GRF 2025 9043663 PI: Chia-Hung CHEN

Uncovering Rare Inherent Resistant Single Cells Through an Automatic Microfluidic Cell Shower Device to Investigate Intratumor Heterogeneity

Researcher(s)

• Chia-Hung CHEN (Principal Investigator / Project Coordinator)Department of Biomedical Engineering
• Ramanuj Dasgupta (Co-Investigator)

Description

One of the major reasons for cancer treatment failure is that inherently different subclonal populations generated by intratumor heterogeneity (ITH) respond differently to chemotherapeutic or even targeted drugs. Resistant clones evolve dynamically in space and time following principles of Darwinian selection, underpinning important emergent features such as cancer reoccurrence and evolvement. Especially for oral squamous cell carcinoma (OSCC) and colorectal cancer (CRC), ITH has not been adequately characterized to address the biomarkers for treatment options. Notably, in OSCC/CRC, while several drugs are capable of delaying initial tumor growth/progression, the effects are usually temporary. A small portion of rare resistant cells in the tumor inevitably survive and expand to cause cancer recurrence. The sequential use of various drugs usually results in drug resistance, ultimately resulting in patients succumbing to their cancer. Unfortunately, there is currently no high-throughput device for long-term single-cell incubation to analyze their innate drug responses on the necessary scale. It is therefore becoming increasingly clear that a new fluidic device is needed to culture single cells and to isolate critical cells by testing their inherent drug resistance for ITH analysis and precision intervention.Conventional methods for single-cell assay include multiparameter flow cytometry. However, without single-cell compartmentalization for incubation, the cross-interactions between cells make the measurement of single-cell innate resistance difficult. The micro-well devices were developed to isolate single cells for culturing and drug testing, while the throughput was limited. Recently, droplet microfluidic technology was investigated to encapsulate single cells in water-in-oil emulsions to measure their inherent tumor properties. However, this technology was disadvantaged by the short single-cell incubation period (<~1 day) that was possible without proving additional nutrition and exchanging medium to remove metabolic wastes in the droplets, restricting its applications in long-term incubation to characterize single-cell drug resistance. Notably, picowasher was previously investigated to refresh the droplet medium through picoinjections, but the medium exchange efficiency was limited. The challenges for high throughput long-term single-cell culture and drug test to investigate ITH remained.Here, we aim to develop a novel automatic microfluidic cell shower device integrated with a multiple-phase deterministic lateral displacement array (M-DLD) to exchange the droplet fluids, enabling long-term single-cell incubation within droplets for high-throughput innate drug resistance analysis. There are three innovative steps involved: 1. The single cells and the drugs will be co-encapsulated in the water-in-oil droplets for incubation. 2. The droplet medium and drugs will be refreshed through a M-DLD. The empty droplets will be subsequently removed by an AI sorter. 3. Long-term single-cell incubations will be approached for drug resistant tests by exchanging the droplet fluids every 12 hours for 14 days. Afterward, all cells will be extracted from the droplets for screening via flow cytometry to isolate the resistant cells. This research will be conducted in three parts to investigate the inherent ITH of OSCC/CRC. First, an automatic microfluidic cell shower device will be developed. With this device, the droplet culture medium will be exchanged on-chip with a highly efficient (~100%). The nutrient will be therefore continuously provided, and the metabolic wastes will be washed out to ensure cell activities and high survivability in the droplets. Second, long-term single-cell drug resistance analysis will be approached by refreshing the medium and drug in the droplets every 12 hours, for 14 days. Notably, an AI sorter will be conducted to remove the empty droplets to ensure high cell encapsulation rate (~90%) in each droplet refreshing cycle. After long-term drug test in the droplets, all cells will be extracted from the droplets for resistant cell sorting by using a flow cytometry with a throughput of ~103 cells per second. Third, this device will be used to screen single cells in patient-derived primary cell (PDPC) models of OSCC/CRC stored in hospitals to uncover rare cells that are resistant to specific clinically relevant drugs, such as cisplatin and oxaliplatin. The treatment predictive biomarkers will be identified by transcriptionally characterizing isolated resistant cells to reveal ITH profiles. The resistant cell lines will be constructed for functional analysis. Overall, the successful completion of the proposed project will allow us to rapidly isolate critical innate resistant cells to indicate the optimal treatment regimens toward precision OSCC/CRC therapeutics. 

Detail(s)