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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.

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

BackgroundFollicular lymphoma (FL), the most common indolent non-Hodgkin’s Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses.MethodsTo capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability.ResultsFL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3.ConclusionsIn summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.

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 toward personalized therapeutic approaches.

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.

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.

Tumor microenvironment (TME) constituents, including tumor-associated macrophages (TAMs), are now well known to have a significant impact on tumor development. TAMs can be predominant cells in the TME, able to promote cancer cell proliferation, resistance to treatments and immunosuppression. Inactivating these TAMs in tumors by their depletion or their repolarization into a specific anti-tumor, inflammatory phenotype constitutes a significant challenge in immuno-oncology. Several tools and methods to target TAMs have been proposed but they often show low specificity for pro-tumoral macrophages and target myeloid cells too broadly. This research area is therefore in full expansion and warrants the development of appropriate study models. Given the current global effort to reduce the use of in vivo approaches, developing in vitro models that mimic the behavior of TAMs in the TME has become a priority. In this study, we focused on targeting pro-tumoral TAMs in a simple in vitro model recapitulating cancer cell proliferation and TAMs specific phenotype. We developed a 3D model consisting of cancer cells and pro-tumoral macrophages, in which these macrophages promote tumor cell proliferation, maintaining an immunosuppressive phenotype. We showed that, in this model, macrophages can be easily targeted and killed with specific antibodies even in the central regions of the spheroid. This model could be a first in vitro approach to screen new TAM depletion or depolarization tools before application in in vivo models.