SUPPLEMENTAL FIGURE LEGENDS

Supplemental Figure 1. The extracellular intratumoral pH of human glioma xenografts is acidic. The pH of subcutaneous human glioma xenografts (5 mice) was measured using a pH electrode at the edge and center of the tumor as well as in non-tumor tissue as a control.

Supplemental Figure 2. GSC markersare elevated in acidic D456MG GSC enriched cultures.CD133+ cells were isolated from D456MG xenografts and subsequently treated with acidic (ph 6.5) or normal (pH 7.5) cell culture media for four 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 the cancer stem cell markers Olig2 (A) and Nanog (B) are elevated in D456MG GSC enriched cultures.

Supplemental Figure 3. FBS induced differentiation of GSC cultures is inhibited by acidic stress.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 pH 6.5.

Supplemental Figure 4. Low pH promotes expression of GSC markers in GSC depleted cultures and this effect can be reverted by increasing pH. CD133- cells isolated from D456MG xenografts were exposed to acidic (pH 6.5) or typical (pH 7.5) cell culture media for a total of ten days. After the initial six days, cells exposed to acidic pH were subsequently exposed to pH 7.5 (6.57.5) for four 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 the cancer stem cell markers Olig2 (A) and Nanog (B) are elevated in cells treated with acidic stress, but restoration to pH 7.5 decreases levels of these markers.

Supplemental Figure 5. Immunofluorescence of GSC depleted cultures exposed to acidic stress. Immunofluorescence for the astrocyte marker glial fibrillary protein (GFAP, green), Nanog (red), and the nuclear stain DAPI (blue) demonstrates the presence of the stem cell marker Nanog in cells cultured at pH 6.5 but not pH 7.5. 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.

Supplemental Figure 6. Fluorescence activated cell sorting demonstrates the percentage of CD133+ cells increases in a bulk primary glioma specimen with exposure to acidic stress. Acutely dissociated cells from a primary recurrent glioblastoma human patient specimen were cultured for fourteen days at standard pH (7.5) or exposed to acidic stress (pH 6.5) and subsequently harvested for FACS analysis with AC133-APC antibody. The percentage of CD133+ cells significantly increased by approximately 15%.

Supplemental Figure 7. CD31 staining in sections of intracranial xenografts originated from cells exposed to different pHs. Frozen sections of intracranial tumors initiated by injection of GSC depleted cultures exposed to pH 7.5 or pH 6.5 were stained with anti-CD31 (BD Pharmingen) to image blood vessels.

Supplemental Figure 8. Angiogenic regulators are elevated in GSC enriched cultures exposed to low pH.CD133+ cells were isolated from D456MG xenografts and subsequently treated with acidic (ph 6.5) or normal (pH 7.5) cell culture media for four 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 VEGF (A) and its regulator HIF2(B), but not HIF1 (C)are elevated by acidic stress.