S24 - Lymphatic Mimicry and Lymphangiogenesis in the Metastatic Potential of Aggressive Adenocarcinoma

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Grace Fluskey

Undergraduate Student
University of Notre Dame
East Amherst, New York, United States

Co-Author(s)

N. Keilany Lightsey

Ph.D. Bioengineering Graduate Student
University of Notre Dame
Notre Dame, Indiana, United States
HY

Heejeong Youn

Graduate student
University of Notre Dame, United States

Primary Investigator(s)

Donny Hanjaya-Putra

Assistant Professor
University of Notre Dame
Notre Dame, Indiana, United States

Introduction: : Lymphangiogenesis (i.e., the process of forming new lymphatic vessels) and the increased presence of lymphatic markers in tumors have been linked to higher metastatic occurrence and a lower survival rate in cancer patients.[1,2] Previous studies have found that tumors induced lymphangiogenesis locally, with growth factors recruiting lymphatic vessels comprised solely of endothelial cells.[1,2] However, recent studies have found that highly metastatic tumors can form mosaic-like vessels through a process known as lymphatic mimicry (LM). The formation of these vessels provides a second mechanism for metastasis, which can decrease life expectancy.[2] Additionally, recent findings have shown that tumors can induce changes in distant organs that create niches for cancer cells before they metastasize. These pre-metastatic niches (PMNs) can involve induced lymphangiogenesis in distant organs and improve the success of metastasis.[3] Combined, these findings suggest that aggressive tumors have the potential to establish PMNs by promoting lymphangiogenesis and the release of lymphatic factors in distant organs while mimicking the lymphatic system themselves, increasing the rate at which secondary tumors become established and metastasize. In this study, the effects of indirect and direct co-culture of the aggressive cancer line OVCAR5 with lymphatic endothelial cells (LECs) aims to elucidate how LM can be induced in the presence of lymphatic growth factors and how the introduction of the lymphatic system affects the relative proliferation rates of an aggressive cancer. Understanding interactions between aggressive cancers and the lymphatic system may provide insight into mechanisms of metastasis and aid in the identification of novel therapeutic targets.

Materials and

Methods: : Four different co-culture conditions were plated and cultured for four days. For the control, OVCAR5 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM). For the direct co-culture, OVCAR5 cells were cultured with LECs in a 1:1 ratio in DMEM.[4] For the indirect co-culture, OVCAR5 cells were cultured in 80% DMEM and 20% Endothelial Growth Medium (MV2); the MV2 was used in previous experiments where LECs were exposed to oscillatory shear stress that has been shown to potentially have inflammatory effects, as it mimics certain vessel physiology.[5] To explore a combination of direct and indirect co-culture, OVCAR5 cells and LECs were plated in a 1:1 ratio and cultured in the same 80% DMEM and 20% MV2 mixture used in the indirect co-culture.
For all experiments, LECs were used between 6 and 10 passages. Cells were plated at a density of 1.8 x 10⁵ cells per well. All experiments were completed in triplicate.
Cells were fixed in 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA. Primary antibodies (1:200 diluted rabbit anti-PROX1 and 1:1600 diluted mouse anti-KI67) and secondary antibodies (anti-mouse Alexa Fluor 647 and anti-rabbit Alexa Fluor 488) were added. Cells were also stained with 300nM DAPI. Cells were stained for PROX1 to quantify LECs and OVCAR5 cells exhibiting lymphatic characteristics.[6] KI67 staining was used to assess the proliferation rates of the cancer and lymphatic cells. Images of each well were taken on ECHO Revolve MICROSCOPE at 10X magnification for n=3.

