3D stem-like spheroids-on-a-chip for personalized combinatorial drug testing in oral cancer

Abstract

Background: Functional drug testing (FDT) with patient-derived tumor cells in microfluidic devices is gaining popularity. However, the majority of previously reported microfluidic devices for FDT were limited by at least one of these factors: lengthy fabrication procedures, absence of tumor progenitor cells, lack of clinical correlation, and mono-drug therapy testing. Furthermore, personalized microfluidic models based on spheroids derived from oral cancer patients remain to be thoroughly validated. Overcoming the limitations, we develop 3D printed mold-based, dynamic, and personalized oral stem-like spheroids-on-a-chip, featuring unique serpentine loops and flat-bottom microwells arrangement.

Results: This unique arrangement enables the screening of seven combinations of three drugs on chemoresistive cancer stem-like cells. Oral cancer patients-derived stem-like spheroids (CD 44⁺) remains highly viable (> 90%) for 5 days. Treatment with a well-known oral cancer chemotherapy regimen (paclitaxel, 5 fluorouracil, and cisplatin) at clinically relevant dosages results in heterogeneous drug responses in spheroids. These spheroids are derived from three oral cancer patients, each diagnosed with either well-differentiated or moderately-differentiated squamous cell carcinoma. Oral spheroids exhibit dissimilar morphology, size, and oral tumor-relevant oxygen levels (< 5% O₂). These features correlate with the drug responses and clinical diagnosis from each patient's histopathological report.

Conclusions: Overall, we demonstrate the influence of tumor differentiation status on treatment responses, which has been rarely carried out in the previous reports. To the best of our knowledge, this is the first report demonstrating extensive work on development of microfluidic based oral cancer spheroid model for personalized combinatorial drug screening. Furthermore, the obtained clinical correlation of drug screening data represents a significant advancement over previously reported personalized spheroid-based microfluidic devices. Finally, the maintenance of patient-derived spheroids with high viability under oral cancer relevant oxygen levels of less than 5% O₂ is a more realistic representation of solid tumor microenvironment in our developed device.

Keywords: 3D printing; Combinatorial drug testing; Functional drug testing; Microfluidics; Personalized medicine; Spheroids-on-chips.

Fig. 1

A Schematic of the bottom layer for cell culture with drug perfusion scheme. B Schematic of top layer for fluid exchange. C Mixing simulation in the serpentine loop of 57 mm length showing complete mixing at the end of the loop along a 300 µm vertical line with inlet concentration of species: 1.3% (v/v). D Velocity profile in channel network showing the V_max in inlet and outlet cell culture area. E Velocity profile with velocity streamlines in microwells. F Transport of drugs into microwells with time: (i) 200 s, (ii) 800 s. Mass transport simulation was performed by considering arbitrary 1% (v/v) concentration of species from an inlet

Fig. 2

A Patient 2 derived primary oral stem-like tumor cells are seeded with 12 µl/min flow rate. Brightfield images of spheroids were captured on day 3 (left panel scale bar = 200 µm and right magnified panel scale bar = 50 µm). B Spheroid size variation with time measured in ImageJ (scale bar = 50 µm). (Mean ± SD, Student’s t-test, p < 0.001 (***) with day 1, n = 49 spheroids) (C) Spheroid circularity with time measured in ImageJ (Mean ± SD, n = 49 spheroids). D Live/dead staining on day 5 showing live cells in green color and dead cells in red color (scale bar in left panel = 100 µm and right magnified panel = 50 µm). E Quantification of live/dead staining on day 5. (Mean ± SD, Student’s t-test, p < 0.001 (***), n = 49 spheroids). F Immunofluorescence with CD 44 marker, showing green cells positive for CD 44 and blue color showing DAPI staining (scale bar = 100 µm and magnified image scale bar = 50 µm). The spheroids cultured in left-most and right-most microwell arrays have been represented in the horizontal orientation

Fig. 3

Live/dead staining after drug perfusion at 2 µl/min for 6 h and 2 h static incubation on day 3: A Patient 1 spheroids showing live cells in green and dead cells in red color. B Patient 2 spheroids showing live cells in green and dead cells in red color. C Patient 3 spheroids showing live cells in green and dead cells in red color. D Inter-patient comparison. (Reduced chemosensitivity in a few spheroids in patient 2 and 3 has been shown by small colored squares in the fluorescent images and arrows showing high standard deviation in the bar graph). (All data normalized to control, data presented as Mean ± SD, A one-way ANOVA, Tukey’s post hoc test, n = 7 spheroids measured for each treatment group, ‘&’ shows p < 0.001 with control, ‘#’ shows p < 0.05 with control) The spheroids cultured in left-most and right-most microwell arrays have been shown in the horizontal orientation. Controls were treated with 0.1% DMSO. Scale bars in left panels are 100 µm and right magnified panels are 50 µm

Fig. 4

A Size comparison of patient-derived spheroids on day 1 (Mean ± SD, A one-way ANOVA, Tukey’s post hoc test, p < 0.001 (***), n = 49 spheroids). B Brightfield images showing morphological changes in patient 1 and patient 3 spheroids from day 1 to day 3 (scale bar = 50 µm and enlarged image scale bar = 10 µm). C Image iT- green hypoxia reagent expression in patient 1 spheroids on day 3 (the dye detects oxygen level < 5% and its intensity is inversely proportional to the oxygen concentration). D Hypoxia dye expression in patient 2 spheroids (day 3) (E) Hypoxia dye expression in patient 3 spheroids (day 3). F Quantification of fluorescence intensity of hypoxia dye (data presented as mean ± SEM, A one-way ANOVA, Tukey’s post hoc test, p < 0.01 (**), n = 3 spheroids). C–E: All scale bars in left images are 100 µm and right magnified images are 50 µm