Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer

Soojung Lee, Nicolai J. Toft, Trine V. Axelsen, Maria Sofia Espejo, Tina M. Pedersen, Marco Mele, Helene L. Pedersen, Eva Balling, Tonje Johansen, Mark Burton, Mads Thomassen, Pernille Vahl, Peer Christiansen, Ebbe Boedtkjer*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

Background: Carbonic anhydrases catalyze CO2/HCO3 buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid–base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear. Methods: We combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas. Results: Carbonic anhydrases—particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14—undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis. Conclusions: We conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

Original languageEnglish
Article number46
JournalBreast Cancer Research
Volume25
Number of pages27
ISSN1465-542X
DOIs
Publication statusPublished - 25. Apr 2023

Keywords

  • Acetazolamide
  • Acidosis
  • Breast cancer
  • Carbonic anhydrases
  • ErbB2
  • HER2
  • Immuno-oncology
  • Metabolism
  • Perfusion
  • Tumor microenvironment
  • Triple Negative Breast Neoplasms
  • Humans
  • Carbonic Anhydrases/genetics
  • Animals
  • Proteomics
  • Antigens, Neoplasm/genetics
  • Mice
  • Acetazolamide/pharmacology
  • Receptor, ErbB-2
  • Tumor Microenvironment/genetics
  • Hydrogen-Ion Concentration

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