Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race.

BACKGROUND: Cancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host's heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation. METHODS: We investigate this "evolutionary arms race" through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round. RESULTS: The evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice. CONCLUSION: Host species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

T-cells produce acidic niches in lymph nodes to suppress their own effector functions.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation.

Translating preclinical MRI methods to clinical oncology.

The complexity of modern in vivo magnetic resonance imaging (MRI) methods in oncology has dramatically changed in the last 10 years. The field has long since moved passed its (unparalleled) ability to form images with exquisite soft-tissue contrast and morphology, allowing for the enhanced identification of primary tumors and metastatic disease. Currently, it is not uncommon to acquire images related to blood flow, cellularity, and macromolecular content in the clinical setting. The acquisition of images related to metabolism, hypoxia, pH, and tissue stiffness are also becoming common. All of these techniques have had some component of their invention, development, refinement, validation, and initial applications in the preclinical setting using in vivo animal models of cancer. In this review, we discuss the genesis of quantitative MRI methods that have been successfully translated from preclinical research and developed into clinical applications. These include methods that interrogate perfusion, diffusion, pH, hypoxia, macromolecular content, and tissue mechanical properties for improving detection, staging, and response monitoring of cancer. For each of these techniques, we summarize the 1) underlying biological mechanism(s); 2) preclinical applications; 3) available repeatability and reproducibility data; 4) clinical applications; and 5) limitations of the technique. We conclude with a discussion of lessons learned from translating MRI methods from the preclinical to clinical setting, and a presentation of four fundamental problems in cancer imaging that, if solved, would result in a profound improvement in the lives of oncology patients. Level of Evidence: 5 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2019;50:1377-1392.

Spatial Heterogeneity and Evolutionary Dynamics Modulate Time to Recurrence in Continuous and Adaptive Cancer Therapies.

Treatment of advanced cancers has benefited from new agents that supplement or bypass conventional therapies. However, even effective therapies fail as cancer cells deploy a wide range of resistance strategies. We propose that evolutionary dynamics ultimately determine survival and proliferation of resistant cells. Therefore, evolutionary strategies should be used with conventional therapies to delay or prevent resistance. Using an agent-based framework to model spatial competition among sensitive and resistant populations, we applied antiproliferative drug treatments to varying ratios of sensitive and resistant cells. We compared a continuous maximum-tolerated dose schedule with an adaptive schedule aimed at tumor control via competition between sensitive and resistant cells. Continuous treatment cured mostly sensitive tumors, but with any resistant cells, recurrence was inevitable. We identified two adaptive strategies that control heterogeneous tumors: dose modulation controls most tumors with less drug, while a more vacation-oriented schedule can control more invasive tumors. These findings offer potential modifications to treatment regimens that may improve outcomes and reduce resistance and recurrence.Significance: By using drug dose modulation or treatment vacations, adaptive therapy strategies control the emergence of tumor drug resistance by spatially suppressing less fit resistant populations in favor of treatment sensitive ones. Cancer Res; 78(8); 2127-39. ©2018 AACR.

Hypoxia and acidosis: immune suppressors and therapeutic targets.

Due to imbalances between vascularity and cellular growth patterns, the tumour microenvironment harbours multiple metabolic stressors including hypoxia and acidosis, which have significant influences on remodelling both tumour and peritumoral tissues. These stressors are also immunosuppressive and can contribute to escape from immune surveillance. Understanding these effects and characterizing the pathways involved can identify new targets for therapy and may redefine our understanding of traditional anti-tumour therapies. In this review, the effects of hypoxia and acidosis on tumour immunity will be summarized, and how modulating these parameters and their sequelae can be a useful tool for future therapeutic interventions is discussed.

Tris-base buffer: a promising new inhibitor for cancer progression and metastasis.

Neutralizing tumor external acidity with oral buffers has proven effective for the prevention and inhibition of metastasis in several cancer mouse models. Solid tumors are highly acidic as a result of high glycolysis combined with an inadequate blood supply. Our prior work has shown that sodium bicarbonate, imidazole, and free-base (but not protonated) lysine are effective in reducing tumor progression and metastasis. However, a concern in translating these results to clinic has been the presence of counter ions and their potential undesirable side effects (e.g., hypernatremia). In this work, we investigate tris(hydroxymethyl)aminomethane, (THAM or Tris), a primary amine with no counter ion, for its effects on metastasis and progression in prostate and pancreatic cancer in vivo models using MRI and bioluminescence imaging. At an ad lib concentration of 200 mmol/L, Tris effectively inhibited metastasis in both models and furthermore led to a decrease in the expression of the major glucose transporter, GLUT-1. Our results also showed that Tris-base buffer (pH 8.4) had no overt toxicity to C3H mice even at higher doses (400 mmol/L). In conclusion, we have developed a novel therapeutic approach to manipulate tumor extracellular pH (pHe) that could be readily adapted to a clinical trial.

