Stromal uptake and transmission of acid is a pathway for venting cancer cell-generated acid

Abstract

Proliferation and invasion of cancer cells require favorable pH, yet potentially toxic quantities of acid are produced metabolically. Membrane-bound transporters extrude acid from cancer cells, but little is known about the mechanisms that handle acid once it is released into the poorly perfused extracellular space. Here, we studied acid handling by myofibroblasts (colon cancer-derived Hs675.T, intestinal InMyoFib, embryonic colon-derived CCD-112-CoN), skin fibroblasts (NHDF-Ad), and colorectal cancer (CRC) cells (HCT116, HT29) grown in monoculture or coculture. Expression of the acidloading transporter anion exchanger 2 (AE2) (SLC4A2 product) was detected in myofibroblasts and fibroblasts, but not in CRC cells. Compared with CRC cells, Hs675.T and InMyoFib myofibroblasts had very high capacity to absorb extracellular acid. Acid uptake into CCD-112-CoN and NHDF-Ad cells was slower and comparable to levels in CRC cells, but increased alongside SLC4A2 expression under stimulation with transforming growth factor β1 (TGFβ1), a cytokine involved in cancer-stroma interplay. Myofibroblasts and fibroblasts are connected by gap junctions formed by proteins such as connexin-43, which allows the absorbed acid load to be transmitted across the stromal syncytium. To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFβ1. In contrast, acid transmission was absent between CRC cells, even after treatment with TGFβ1. Thus, stromal cells have the necessary molecular apparatus for assembling an acidventing route that can improve the flow of metabolic acid through tumors. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities.colorectal cancer | myofibroblast | anion exchanger 2 | gap junctions | TGFβ1

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm

“…Based on this biophysical principle, it is possible to describe the diffusive properties of cytoplasmic buffers from measurements of H + dynamics in intact cells1825. To determine D H app , a local source of H + ions was produced in cytoplasm by photolytic uncaging from the membrane-permeant H + -donor 6-nitroveratraldehyde (NVA)23 once every 0.13 s. This generates an acidic microdomain which dissipates at a rate determined by the buffers’ concentrations, reaction kinetics with H + ions, and diffusion coefficients. To follow the progress of buffer-facilitated H + diffusion, pH-sensitive cSNARF1 fluorescence was imaged confocally across the RBC’s horizontal plane at intervals between H + uncaging (e.g.…”

“…1C,D)20. Provided that membrane transport of H + and HCO 3 − ions is slow or inactivated, best-fitting these spatio-temporal [H + ] data with a diffusion equation (derived previously182326) gives a robust measure of D H app .…”

“…Photolytic uncaging of H + ions from 6-nitroveratralhyde23 (NVA; added to solutions at 1 mM) was evoked by scanning a region of interest (ROI) at one end of the RBC (ROI width equal to 1/10 th of RBC diameter) with 405 nm laser light, as shown in Fig. 1C,D.…”

“…This measurement method is based on the observation that cytoplasmic H + ions are heavily buffered20, and therefore diffuse only as fast as the buffers there are bound to (with essentially no free ion movement)2122. Thus, by determining the CO 2 /HCO 3 − -dependent component of buffer-facilitated H + diffusion171823, it is possible to quantify D CO2 . Our measurements on human RBCs demonstrate substantially restricted CO 2 diffusivity, to 5% of the rate in water, which is an order of magnitude slower than estimates made previously in cell-free hemoglobin solutions.…”

Normoxic cells remotely regulate the acid‐base balance of cells at the hypoxic core of connexin‐coupled tumor growths

“…Various mechanisms for assisting cancer cell acid-base homeostasis have been proposed, such as acid uptake by juxtaposed stromal cells (16), but these do not involve the direct provision of HCO 3 2 ions to cancer cell cytoplasm. Although carbonic anhydrase (CA)-catalyzed intracellular CO 2 hydration produces HCO 3 2 ions (17), it also loads cytoplasm with H + ions, which counters the objective of acid extrusion.…”