Search

Mantle cell lymphoma (MCL), a rare and aggressive B cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T cells self-organize in disc-shaped structures, where B and T cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing towards personalized therapeutic approaches.

HIV integration occurs in chromatin sites that favor the release of high levels of viral progeny; alternatively, the virus is also able to discreetly coexist with the host. The viral infection perturbs the cellular environment inducing the remodelling of the nuclear landscape. Indeed, HIV-1 triggers the nuclear clustering of the host factor CPSF6, but the underlying mechanism is poorly understood. Our data indicate that HIV usurps a recently discovered biological phenomenon, called liquid–liquid phase separation, to hijack the host cell. We observed CPSF6 clusters as part of HIV-induced membraneless organelles (HIV-1 MLOs) in macrophages, one of the main HIV target cell types. We describe that HIV-1 MLOs follow phase-separation rules and represent functional biomolecular condensates. We highlight HIV-1 MLOs as hubs of nuclear reverse transcription, while the double-stranded viral DNA, once formed, rapidly migrates outside these structures. Transcription-competent proviruses localize outside but near HIV-1 MLOs in LEDGF-abundant regions, known to be active chromatin sites. Therefore, HIV-1 MLOs orchestrate viral events prior to the integration step and create a favorable environment for the viral replication. This study uncovers single functional host–viral complexes in their nuclear landscape, which is markedly restructured by HIV-1.

Background Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters. Results In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact. Conclusions Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.

Simple Summary Metastases formation involves the formation, circulation and seeding of cohesive group of tumor cells called circulating tumors cells clusters at distant organs from the primary tumor. These clusters have a much higher metastatic potential than individual circulating tumor cell, it is therefore important to understand the molecular mechanisms involved in their formation. To this aim, in this study, from the analysis of the relationship between in vitro aggregation quantitative characterization of 25 cancer cell lines and their expression data, we identified genes significantly associated with aggregation. Interestingly, we found that these genes were strongly correlated with the transcriptional signature induced by HDAC inhibitors treatment and we finally showed experimentally that two HDAC inhibitors inhibits tumor cells cluster formation in vitro. These results open new therapeutic perspectives to prevent metastasis formation. Abstract Characterization of the molecular mechanisms involved in tumor cell clustering could open the way to new therapeutic strategies. Towards this aim, we used an in vitro quantitative procedure to monitor the anchorage-independent cell aggregation kinetics in a panel of 25 cancer cell lines. The analysis of the relationship between selected aggregation dynamic parameters and the gene expression data for these cell lines from the CCLE database allowed identifying genes with expression significantly associated with aggregation parameter variations. Comparison of these transcripts with the perturbagen signatures from the Connectivity Map resource highlighted that they were strongly correlated with the transcriptional signature of most histone deacetylase (HDAC) inhibitors. Experimental evaluation of two HDAC inhibitors (SAHA and ISOX) showed that they inhibited the initial step of in vitro tumor cell aggregation. This validates our findings and reinforces the potential interest of HDCA inhibitors to prevent metastasis spreading.

Simple Summary Follicular lymphoma is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Although huge efforts have been made in the last 10 years, this pathology is still considered as incurable, leaving open the discovery and testing of new therapeutic targets requiring relevant preclinical models. Here, we report a realistic 3D model of t (14;18)-positive B-NHL cell culture (ultra-low attachment (ULA)-multicellular aggregates of lymphoma cells (MALC)), which monitored by state-of-the-art 2D and 3D imaging, allows more robust drug testing. Abstract Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

International audience; As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

International audience; Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.

Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVgamma9Vdelta2 gamma delta T lymphocytes and high expression of gamma delta cytolytic markers. In a 3D co-culture assay (MALC), gamma delta T cells could mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating gamma delta T lymphocytes with ADCC capacity, impaired these functions. In conclusion, we identify a PD1-regulated gamma delta T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting gamma delta anti-tumor function in FL. Disclosures Morin: IMACTIV-3D: Employment. Lagarde:IMACTIV-3D: Employment. Savina:Institut Roche: Employment. Klein:F. Hoffmann-La Roche Ltd.: Employment, Other: stock, Patents & Royalties.

International audience; High resolution medical ultrasound (US) imaging is an ongoing challenge in many diagnosis applications and can be achieved either by instrumentation or by post-processing. Though many works have considered the issue of resolution enhancement in optical imaging, very few works have investigated this issue in US imaging. In optics, several algorithms have been proposed to achieve super-resolution (SR) image reconstruction, which consists of merging several low resolution images to create a higher resolution image. However, the straightforward implementation of such techniques for US imaging is unsuccessful, due to the interaction of ultrasound with tissue and speckle. We show how to overcome the limit of SR in this framework by refining the registration part of common multiframe techniques. For this purpose, we investigate motion estimation methods adapted to US imaging. Performance of the proposed technique is evaluated on both realistic simulated US images (providing an estimated best-case performance) and real US sequences of phantom and in-vivo thyroid images. Compared to classical SR methods, our technique brings both quantitative and qualitative improvements. Resolution gain was found to be 1.41 for the phantom sequence and 1.12 for the thyroid sequence and a quantitative study using the phantom further confirmed the spatial resolution enhancement. Furthermore, the contrast-to-noise ratio was increased by 27% and 13% for simulated and experimental US images, respectively.

International audience; In the field of ultrasound imaging, resolution enhancement is an up-to-date challenging task. Many device-based approaches have been proposed to overcome the low resolution nature of ultrasound images but very few works deal with post-processing methods. This paper investigates a novel approach based on semi-blind deconvolution formulation and alternating direction method framework in order to perform the ultrasound image restoration task. The algorithm performance is addressed using optical images and synthetic ultrasound data for a various range of criteria. The results demonstrate that our technique is more robust to uncertainties in the a priori ultrasonic pulse than classical non-blind deconvolution methods.

International audience; Ultrasound image resolution enhancement is an ongoing challenge to date. Though many works have been performed using device-based approach, there exists few works dealing with post-processing methods. This paper investigates a technique based on the Alternating Direction Method of Multipliers for the resolution enhancement in ultrasound imaging, which includes the deblurring and denoising tasks. We here point out the characteristics of the proposed technique and thereby underline the features that must be dealt with for successful ultrasound image restoration. Synthetic and in vivo ultrasound images are processed in order to asses its performances.

International audience; High resolution medical ultrasound imaging is an ongoing challenge in many diagnosis applications and can be achieved by instrumentation. Very few works have investigated ultrasound image resolution enhancement whereas many works regarded general purpose optical image or video fields. Many algorithms were proposed within these fields to achieve the "super-resolution" (SR), which consists in merging several low resolution images to create a higher resolution image. However, the straightforward implementation of such techniques for ultrasound imaging is unsuccessful, due to the intrinsic nature of ultrasound motions and speckle. We show how to overcome the intrinsic limit of super-resolution in this framework by refining the registration part of common multi-frame techniques. Classic super-resolution algorithms were implemented and evaluated using sequences of ultrasound images. Such methods not only fail to estimate the true elastic motion but also break the speckle characteristics, resulting in a degradation of the original image. Knowing that a registration error of only 1 pixel leads to a high-resolution image worse than an interpolation, the registration must be adapted to the framework of ultrasound imaging. For this purpose, we investigate different motion estimations. The process described above was evaluated on ultrasound sequences containing up to 15 phantom images with an inclusion scanned with a 7.5 MHz linear probe. Qualitative improvements were observable as soon as at least 5 low-resolution images were used. Ultrasound B-mode profiles of radio-frequency lines were studied and the inclusion was more accurately identified. The Contrast-to-Noise Ratio was increased by approximately 13%.