Characterization of the Intracellular Acidity Regulation of Brain Tumor Cells and Consequences for Therapeutic Optimization of Temozolomide.

A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered.

Efficient deformation mechanisms enable invasive cancer cells to migrate faster in 3D collagen networks.

Cancer cell migration is a widely studied topic but has been very often limited to two dimensional motion on various substrates. Indeed, less is known about cancer cell migration in 3D fibrous-extracellular matrix (ECM) including variations of the microenvironment. Here we used 3D time lapse imaging on a confocal microscope and a phase correlation method to follow fiber deformations, as well as cell morphology and live actin distribution during the migration of cancer cells. Different collagen concentrations together with three bladder cancer cell lines were used to investigate the role of the metastatic potential on 3D cell migration characteristics. We found that grade-3 cells (T24 and J82) are characterized by a great diversity of shapes in comparison with grade-2 cells (RT112). Moreover, grade-3 cells with the highest metastatic potential (J82) showed the highest values of migration speeds and diffusivities at low collagen concentration and the greatest sensitivity to collagen concentration. Our results also suggested that the small shape fluctuations of J82 cells are the signature of larger migration velocities. Moreover, the displacement fields generated by J82 cells showed significantly higher fiber displacements as compared to T24 and RT112 cells, regardless of collagen concentration. The analysis of cell movements enhanced the fact that bladder cancer cells were able to exhibit different phenotypes (mesenchymal, amoeboid). Furthermore, the analysis of spatio-temporal migration mechanisms showed that cancer cells are able to push or pull on collagen fibers, therefore producing efficient local collagen deformations in the vicinity of cells. Our results also revealed that dense actin regions are correlated with the largest displacement fields, and this correlation is enhanced for the most invasive J82 cancer cells. Therefore this work opens up new routes to understand cancer cell migration in soft biological networks.

Noninvasive monitoring of liver metastasis development via combined multispectral photoacoustic imaging and fluorescence diffuse optical tomography.

Rationale:In vivo molecular imaging in preclinical animal models is a tool of choice for understanding the pathophysiological mechanisms involved in cancer development and for conducting drug development research. Moreover, combining several imaging modalities can provide multifaceted, complementary and cross-validated information. Photoacoustic imaging (PAI) is a promising imaging modality that can reflect blood vasculature and tissue oxygenation as well as detect exogenous molecules, but one shortcoming of PAI is a lack of organic photoacoustic contrast agents capable of providing tumor contrast. Methods: In the present study, we designed an animal model of liver metastases from colon cancer and monitored metastasis development by in vivo bioluminescence and X-ray microcomputed tomography. Contrast-agent-free PAI was used to detect the respective amounts of oxy- and deoxyhemoglobin and, thus, liver tissue oxygenation. two contrast agents, Angiostamp800 and indocyanin green (ICG), respectively with and without tumor targeting specificity, were then evaluated for their dual fluorescence and photoacoustic detectability and were then used for combined PAI and fluorescence diffuse optical tomography (fDOT) at various disease development stages. Findings: Contrast-agent-free PAI reflected tumor angiogenesis and gradual hypoxia during metastasis development. Multispectral PAI enabled noninvasive real-time monitoring of ICG blood pharmacokinetics, which demonstrated tumor-related liver dysfunction. Both PAI and fluorescence ICG signals were clearly modified in metastasis-bearing livers but did not allow for differentiation between different disease stages. In contrast, there was a significant improvement achieved by using the tumor-specific marker Angiostamp800, which provided gradually increasing PAI and fDOT signals during metastasis development. Conclusion: We demonstrated for the first time the value of using Angiostamp800 as a bimodal tumor-targeting contrast agent for combined PAI and fluorescence imaging of liver metastasis progression in vivo.

High-Resolution Shortwave Infrared Imaging of Vascular Disorders Using Gold Nanoclusters.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.

Displacement fields using correlation methods as a tool to investigate cell migration in 3D collagen gels.

Living cells embedded in a complex extra-cellular matrix migrate in a sophisticated way thanks to adhesions to matrix fibres and contractility. It is important to know what kind of forces are exerted by the cells. Here, we use reflectance confocal microscopy to locate fibres accurately and determine displacement fields. Correlation techniques are used to this aim, coupled with proper digital image processing. Benchmark tests validate the method in the case of shear and stretching motions. Finally, the method is tested successfully for studying cancer cells migrating in collagen gels of different concentration.

New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation.

Co-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial's structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner's dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice.

Motor-driven marginal band coiling promotes cell shape change during platelet activation.

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.

Orientation towards prey in antlions: efficient use of wave propagation in sand.

Substrate-borne vibration for locating mates, predators and prey is widespread in the animal kingdown. Antlion larvae dig funnel-shaped traps to catch ants and they are totally immersed in dry sand. We used a playback setup reproducing an ant walking on sand to clearly demonstrate that antlions use sand-borne vibrations to locate their prey. Half the tested animals moved towards the stimulus source. The shoot angle of sand tossing was very close to the target angle, indicating excellent ability to perceive stimulus direction. We also discuss orientation mechanisms in sand, a medium with highly unusual wave propagation properties.

Efficiency of antlion trap construction.

Assessing the architectural optimality of animal constructions is in most cases extremely difficult, but is feasible for antlion larvae, which dig simple pits in sand to catch ants. Slope angle, conicity and the distance between the head and the trap bottom, known as off-centring, were measured using a precise scanning device. Complete attack sequences in the same pits were then quantified, with predation cost related to the number of behavioural items before capture. Off-centring leads to a loss of architectural efficiency that is compensated by complex attack behaviour. Off-centring happened in half of the cases and corresponded to post-construction movements. In the absence of off-centring, the trap is perfectly conical and the angle is significantly smaller than the crater angle, a physical constant of sand that defines the steepest possible slope. Antlions produce efficient traps, with slopes steep enough to guide preys to their mouths without any attack, and shallow enough to avoid the likelihood of avalanches typical of crater angles. The reasons for the paucity of simplest and most efficient traps such as theses in the animal kingdom are discussed.