A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics

Abstract

The heterogeneity of cellular microenvironments in tumors severely limits the efficacy of most cancer therapies. We have designed a microfluidic device that mimics the microenvironment gradients present in tumors that will enable the development of more effective cancer therapies. Tumor cell masses were formed within micron-scale chambers exposed to medium perfusion on one side to create linear nutrient gradients. The optical accessibility of the PDMS and glass device enables quantitative transmitted and fluorescence microscopy of all regions of the cell masses. Time-lapse microscopy was used to measure the growth rate and show that the device can be used for long-term efficacy studies. Fluorescence microscopy was used to demonstrate that the cell mass contained viable, apoptotic, and acidic regions similar to in vivo tumors. The diffusion coefficient of doxorubicin was accurately measured, and the accumulation of therapeutic bacteria was quantified. The device is simple to construct, and it can easily be reproduced to create an array of in vitro tumors. Because microenvironment gradients and penetration play critical roles controlling drug efficacy, we believe that this microfluidic device will be vital for understanding the behavior of common cancer drugs in solid tumors and designing novel intratumorally targeted therapeutics.

Figure 1. Microenvironments in the microfluidic device mimic those surrounding blood vessels in tumors

A) Nutrient and waste gradients away from vessels creates regions of proliferating (green), quiescent (transition), and necrotic (red) tissue. Drugs have varying penetration capabilities. Some penetrate deeply (blue stars), while others do not (purple crosses). Engineered bacteria (green ovals) have the potential to penetrate to therapeutically resistant regions. B) The linear, observable microenvironment gradients in the microfluidic device have a similar pattern to those surrounding blood vessels in tumors: proliferating (green), quiescent (yellow), and necrotic (red). C) Conceptual concentration profiles of nutrients (green), drugs (green), and wastes (blue) around blood vessels that are emulated by the device. Regions far from blood or culture medium are low in nutrients and drugs and high in wastes.

Figure 2. Design of the microfluidic tumor device

A) Top-view of the device showing the arrangement of the medium/cell inlet, the packing outlet, and the medium outlet. Both the spheroid-containing, packing syringe and the medium syringe were attached to the inlet. A check valve was attached to the packing outlet. B) Working device with flow inlets, outlets and check valve attached. C) Cross-section view of the device showing holes through the microscope slide used to connect to the fluid flow. D) Expanded image of the packing chamber in the center of the device in (A). The adjusted dimensions of the chamber were width, depth, and channel width. The cell retention filter is shown at the distal end. E) Adjusted dimensions of the cell retention filter: post width, post length, and gap width.

Figure 3. Tumor tissue chamber and cell-retention filter

A) Bright field image of the chamber and filter. Scale bar is 100 μm. B) Bright field image of tissue packed into the chamber. Scale bar is 100 μm. C) Scanning electron microscope image of the filter posts. Scale bar is 50 μm.

Figure 4. Effects of packed spheroid size and spheroid growth

A) Fill fraction increased as a function of the age of spheroid cultures prior to packing. Beneath 8 days old, spheroids were too small to be retained by the filter. Greater than 21 days old, spheroids were too fragile and broke apart during the packing process. B) Packed 11 day old spheroid. C) Packed 18 day old spheroid. D) Bright field images of tumor mass growing in the microfluidic device, acquired at 0, 24, and 43 hours. Scale bars are 100 μm.

Figure 5. Microenvironment gradients in tumor tissue constrained by the device

A) Viability staining. Viable and dead cells are cells are stained green and red, respectively. Unstained cells at the distal end of the chamber were visibly necrotic in transmitted light images. White arrows indicate a region of newly formed dead cells bordering the edge of the chamber. B, C) Apoptosis staining. Cells indicated in red (B) and white (C) have active caspase-3, indicating commitment to programmed cell death. D) Cellular pH. Acidic and alkaline regions are indicated red and blue, respectively, and as designated by the scale. Scale bars are 100 μm.

Figure 6. Diffusion and penetration of doxorubicin and therapeutic Salmonella bacteria

A) Bright field image of tissue used to measure drug diffusion, 24 hours after packing. B) Quantitative fluorescence images of doxorubicin diffusing into tumor tissue. C) Normalized concentration profiles derived from (B). Model fits (black) were calculated using the average determined diffusion coefficient and closely fit experimental values. D, E) Bacterial accumulation in the device following inoculation with GFP-expressing Salmonella typhimurium. Images were acquired (D) at 28.5 after 20 hours of bacterial delivery and (E) at 45 hours after 16.5 hours of bacteria-free medium delivery. White arrows indicate a growing bacterial colony at the distal end of the chamber. Scale bars are 100 μm.