Acidosis-mediated inhibition of cancer cell dissemination is restored by hypoxia

Introduction: Inadequate blood flow and altered metabolism in solid tumors result in hypoxia and acidosis, respectively. While hypoxia may contribute to a locally acidic microenvironment, tumor acidosis may occur independently of hypoxia because of the Warburg effect, according to which cancer cells undergo a metabolic shift toward glycolysis and lactic acid production even in non-hypoxic conditions. This results in an acidic tumor microenvironment with an extracellular pH (pHe) ranging from 6.2 to 6.9, while the pHe in normal tissues is approximately 7.4. Extracellular acidosis, like hypoxia, is commonly thought to promote an aggressive cancer cell phenotype. Indeed, a more invasive metastatic potential for breast, colon, prostate, and lung cancer cells has been reported following exposure to extracellular acidosis. However, more evidence suggests that when cells are exposed to an acidic environment with a pHe of 6.4, the migration of lung cancer cells and the dissociation of melanoma cells from 3D spheroids is inhibited. Because of these contradictory results, we aimed to investigate how extracellular acidosis affects cancer cell behavior through in vitro and in vivo experiments. In particular, we focused on examining the potential contribution of sodium-hydrogen exchanger isoform-1 (NHE1), given its function in regulating intracellular pH (pHi), cell cytoskeleton dynamics, and cell migration.

Materials and

Methods: We employed PDMS microfluidic devices with confining channels (Width=20,10,6, or 3 µm, and Height=10 µm) to assess the migratory potential in confinement of MDA-MB-231 or SUM 159 cells subjected to physiological (7.4) or acidic (6.4) extracellular pH. Moreover, we generated 3D collagen spheroids to further our findings to invasion assays. Moreover, we utilized an ex ovo chick embryo cancer cell extravasation model to assess the extravasation of MDA-MB-231 or HT1080 cells preconditioned at pHe= 7.4 or 6.4 for 24h. MDA-MB-231 preconditioned at pHe of 7.4 or 6.4 for 24 h or 23 days, were injected in mice via tail vain injection and the colony formation in the lungs after 48 h or 7 days was assessed.

Results, Conclusions, and Discussions: Our findings reveal that exposing cancer cells to an acidic pHe of 6.4 leads to the suppression of cell proliferation, cell dissociation from tumor spheroids and migration in vitro as well as lower extravasation in vivo using the chick embryo and mouse models In the acute phase (within ~2h), acidosis rapidly inhibits cell motility by disrupting NHE1 polarization and reducing NHE1 activity, which in turn suppresses the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt). This impaired migratory potential in acidic conditions can be reversed by the presence of constitutively active Akt, highlighting the bi-directional crosstalk between NHE1 and PI3K/Akt pathway. At later time points, the inhibition of PI3K/Akt prevents Yes-associated protein (YAP) translocation to the nucleus, resulting in decreased expression of NHE1. While cells can adapt within minutes their pHi in response to changes in pHe, prolonged (≥24 h) exposure to acidosis leads to decreased NHE1 expression levels . However, upon switching the cell culture medium back to physiological pHe, NHE1 expression is restored in a delayed fashion. This lag recovery phase can explain the decreased extravasation of cancer cells that are preconditioned at an acidic pHe prior to their injection into chick embryos and mice. Importantly, the diminished NHE1 expression and migratory potential induced by acidosis are reversed by hypoxia. Our novel findings suggest that the aggressive nature of cancerous cells present in an acidic microenvironment is attributed to the local co-existence of hypoxia. As such, acidosis alone does not promote aggressive tumor phenotypes, but is instead an epiphenomenon of tumor growth.

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