Acidification of Tumor at Stromal Boundaries Drives Transcriptome Alterations Associated with Aggressive Phenotypes

Abstract

Acidosis is a fundamental feature of the tumor microenvironment, which directly regulates tumor cell invasion by affecting immune cell function, clonal cell evolution, and drug resistance. Despite the important association of tumor microenvironment acidosis with tumor cell invasion, relatively little is known regarding which areas within a tumor are acidic and how acidosis influences gene expression to promote invasion. Here, we injected a labeled pH-responsive peptide to mark acidic regions within tumors. Surprisingly, acidic regions were not restricted to hypoxic areas and overlapped with highly proliferative, invasive regions at the tumor-stroma interface, which were marked by increased expression of matrix metalloproteinases and degradation of the basement membrane. RNA-seq analysis of cells exposed to low pH conditions revealed a general rewiring of the transcriptome that involved RNA splicing and enriched for targets of RNA binding proteins with specificity for AU-rich motifs. Alternative splicing of Mena and CD44, which play important isoform-specific roles in metastasis and drug resistance, respectively, was sensitive to histone acetylation status. Strikingly, this program of alternative splicing was reversed in vitro and in vivo through neutralization experiments that mitigated acidic conditions. These findings highlight a previously underappreciated role for localized acidification of tumor microenvironment in the expression of an alternative splicingdependent tumor invasion program.Significance: This study expands our understanding of acidosis within the tumor microenvironment and indicates that acidosis induces potentially therapeutically actionable changes to alternative splicing.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery

“…pHLIP peptides tumor targeting has been shown to be enhanced in tumors that are further acidified by co-injection of glucose 12 or by overexpression of CAIX at the surface of HCT116 cancer cells 10 . Alternatively, pHLIP peptides tumor targeting was shown to be reduced by alkalization of tumors in mice fed with bicarbonate drinking water 13 . Micro-metastases near primary tumors and in distant organs were shown to be very well targeted by pHLIP peptides 12 , 14 – 16 .…”

“…In multiple studies, it has been found that non-protonatable (at low pH) pHLIP variants, where some or all Asp/Glu residues were replaced by Lys, did not target tumors and did not exhibit pH-dependent membrane insertion. pHLIP peptides distribution within tumors, cellular localization and correlation with a variety of markers have been intensively investigated 12 , 13 , 17 , 18 .

Figure 1 Characterization of pHLIP ICG and blood clearance in mice and dogs.

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Cellular and Extracellular Components in Tumor Microenvironment and Their Application in Early Diagnosis of Cancers

“…Additionally, tumor cells possess all enzyme systems to adjust to the acid environment that plays a crucial role in cancer progression [6,64]. It is reported that acidic regions are not only restricted to hypoxic areas but overlapped at the tumor-stroma interface which plays a crucial role in tumor proliferation and invasion [69]. Some people explained that neighbor normal stromal cells can absorb the large amount of lactic acid released from tumor cells to regenerate pyruvate and restrict extracellular overacidification [70].…”

A pH ratiometrically responsive surface enhanced resonance Raman scattering probe for tumor acidic margin delineation and image-guided surgery

“…5 Gertler et al showed that acidication in tumor boundaries drove transcriptome dynamics to promote the acquisition of invasive phenotypes. 6 Therefore, intra-operative determination of acidities in tumor cutting edges holds promise in locating and excising the inltrative region with high malignancy.…”