Defining Cancer Subpopulations by Adaptive Strategies Rather Than Molecular Properties Provides Novel Insights into Intratumoral Evolution.

Ongoing intratumoral evolution is apparent in molecular variations among cancer cells from different regions of the same tumor, but genetic data alone provide little insight into environmental selection forces and cellular phenotypic adaptations that govern the underlying Darwinian dynamics. In three spontaneous murine cancers (prostate cancers in TRAMP and PTEN mice, pancreatic cancer in KPC mice), we identified two subpopulations with distinct niche construction adaptive strategies that remained stable in culture: (i) invasive cells that produce an acidic environment via upregulated aerobic glycolysis; and (ii) noninvasive cells that were angiogenic and metabolically near-normal. Darwinian interactions of these subpopulations were investigated in TRAMP prostate cancers. Computer simulations demonstrated invasive, acid-producing (C2) cells maintain a fitness advantage over noninvasive, angiogenic (C3) cells by promoting invasion and reducing efficacy of immune response. Immunohistochemical analysis of untreated tumors confirmed that C2 cells were invariably more abundant than C3 cells. However, the C2 adaptive strategy phenotype incurred a significant cost due to inefficient energy production (i.e., aerobic glycolysis) and depletion of resources for adaptations to an acidic environment. Mathematical model simulations predicted that small perturbations of the microenvironmental extracellular pH (pHe) could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200 mmol/L NaHCO3 added to the drinking water of 4-week-old TRAMP mice increased the intraprostatic pHe by 0.2 units and promoted proliferation of noninvasive C3 cells, which remained confined within the ducts so that primary cancer did not develop. A 0.2 pHe increase in established tumors increased the fraction of C3 cells and signficantly diminished growth of primary and metastatic tumors. In an experimental tumor construct, MCF7 and MDA-MB-231 breast cancer cells were coinjected into the mammary fat pad of SCID mice. C2-like MDA-MB-231 cells dominated in untreated animals, but C3-like MCF7 cells were selected and tumor growth slowed when intratumoral pHe was increased. Overall, our data support the use of mathematical modeling of intratumoral Darwinian interactions of environmental selection forces and cancer cell adaptive strategies. These models allow the tumor to be steered into a less invasive pathway through the application of small but selective biological force. Cancer Res; 77(9); 2242-54. ©2017 AACR.

Exploiting evolutionary principles to prolong tumor control in preclinical models of breast cancer.

Conventional cancer treatment strategies assume that maximum patient benefit is achieved through maximum killing of tumor cells. However, by eliminating the therapy-sensitive population, this strategy accelerates emergence of resistant clones that proliferate unopposed by competitors-an evolutionary phenomenon termed "competitive release." We present an evolution-guided treatment strategy designed to maintain a stable population of chemosensitive cells that limit proliferation of resistant clones by exploiting the fitness cost of the resistant phenotype. We treated MDA-MB-231/luc triple-negative and MCF7 estrogen receptor-positive (ER(+)) breast cancers growing orthotopically in a mouse mammary fat pad with paclitaxel, using algorithms linked to tumor response monitored by magnetic resonance imaging. We found that initial control required more intensive therapy with regular application of drug to deflect the exponential tumor growth curve onto a plateau. Dose-skipping algorithms during this phase were less successful than variable dosing algorithms. However, once initial tumor control was achieved, it was maintained with progressively smaller drug doses. In 60 to 80% of animals, continued decline in tumor size permitted intervals as long as several weeks in which no treatment was necessary. Magnetic resonance images and histological analysis of tumors controlled by adaptive therapy demonstrated increased vascular density and less necrosis, suggesting that vascular normalization resulting from enforced stabilization of tumor volume may contribute to ongoing tumor control with lower drug doses. Our study demonstrates that an evolution-based therapeutic strategy using an available chemotherapeutic drug and conventional clinical imaging can prolong the progression-free survival in different preclinical models of breast cancer.

Application of Evolutionary Principles to Cancer Therapy.

