Acidic extracellular pH of tumors induces octamer-binding transcription factor 4 expression in murine fibroblasts in vitro and in vivo

Octamer-binding transcription factor 4 (OCT-4) is an important marker of cellular de-differentiation that can be induced by environmental stressors, such as acidity. Here we demonstrate that chronic acidic stress in solid tumors induced OCT-4 expression in fibroblasts and other stromal cells in four tumor models. The results have implications for how tumors utilize pH modulation to recruit associated stromal cells, induce partial reprogramming of tumor-associated stromal cells, and respond to therapy.

Scientific RepoRts | 6:27803 | DOI: 10.1038/srep27803 absence of cancer cells, OCT-4 expression in the 3T3 cells did not increase over time (Fig. 1a). In contrast, co-culture of these fibroblasts with tumor cells at pH 7.4 resulted in increased OCT-4 expression (Fig. 1b,c). The enhanced nuclear localization of OCT-4 indicates that tumor cells can facilitate OCT-4 expression in surrounding healthy tissue under normal physiological pH.
A previous study showed that cells cultured in a highly acidic environment (pH 5) showed increased OCT-4 expression 15 . Because the extracellular pH of solid tumors rarely falls below pH 6, we investigated whether OCT-4 expression could be induced in cells cultured in a more physiologically relevant, mildly acidic medium (pH 6.5), similar to the extracellular pH of solid tumors. GFP transfected 3T3 fibroblast cells were cultured in acidic (pH 6.5) media for 7 days in the presence or absence of tumor cells. A high level and intense nuclear localization of OCT-4 expression was observed in both the presence and absence of tumor cells (Fig. 1d-f), suggesting the possibility of cell reprogramming under these conditions. Co-staining with vimentin shows strong cytoplasmic vimentin positivity confirming the fibroblast origin of the cells (Fig. 1). The percentage of OCT-4 positive cells sequentially increased over time (Fig. 2), indicating that chronic exposure of these cells to acidic environments such as those found in some solid tumors, provides a potential pathway to induce cellular stress to surrounding healthy cells. In addition, there is an inverse correlation of OCT-4 expression with acidity, OCT-4 intensity increasing as pH decreases from 7.4 to 6.5 (Supplementary Figure 2). Very few BxPC-3 and MDA-MB-231 (GFP-negative) cells were observed because these cells do not remain adherent during staining. Similar results were obtained when cells were stained using a second OCT-4 antibody (Supplementary Figure 1). Incubation with non-tumor cell lines, such as primary mammary epithelial cells (PMEC), under acidic conditions showed OCT-4 expression in both cell lines, while no OCT-4 expression was seen under non-acidic conditions. (Supplementary Figure 3).

Ex vivo expression of OCT-4 in tumor tissue. We next investigated OCT-4 expression in xenografts
from three tumor models: 4T1luc, EMT-6, and BxPC-3. Cardiac muscle was used as an in vivo negative control due to the low propensity of that tissue to stem cell activity. Expectedly, we did not detect OCT-4 expression in heart tissue (Supplementary Figure 6). Tumors were 0.5 cm to 1.5 cm in size on excision. The average pH level in 4T1 tumor models was 6.8+ /− 0.1 pH units as measured by an invasive 5 mm probe. This is consistent with previous studies, which reported acidity in BxPC-3 and EMT-6 tumor models and the importance of the pH for chemotherapy resistance 19 . In contrast, OCT-4 expression was detected in all three tumor tissues (Fig. 3) and was localized to certain cells in and around the tissue section. The entirety of the section was considered to be tumor. These results suggest that OCT-4 expression spans multiple human and murine tumor types. OCT-4 expression, however, seems to be sparse, with only a few tissue areas showing positive staining.

Identification of fibroblasts expressing OCT-4 in tumor tissue.
Fibroblasts are an important and common cell type in cancer stromal tissue. To verify that the OCT-4 positive cells were fibroblasts, we co-stained tissues for the fibroblast marker vimentin and OCT-4 ( Fig. 4). Co-staining indicated that OCT-4 localized in the vicinity of vimentin positive cells, with several instances of co-localization (yellow, Fig. 4), suggesting that these cells are indeed fibroblasts. Our data suggest the presence of three populations of cells: OCT-4 + , vimentin + , and a subset of cells positive for both OCT-4 and vimentin. MDA-MB-231 is a human breast cell carcinoma cell line that has high intrinsic expression of OCT-4. We did not evaluate the subcellular distribution of OCT-4 or vimentin in the tissue sections.
Literature values and independent pH measurements in our laboratory show that the pH in the tumor area ranges from 6.5-7.0. By chronically maintaining the extracellular pH below 7, cancer cells can stimulate non-tumor cells to reprogram via pH-related stress. This process could aid the recruitment of surrounding cells into the tumor matrix.

