:  Tumor spheroids formed via hanging drop culture from the human lung carcinoma cell line (H1299), were seeded onto excised rat mesentery tissues from adult male Wistar rats. Tissues were cultured for 3-5 days with MEM or MEM supplemented with 400 μg/mL 1-MT. After 3-5 days of culture, tissues were fixed in methanol and then stained to label for PECAM, and LYVE-1 to identify blood and lymphatic vessels respectively. Angiogenesis was quantified at Day 3 by measuring the number of segments and sprouts per vascular length. Lymphangiogenesis was quantified at Day 5 by measuring the number of sprouts per lymphatic vascular length. Plasticity was quantified at Day 5 by measuring the number of gaps in the lymphatic patterning and lymphatic/blood vessel connections per lymphatic vascular length. Image processing and analysis was done using Image J. Analysis was blinded and groups were compared using a two-tailed unpaired Welch’s t-test.

Results, Conclusions, and Discussions: Inhibition of IDO via 1-MT in day 3 tissues did not significantly impact angiogenic growth (Figure 1A-B). On day 5 tissues, 1-MT tissues did not significantly decrease lymphangiogenic growth (Figure 1C). Day 5 tissues cultured with only MEM displayed increased lymphatic/blood vessel plasticity compared to tissues cultured in MEM supplemented with 400 μg/mL 1-MT, as supported by quantification of gaps in lymphatic patterning and lymphatic/blood vessel connections (Figure 1F and 1I).
The results for angiogenic and lymphangiogenic growth did not align with the initial expectations. However, the results on lymphatic/blood vessel connections and the gaps on lymphatic patterning did support the hypothesis that inhibiting IDO decreases vessel plasticity and remodeling. This suggests that the assumption that lymphatic/blood vessel connections and vessel growth occur together in the tumor microenvironment is not always accurate. This opens the possibility that angiogenesis and lymphangiogenesis are processes belonging to different stages in the tumor progression timeline.
This study highlights the need for further research into the mechanisms and functionality of lymphatic/blood vessel plasticity in the tumor microenvironment.

Acknowledgements (Optional):  Thank you to the American Heart Association (AHA), grant number: UF AHA CRSSTP (24IAUST1247377).