Abstract B022: pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs
Authors: Matsui Toma , Toda Yuki , Mosnikova Anna , Andreev Oleg A. , Hosogi Shigekuni , Reshetnyak Yana K. , Ashihara Eishi 
Published: January 2023

Abstract
Background: Pre-metastatic niche refers to the environment prepared for efficient colonization of disseminated cancer cells in specific organs. Primary tumor secretome facilitates pre-metastatic niche through a series of molecular and cellular changes. This study focuses on interstitial pH in organs with pre-metastatic niche because dysregulated pH homeostasis plays a well-established role in tumor metastasis. In primary tumor, extracellular acidification directly promotes invasion/migration of cancer cells for extravasation. Meanwhile, little is known regarding interstitial pH in organs that are distant from primary tumor. Methods: BALB/c mice were subcutaneously inoculated with 4T1.2 cells or 66cl4 cells (mouse breast cancer cell lines) with resistance against 6-thioguanine. Absence of metastatic cells in their lungs was confirmed by long-termed culture of dissociated pulmonary cells in presence of 6-thioguanine; it defines Day 21 from inoculation as a pre-metastatic phase. Alternatively, 4T1.2 cell-conditioned media (4T1.2-CM) or control media was intraperitoneally administered every day for 21 consecutive days. pH-low insertion peptide (pHLIP® peptide) stably labels cells exposed to acidic conditions, which is feasible to evaluate interstitial acidity in each organ. Alexa Fluor® 750-labelled pHLIP® peptide was injected into mice, and fluorescent signals in harvested organs were detected by IVIS Lumina XRMS Series III. For a metastasis model, mice first received 4T1.2-CM or in combination with sodium oxamate (a lactate dehydrogenase inhibitor) followed by an intravenous injection of 66cl4 cells expressing luciferase. Results: At the pre-metastatic phase, 4T1.2 tumor-bearing mice showed great accumulation of pHLIP® peptide and high amounts of lactate in their lungs. By contrast, there was no significant fluorescence from pHLIP® peptide in lungs of low-metastatic 66cl4 cell-inoculated mice. Notable accumulation of pHLIP® peptide was also observed in the lungs of 4T1.2-CM-given mice. Intravenously injected 66cl4 cells colonized more aggressively in the acidic lungs than in control ones. Suppression of the lung acidification by sodium oxamate attenuated the metastatic burden, which significantly improved survival of the mice. In the acidic lungs, pHLIP® peptide was localized in alveolar type II (AT2) cells expressing surfactant protein C. Sorted AT2 cells from the acidic lungs strongly expressed glycolysis-related proteins. Conclusions: This study using pHLIP® peptide indicates a highly glycolytic activity with increased lactic acid in the lungs of 4T1.2 tumor-bearing mice at the pre-metastatic phase. Mechanistically, soluble factors derived from 4T1.2 cells could transform AT2 cells into acidic cells, which might provide metastasis-promoting function. This study will shed light on understanding the whole picture of pre-metastatic niche and give scientific insights to develop novel diagnosis and therapy for tumor metastasis.
Citation Format: Toma Matsui, Yuki Toda, Anna Mosnikova, Oleg A. Andreev, Shigekuni Hosogi, Yana K. Reshetnyak, Eishi Ashihara. pH-low insertion peptide detects lactic acidosis contributing metastatic niche formation in lungs [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B022.