Discussion
The extracellular pH of tumors has been implicated in the metastasis and behavior of tumor cells, and changes in this pH could modify the downstream behavior of cancer 18 . To our knowledge, this is the first report of the expression of OCT-4 in the vicinity of tumors, particularly in supporting stromal cells. Stress based in vitro OCT-4 expression has been reported previously for stem cells 14,21 . Our data suggests that chronic mild acidity, which is present in the tumor environment along with other stressors, is capable of inducing OCT-4 expression in murine fibroblasts and may be sufficient to induce OCT-4 expression in vivo. These observations suggest that tumor cells in vivo may be capable of reprogramming surrounding stromal cells using environmentally mediated factors.
There are a few caveats to the above platform. The tumors were all xenografts and it is not known if this phenomenon occurs in spontaneous tumor models. We focused primarily on murine fibroblasts to facilitate in vivo Ex vivo histology showed expression of OCT-4 in cells that are not fibroblasts. Other than MDA MB-231 tumors, which had high intrinsic expression of OCT-4, the positive cells did not make up the bulk of the tumor mass. We posit that these cells may be endothelial or other epithelial cells in the stromal region, or perhaps some small subset of cancer cells. This hypothesis is supported by in vitro expression of OCT4 + in PMEC cells, a primary mammary epithelial cell line, under acidic conditions. This report offers observational evidence that mild acidity does induce some degree of OCT-4 expression in vivo, therefore confirming previous findings about stress based OCT-4 expression. However, it is not clear how  long these cells maintain the OCT-4 expression nor if the expression induces any other pluripotent stem cell phenotype. To rule out the possibility of artifact, we tested both a mouse monoclonal and a rabbit polyclonal anti-OCT-4 antibody. We observed similar results with both antibodies, although OCT-4 localization at times alternated between cytoplasmic (murine monoclonal) and nuclear (rabbit polyclonal), depending on the type of antibody used (Fig. 1, Supplementary Figure 1).
Fibroblasts are traditionally considered to be strong supporting actors in the tumor microenvironment. The concept that chronic physiological extracellular acidity can have significant effects on the expression of reprogramming markers, such as OCT-4 both in vitro and in vivo, suggests a possible mechanism for the phenotypic changes in cancer associated fibroblasts. One report indicates that acidity may actually transform fibroblasts into cancerous tissue 22 . Some preliminary data (Supplementary Figure 4) demonstrate that implantation of these acid treated cells induces tumor growth in athymic nude mice. For this study, fibroblast cells were not co-cultured with tumor cells. Further, the concept that acid mediated stress can induce the presence of factors that cause reprogramming in vivo raises the question of what happens if intratumoral pH is changed in vivo. Indeed, pH changes in vivo can inhibit tumor growth 23 .
Overall, this study reveals that the stroma of tumors express OCT-4. Based on in vitro data, it is likely that chronic mild acidic stress could facilitate cell reprogramming. Given the ubiquity of extracellular acidic environments in some solid cancers, this process could be a common occurrence in vivo. We demonstrated that pH evidently has an important effect on both cancer cells and the supporting stroma. These results further support the relevance of the extracellular matrix and its environment to cancer behavior, prognosis, and therapy. In particular, the increasing evidence that tumors actively maintain an extracellular acidic environment implies a causative role for pH in tumor survival and proliferation. There is thus an intersection between the significant work involved in cancer metabolomics, the Warburg effect, and the tumor microenvironment. Future work will focus on replicating this observed phenomenon in vivo in human subjects. Tumor model growth. All the animal experiments were conducted in accordance with the approved guidelines for the care and use of laboratory animals in research, and the protocol was approved by the Washington University Animal Welfare Committee. BxPC-3, MDA-MB-231, and EMT-6 cells were injected subcutaneously into the dorsal flanks of athymic nude mice, while 4T1luc cells were injected into Balb/c mice. Mice were sacrificed when the tumors were between 0.5 and 1.5 cm in diameter. In certain cases, tumors may have had exposure to the near-infrared tumor imaging agent cypate prior to excision. Tumors were excised based on gross appearance and frozen in OCT media. The average pH in 4T1 tumor models was 6.8+ /− 0.1 pH units, as measured by an invasive probe (5 mm probe). The average pH level in EMT-6 and BxPC-3 tumors was not measured, previous studies reported acidity in BxPC-3 and EMT-6 tumor models and the importance of the pH for chemotherapy resistance 19 . Tissue sections were cryosectioned at 10 µm thickness. Tissue was visualized with an Olympus BX51 upright epifluorescence microscope (Olympus America, Center Valley, PA) using excitation/emission filters of 480 nm/535 nm, and 620 nm/700 nm.
Longitudinal study. Cells were cultured as described above in normal media overnight and then switched to acidic conditions at day 0, 1, 3, and 5 and incubated until day 7 prior to immunostaining. 3T3 Fibroblast growth in nude mice. 3T3-GFP Fibroblast cells were grown in T75 flasks under either HCl titrated, pH 6.5 conditions, as described above, or in pH 7.4 media for 7 days, or grown in T75 flasks under normal conditions overnight. Cells were trypsinized and injected in a 50% matrigel/PBS matrix. For each "tumor" injection site, 1 million cells from the above three categories were used. Mouse 1 received the acid treated cells on its left flank and 7 day pH 7.4 treated cells in its right flank. Mouse 2 received the acid treated cells on its left flank and 1 day pH 7.4 treated cells in its right flank. Mouse 3 received 7 day control cells on its left flank and 1 day pH 7.4 treated cells in its right flank. GFP images were taken 2 weeks later using the ART Optix MX3 system.