Transition to invasion in breast cancer: a microfluidic in vitro model enables examination of spatial and temporal effects

Abstract

The transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer progression. We introduce a simple microfluidic 3D compartmentalized system in which mammary epithelial cells (MCF-DCIS) are co-cultured with human mammary fibroblasts (HMFs), which promotes a transition from DCIS to IDC in vitro. The model enables control of both spatial (distance-dependence) and temporal (transition from larger clusters) aspects within the microenvironment, allowing recapitulation of the in vivo environment in ways not practical with existing experimental models. When HMFs were cultured some distance (0.5-1.5 mm) from the MCF-DCIS cells, we observed an initial morphological change, suggesting soluble factors can begin the transition. However, cell-cell contact with HMFs allowed the MCF-DCIS cells to complete the transition to invasion. Uniquely, the compartmentalized platform enables the analysis of the intrinsic second harmonic generation signal of collagen, providing a label-free quantitative analysis of DCIS-associated collagen remodeling. The arrayed microchannel-based model is compatible with existing infrastructure and, for the first time, provides a cost effective approach to test for inhibitors of pathways involved in DCIS progression to IDC allowing a screening approach to the identification of potential therapeutic targets. Importantly, the model can be easily adapted and generalized to a variety of cell-cell signaling studies.

Fig 1

3D compartmentalization and the invasive transition of MCF-DCIS cells. (a) Passive pumping allows for the loading and compartmentalization of 3D cultures. Drops of cell containing polymer solutions are loaded onto the inlet ports. Laminar flow leads to two side-by-side 3D compartments. (b) Yellow and green food coloring solutions are pumped into Y-shaped PDMS microchannels to demonstrate compartmentalization by passive pumping. Scale bar represents 6mm. Y-shaped channels are arrayed (4×3 array). Scale bar is 2cm. (c) The invasive transition of MCF-DCIS cells in compartmentalized culture is observed at interface. Cells in control side retained noninvasive morphology. These cells were imaged after 12 days of culture. Scale bar represents 30μm. (d) Representative morphology of MCF-DCIS cells after 7 days culture with a spacer gel of specific width between MCF-DCIS and HMF compartments. Scale bar represents 150μm. Bar graph presents the values from shape descriptor analysis, showing decreasing circularity and roundness and increasing aspect ratio as gap distance decreases. Error bars represent standard error. *: p<0.005 compared with the control group. **: p<0.0005 compared with gap ‘0’ group.

Fig 2

Co-injection and Sequential injection processes. (a) MCF-DCIS cells (left) and HMF cells (right) are co-injected and compartmentalized. The inset shows MCF-DCIS cells imaged 4 hours after loading, showing the size of cells from which the transition process was initiated. (b) The aqueous compartment filled with culture media (right) is created next to the MCF-DCIS cells containing gel compartment, which is replaced by HMF cells containing gel after 6 days' mono-culture of MCF-DCIS cells. The inset shows MCF-DCIS clusters cultured for 6 days, showing the size of cell clusters from which the transition process was initiated. Scale bars in A and B represent 0.5mm, and the ones in insets are 30μm. The invasive transition of MCF-DCIS cells in co-injected culture model (c) and sequentially injected culture model (d), showing more widely spread invasive transition in the sequentially injected culture model after a total of 10 days' culture (6 days' mono-culture, followed by 4 days' co-culture). Co-injected cells were cultured together for 10 days. Scale bars in c and d represent 30μm.

Fig 3

Invasion marker validation for in vitro and in vivo models. (a) In vitro model validation. Co-injected cells were cultured for 10 days and fixed, and Sequentially injected cells were cultured a total of 10 days (6 days mono-culture and 4 days co-culture). Cell nuclei (blue) are labeled with TO-PRO-3. Areas of dotted square shows cells losing collagen IV (green) and E-cadherin (red) expression. HMF were GFP labeled, and are shown in collagen IV image on the right side (marked with arrows). The control (Ctrl) cluster was cultured at the control side of co-injected culture and fixed after 10 days culture. Scale bar is 50 μm. (b) F-actin structure analysis.(i) F-actin structure of co-cultured MCF-DCIS cells for 8 days (6 days mono-culture and 2 days co-culture), showing highly condensed actin at the invading edge.. (ii) F-actin structure of MCFDCIS cells mono-cultured for 8 days. Scale bar represent 30 um. (c) In vivo model validation. H & E staining showed that lesions resembling high-grade, comedo-type DCIS developed approximately 2 to 3 weeks after inoculation. Partial loss of collagen IV and E-cadherin observed in both in vivo and in vitro models mark transition to invasive phenotype. Scale bar is 200 μm.

