High-throughput method to analyze the cytotoxicity of CAR-T Cells in a 3D tumor spheroid model using image cytometry.

Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Characterization of CAR T cell expansion and cytotoxic potential during Ex Vivo manufacturing using image-based cytometry.

Since the FDA approval of two Chimeric Antigen Receptor (CAR) T cell therapies against CD19+ malignancies, there has been significant interest in adapting CAR technology to other diseases. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products by a single instrument would likely accelerate the development of candidate therapies. Here, we demonstrate that image-based cytometry yields high-throughput measurements of CAR T cell proliferation and size, and captures the kinetics of in vitro antigen-specific CAR T cell-mediated killing. The data acquired and analyzed by the image cytometer are congruent with results derived from conventional technologies when tested contemporaneously. Moreover, the use of bright-field and fluorescence microscopy by the image cytometer provides kinetic measurements and rapid data acquisition, which are direct advantages over industry standard instruments. Together, image cytometry enables fast, reproducible measurements of CAR T cell manufacturing criteria and effector function, which can greatly facilitate the evaluation of novel CARs with therapeutic potential.

Borrelia burgdorferi RNA induces type I and III interferons via Toll-like receptor 7 and contributes to production of NF-κB-dependent cytokines.

Borrelia burgdorferi elicits a potent cytokine response through activation of multiple signaling receptors on innate immune cells. Spirochetal lipoproteins initiate expression of NF-κB-dependent cytokines primarily via TLR2, whereas type I interferon (IFN) production is induced through the endosomal receptors TLR7 and TLR9 in human dendritic cells and TLR8 in monocytes. We demonstrate that DNA and RNA are the B. burgdorferi components that initiate a type I IFN response by human peripheral blood mononuclear cells (PBMCs). IFN-α protein and transcripts for IRF7, MX1, and OAS1 were induced by endosomal delivery of B. burgdorferi DNA, RNA, or whole-cell lysate, but not by lysate that had been treated with DNase and RNase. Induction of IFN-α and IFN-λ1, a type III IFN, by B. burgdorferi RNA or live spirochetes required TLR7-dependent signaling and correlated with significantly enhanced transcription and expression of IRF7 but not IRF3. Induction of type I and type III IFNs by B. burgdorferi RNA could be completely abrogated by a TLR7 inhibitor, IRS661. In addition to type I and type III IFNs, B. burgdorferi RNA contributed to the production of the NF-κB-dependent cytokines, IFN-γ, interleukin-10 (IL-10), IL-1ß, IL-6, and tumor necrosis factor alpha (TNF-α), by human PBMCs. Collectively, these data indicate that TLR7-dependent recognition of RNA is pivotal for IFN-α and IFN-λ1 production by human PBMCs, and that RNA-initiated signaling contributes to full potentiation of the cytokine response generated during B. burgdorferi infection.