Switchable CAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a disease of unmet medical need. While immunotherapy with chimeric antigen receptor T (CAR-T) cells has shown much promise in haematological malignancies, their efficacy for solid tumours is challenged by the lack of tumour-specific antigens required to avoid on-target, off-tumour effects. Switchable CAR-T cells whereby activity of the CAR-T cell is controlled by dosage of a tumour antigen-specific recombinant Fab-based 'switch' to afford a fully tunable response may overcome this translational barrier. DESIGN: In this present study, we have used conventional and switchable CAR-T cells to target the antigen HER2, which is upregulated on tumour cells, but also present at low levels on normal human tissue. We used patient-derived xenograft models derived from patients with stage IV PDAC that mimic the most aggressive features of PDAC, including severe liver and lung metastases. RESULTS: Switchable CAR-T cells followed by administration of the switch directed against human epidermal growth factor receptor 2 (HER2)-induced complete remission in difficult-to-treat, patient-derived advanced pancreatic tumour models. Switchable HER2 CAR-T cells were as effective as conventional HER2 CAR-T cells in vivo testing a range of different CAR-T cell doses. CONCLUSION: These results suggest that a switchable CAR-T system is efficacious against aggressive and disseminated tumours derived from patients with advanced PDAC while affording the potential safety of a control switch.

The miR-25-93-106b cluster regulates tumor metastasis and immune evasion via modulation of CXCL12 and PD-L1.

The stromal microenvironment controls response to injury and inflammation, and is also an important determinant of cancer cell behavior. However, our understanding of its modulation by miRNA (miR) and their respective targets is still sparse. Here, we identified the miR-25-93-106b cluster and two new target genes as critical drivers for metastasis and immune evasion of cancer cells. Using miR-25-93-106b knockout mice or antagomiRs, we demonstrated regulation of the production of the chemoattractant CXCL12 controlling bone marrow metastasis. Moreover, we identified the immune checkpoint PD-L1 (CD274) as a novel miR-93/106b target playing a central role in diminishing tumor immunity. Eventually, upregulation of miR-93 and miR-106b via miR-mimics or treatment with an epigenetic reader domain (BET) inhibitor resulted in diminished expression of CXCL12 and PD-L1. These data suggest a potential new therapeutic rationale for use of BET inhibitors for dual targeting of cancers with strong immunosuppressive and metastatic phenotypes.

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.

Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to combine mitochondrial Ca(2+) measurements with nuclear translocation. In conclusion, this method enables the high throughput study of spatiotemporal patterns of Ca2(+) signals in T cells responding to different stimuli.

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution.

Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.

A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution.

Labeling nonquiescent cells with carboxyfluorescein succinimidyl ester (CFSE)-like dyes gives rise to a population width exceeding the threshold for resolving division peaks by flow cytometry. Width is a function of biological heterogeneity plus extrinsic and intrinsic error sources associated with the measurement process. Optimal cytometer performance minimizes extrinsic error, but reducing intrinsic error to the point of facilitating peak resolution requires careful fluorochrome selection and fluorescent cell sorting. In this study, we labeled the Jurkat and A549 cell lines with CFSE, CellTraceViolet (CTV), and eFluor 670 proliferation dye (EPD) to test if we could resolve division peaks in culture after reducing the labeled input widths by cell sorting. Reanalysis of the sorted populations to ascertain the level of reduction achieved always led to widths exceeding the gated limits due to the contribution of errors. Measuring detector-specific extrinsic error by sorting uniform fluorescent particles with similar spectral properties to the tracking dyes allowed us to determine the intrinsic error for each dye and cell type using a simple mathematical approach. We found that cell intrinsic error ultimately dictated whether we could resolve division peaks, and that as this increased, the required sort gate width to resolve any division peaks decreased to the point whereby issues with yield made A549 unsuitable for this approach. Finally, attempts to improve yields by setting two concurrent sort gates on the fluorescence distribution enriched for cells in different stages of the cell cycle that had nonequivalent proliferative properties in culture and thus should be practiced with caution.