Acidic stress promotes a glioma stem cell phenotype

Abstract

Malignant gliomas are lethal cancers that display cellular hierarchies with cancer stem cells at the apex. Glioma stem cells (GSCs) are not uniformly distributed, but rather located in specialized niches, suggesting that the cancer stem cell phenotype is regulated by the tumor microenvironment. Indeed, recent studies show that hypoxia and its molecular responses regulate cancer stem cell maintenance. We now demonstrate that acidic conditions, independent of restricted oxygen, promote the expression of GSC markers, self-renewal and tumor growth. GSCs exert paracrine effects on tumor growth through elaboration of angiogenic factors, and low pH conditions augment this expression associated with induction of hypoxia inducible factor 2α (HIF2α), a GSC-specific regulator. Induction of HIF2α and other GSC markers by acidic stress can be reverted by elevating pH in vitro, suggesting that raising intratumoral pH may be beneficial for targeting the GSC phenotype. Together, our results suggest that exposure to low pH promotes malignancy through the induction of a cancer stem cell phenotype, and that culturing cancer cells at lower pH reflective of endogenous tumor conditions may better retain the cellular heterogeneity found in tumors.

Figure 1

Cancer stem cell markers are maintained in acidic GSC-enriched cultures. (a–c) Expression of cancer stem cell markers was evaluated in CD133+ cells isolated from three different human glioma xenografts and subsequently treated with acidic (pH 6.5) or normal (pH 7.5) cell culture media for 6 days. RNA was collected using Qiagen RNAeasy kits, reverse transcribed and analyzed for the specific genes indicated using real-time PCR. n=2 for each tumor type. (a) Treatment with low pH increased expression of Olig2 mRNA in T1863, T4302 and T4121 GSC-enriched cultures. (b) Exposure to reduced pH increased expression of Oct4 mRNA in T1863, T4302 and T4121 GSCs. (c) Acidic stress increased expression of Nanog mRNA in T1863, T4302 and T4121 GSCs. (d) All GSCs shown were cultured in the presence of serum to induce differentiation. Immunofluorescence for the astrocyte marker glial fibrillary protein (GFAP, green) and the nuclear stain DAPI (blue) demonstrates the presence of the differentiation cell marker GFAP in glioma stem cells cultured at pH 7.5. In contrast, GFAP is rarely expressed in cells exposed to acidic stress

Figure 2

Cancer stem cell markers increase in GSC-depleted cultures after treatment with low pH. (a–c) Expression of cancer stem cell markers was evaluated in CD133− cells isolated from three different human glioma xenografts and subsequently cultured under low pH (6.5) or typical cell culture conditions (pH 7.5) for 6 days. RNA was collected using Qiagen RNAeasy kits, reverse transcribed, and analyzed for the specific genes indicated using real-time PCR. n=2 for each tumor type. (a) Treatment with low pH increased expression of Olig2 mRNA in T1863, T4302 and T4121 GSC-depleted cultures. (b) Exposure to low pH increased expression of Oct4 mRNA in T1863, T4302 and T4121 GSC-depleted cultures. (c) Acidic stress increased expression of Nanog mRNA in T1863, T4302 and T4121 GSC-depleted cultures. (d) Immunofluorescence for the stem cell marker Nanog (green), astrocyte marker glial fibrillary protein (GFAP, red) and the nuclear stain DAPI (blue) demonstrates the presence of Nanog in cells cultured at pH 6.5. In contrast, the differentiation marker GFAP is highly present in cells cultured at pH 7.5, but rarely when cells were exposed to pH 6.5

Figure 3

Reduced differentiation marker expression in GSC-depleted cultures after exposure to acidic stress. (a) Immunofluorescence for the neuronal differentiation marker class III β-tubulin (green) and the nuclear stain Hoescht (blue) demonstrates that class III β-tubulin is highly present in cells cultured at pH 7.5, but rarely present when CD133− D456 MG cells were exposed to pH 6.5. Immunofluorescence for β III tubulin (green) and the nuclear stain DAPI (blue) in T1863 (b), T4302 (c), and T4121 (d) CD133− cells demonstrates reduced neuronal differentiation marker expression after acidic stress

