Microfluidic platform for chemotaxis in gradients formed by CXCL12 source-sink cells

Abstract

Chemokine CXCL12 promotes CXCR4-dependent chemotaxis of cancer cells to characteristic organs and tissues, leading to metastatic disease. This study was designed to investigate how cells expressing CXCR7 regulate chemotaxis of a separate population of CXCR4 cells under physiologic conditions in which cells are exposed to gradients of CXCL12. We recapitulated a cancer-stroma microenvironment by patterning CXCR4-expressing cancer cells in microchannels at spatially defined positions relative to CXCL12-producing cells and CXCR7-expressing cells. CXCR7 scavenges and degrades CXCL12, which has been proposed to facilitate CXCR4-dependent chemotaxis through a source-sink model. Using the microchannel device, we demonstrated that chemotaxis of CXCR4 cells depended critically on the presence and location of CXCR7 cells (sink) relative to chemokine secreting cells (source). Furthermore, inhibiting CXCR4 on migrating cells or CXCR7 on sink cells blocked CXCR4-dependent chemotaxis toward CXCL12, showing that the device can identify new therapeutic agents that block migration by targeting chemoattractant scavenging receptors. Our system enables efficient chemotaxis under much shallower yet more physiological chemoattractant gradients by generating an in vitro microenvironment where combinations of cellular products may be secreted along with formation of a chemoattractant gradient. In addition to elucidating mechanisms of CXCL-12 mediated chemotaxis, this simple and robust method can be broadly useful for engineering multiple microenvironments to investigate intercellular communication.

Fig. 1

Microfluidic system for patterning source, sink, and migrating cells in defined positions. (A) Schematic illustration of the device. Two layers of PDMS channels are separated by a semi-permeable membrane. The top layer is a straight channel with a dead end where cells are patterned. (B) The cellular seeding process; three types of cells are sequentially introduced into each inlet of the top channel. Cells are patterned over select bottom channels by keeping the outlet for that respective channel open and the other two bottom channel outlets closed. (C) Time lapse images of X4 cell migration. X4 cells (blue) were patterned between L12 cells (red) and X7 cells (green) with 200 mm gaps and co-cultured in a device. Fluorescent images were taken 1 h, 10 h, and 20 h after seeding the cells. Scale bar: 200 μm.

Fig. 2

Migration of CXCR4 expressing cells is modulated by CXCR7 expressing cells. X4 cells were co-cultured with L12 cells but without X7 cells (A), with both L12 cells and X7 cells but with the positions of the X4 and X7 cells inverted (B), with wider gaps (400 mm) between the X4 cells and both the L12 cells and X7 cells (C), or with X7 cells replaced with GFP cells that do not express CXCR7 (D). Fluorescent images were taken 20 h after seeding cells. (E) Quantification of X4 cell migration under each condition (n = 9–16 independent experiments for each condition). (1C) shows the result of Fig. 1C at 20 h. The X-axis shows distance from the center of the middle bottom channel where X4 cells are initially positioned. Scale bars: 200 μm. * p < 0.001, ** p < 0.0001.

Fig. 3

Effect of closely positioning CXCR4 and CXCR7 expressing cells together. (A, B) X4 cells (blue) and X7 cells (green) were co-patterned over the middle channel of the lower layer by either randomly mixing together (A) or by patterning side-by-side (B). In (C) MDA-MB-231 cells stably expressing both CXCR4 and CXCR7 (green) were patterned over the middle channel of the lower layer. For comparison, X4 cells (blue) were patterned side-by-side to X7 cells (green) without L12 cells (red) in (D) and X4 cells (blue) and GFP cells that do not express CXCR7 (green) were co-patterned over the middle channel of the lower layer in a side-by-side manner in (E). Fluorescent images were taken 1 h and 20 h after seeding the cells. (F) Quantification of X4 cell migration under each condition (n = 5–10 independent experiments for each condition). The X-axis shows distance from the center of the initial position of X4 cells. Scale bars: 200 μm.

Fig. 4

CXCL12 gradients generated by microfluidically patterned source and sink cells. (A) Simulated gradient profiles of CXCL12 in channels after 20 h in culture for the various cellular patterns generated. The X axis shows distance from the center of the middle bottom channel where X4 cells are typically seeded. (B) Estimated specific gradients over X4 cells for the different cellular patterns. The magnitude of the specific gradient matches well the observed trends in degree of migration except for condition 3C.

Fig. 5

Evaluation of inhibitors on CXCL-12-dependent chemotaxis. CXCR4 inhibitor (1 mM AMD3100) (A) or CXCR7 inhibitor (100 nM CCX733) (B) was added in the device right after seeding the cells. Fluorescent images were taken 20 h after seeding cells. (C) Quantification of X4 cell migration (n = 9–16 independent experiments for each condition). (1C) shows the result of Fig. 1C at 20 h. X-axis shows distance from the center of the middle bottom channel. Scale bars: 200 mm. * p o 0.001, ** p o 0.0001.