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.

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.

Background Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. Methods We used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering. Results We first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer. Conclusions Our results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells. Electronic supplementary material The online version of this article (10.1186/s12885-018-4148-5) contains supplementary material, which is available to authorized users.

Purpose: Cancer cell cluster formation is a key process involved during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. We previously reported that cell-cell adhesion proteins such as E-Cadherin and desmosomal proteins are involved in cell aggregation independently of cell migration or matrix adhesion (Saias et al. 2015). Here we investigate the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. Experimental Design: Live cell imaging and image processing was used to evaluate MCF7 clustering in the presence of pharmacological inhibitors and to perform a LOPAC® small bioactive compound library screening. Flow cytometry was performed to monitor calcein transfer assay and to evaluate GJIC. Results: We first demonstrate that functional GJIC are established in the early phase of aggregation. We next show that pharmacological inhibition of GJIC using Tonabersat and Meclofenamate both reduces calcein transfer and delays clustering of MCF7 cells. We found that treatment with Latrunculin A, an actin cytoskeleton-disrupting agent, inhibits MCF7 clustering and impairs calcein transfer. In a screen for inhibitors of cell aggregation using a small-compound chemical library we identified Brefeldin A, an inhibitor of vesicular trafficking, and showed that it also impairs calcein transfer. Conclusion: Our results demonstrate that GJIC are involved at the earliest stages of the anchorage-independent clustering process. They shed new light on the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells. References: Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H et al: Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014, 158(5):1110-1122. Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V: Cell-Cell adhesion and cytoskeleton tension are key but opposing regulators of tumor cell aggregation. Cancer Research 2015, 75(12):1-8. Gava, F., Rigal, L ., Mondesert, O., Pesce, E., Ducommun, B. and Lobjois, V. Gap junctions contribute to anchorage-independent clustering of breast cancer cells (Submitted). Citation Format: Fabien Gava, Lise Rigal, Odile Mondesert, Elise Pesce, Bernard Ducommun, Valérie Lobjois. Gap junctions contribute to anchorage-independent clustering of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4023.

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery, but the prognosis of patients with metastatic disease remains very poor. The current treatment of metastatic CRC (mCRC) relies on therapy combining chemotherapy and targeted therapies. However, relapses are observed in most cases due to the occurrence of drug resistance. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies. We previously showed that Claudin-1 (CLDN1), a major constituent of tight junctions, is overexpressed at the membrane of CRC cells which makes it a good target for antibodies. Besides, CLDN1 is differentially expressed in the new CRC molecular subtypes. We have then developed a monoclonal antibody (mAb) against CLDN1, called 6F6, which targets the extracellular part of CLDN1. Results showed that the 6F6 mAb significantly decreased cell growth, survival, and migration in vitro as well as in vivo. Furthermore, we demonstrated the cytostatic effect of the 6F6 mAb through proliferation assays. We are now deciphering the role of CLDN1 on 3D spheroids, a model that reflects the physiopathology. Using immunofluorescence staining on frozen sections we are exploring the impact of CLDN1 on spheroids proliferation. We are also investigating on the distribution of tight junction proteins implicated on cell proliferation in the presence or absence of CLDN1. CLDN1 is a new promising therapeutic target in CRC, it is now interesting to elucidate its role in colorectal cancer cell proliferation. Keywords: Colorectal, Cancer, Claudin-1, monoclonal antibodies, spheroids, proliferation Citation Format: Sara Cherradi, Adeline Ayrolles-Torro, Nadia Vezzo-Vié, Eve Combes, Lucile Canterel-Thouennon, Fabien Gava, Valérie Lobjois, Bernard Ducommun, Celine Gongora, Maguy Del Rio. Targeting the tight junction protein claudin-1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2902.