Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer tumor microenvironment

Abstract Background: Unfavorable vasculogenesis, such as lymphangiogenesis and hemoangiogenesis are known to be pivotal of cancer progression. Our group reported that S1P pathway link chronic inflammation, such as obesity, and cancer progression by inducing lymphangiogenesis and hemoangiogenesis in breast cancer. During the past decade, complex cytokine network interactions between cancer cells and stromal cells have been found to mediate tumor-associated lymphangiogenesis and hemoangiogenesis. Thus, it is crucial to understand cancer biology as a tumor microenvironment, rather than focusing solely on cancer cells. To this end, we hypothesized that amount of intratumoral lymphatic endothelial cells (iLEC) reflect lymphangiogenesis and associate with poor clinical characteristics and outcomes. Method: We analyzed a total of 4145 breast cancer patient’s clinical and transcriptome data from The Cancer Genome Atlas (TCGA), and GSE96058 datasets. Intratumoral lymphatic endothelial cells (iLEC) and all the other immune cells were determined by xCell algorithm, and a median cutoff was adopted to divide high and low groups. Result: First we performed gene set enrichment assay using Hallmark and PID collections from MSigDB and found that high iLEC breast cancer enriched Lymphangiogenesis-related gene sets (lymph angiogenesis, S1P meta, S1P1, and S1P3 pathway) and hemoangiogenesis-related gene sets (Angiogenesis, VEGFR1 pathway, and VEGFR2 pathway) (all FDR<0.25). Microvascular endothelial cells were also predominant in high iLEC tumors (p < 0.001). Even though iLEC was significantly higher in patients with lymph node metastasis consistently in both TCGA and GSE96058 cohorts, there was no survival difference by the amount of iLEC in neither of the cohorts. High iLEC breast cancer also enriched cancer stem cell-related gene sets (notch signaling, hedgehog signaling, epithelial mesenchymal transition, and Wnt β catenin signaling) and metabolic pathways (bile acid metabolism, fatty acid metabolism), and S1P2 pathway (all FDR<0.25). Stromal cells such as Adipocytes, Preadipocytes and Fibroblasts, were also more prominent in the high iLEC group (all p < 0.001). The immune-related gene sets (allograft rejection, IL2 STAT5 pathway, IL6 Jak STAT3 signaling, inflammatory response, TNFα signaling via NFkB, and complement) and cytolytic activity, reflecting anti-tumor immunity, were elevated in high iLEC group of both cohorts (all FDR<0.25, p < 0.001), but anti-cancer immune cells were not significantly infiltrated. Interestingly, cell proliferation-related gene set (E2F target, G2M checkpoint, MYC target v1) and mTORc1 signaling gene set was strongly enriched in low iLEC breast cancer (all FDR<0.25). Furthermore, Ki67 expression and histological grade as well as aggressive cancer subtype were negatively correlated with iLEC fraction (all p<0.002, r <-0.29). Finally, we found that in the TCGA cohort, low iLEC breast cancer was significantly associated with intratumor heterogeneity, silent and nonsilent mutation rate, fraction altered, and homologous recombination deficiency, based on scoring by Thorrson et al (all p < 0.001). Despite the association with high TIL regional fraction and IFN-γ Response, low iLEC breast cancer was infiltrated by pro-cancer immune cells, such as Th2, Treg, and B-cells (all p < 0.001). Conclusion: High iLEC breast cancer reflected lymphangiogenesis and were associated with cancer stem cell-related genes and lymph node metastasis, while low iLEC breast cancers were associated with accelerated cell proliferation and pro-cancer immune cell infiltrations. Unfavorable cancer characteristics on both high and low iLEC groups may explain the lack of difference in patient survival. Citation Format: Rongrong Wu, Masanori Oshi, Mariko Asaoka, Akimitsu Yamada, Yamato Takabe, Li Yan, Itaru Endo, Takashi Ishikawa, Kazuaki Takabe. Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-03.