The dynamic cancer ecosystem, with its rich temporal and spatial diversity in environmental conditions and heritable cell phenotypes, is remarkably robust to therapeutic perturbations. Even when response to therapy is clinically complete, adaptive tumor strategies almost inevitably emerge and the tumor returns. Although evolution of resistance remains the proximate cause of death in most cancer patients, a recent analysis found that evolutionary terms were included in less than 1% of articles on the cancer treatment outcomes, and this has not changed in 30 years. Here, we review treatment methods that attempt to understand and exploit intratumoral evolution to prolong response to therapy. In general, we find that treating metastatic (i.e., noncurable) cancers using the traditional strategy aimed at killing the maximum number of tumor cells is evolutionarily unsound because, by eliminating all treatment-sensitive cells, it enables rapid proliferation of resistant populations-a well-known evolutionary phenomenon termed "competitive release." Alternative strategies, such as adaptive therapy, "ersatzdroges," and double-bind treatments, shift focus from eliminating tumor cells to evolution-based methods that suppress growth of resistant populations to maintain long-term control.

Structures of glycans bound to receptors from saturation transfer difference (STD) NMR spectroscopy: quantitative analysis by using CORCEMA-ST.

Glycan-receptor interactions are of fundamental relevance for a large number of biological processes, and their kinetics properties (medium/weak binding affinities) make them appropriated to be studied by ligand observed NMR techniques, among which saturation transfer difference (STD) NMR spectroscopy has been shown to be a very robust and powerful approach. The quantitative analysis of the results from a STD NMR study of a glycan-receptor interaction is essential to be able to translate the resulting spectral intensities into a 3D molecular model of the complex. This chapter describes how to carry out such a quantitative analysis by means of the Complete Relaxation and Conformational Exchange Matrix Approach for STD NMR (CORCEMA-ST), in general terms, and an example of a previous work on an antibody-glycan interaction is also shown.

Assembling different antennas of the gp120 high mannose-type glycans on gold nanoparticles provides superior binding to the anti-HIV antibody 2G12 than the individual antennas.

In order to re-build Man9GlcNAc2 clusters of the HIV gp120 glycoprotein, ∼2 nm gold glyconanoparticles (GNPs) were coated with the synthetic partial structures of Man9, the tetramannoside Manα1-2Manα1-2Manα1-3Manα1- and the pentamannoside Manα1-2Manα1-3[Manα1-2Manα1-6]Manα1-. Their interactions with the anti-HIV broadly neutralizing antibody 2G12 were studied by surface plasmon resonance (SPR)-based biosensors and saturation transfer difference (STD)-NMR spectroscopy. A synergistic effect of the tetra- and pentamannosides multimerized on a same GNP was observed. The assembly of these antennas of the gp120 high-mannose type glycan on GNPs provided superior binding to the anti-HIV antibody 2G12 with respect to GNPs carrying only the individual oligomannosides. The results presented in this work provide new molecular information on the interactions between clusters of oligomannosides and 2G12 that could help in the design of a carbohydrate-based vaccine against HIV.

STD NMR study of the interactions between antibody 2G12 and synthetic oligomannosides that mimic selected branches of gp120 glycans.

The human immunodeficiency virus type-1 (HIV-1) is able to shield immunogenic peptide epitopes on its envelope spike (a trimer of two glycoproteins, gp120 and gp41) by presenting numerous host-derived N-linked glycans. Nevertheless, broadly neutralizing antibodies against gp120 and gp41 have been isolated from HIV-1-infected patients and provide protection against viral challenge in animal models. Among these, the monoclonal antibody 2G12 binds to clusters of high-mannose-type glycans that are present on the surface of gp120. These types of glycans have thus been envisaged as target structures for the development of synthetic agents capable of eliciting 2G12-like antibodies. High-resolution structural studies of 2G12 and chemically defined glycan-type ligands, including crystallographic data, have been performed to gain an insight into this interaction. Further studies are still required to design a carbohydrate-based vaccine for HIV. Our previous NMR studies highlighted different recognition modes of two branched synthetic oligosaccharides, a penta- and a heptamannoside, by 2G12 in solution. In order to clarify the underlying structural reasons for such different behaviors, we have herein "dissected" the branches into the linear tri- and tetra- oligomannosides by chemical synthesis and studied their interactions with 2G12 in solution by saturation transfer difference (STD) NMR spectroscopy. The results confirm the distinct preferences of 2G12 for the studied branches and afford explanations for the observed differences. This study provides important structural information for further ligand optimizations. Possible effects of structural modifications on the solvent-exposed end of the ligands are also discussed.

