Droplet microfluidics for functional temporal analysis and cell recovery on demand using microvalves: application in immunotherapies for cancer.

Most common methods of cellular analysis employ the top-down approach (investigating proteomics or genomics directly), thereby destroying the cell, which does not allow the possibility of using the same cell to correlate genomics with functional assays. Herein we describe an approach for single-cell tools that serve as a bottom-up approach. Our technology allows functional phenotyping to be conducted by observing the cytotoxicity of cells and then probe the underlying biology. We have developed a droplet microfluidic device capable of trapping droplets in the array and releasing the droplet of interest selectively using microvalves. Each droplet in the array encapsulates natural killer cells (NK cells) and tumour cells for real-time monitoring of burst kinetics and spatial coordination during killing by single NK cells. Finally, we use the microvalve actuation to selectively release droplets with the desired functional phenotype such as for fast and serial killing of target tumour cells by NK cells. From this perspective, our device allows for investigating first interactions and real-time monitoring of kinetics and later cell recovery on demand for single-cell omic analysis such as single-cell RNA sequencing (scRNA), which to date, is primarily based on in-depth analyses of the entire transcriptome of a relatively low number of cells.

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers.

Droplet microfluidics has revolutionized the biomedical and drug development fields by allowing for independent microenvironments to conduct drug screening at the single cell level. However, current microfluidic sorting devices suffer from drawbacks such as high voltage requirements (e.g., >200 Vpp), low biocompatibility, and/or low throughput. In this article, a single-phase focused transducer (SPFT)-based acoustofluidic chip is introduced, which outperforms many microfluidic droplet sorting devices through high energy transmission efficiency, high accuracy, and high biocompatibility. The SPFT-based sorter can be driven with an input power lower than 20 Vpp and maintain a postsorting cell viability of 93.5%. The SPFT sorter can achieve a throughput over 1000 events per second and a sorting purity up to 99.2%. The SPFT sorter is utilized here for the screening of doxorubicin cytotoxicity on cancer and noncancer cells, proving its drug screening capability. Overall, the SPFT droplet sorting device shows great potential for fast, precise, and biocompatible drug screening.

Plasma-enhanced protein patterning in a microfluidic compartmentalized platform for multi-organs-on-chip: a liver-tumor model.

A microfluidic technique is presented for micropatterning protein domains and cell cultures within permanently bonded organs-on-chip devices. This method is based on the use of polydimethylsiloxane layers coupled with the plasma ablation technique for selective protein removal. We show how this technique can be employed to generate a multi-organin vitromodel directly within a microscale platform suitable for pharmacokinetic-based drug screening. We miniaturized a liver model based on micropatterned co-cultures in dual-compartment microfluidic devices. The cytotoxic effect of liver-metabolized Tegafur on colon cancer cell line was assessed using two microfluidic devices where microgrooves and valves systems are used to model drug diffusion between culture compartments. The platforms can reproduce the metabolism of Tegafur in the liver, thus killing colon cancer cells. The proposed plasma-enhanced microfluidic protein patterning method thus successfully combines the ability to generate precise cell micropatterning with the intrinsic advantages of microfluidics in cell biology.

High throughput microfluidic system with multiple oxygen levels for the study of hypoxia in tumor spheroids.

Replication of physiological oxygen levels is fundamental for modeling human physiology and pathology inin vitromodels. Environmental oxygen levels, applied in mostin vitromodels, poorly imitate the oxygen conditions cells experiencein vivo, where oxygen levels average ∼5%. Most solid tumors exhibit regions of hypoxic levels, promoting tumor progression and resistance to therapy. Though this phenomenon offers a specific target for cancer therapy, appropriatein vitroplatforms are still lacking. Microfluidic models offer advanced spatio-temporal control of physico-chemical parameters. However, most of the systems described to date control a single oxygen level per chip, thus offering limited experimental throughput. Here, we developed a multi-layer microfluidic device coupling the high throughput generation of 3D tumor spheroids with a linear gradient of five oxygen levels, thus enabling multiple conditions and hundreds of replicates on a single chip. We showed how the applied oxygen gradient affects the generation of reactive oxygen species (ROS) and the cytotoxicity of Doxorubicin and Tirapazamine in breast tumor spheroids. Our results aligned with previous reports of increased ROS production under hypoxia and provide new insights on drug cytotoxicity levels that are closer to previously reportedin vivofindings, demonstrating the predictive potential of our system.

Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction.

The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8+ T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8+ T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8+ T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations of CD8+ T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8+ T cells.

Lymphatic endothelium contributes to colorectal cancer growth via the soluble matrisome component GDF11.

Colorectal cancer (CRC) is one of the most malignant tumors worldwide. Stromal cells residing in the tumor microenvironment strongly contribute to cancer progression through their crosstalk with cancer cells and extracellular matrix. Here we provide the first evidence that CRC-associated lymphatic endothelium displays a distinct matrisome-associated transcriptomic signature, which distinguishes them from healthy intestinal lymphatics. We also demonstrate that CRC-associated human intestinal lymphatic endothelial cells regulate tumor cell growth via growth differentiation factor 11, a soluble matrisome component which in CRC patients was found to be associated with tumor progression. Our data provide new insights into lymphatic contribution to CRC growth, aside from their conventional role as conduits of metastasis.