Cancer cells tend to turn their extracellular environment acidic, thus helping these rogue cells thrive while creating a hostile environment for normal ones. Acidosis promotes tumor growth, metastasis and drug resistance, while helping tumor cells evade the immune system. But it’s not clear exactly how this occurs. Now, a recent study in Cancer Research provides vibrant visual evidence showing exactly where acidosis occurs in tumors and offers clues about how changes in gene expression might drive tumor cells to invade surrounding tissues, a precursor to metastasis.
“This is a really fascinating study with a lot of new discovery,” says Diane Barber, a cell biologist at the University of California San Francisco who was not involved with the research. “It will open new directions for investigating adaptive features of cancer cells and how they can be therapeutically targeted.”
To investigate how the acidic tumor microenvironment might encourage cells to invade tissues, the researchers first sought to pinpoint exactly where acidosis occurs in tumors. Using a fluorescently-labeled peptide called pHLIP that only inserts itself into the membranes of cells under acidic conditions, they mapped the acidic regions of mammary tumors produced by human breast cancer cell lines in mice. The pH probe provided striking images, for the first time at cell-level resolution, that confirmed what previous methods had suggested: acidosis is not confined to the hypoxic tumor core, but rather extends to the boundary between tumors and healthy tissue. Called the tumor-stroma interface, this boundary is the advancing edge of a malignant tumor, where cells spread aggressively and break into blood vessels and nearby healthy tissues. Again using fluorescent markers, the researchers found that the acidic regions displayed signatures of tumor invasiveness: increased expression of matrix metalloproteases in the cells at the acidic tumor-stroma interface as well as degradation of the basement membrane surrounding healthy tissues.
To further examine the triggers for acidosis, the researchers employed a fluorescent marker for the enzyme lactate dehydrogenase-A. It revealed that the majority of cells at the interface were carrying out glycolysis, a type of metabolism that perpetuates an acidic environment. Hypoxia, or low oxygen, can force cells to resort to anaerobic glycolysis; however, glycolysis was occurring at the tumor-stroma interface even in the presence of oxygen. Together, the results suggest that aerobic glycolysis may be a primary driver of acidosis at the interface, spurring cells to invade. “The next logical steps would be looking into the causes of tumor acidity that are independent of glycolysis and hypoxia because it’s still relatively unknown,” says Ian Robey, a tumor biologist at the University of Arizona Cancer Center who was not involved with the research.
The researchers also found changes in gene expression associated with the acidic environment. RNA sequencing of mouse and human breast cancer cells exposed to an acidic environment in vitro revealed changes in the expression or post-transcriptional processing, binding, and splicing of around 3,000 genes, many of which are known or suspected to be involved in cell invasion, metastasis, and drug resistance.
Two of particular interest were Mena and CD44. The alternatively spliced form of Mena produces a protein that helps tumor cells enter the blood stream during metastasis. The alternative form of CD44 is proposed to play a role in drug resistance. Further experiments in mice demonstrated that an acidic pH causes modifications of cellular chromatin, which in turn affects how RNA transcripts from Mena and CD44 are spliced.
“What this paper shows is how the lower extracellular pH can change the transcriptional program of cancer cells,” Barber says. Though she did see one shortcoming. “I think a major limitation is that they don’t consider or measure intracellular pH dynamics, which are likely major regulators of some of these cellular processes.”
Still, because most solid tumors surround themselves with an acidic environment, the findings have potentially wide-ranging therapeutic applications. “I hypothesize that there will be similar enrichment in acidic compartments near invasive areas of other solid tumor types, but that’s something that remains to be demonstrated experimentally,” says paper coauthor Frank Gertler, a cell biologist at the MIT Koch Institute for Integrative Cancer Research in Cambridge.
In the study, when researchers fed sodium bicarbonate (baking soda) to mice with mammary or lung tumors, the tumor boundaries became less acidic and the proliferative and metastatic signatures of the tumor cells decreased. “It adds to the sense that this pH dynamic is not permanent. It’s reversible,” Robey says. “I think that’s an important addition to an ongoing discussion about the role of pH in tumor behavior.”
While baking soda may not be a realistic treatment for humans, clinical trials are currently testing nanoparticles and other agents that zero in on the low pH environment surrounding solid tumors and raise extracellular pH in an attempt to shut down metastasis. “It’s an exciting possibility to essentially have pH-sensitive therapeutics that that might become active upon encountering the acidity of the microenvironment,” adds Gertler.
Gertler and study coauthor Nazanin Rohani, now a cell biologist at the National Research Council Canada in Montreal, say that future work will focus on investigating whether the pH-induced transcriptional changes their team observed can explain the mechanisms underlying metastasis and possibly identify new therapeutic targets.