Figure 4

Functional indicators of the increase in GSC-depleted cultures after treatment with low pH. Expression of cancer stem cell markers was evaluated in CD133− cells isolated from three different human glioma xenografts and subsequently cultured with acidic (pH 6.5) or normal (pH 7.5) media for 14 days. (a) Representative images of D456 MG and T4121 GSC-depleted cultures with and without low pH. (b) The percentage of wells with neurospheres increases with low-pH treatment. D456 MG cells pretreated with pH 6.5 or pH 7.5 media were plated using flow cytometry with five cells per well and the percentage of wells with neurospheres was determined after 10 days. n=2. (c) Kaplan–Meier Curves demonstrate the survival of mice (n=3 for each arm) bearing human glioma xenografts generated from D456 MG cells pretreated with pH 6.5 or pH 7.5. Survival is significantly reduced when xenografts originate from cells treated with low pH. (d) Growth of subcutaneous human glioma xenografts (n=3 for each arm) is increased with a trend towards statistical significance when cells are exposed to low pH in comparison with standard pH. (e) Representative images of tumors harvested from (c) and subsequently sectioned and stained with hematoxylin and eosin (H&E) or CD31 to indicate blood vessels

Figure 5

Treatment with low pH increases expression of angiogenic factors including VEGF in both GSC-enriched and -depleted cultures. (a) Human Angiogenesis Arrays demonstrate differential expression of angiogenic factors in the conditioned media of GSCs isolated from a T1863 glioma xenograft and pretreated at pH 7.5 or pH 6.5 for 6 days. (b) Image J quantification of the expression of VEGF on the Human Angiogenesis Array in (a). (c) ELISA demonstrates elevated VEGF protein expression in the conditioned media of T1863 GSCs pretreated with media at pH 6.5. n=2. (d) Human Angiogenesis Arrays demonstrate differential expression of angiogenic factors in the conditioned media of GSC-depleted cultures isolated from a T1863 glioma xenograft and pretreated at pH 7.5 or pH 6.5. (e) Image J quantification of the expression of VEGF on the Human Angiogenesis Array in (d). (f) ELISA demonstrates elevated VEGF protein expression in the conditioned media of T1863 GSC-depleted cultures pretreated at pH 6.5. n=2. (g) Real-time PCR demonstrates increased VEGF mRNA in GSC-enriched or -depleted cultures isolated from T1863, T4302 or T4121 xenografts. n=2 for each tumor type. RNA was collected after 6 days of treatments with media at pH 6.5 or pH 7.5 using Qiagen RNAeasy kits. RNA was reverse transcribed and analyzed for VEGF using real-time PCR

Figure 6

Exposure to low pH induces HIF2α mRNA. (a and b) Expression of HIFs was evaluated in CD133+ enriched (a and b) or CD133+ depleted (c and d) cells isolated from three different human glioma xenografts and subsequently treated with acidic (pH 6.5) or normal (pH 7.5) cell culture media for 6 days. RNA was collected using Qiagen RNAeasy kits, reverse transcribed and analyzed for HIF2α (a) or HIF1α (b) using real-time PCR. n=2 for each tumor type. (c) HIF2α mRNA increases in T1863, T4302 and T4121 GSC-depleted cultures exposed to pH 6.5 compared with those cultured at pH 7.5. (d) Exposure to low pH represses HIF1α mRNA in T1863, T4302 and T4121 GSC-depleted cultures. (e) Western analysis demonstrates that HIF2α protein is elevated after treatment with acidic media. Total-cell lysates were collected from the human glioma cell line U87 MG or GSC-depleted cultures isolated from T4302 and T4121 xenografts treated with media at pH 6.5 or pH 7.5 for 14 days. In GSC-depleted cultures, addition of 100 μM of the hypoxia mimetic desferrioxamine (DFX) in pH 7.5 media was included as a positive control for HIF expression. Equivalent amounts of total protein were analyzed with the indicated antibodies. Tubulin expression was used as a loading control

Figure 7

Low pH treatment increases the expression of the HIF2α target genes Glut1 and SerpinB9. mRNA expression was evaluated in CD133+ enriched (a and b) or CD133+ depleted (c and d) cells isolated from three different human glioma xenografts and subsequently treated with acidic (pH 6.5) or normal (pH 7.5) cell culture media for 6 days. RNA was collected using Qiagen RNAeasy kits, reverse transcribed and analyzed for the Glut1α (a and c) or SerpinB9 (b and d) using real-time PCR. n=2 for each tumor type

Figure 8

Promotion of HIF2α and HIF2α target genes by acidic stress can be reverted by increasing pH. CD133− cells isolated from D456 MG xenografts were exposed to acidic (pH 6.5) or typical (pH 7.5) cell culture media for a total of 10 days. After the initial 6 days, cells exposed to acidic pH were subsequently exposed to pH 7.5 (6.5 → 7.5) for 4 days. RNA was collected using Qiagen RNAeasy kits, reverse transcribed and analyzed for the specific genes indicated using real-time PCR. n=3. mRNA expression of HIF2α (a), VEGF (b), Glut1 (c) and SerpinB9 (d) are elevated in cells treated with acidic stress. Restoration to pH 7.5 significantly decreases levels of most HIF2α target genes over the time course evaluated