Our compartmentalized co-culture system has several advantages over conventional co-culture methods (summarized in ). First, the channel design (i.e. arrayed, ) and the loading process (i.e., passive pumping) are compatible with existing high-throughput infrastructure, facilitating rapid adoption and implementation. The platform is operationally robust while allowing control of the matrix structure16, 17
. Additionally, the contraction of collagen-based gels after polymerization and during cell culture provide a path for rapid media exchange, simplifying channel design for 3D cultures28
. Importantly, PDMS is known to absorb hydrophobic molecules and can disrupt paracrine signaling in 2D culture30
and, thus, could be a potential source of bias or artifact. However, based on the validation experiments performed here, we postulate that secreted molecules might be largely retained in a 3D matrix, thus attenuating the absorption influence of PDMS as compared to 2D culture. Importantly, our method is not dependent on elastomeric material properties, and, thus, further increases in sensitivity may be possible by moving to other materials (e.g., polystyrene).
Summary of unique capabilities of the developed in vitro system.
Second, the spatial control of the microenvironment allows recapitulation of aspects of the in vivo environment in vitro, and provides the ability to explore the distance-dependent effects of signaling. Compartmentalization of multiple layers in microfluidic channels is achieved by flowing various solutions simultaneously. This is in contrast to the serial loading process required to create multiple layers in 48 wells in which each layer needs to be completely polymerized (4-6 hours per layer) prior to adding another layer. Furthermore, imaging/monitoring cells through the layered gels is challenging due to the thickness of the gels. Thus, the micro scale system allows efficient study of the distance dependence of the invasive transition. The transition occurred actively around the interface where MCF-DCIS and HMF cells were close to each other and decreased as the distance between the cell types increased. At sufficient distance, no transition to invasive phenotype was observed (Circ: 0.44±0.21, Round: 0.83±0.10, AR: 1.22±0.18). Our observations that the transition to an invasive phenotype is dependent on distance from the stromal cells provides new insights into the signaling process. That is, when both soluble factors and cell-cell contact are present (zero spacing between compartments) the transition is accelerated, when only soluble factor signaling is present (spacer gel present) the transition is incomplete, and when there is minimal soluble factor signaling (distance is large enough) DCIS clusters retain their rounded morphology with no signs of a transition. It is also possible that, in addition to cell-cell contact, matrix rearrangement may have actively occurred at the interface by stromal fibroblasts, causing severe invasive transition. Even though we have not fully characterized whether the transition is dependent on either cell-cell contact or matrix remodeling, these observations illustrate how the compartmentalized system facilitates inquiry into relevant parameters. While it is possible to make similar observations in traditional co-culture systems (Fig. S4
), it is not efficient (for example, observing fibroblast migration in a layered macro system would typically be performed using confocal microscopy, while in the shallow side-by-side micro scale compartments one can readily observe relative cell positions via phase contrast microscopy). The observed distance dependence effects provide insights, which help to shape our thinking about how the transition to invasion is regulated. For example, the data suggests that soluble factors may initiate the transition to invasion, but cell-cell contact (or matrix remodeling by stromal fibroblasts) may be required for invasion to continue.
Third, the temporal control providing the capability to form larger DCIS clusters prior to the introduction of the stromal cells takes an additional step closer to the in vivo organization. For this sequentially loaded culture, we have observed that HMFs encircle the pre-existing DCIS-containing gel due to the collagen contraction that occurs during initial DCIS culture. Additionally, this encasement of DCIS-containing gel may change the soluble factor gradient in the system. While this can be viewed as a step backwards in terms of imaging as the cell types now overlap one another, it is a step forward in terms of creating a structural organization more like the in vivo organization. Importantly, the adaptability of the approach allows for the control of these structural aspects without any fundamental changes to the device concept or operation. For example, a filler gel could be introduced prior to the stromal compartment introduction to maintain the side-by-side organization.
Fourth, the ability to compartmentalize by cell type facilitates readouts from one compartment without image overlap between cell types, thereby improving signal and simplifying image analysis. Here we used SHG signal intensity around the MCF-DCIS clusters to provide a unique quantitative readout of DCIS cell-associated collagen structure and a score of the degree collagen rearrangement near MCF-DCIS clusters. Both intensity profiles and area-based analyses show distinct differences between invasive and noninvasive clusters. In addition, the difference between short (less invasive) and long (more invasive) invasive clusters was detectable. While the two analyses revealed similar tendencies, each method provides unique information. In other words, the intensity profile analysis provides localized information such as heterogeneous collagen density distribution around a cluster and the invading direction of the cluster, while area-based analysis provides extensive information from a specific image (not limited to one cluster). Area-based analysis data not only simplifies the process of obtaining a quantitative measure of invasiveness, but also supports automation of analysis providing a path to high-throughput analysis. While our focus was on SHG signal intensity and collagen rearrangement in this study, the importance of collagen fiber alignment and its effect on the cell invasion process has been highlighted recently. For instance, Provenzano et al have shown that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of mammary epithelial cells31
. In our culture mode, we have, indeed, observed well-aligned collagen structures around invasive clusters generated by MCF-DCIS cells (Fig. S8
). Our in vitro data is consistent with precedent observations and suggests that collagen fiber alignment and its contact guidance is one of the factors in DCIS progression to IDC, thus warranting further investigation. In addition, the platform enables efficient investigation of various ECM conditions that will facilitate further study of matrix concentration and composition - parameters that have been impractical to study using traditional assays.