Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity

Abstract

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Metabolic Heterogeneity of Cancer Cells: An Interplay between HIF-1, GLUTs, and AMPK

“…For example, in hypoxia conditions, tumor cells have been shown to produce more HIF-1-induced IX and XII isoforms of carbonic anhydrase, which catalyzes reversible hydration of carbon dioxide into bicarbonate and protons to contribute to intracellular acidification and tumor cell survival [130]. Moreover, in a mouse model of ductal carcinoma, in situ differences in levels of GLUT1 and carbonic anhydrase IX expression between normal and pre-cancer cells along with heterogeneity in intracellular pH values have been demonstrated [131].…”

Intracellular pH Regulates Cancer and Stem Cell Behaviors: A Protein Dynamics Perspective

“…Using H1299 cells expressing the previously described ( 41 ) genetically encoded and ratiometric pH biosensor mCherry-pHluorin, we observe distinct intercellular differences in pHi when grown in 3D ( Figure 2C ). Distinct pH heterogeneity (including intracellular and extracellular pH) is seen in cancer spheroids ( 55 – 58 ) and a mouse model of breast ductal carcinoma ( 59 ); however, whether this heterogeneity reflects differences in mutational signatures, cell identity, phenotypes, or epithelial or metabolic plasticity remains unresolved. For example, might cells with a lower pHi be stem-like tumor initiating cells?…”

Towards an Integral Therapeutic Protocol for Breast Cancer Based upon the New H+-Centered Anticancer Paradigm of the Late Post-Warburg Era

“…Furthermore, invasive BC cells show a more elevated pHi and higher production and exportation rates of hydrogen ions to the TME than noninvasive cells [ 44 , 48 ]. Most recently, however, other channels, transporters and even certain aquaporins (AQP), mainly AQP1 and AP3, as well as Cl − channels with Ca 2+ influx, have also been shown to be important in the initiation and progression of TNBC [ 49 , 50 ].…”