The notion that invasive cancer is a product of somatic evolution is a well-established theory that can be modelled mathematically and demonstrated empirically from therapeutic responses. Somatic ...evolution is by no means deterministic, and ample opportunities exist to steer its trajectory towards cancer cell extinction. One such strategy is to alter the chemical microenvironment shared between host and cancer cells in a way that no longer favours the latter. Ever since the first description of the Warburg effect, acidosis has been recognised as a key chemical signature of the tumour microenvironment. Recent findings have suggested that responses to acidosis, arising through a process of selection and adaptation, give cancer cells a competitive advantage over the host. A surge of research efforts has attempted to understand the basis of this advantage and seek ways of exploiting it therapeutically. Here, we review key findings and place these in the context of a mathematical framework. Looking ahead, we highlight areas relating to cellular adaptation, selection, and heterogeneity that merit more research efforts in order to close in on the goal of exploiting tumour acidity in future therapies.
Malignancy is enabled by pro-growth mutations and adequate energy provision. However, global metabolic activation would be self-terminating if it depleted tumor resources. Cancer cells could avoid ...this by rationing resources, e.g., dynamically switching between “baseline” and “activated” metabolic states. Using single-cell metabolic phenotyping of pancreatic ductal adenocarcinoma cells, we identify MIA-PaCa-2 as having broad heterogeneity of fermentative metabolism. Sorting by a readout of lactic acid permeability separates cells by fermentative and respiratory rates. Contrasting phenotypes persist for 4 days and are unrelated to cell cycling or glycolytic/respiratory gene expression; however, transcriptomics links metabolically active cells with interleukin-6 receptor (IL-6R)-STAT3 signaling. We verify this by IL-6R/STAT3 knockdowns and sorting by IL-6R status. IL-6R/STAT3 activates fermentation and transcription of its inhibitor, SOCS3, resulting in delayed negative feedback that underpins transitions between metabolic states. Among cells manifesting wide metabolic heterogeneity, dynamic IL-6R/STAT3 signaling may allow cell cohorts to take turns in progressing energy-intense processes without depleting shared resources.
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•PDAC cells sorted by lactic acid efflux capacity yield metabolically distinct sub-populations•The contrast in fermentative rate is short lived and linked to IL-6R/STAT3 signaling•IL-6R/STAT3 activation is transient under delayed negative feedback from SOCS3•The ensuing metabolic heterogeneity may facilitate rationing of tumor resources
Blaszczak et al. describe a mechanism for metabolic heterogeneity in PDAC cells, driven by transient activation of the IL-6R/STAT3 pathway. To separate cells by fermentative rate, the authors design a sorting protocol that sortscells by lactic acid efflux capacity and allows phenotyping of the emergent sub-populations.
Ascorbic acid (vitamin C) has been gaining attention as a potential treatment for human malignancies. Various experimental studies have shown the ability of pharmacological doses of vitamin C alone ...or in combinations with clinically used drugs to exert beneficial effects in various models of human cancers. Cytotoxicity of high doses of vitamin C in cancer cells appears to be related to excessive reactive oxygen species generation and the resulting suppression of the energy production via glycolysis. A hallmark of cancer cells is a strongly upregulated aerobic glycolysis, which elevates its relative importance as a source of ATP (Adenosine 5'-triphosphate). Aerobic glycolysis is maintained by a highly increased uptake of glucose, which is made possible by the upregulated expression of its transporters, such as GLUT-1, GLUT-3, and GLUT-4. These proteins can also transport the oxidized form of vitamin C, dehydroascorbate, permitting its preferential uptake by cancer cells with the subsequent depletion of critical cellular reducers as a result of ascorbate formation. Ascorbate also has a potential to affect other aspects of cancer cell metabolism due to its ability to promote reduction of iron(III) to iron(II) in numerous cellular metalloenzymes. Among iron-dependent dioxygenases, important targets for stimulation by vitamin C in cancer include prolyl hydroxylases targeting the hypoxia-inducible factors HIF-1/HIF-2 and histone and DNA demethylases. Altered metabolism of cancer cells by vitamin C can be beneficial by itself and promote activity of specific drugs.
Fucoidans have been reported to exert anticancer effects with simultaneous low toxicity against healthy tissue. That correlation was observed in several cancer models, however, it has never been ...investigated in head and neck cancer before. To magnify the efficacy of conventional therapy, the administration of agents like fucoidan could be beneficial. The aim of this study was to evaluate the anticancer effect of
(FV) extract alone and with co-administration of cisplatin in head and neck squamous cell carcinoma (HNSCC) in vitro. MTT assay results revealed an FV-induced inhibition of proliferation in all tested cell lines (H103, FaDu, KB). Flow cytometric cell cycle analysis showed an FV-induced, dose-dependent arrest in either S/G2 phase (H103, FaDu) or G1 arrest (KB). Furthermore, a dose-dependent gain in apoptotic fraction was observed. Western blot analysis confirmed the induction of apoptosis. A significant dose-dependent increase in reactive oxygen species (ROS) production was revealed in the H103 cell line, while FaDu cells remained unresponsive. On the contrary, an HPV-positive cell line, KB, demonstrated a dose-dependent decrease in ROS synthesis. Moreover, fucoidan enhanced the response to cisplatin (synergistic effect) in all cell lines with the HPV-positive one (KB) being the most sensitive. These results have been confirmed by flow-cytometric apoptosis analysis. In conclusion, we confirmed that fucoidan exhibits anticancer properties against HNSCC, which are manifested by the induction of apoptosis, regulation of ROS production, cell cycle arrest, and inhibition of proliferation.
Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells ...according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations.
For complete details on the use and execution of this protocol, please refer to Blaszczak et al.1
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•Metabolic activity is a key cellular phenotype, determined by a myriad of variables•Cancer cells with higher lactic acid permeability tend to have higher metabolic rate•Lactate unloading produces alkaline transients that identify cells by metabolic rate•Implementation for FACS generates subpopulations of distinct metabolic phenotype
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations.
Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for ...cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1−/− cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.
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•CRISPR screen identifies 51 genes required for survival at low versus physiological pH•Cancer cell survival at low pH requires NDUFS1 and other OXPHOS genes•OXPHOS inhibitors selectively kill cancer cells at acidic pH, but allow survival at physiological pH•NDUFS1 knockout abrogates tumor xenograft growth in a pH-dependent manner
Alongside hypoxia, acidosis is a chemical signature of many solid tumors. By performing a genome-wide CRISPR-Cas9 screen, Michl et al. find that cancer cells require NDUFS1 and other OXPHOS genes for survival in acidic environments. OXPHOS inhibitors selectively kill cancer cells at acidic pH, but permit survival at physiological pH, which defines a strategy for targeting acidic tumor regions.
Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low ...extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.
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•Screen of 66 cancer cell lines reveals variation in pHi-regulatory phenotypes•Resting pHi is set by the activity of acid-loading anion exchanger 2 (AE2)•Cancer cells adapt to acidic environments by degrading AE2, which raises pHi•Lysosomal degradation of AE2 is triggered by mTORC1 inhibition by low pHi
It remains unclear how cancer cells can maintain a physiological intracellular pH, despite their acidic microenvironment. Michl et al. show that extracellular acidity triggers the lysosomal degradation of anion exchanger 2 (AE2), an acid-loading membrane transporter. Reduced AE2 activity raises intracellular pH toward physiological levels in cells under chronic acidosis.
Aberrant protein acetylation is strongly linked to tumorigenesis, and modulating acetylation through targeting histone deacetylase (HDAC) with small‐molecule inhibitors has been the focus of clinical ...trials. However, clinical success on solid tumours, such as colorectal cancer (CRC), has been limited, in part because the cancer‐relevant mechanisms through which HDAC inhibitors act remain largely unknown. Here, we have explored, at the genome‐wide expression level, the effects of a novel HDAC inhibitor CXD101. In human CRC cell lines, a diverse set of differentially expressed genes were up‐ and downregulated upon CXD101 treatment. Functional profiling of the expression data highlighted immune‐relevant concepts related to antigen processing and natural killer cell‐mediated cytotoxicity. Similar profiles were apparent when gene expression was investigated in murine colon26 CRC cells treated with CXD101. Significantly, these changes were also apparent in syngeneic colon26 tumours growing in vivo. The ability of CXD101 to affect immune‐relevant gene expression coincided with changes in the tumour microenvironment (TME), especially in the subgroups of CD4 and CD8 tumour‐infiltrating T lymphocytes. The altered TME reflected enhanced antitumour activity when CXD101 was combined with immune checkpoint inhibitors (ICIs), such as anti‐PD‐1 and anti‐CTLA4. The ability of CXD101 to reinstate immune‐relevant gene expression in the TME and act together with ICIs provides a powerful rationale for exploring the combination therapy in human cancers.
CXD101 is a novel histone deacetylase inhibitor with potent antitumour activity. We find that CXD101 reinstates immune‐relevant gene expression in tumours, which includes major histocompatibility complex class I and class II genes. This enables CXD101 to enhance the activity of immune checkpoint therapies, such as anti‐PD‐1, on tumours that would otherwise be poorly responsive and coincides with increased T lymphocyte infiltration into the tumour microenvironment.
Diversity in research teams ties alternative perspectives into research projects, and this can fast‐forward scientific progress. Concerted efforts have been aimed at encouraging and supporting women ...to pursue a career in science, yet a gender disparity can still be observed at senior positions, with fewer women in leadership roles. To get insight into how the current landscape for women in science is perceived by different career stages, we interviewed female authors of Molecular Oncology from diverse career stages and disciplines about their inspiration, challenges they have faced as scientists as well as their thoughts on how gender diversity can be further enhanced.
Despite funding schemes and support networks addressing gender imbalance, female researchers still occupy considerably fewer senior positions than male scientists. We interviewed female authors of Molecular Oncology about their motivation, challenges faced as scientists, and their perspectives on reinforcing gender diversity. Responses suggested joint efforts from researchers and academic bodies are needed to even the scales.
A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to ...quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.
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