(J-385) Developing a Label-Free, Non-Invasive Potency-on-a-chip Assay for CAR T Therapy

Chimeric antigen receptor (CAR) T-cell therapy  is a promising cancer treatment that  has  proven effective for many hematologic malignancies, but its efficacy in solid tumor cancers remains limited. CAR T cells engineered to target GD-2 antigen is a promising treatment for neuroblastoma. In vitro CAR T cell potency can be evaluated via coculture with  target tumor cells; however there is currently a lack of high-throughput co-culture model to track cell-cell interactions at different ratios of CAR T cells (effector cells) to tumor cells (target cells). The Lu Lab has developed a microfluidic device to study the effects of varying effector-to-target (E:T) ratios on CAR T cell functions while tracking the same cell-cell interaction over 48 hours. This device was designed for hematologic cancer cells so the process needed to be optimized for GD-2 CAR T cells and CHLA-20 neuroblastoma cells. Optical Metabolic Imaging (OMI) is a powerful, label-free, non-invasive method  to study single cell  metabolism based on  the fluorescence intensities and lifetimes of two metabolic coenzymes NAD(P)H and FAD. By measuring metabolic changes in single cells, OMI is sensitive to chemotherapy efficacy  in several cancer models. However, the application of OMI on this specific microfluidic device for CAR T and adherent tumor cell coculture has yet to be demonstrated. In this project, we optimized the microfluidic device setup and established the OMI imaging workflow to investigate the metabolic changes in tumor cells and CAR T cells in coculture.  

200uL 0.1mg/mL fibronectin was  added into the inlet tube and left for 24 hours to coat the microfluidic device. CHLA-20 neuroblastoma cell suspension in DMEM (10% FBS, 1% Pen-Strep) was loaded into the microfluidic device inlet to flow into individual chambers for roughly 4 hours. In parallel, 1x10⁶ CHLA-20s were also plated into 35mm glass-bottom imaging dishes with and without fibronectin coating. Once CHLA-20 evenly distributed into all chambers, the outlet tube was  clamped to halt flow. CHLA-20 cells were cultured in the microfluidic device  for 24 hours, after which CAR T-cells were loaded into the device inlet to distribute into individual chambers for 4 hours. OMI  was performed 4 hours following co-culture using a stage top incubator to keep the culture at 37C and 5% CO2 . NAD(P)H and FAD were excited at 750nm and 890nm and their emissions were collected with 440/80nm and 550/50nm filter cubes, respectively. The same chambers were identified and imaged again with OMI at 24 hours post culture. Additionally, CHLA-20 cells cultured in fibronectin-coated and non-coated dishes were also imaged at 24 and 48 hours. 

Fibronectin coating in the microfluidic device and imaging dish promoted cell adherence. Cell morphology indicated more spreading in the presence of fibronectin. CHLA-20 cultured on fibronectin coated dishes had significantly higher NAD(P)H mean lifetime (NAD(P)H Tm) (Figure 1B). This demonstrated the benefits of using fibronectin in supporting cell adherence. Since the microfluidic flow rate and direction were controlled based on the differential gravitational force between the inlet and outlet tubes, we optimized the microfluidic setup to ensure even distribution of cells throughout the whole device. We determined that a 4 inch height difference between the inlet and outlet tubes allowed cells entering the chambers while minimizing cell escape through chamber legs. The number of legs in each chamber controlled its specific flow force and, hence, the number of captured cells, leading to  a variety of E:T  ratios (Figure 1D). CAR T cells and CHLA-20 were manually segmented based on their distinct morphologies. After 24 hours of co-culture, we observed an increase in E:T ratio in 65% (5/8)  of the chambers, which demonstrated CAR T-cell’s cytotoxic functions towards CHLA-20. Additionally, metabolic changes in CHLA-20 were observed in each chamber, varying in trend between different unique chambers. (Figure 1C)

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