Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Two types of biopsy needles were used in this work.Needle #16 (11 mm sample groove) was used in mice samples.Needle #18 (18 mm sample groove) was used in clinical samples.As shown in Fig. S3a, about 1.5×10 4 cells were obtained from the xenograft tumor on mice.To quantify the cell numbers obtained from different stages of cancer progress, we obtained samples from three liver cancer patients with two at early stage and one at late stage.As shown in Fig. S3b, the cell numbers do not correlated with the stage of tumors, ranging from 3.8×10 4 cells to 6.6×10 4 cells, with an average cell number of 5×10 4 .This is reasonable because the obtained cell number depends on the grove volume on the biopsy needle, not the sample stage.
On DMF chip, we normally used about 300 cells in each droplet for drug screening, but this may not be the lowest number that can provide a reliably drug screening results.To test the limit of cell numbers for valid drug screening, we ran a serial dilution of cell numbers from 100 to 100,000 in the presence of drug in a 96-well microplate or on-chip.MDA-MB-231 was used as the cell model and EP as the drug model.AlamarBlue® assay was used for the cell toxicity quantification on 96-well plates.Briefly, a serious of cell numbers (1.0×10 5 cells, 1.0×10 4 cells, 5.0×10 3 cells, 10 3 cells, 5.0×10 2 cells, 1.0×10 2 cells, the total volume was 100 μl) per well were seeded in a 96-well plate in the DMEM cell culture medium.They were then treated with various concentrations of EP (with 0.1% (v/v) dimethyl sulfoxide (DMSO) treatment as a negative control and a cell culture medium without cells as a blank control) for 24 hours.Then, 10 μl of alamarBlue solution was added to each well and incubated for 2 h.
All experiments were performed in triplicate.Finally, 585 nm emission was measured by a microplate reader (with the fluorescence excitation wavelength of 555 nm).The emission values were reduced by the blank and normalized to the control wells.Graphs were plotted as the drug concentration versus the percentage of viable cells.As shown in Fig. S3c, the cell viability decreased with increasing the drug concentration in each group of different cell numbers.The IC 50 values were comparable to each other when the cell number was more than 1000.When the cell number was lowered to 500 cells per sample, the deviation was obviously enlarged and the IC 50 value increased a lot.The reliable cell required for 96-well plate is about 1000 cells.Compared to the 100 L solution volume in 96-well plate, 384-well plate takes 50 L solutions per sample.The biopsied cells may be enough for a screening of one drug with 6 conditions in a 384-well plate.One drug screening would not provide useful information for precision medicine.
We further tested the drug screening with 100 cells in a droplet on DMF chip.No higher cell numbers were tested due to the droplet accommodation.As shown in Fig S3d, the cell viability curve was similar as that with 300 cells.
As shown in Fig. S3c and Fig. S3d, there are subtle different responses for on-chip and off-chip to the same increased concentration of drugs, which may be attributed to the different platforms.However, the IC 50 for on-chip and off-chip were both around 25 µM under optimal conditions, validating the on-chip drug screening indication.
The gene mutation for the patients was detected by Whole-exome sequencing (WES).The exactly mutation for Patient #1 and Patient #5 was shown in following Fig.S5.To avoid hypoxia, the requirements are to maintain sufficient oxygen supply and eliminate factors that consume oxygen.Primary cells can be exposed to hypoxic conditions for a certain period of time before damage occurs, but this varies depending on cell type and experimental conditions.Generally speaking, the longer the exposure time, the greater the risk of damage to the cells.
To figure out that how long can primary tumor cells be exposed to hypoxic conditions before damage sets in, we designed an experiment.A relatively large tumor tissue with good initial activity was chosen and put in a 50 ml tube with no air in the tube to create hypoxic conditions (Fig. S8a).Then we cut off a piece of tumor tissues at different time points (0 h, 6 h, 12 h), dissociated them into single cells, and checked the cell activity.As shown in Fig. S8b, the cell viability remained at about 70% when the primary liver cancer cells were kept in a relatively hypoxic environment for 6 hours.There was no big difference between 0 hour and 6 hours.However, cell viability decreased sharply after 6 hours and was less than 10% at 12 hours (Fig. S8c).This indicates that damage occurs after 6 hours of hypoxia condition.
For the highest cell viability in drug screening, the most fresh cells the best results.The longest period for the tumor to be kept without any treatment was 6 hours.To characterize what was obtained from the biopsies of mice, we did HE staining analysis.
The paraffin-embedded tumor slides with 5 μm thickness were analyzed by immunohistochemical method.The tissues were deparaffinized in xylene, rehydrated with a graded series of ethanol, and rinsed in water, following by staining with Hematoxylin stain (Harris) for 3-5 min, and rinsed in water for 1-2 min, differentiation with 0.8% ~ 1% hydrochloric acid alcohol and rinsing with water.Subsequently, the tissues were stained with eosin stain (alcohol soluble) for 1-2 sec, and dehydrated with 95% ethanol and anhydrous ethanol for 1-2 minutes.Finally, the tissues were trans parented with xylene, sealed, and observed with microscopy.The results in Fig. S12 suggested almost all of the cells were tumor cells, with the evidence of the similar morphology of cells in the slides and big nuclear to cytoplasmic ratio of the cells.
Figure.S1.The scheme and work principle of the portable digital microfluidic system and electrodes

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Fig.S4showed the detailed drug screening results of Dox alone, Cur alone, and the combination of 10 M Dox and various concentration of Cur.As can be seen, Dox alone worked better than Cur alone in most cases with lower cell viabilities.The combination of two drugs always showed better effect than single drug, with lower cell viabilities.
Figure.S4 On-chip single drug and combinational drug screening results of biospy samples from 15 individual mice.The drugs are Doxorubicin (Dox), Curcumol and Doxorubicin (Dox) plus Curcumol.For

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Figure.S5.The exact mutation for the patients.

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Figure.S6.Histological image results for patients #1 to #5.Cancer cells are highlighted with yellow area.

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Figure.S8.Cell viability measurement of the primary tumor cells exposed to hypoxic conditions.(a).Picture of tumor tissue from patient #9 in hypoxia condition.(b).Corresponding chart showed cell viability from primary liver cancer samples after exposed to hypoxia condition for 0-12 h.n=10 independent experiments.(c).Fluorescent image results of dissociated cells from primary liver cells after exposed to

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Figure.S9The image picture of portable digital microfluidic system and its connection with digital microfluidic chip to provide power supply for droplet actuation.The digital microfluidic system mainly includes four parts: transformer, signal generator, button array and signal output interface.The portable digital microfluidic system is connected with Digital Microfluidic (DMF) chip via alligator clip.

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Figure.S10.The image picture for droplet operation on Digital Microfluidic (DMF) chip.Microscope was used to observe the droplet operation on DMF chip charged by the portable digital microfluidic system.The digital microfluidic system mainly includes four parts: transformer, signal generator, button array and signal output interface.The portable digital microfluidic system is connected with DMF chip via alligator clip.

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Figure.S11.The image result of printed circuit board (PCB) substrate observed under natural light (a) and the enlarged substrate area observed under microscopy (b).

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Figure.S12.HE staining results of the biopsy samples from two mice, n=2 independent experiments.