Results, Conclusions, and Discussions:: In the images, there is an increase in KI67 when direct co-culture is introduced. This suggests increased proliferation rates and metastatic activity in the direct-co-culture condition cells.[7,8] However, KI67 expression seems to remain unchanged with the introduction of the indirect co-culture media. This could be due to the MV2 ratio (while OVCAR5 cells and LECs were plated in a 1:1 ratio, a ratio of only 1:5 MV2 to DMEM was used in the indirect co-culture) or could indicate that direct LEC contact is needed to increase proliferation. Alternatively, cells could be undergoing epithelial-to-mesenchymal transition (EMT) rather than increased proliferation.[9]
LECs typically stain for PROX1 in the nucleus, but nuclear PROX1 was rarely found in the direct co-culture plates.[6,10] This could be because the 20% MV2/80% DMEM mixture did not provide enough nutrients for the LECs to survive; however, their presence still influenced cancer cell growth as evidenced by the increase in KI67 expression. The lack of nuclear staining could also be due to LECs losing identifying characteristics in the presence of the cancer cells, possibly due to increased TGF-ß (released by cancer cells during de-differentiation) inducing an endothelial-to-mesenchymal transition (EndMT) in the LECs.[11–13]
Despite the lack of nuclear staining, PROX1 is visible in the cytoplasm of cells in the direct co-culture conditions. This may result from changes in the cancer cells induced by the presence of lymphatic growth factors. The mislocated PROX1 expression in the presence of lymphatic cells suggests that these cells could be undergoing lymphatic mimicry.[2] Although PROX1 is associated with LECs, it is unlikely these cells are lymphatic, as previous studies have found PROX1 consistently located in the nucleus of lymphatic cells.[14]
Overall, these initial results suggest that lymphatic system factors may alter the characteristics and growth rate of ovarian adenocarcinoma cells. By modifying the co-culture environment and testing other markers (LYVE-1, PDPN, TGF-ß), the effects of lymphatic-ovarian adenocarcinoma cell interactions on lymphatic mimicry and metastasis can be better understood. [11,14]

Acknowledgements and/or References (Optional): : 1. Stacker, S. A. et al. Lymphangiogenesis and lymphatic vessel remodelling in cancer. Nat. Rev. Cancer 14, 159–172 (2014).
2. Karinen, S. et al. Tumour cells express functional lymphatic endothelium-specific hyaluronan receptor in vitro and in vivo: Lymphatic mimicry promotes oral oncogenesis? Oncogenesis 10, 1–11 (2021).
3. Rabas, N., Ferreira, R. M. M., Di Blasio, S. & Malanchi, I. Cancer-induced systemic pre-conditioning of distant organs: building a niche for metastatic cells. Nat. Rev. Cancer 24, 829–849 (2024).
4. Oliveira-Ferrer, L. et al. Mechanisms of Tumor-Lymphatic Interactions in Invasive Breast and Prostate Carcinoma. Int. J. Mol. Sci. 21, 602 (2020).
5. Pujari, A. et al. Lymphatic Valves Separate Lymph Flow Into a Central Stream and a Slow-Moving Peri-Valvular Milieu. J. Biomech. Eng. 142, 100805 (2020).
6. Bernier-Latmani, J. & Petrova, T. V. Intestinal lymphatic vasculature: structure, mechanisms and functions. Nat. Rev. Gastroenterol. Hepatol. 14, 510–526 (2017).
7. Van de Velde, M. et al. Tumor exposed-lymphatic endothelial cells promote primary tumor growth via IL6. Cancer Lett. 497, 154–164 (2021).
8. Li, L. T., Jiang, G., Chen, Q. & Zheng, J. N. Ki67 is a promising molecular target in the diagnosis of cancer (Review). Mol. Med. Rep. 11, 1566–1572 (2015).
9. Gao, J., Zhu, Y., Nilsson, M. & Sundfeldt, K. TGF-β isoforms induce EMT independent migration of ovarian cancer cells. Cancer Cell Int. 14, 72 (2014).
10. Wilting, J. et al. The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues. FASEB J. 16, 1271–1273 (2002).
11. Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial–mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178–196 (2014).
12. Yoshimatsu, Y. et al. TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals. PLoS ONE 15, e0232356 (2020).
13. Xu, J., Lamouille, S. & Derynck, R. TGF-β-induced epithelial to mesenchymal transition. Cell Res. 19, 156–172 (2009).
14. Miettinen, M. & Wang, Z.-F. PROX1 TRANSCRIPTION FACTOR AS A MARKER FOR VASCULAR TUMORS – EVALUATION OF 314 VASCULAR ENDOTHELIAL AND 1086 NONVASCULAR TUMORS. Am. J. Surg. Pathol. 36, 351–359 (2012).