Early ducts of breast tumors are unequivocally acidic. High rates of glycolysis combined with poor perfusion lead to a congestion of acidic metabolites in the tumor microenvironment, and pre-malignant cells must adapt to this acidosis to thrive. Adaptation to acidosis selects cancer cells that can thrive in harsh conditions and are capable of outgrowing the normal or non-adapted neighbors. This selection is usually accompanied by phenotypic change. Epithelial mesenchymal transition (EMT) is one of the most important switches correlated to malignant tumor cell phenotype and has been shown to be induced by tumor acidosis. New evidence shows that the EMT switch is not a binary system and occurs on a spectrum of transition states. During confirmation of the EMT phenotype, our results demonstrated a partial EMT phenotype in our acid-adapted cell population. Using RNA sequencing and network analysis we found 10 dysregulated network motifs in acid-adapted breast cancer cells playing a role in EMT. Our further integrative analysis of RNA sequencing and SILAC proteomics resulted in recognition of S100B and S100A6 proteins at both the RNA and protein level. Higher expression of S100B and S100A6 was validated in vitro by Immunocytochemistry. We further validated our finding both in vitro and in patients' samples by IHC analysis of Tissue Microarray (TMA). Correlation analysis of S100A6 and LAMP2b as marker of acidosis in each patient from Moffitt TMA approved the acid related role of S100A6 in breast cancer patients. Also, DCIS patients with higher expression of S100A6 showed lower survival compared to lower expression. We propose essential roles of acid adaptation in cancer cells EMT process through S100 proteins such as S100A6 that can be used as therapeutic strategy targeting both acid-adapted and malignant phenotypes.

Many invasive cancers emerge through a years-long process of somatic evolution, characterized by an accumulation of heritable genetic and epigenetic changes and the emergence of increasingly aggressive clonal populations. In solid tumors, such as breast ductal carcinoma, the extracellular environment for cells within the nascent tumor is harsh and imposes different types of stress on cells, such as hypoxia, nutrient deprivation, and cytokine inflammation. Acidosis is a constant stressor of most cancer cells due to its production through fermentation of glucose to lactic acid in hypoxic or normoxic regions (Warburg effect). Over a short period of time, acid stress can have a profound effect on the function of lysosomes within the cells exposed to this environment, and after long term exposure, lysosomal function of the cancer cells can become completely dysregulated. Whether this dysregulation is due to an epigenetic change or evolutionary selection has yet to be determined, but understanding the mechanisms behind this dysregulation could identify therapeutic opportunities.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO₂, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer–magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

After an initial response to chemotherapy, tumor relapse is frequent. This event is reflective of both the spatiotemporal heterogeneities of the tumor microenvironment as well as the evolutionary propensity of cancer cell populations to adapt to variable conditions. Because the cause of this adaptation could be genetic or epigenetic, studying phenotypic properties such as tumor metabolism is useful as it reflects molecular, cellular, and tissue-level dynamics. In triple-negative breast cancer (TNBC), the characteristic metabolic phenotype is a highly fermentative state. However, during treatment, the spatial and temporal dynamics of the metabolic landscape are highly unstable, with surviving populations taking on a variety of metabolic states. Thus, longitudinally imaging tumor metabolism provides a promising approach to inform therapeutic strategies, and to monitor treatment responses to understand and mitigate recurrence. Here we summarize some examples of the metabolic plasticity reported in TNBC following chemotherapy and review the current metabolic imaging techniques available in monitoring chemotherapy responses clinically and preclinically. The ensemble of imaging technologies we describe has distinct attributes that make them uniquely suited for a particular length scale, biological model, and/or features that can be captured. We focus on TNBC to highlight the potential of each of these technological advances in understanding evolution-based therapeutic resistance.

When cancer research advanced into the post-genomic era, it was widely anticipated that the sought-after cure will be delivered promptly. Instead, it became apparent that an understanding of cancer genomics, alone, is unable to translate the wealth of information into successful cures. While gene sequencing has significantly improved our understanding of the natural history of cancer and identified candidates for therapeutic targets, it cannot predict the impact of the biological response to therapies. Hence, patients with a common mutational profile may respond differently to the same therapy, due in part to different microenvironments impacting on gene regulation. This complexity arises from a feedback circuit involving epigenetic modifications made to genes by the metabolic byproducts of cancer cells. New insights into epigenetic mechanisms, activated early in the process of carcinogenesis, have been able to describe phenotypes which cannot be inferred from mutational analyses per se. Epigenetic changes can propagate throughout a tumor via heritable modifications that have long-lasting consequences on ensuing phenotypes. Such heritable epigenetic changes can be evoked profoundly by cancer cell metabolites, which then exercise a broad remit of actions across all stages of carcinogenesis, culminating with a meaningful impact on the tumor's response to therapy. This review outlines some of the cross-talk between heritable epigenetic changes and tumor cell metabolism, and the consequences of such changes on tumor progression.

Evolutionary dynamics can be used to control cancers when a cure is not clinically considered to be achievable. Understanding Darwinian intratumoral interactions of microenvironmental selection forces can be used to steer tumor progression towards a less invasive trajectory. Here, we approach intratumoral heterogeneity and evolution as a dynamic interaction among subpopulations through the application of small, but selective biological forces such as intracellular pH (pHi) and/or extracellular pH (pHe) vulnerabilities. Increased glycolysis is a prominent phenotype of cancer cells under hypoxia or normoxia (Warburg effect). Glycolysis leads to an important aspect of cancer metabolism: reduced pHe and higher pHi. We recently showed that decreasing pHi and targeting pHi sensitive enzymes can reverse the Warburg effect (WE) phenotype and inhibit tumor progression. Herein, we used diclofenac (DIC) repurposed to control MCT activity, and Koningic acid (KA) that is a GAPDH partial inhibitor, and observed that we can control the subpopulation of cancer cells with WE phenotype within a tumor in favor of a less aggressive phenotype without a WE to control progression and metastasis. In a 3D spheroid co-cultures, we showed that our strategy can control the growth of more aggressive MDA-MB-231 cells, while sparing the less aggressive MCF7 cells. In an animal model, we show that our approach can reduce tumor growth and metastasis. We thus propose that evolutionary dynamics can be used to control tumor cells’ clonal or sub-clonal populations in favor of slower growth and less damage to patients. We propose that this can result in cancer control for tumors where cure is not an option.