A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture

Abstract

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

Figure. 1.

Microfluidic CoC platform design and overview. A, Top view of the microfluidic chip illustrating its components: the PDMS film in which the microfluidics are embedded, the silicon frame, which includes the inlet and outlet to the channels in the film. Scale bar, 2.5 mm. B, Vertical cross-section of the microfluidic chip. The PDMS film with the microfluidic channel is supported by a silicon frame, which includes a well facing the PDMS layer. The microchannel and the well are separated by a microporous PDMS membrane (4-μm pore size). Scale bar, 100 μm. C, Representation of the CoC platform. The platform, which consists of a bottom and a top plate, houses the microfluidic chip and allows for its connection to external fluidics. The ring is used to seal the system, to maintain adequate pressure for the controlled flow within the fluidic channel, and to minimize leakage. Scale bar, 2 cm. D, Accessories of the CoC platform with two different top plate designs. We have used double flow for this study. E, Cross-section of CoC illustrating the diffusion and perfusion toward the tissue slice. First the tissue slice is added to the microfluidic chip, which is in turn sandwiched between the top and bottom plates and connected to the external pump. Breast PDX tissue slices were perfused with an inlet flowrate of 5 μL/minute through the top and bottom channels. F, CoC platform connected to Fluigent Microfluidic flow control system that was further connected to flowrate sensors (Fluigent FLOW UNIT-S) using Fluigent MAT for the entire culture period.

Figure 2.

Prediction of therapy response using cisplatin-sensitive and -resistant PDX in ex vivo and CoC platform. Three cisplatin-sensitive and three cisplatin-resistant breast PDX tumors were used in three independent experiments to study cisplatin response in ex vivo 6-well plate culture and CoC platform. Cisplatin-sensitive and -resistant PDX tumor tissue slices were exposed to 5 μg/mL cisplatin for 3 days and evaluated for cell proliferation (EdU incorporation, red nuclei) and apoptosis (TUNEL staining, green nuclei). DAPI stains all nuclei blue. A, Representative EdU and TUNEL staining of cisplatin-sensitive breast PDX. B, Quantification of the fraction of EdU-positive and TUNEL-positive cells showing breast PDXs (n = 3) were sensitive to cisplatin. C, Representative EdU and TUNEL staining of cisplatin-resistant breast PDX. D, Quantification of the fraction of EdU-positive and TUNEL-positive cells showing breast PDXs (n = 3) were insensitive to cisplatin therapy, thereby validating the application of CoC for therapy response for patient tumors. Ten fields of view were quantified from each breast PDX slice. Each point in the graph represents one breast PDX sample and SEM is indicated for all three tumors in the three independent experiments. Scale bar, 50 μm. E, Analysis of DNA damage response in cisplatin-sensitive and -resistant PDX treated with cisplatin. Cisplatin treatment induced more double-strand breaks in cisplatin-sensitive PDX than in cisplatin-resistant PDX. Scale bar 50 μmol/L. F, Scatter plot showing 53BP1 foci count per cell in cisplatin-sensitive and -resistant PDX. Averages and SEM are indicated. Cis, cisplatin; cis res, cisplatin-resistant; cis sen, cisplatin-sensitive; H&E, hematoxylin and eosin. NS, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

Response to apalutamide treatment in CoC platform. A, PC82 tumor tissue slices cultured with apalutamide for 7 days and evaluated for AR expression, cell proliferation (EdU incorporation, red nuclei), and apoptosis (TUNEL staining, green nuclei). AR staining for PC82 tissue slice sections at day 0, day 7 ex vivo culture condition, and CoC platform with and without apalutamide treatment. DAPI stains all nuclei blue. H&E, hematoxylin and eosin. Scale bar, 50 μm. B, Quantification of the AR expression in prostate PDX slices cultured in ex vivo and CoC platform. C and D, Apalutamide treatment showed a significant increase in TUNEL-positive cells and significant decrease in EdU-positive cells when compared with untreated tumor slices in our CoC platform. Ten fields of view were quantified per prostate PDX slice. Each point in the graph represents one image field and SEM is indicated for 10 fields. D7, day 7; D0, day 0. ***, P < 0.001; ****, P < 0.0001.

Figure 4.

Breast PDX tumor tissue slices cultured in ex vivo condition and in CoC platform for up to 14 days. A, We used five independent breast PDX tumors in five independent experiments to establish optimized culture condition for tumor slices in our CoC platform. From each breast PDX tumor, tissue slices were cultured in ex vivo 6-well plate and CoC device for up to 14 days and evaluated for tissue morphology [hematoxylin and eosin (H&E) staining], DAPI (blue nuclei), proliferation (EdU incorporation, red nuclei), Ki67 (brown nuclei), and apoptosis (TUNEL staining, green nuclei). Scale bar, 50 μm. B and C, Quantification of the fraction of EdU-positive and TUNEL-positive cells for 5 breast PDX tissue slices (derived from individual PDX tumors) cultured for up to day 7 (B) and day 14 (C). For each breast PDX tumor and each condition, tissue slices were imaged, and ten random fields of view were quantified from each breast PDX slice. Each data point in the graph represents one image field. Error bar represents the SEM for the five independent tumors performed in five independent experiments. D, Representative image showing breast PDX tumors (n = 3) labeled with geminin (red nuclei) and DAPI (blue nuclei). Scale bar, 50 μm. E, QIBC analysis of three independent breast PDX tumors with more than 3,000 cells analyzed for each are shown in each condition. F, Quantification of geminin-positive cells showed CoC at day 7 had similar cell proliferation profile as in day 0 than ex vivo condition. Error bar, SEM. NS, not significant; D0, day 0; D7, day 7, D14, day 14. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

Gene expression analysis of breast PDX tumor cultured in ex vivo and CoC platform. Three independent breast PDX tumors (P1, P2, and P3) were used for analysis of pathway activity using OncoSignal and RNA-seq for whole mRNA expression analysis. A, Seven major pathways were analyzed (ER, AR, PI3K, and MAPK growth factor pathways, HH, Notch, and TGFβ cell signaling pathways) using OncoSignal. Pathway activity scores are presented on a normalized scale of 0 to 100. Error bars, SEM. Mann–Whitney test of seven pathway activity across PDX tumor slices (day 0, day 7, day 14) in ex vivo and CoC condition did not show any statistically significant change. B, Venn diagram showing differentially expressed human (ex vivo-D7, CoC-D7, and CoC-D14) genes (log_FC > 1.5 or log_FC ≤ 1.5, FDR < 0.1) in PDX tumors at day 7 and day 14 in ex vivo and CoC platform when compared with day 0 PDX tumors. C, Heatmap diagram showing IFN signaling pathway gene expression at different days in ex vivo and in CoC platform culture condition. D, Network diagram showing top ranked networks significantly enriched in immune signaling and tumor microenvironment (P < 0.05) exclusively in day 7 ex vivo culture condition. Orange, predicted activation of the pathways (Z score > 2); blue, predicted inhibition (Z score ≤2). E, Representative image showing 53BP1 staining in breast PDX tumors cultured at day 0, day 7 ex vivo and CoC condition. DAPI staining is shown as blue nuclei. Scale bar, 50 μm. F, Bar chart showing the percentage of cells with 53BP1 foci. Error bar, SEM for the three independent PDX tumor samples. D0, day 0; D7, day 7, D14, day 14. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.