Fig 3

Invasion marker validation for in vitro and in vivo models. (a) In vitro model validation. Co-injected cells were cultured for 10 days and fixed, and Sequentially injected cells were cultured a total of 10 days (6 days mono-culture and 4 days co-culture). Cell nuclei (blue) are labeled with TO-PRO-3. Areas of dotted square shows cells losing collagen IV (green) and E-cadherin (red) expression. HMF were GFP labeled, and are shown in collagen IV image on the right side (marked with arrows). The control (Ctrl) cluster was cultured at the control side of co-injected culture and fixed after 10 days culture. Scale bar is 50 μm. (b) F-actin structure analysis.(i) F-actin structure of co-cultured MCF-DCIS cells for 8 days (6 days mono-culture and 2 days co-culture), showing highly condensed actin at the invading edge.. (ii) F-actin structure of MCFDCIS cells mono-cultured for 8 days. Scale bar represent 30 um. (c) In vivo model validation. H & E staining showed that lesions resembling high-grade, comedo-type DCIS developed approximately 2 to 3 weeks after inoculation. Partial loss of collagen IV and E-cadherin observed in both in vivo and in vitro models mark transition to invasive phenotype. Scale bar is 200 μm.

Fig 3

Invasion marker validation for in vitro and in vivo models. (a) In vitro model validation. Co-injected cells were cultured for 10 days and fixed, and Sequentially injected cells were cultured a total of 10 days (6 days mono-culture and 4 days co-culture). Cell nuclei (blue) are labeled with TO-PRO-3. Areas of dotted square shows cells losing collagen IV (green) and E-cadherin (red) expression. HMF were GFP labeled, and are shown in collagen IV image on the right side (marked with arrows). The control (Ctrl) cluster was cultured at the control side of co-injected culture and fixed after 10 days culture. Scale bar is 50 μm. (b) F-actin structure analysis.(i) F-actin structure of co-cultured MCF-DCIS cells for 8 days (6 days mono-culture and 2 days co-culture), showing highly condensed actin at the invading edge.. (ii) F-actin structure of MCFDCIS cells mono-cultured for 8 days. Scale bar represent 30 um. (c) In vivo model validation. H & E staining showed that lesions resembling high-grade, comedo-type DCIS developed approximately 2 to 3 weeks after inoculation. Partial loss of collagen IV and E-cadherin observed in both in vivo and in vitro models mark transition to invasive phenotype. Scale bar is 200 μm.

Fig 4

SHG signal intensity profiles around MCF-DCIS cell clusters. Two co-cultured clusters (co-A and co-B) show heterogeneous intensity profiles around clusters, while two mono-cultured clusters (mono-A and mono-B) show relatively homogeneous intensity profiles around clusters. Cluster outlines were found after F-actin staining of clusters. Collagen structures (green) were visualized by SHG signal and merged with F-actin-stained cells (red). Seven points were randomly selected around clusters, and lines (approximately 50 μm) were radially extended out to the surrounding collagen matrix from the points. Intensity profiles on the lines were measured. Scale bar represents 25 μm.

Fig 5

Area-based SHG signal analysis provides quantitative estimation of the degree of invasive transition of MCF-DCIS cells within a specific area. (a) Three sequential steps involved in image processing for an area-based analysis. The original images (OG) were first thresholded with the value obtained from a blank gel, and the thresholded images (TH) were then converted to binary (BN) for pixel counting. The percentage of affected area in mono-culture (mono-A in Fig 4) is 2.32%, whereas that in co-culture (co-B in Fig 4) is 18.80%. SHG signal values from a blank gel (containing no cells) were applied for thresholding to distinguish SHG signals altered by cells. The numbers represent the percentage of affected collagen area (PAC) in an image. (b) The bar graph is generated after analyzing 150 images each (identical sizes of 1.5 mm²) for both mono- and co-cultured clusters. The bar graph indicates the number of images in a specific range, showing that most of mono-cultured clusters fall into a range between 0 and 3, whereas co-cultured clusters show a range between 1 and 17. Schematic illustration above the bar graph shows representative appearance of clusters in an area corresponding to the PAC range. Circularity and roundness decreases approximately from 1 to 0, while aspect ratio increases from 1 to 8 as PAC increases. Noninvasive clusters apart produce a value close to ‘0’, and more invasive clusters yield a value close to ‘17’.