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.
Highlights
Ductal carcinoma in situ (DCIS) accounts for 27% of all breast cancers diagnosed in women over 50 (DeSantis et al, 2014), and since the 1970’s it has been accepted as the non-obligate precursor of invasive ductal carcinoma (Cowell et al, 2013)
We showed the presence of a subset of GLUT1 and CAIX positively stained MINO cells indicating that MINO tissue might be metabolically heterogeneous (Figure 1)
CAIX is a hypoxia inducible protein that regulates cell pH (Airley et al, 2003). Increased expression of both GLUT1 and CAIX in breast cancer has been found to correlate to metastasis, invasion, and poor survival outcomes (Pinheiro et al, 2011; Lock et al, 2012). 2-NBDG uptake in individual normal mammary epithelial cells (nMECs), MINO precancer, and Met1 cancer cells (Figure 2) was different, and the wide range of distribution in the MINO cells suggests that MINO cells are microanatomically and functionally heterogeneous (Maglione et al, 2004; Namba et al, 2004, 2006; Damonte et al, 2008) but are metabolically heterogeneous
Summary
Ductal carcinoma in situ (DCIS) accounts for 27% of all breast cancers diagnosed in women over 50 (DeSantis et al, 2014), and since the 1970’s it has been accepted as the non-obligate precursor of invasive ductal carcinoma (Cowell et al, 2013). The MINO model is derived from the Polyoma virus middle-T (PyVmT) transgene mouse model and consists of the transplantation of MINO from transgenic mice, FVB/NTg(MMTV-PyVmT) on an FVB background, to syngeneic immune-intact FVB mice (Maglione et al, 2001, 2004) Both DCIS and MINO are heterogeneous in cell type and function compared with corresponding invasive carcinomas (Damonte et al, 2008; Cowell et al, 2013). Promoter DNA methylation has not been found to be significantly different between DCIS and IBC tumors suggesting that methylation changes may be early events in carcinogenesis rather than essential events in the transition to invasive disease (Moelans et al, 2011; Verschuur-Maes et al, 2012). There is considerable evidence that intracellular and extracellular pH can alter malignant cell survival and invasion (Gatenby et al, 2006; Rofstad et al, 2006; Webb et al, 2011)
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