Abstract 1476: Vessel normalization following LDH-A knockdown in murine breast tumors
Authors: Serganova Inna , Vemuri Kiranmayi , Cohen Ivan , Lubin Matthew , Roberts Sheryl , Maeda Masatomo , Mane Mayuresh , Mann Henry , Reiner Thomas , Chan Eric , Yarilin Dmitry , Manova-Todorova Katia , Zappasodi Roberta , Merghoub Taha , Koutcher Jason , Blasberg Ronald 
Published: September 2020

Abstract
Abnormal vessel formation plays a major role in tumor progression. Previously, we showed that lactate dehydrogenase A (LDH-A) knockdown (KD) reduces orthotopic 4T1 breast tumor lactate, and has a robust anti-tumor effect through changes in the tumor microenvironment (TME) [1, 2]. Tumors with LDH-A downregulation have lower VEGF-A secretion and reduced formation of new blood vessels [2]. In addition, LDH-A KD cells/tumors displayed reduced HIF-1 signaling, as assessed by BLI using HRE-exGLuc reporter. Changes in the bioluminescence signal corresponded with changes in HIF-1α staining. LDH-A KD tumors also showed less HIF-1α staining compared to control tumors, consistent with less pimonidazole (hypoxia) staining [2]. Murine 4T1 breast cancer cells and tumors with LDH-A knockdown were developed and described [2]. To evaluate the degree of tumor neovascularization and its normalization, we stained tumors for CD31 (endothelial marker) and NG2 (pericytes marker). For pericyte coverage quantification, vessels were counted, and percentage of vessels covered by pericytes were calculated. Blood vessel permeability was assessed by intravenous injection of Evans Blue dye and tumor dye extravasation was quantified. To assess in vivo vascular network over time, a raster-scan optoacoustic mesoscopy (RSOM) was performed. Vessel structure in 4T1 murine tumors changes significantly following LDH-A KD. Normalized LDH-A KD tumor neovascularization was shown using the double immunofluorescence staining for CD31 and NG2, including a more normal vascular morphology, with shorter, more linear (less branched) vessel structure, with a higher percentage of vessels covered by pericytes compared to control tumors. Evans Blue dye extravasation was significantly less (lower vascular permeability) in LDH-A KD tumors compared to control tumors. Applying RSOM to follow the development of the vascular network during tumor growth, we showed better vascularization of LDH-A KD tumors. Furthermore, the number of circulating tumor cells (CTCs) was also significantly lower in blood collected from mice bearing LDH-A KD tumors. We show that LDH-depletion leads to tumor vessel normalization that includes several unique features: structural changes, greater coverage of blood vessel by pericytes and reduced vessel leakiness, leading to changes in the tumor microenvironment, inhibition of metastases formation and prolongation of survival. 1. Rizwan, A., et al., Relationships between LDH-A, lactate, and metastases in 4T1 breast tumors. Clin Cancer Res, 2013. 19(18): p. 5158-69. 2. Serganova, I., et al., LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLOS ONE, 2018. 13(9): p. e0203965.
Citation Format: Inna Serganova, Kiranmayi Vemuri, Ivan Cohen, Matthew Lubin, Sheryl Roberts, Masatomo Maeda, Mayuresh Mane, Henry Mann, Thomas Reiner, Eric Chan, Dmitry Yarilin, Katia Manova-Todorova, Roberta Zappasodi, Taha Merghoub, Jason Koutcher, Ronald Blasberg. Vessel normalization following LDH-A knockdown in murine breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1476.