Gold nanoparticles coated with oligomannosides of HIV-1 glycoprotein gp120 mimic the carbohydrate epitope of antibody 2G12.

After three decades of research, an effective vaccine against the pandemic AIDS caused by human immunodeficiency virus (HIV) is not still available, and a deeper understanding of HIV immunology, as well as new chemical tools that may contribute to improve the currently available arsenal against the virus, is highly wanted. Among the few broadly neutralizing human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies, 2G12 is the only carbohydrate-directed one. 2G12 recognizes a cluster of high-mannose glycans on the viral envelope glycoprotein gp120. This type of glycan has thus been envisaged as a target to develop an HIV vaccine that is capable of eliciting 2G12-like antibodies. Herein we show that gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides [manno-gold glyconanoparticles (GNPs)], which are present in gp120, are able to bind 2G12 with high affinity and to interfere with 2G12/gp120 binding, as determined by surface plasmon resonance and saturation transfer difference NMR spectroscopy. Cellular neutralization assays demonstrated that GNPs coated with a linear tetramannoside could block the 2G12-mediated neutralization of a replication-competent virus under conditions that resemble the ones in which normal serum prevents infection of the target cell. Dispersibility in water and physiological media, absence of cytotoxicity, and the possibility of inserting more than one component into the same nanoparticle make manno-GNPs versatile, polyvalent, and multifunctional systems that may aid efforts to develop new multifaceted strategies against HIV.

A solution NMR study of the interactions of oligomannosides and the anti-HIV-1 2G12 antibody reveals distinct binding modes for branched ligands.

The structural and affinity details of the interactions of synthetic oligomannosides, linear (di-, tri-, and tetra-) and branched (penta- and hepta-), with the broadly neutralizing anti-HIV-1 antibody 2G12 (HIV=human immunodeficiency virus) have been investigated in solution by using ligand-based NMR techniques, specifically saturation transfer difference (STD) NMR spectroscopy and transferred NOE experiments. Linear oligomannosides show similar binding modes to the antibody, with the nonreducing terminal disaccharide Manα(1→2)Man (Man=mannose) making the closest protein/ligand contacts in the bound state. In contrast, the branched pentamannoside shows two alternate binding modes, involving both ligand arms (D2- and D3-like), a dual binding description of the molecular recognition of this ligand by 2G12 in solution that differs from the single binding mode deduced from X-ray studies. On the contrary, the antibody shows an unexpected selectivity for one arm (D1-like) of the other branched ligand (heptamannoside). This result explains the previously reported lack of affinity enhancement relative to that of the D1-like tetramannoside. Single-ligand STD NMR titration experiments revealed noticeable differences in binding affinities among the linear and branched ligands in solution, with the latter showing decreased affinity. Among the analyzed series of ligands, the strongest 2G12 binders were the linear tri- and tetramannosides because both show similar affinity for the antibody. These results demonstrate that NMR spectroscopic techniques can deliver abundant structural, dynamics, and affinity information for the characterization of oligomannose-2G12 binding in solution, thus complementing, and, as in the case of the pentamannoside, extending, the structural view from X-ray crystallography. This information is of key importance for the development of multivalent synthetic gp120 high-mannose glycoconjugate mimics in the context of vaccine development.

Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates.

The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

Carbohydrate-carbohydrate interaction prominence in 3D supramolecular self-assembly.

Self-association in water of biologically significant carbohydrate molecules is a controversial topic due to the strong solvation of these molecules in this solvent and the difficulty to experimentally detect these very weak intermolecular forces by biophysical techniques. Herein we report the tremendous ability of amphiphilic carbohydrate molecules to form complex three-dimensional architectures. We have experimentally observed the 3D self-assembly into multilayers of disaccharide neoglycolipid dimers on graphite by means of noncontact AFM and we have also theoretically modeled the interaction between two dimers in order to learn about the structure and composition of these layers. A simple bilayer structure as observed for many amphiphilic lipids was discarded by the experiments. Instead, based on the good agreement between experiments and calculations, we propose that multilayer formation takes place through the assembly of building blocks consisting of two dimers each. The fundamental key in the formation of this supramolecular structure is the complementarity between the van der Waals surfaces of the amphiphilic carbohydrate molecules, a result which differs from the most common idea that H-bonding interactions are prominent in carbohydrate-mediated interactions.