(C-105) Exploring the Role of Collagen Fiber Alignment Gradients as a Taxis Cue in Directing Cell Migration

Introduction:: Within the tumor microenvironment, guidance cues are essential for directed migration, enabling cancer cells to effectively navigate and invade the surrounding extracellular matrix. These cues consist of chemical signals such as growth factors and chemokines, as well as biophysical cues like extracellular matrix stiffness, porosity, and fiber density. Cancer cells can sense and react to these cues, allowing them to traverse the tissue architecture, enter blood or lymphatic vessels, and seed new tumor sites in distant locations.

In addition to these guidance cues, research has shown that cells can reorganize surrounding collagen I fibers, resulting in local fiber alignment that decreases with distance away from the cell boundary, resulting in spatially graded fiber alignment. Although gradients in biophysical properties are recognized as important guidance cues, the influence of local fiber alignment gradients on cellular migration has not been widely investigated.

In our prior work, we developed a platform to engineer collagen I fiber alignment gradients using a microfluidic channel with constricting and expanding geometry. This approach allowed control over the range of fiber alignment and the slope of the alignment gradient. In the current study, we provide preliminary evidence that cells respond to subtle gradients in fiber alignment by moving preferentially towards higher fiber alignment. This work thus suggests a role for gradations in fiber alignment as a new taxis cue.


Materials and Methods:: Figure 1A shows a 200 µm tall PDMS sheet with a through-cut channel geometry that was plasma bonded to a coverslip. A PDMS lid with injection ports was sealed to the PDMS sheet and bovine methacrylate collagen (Advanced Biomatrix) was prepared at a concentration of 2.7 mg/ml. After priming the microchannel with neutralized collagen at 10 µl/min, the collagen solution was injected at 500 µl/min and then incubated at 37°C to facilitate gel formation.

The resulting collagen I fibers were imaged using confocal reflectance microscopy and analyzed using LOCI CT-Fire to determine the coefficient of alignment (CoA), defined as the ratio of the number of fibers within ±15° of the mode on the histogram to the total number of fibers. After polymerization, the PDMS lids were removed (Figure 1B), media was added to the collagen gel and incubated overnight. A PDMS sheet with a patterning window was placed on the collagen gel to provide direct access to a defined region (Figure 1C) of the gel.

A cell aggregate was added to the patterning region and settled onto the collagen gel at a defined location (Figure 1D). The migration of the cells was tracked by measuring the position of the cell front as a function of time. The center point of the patterning window was used as the reference position. Brightfield images were processed using convolution filter and followed by a variance filter to identify cells. The images were then was converted to binary using the ImageJ Otsu threshold filter.


Results, Conclusions, and Discussions:: Cells were added within the patterning window on an underlying alignment gradient, with the CoA increasing to the left and decreasing to the right. Cells on the left edge experienced a CoA of 0.6, while those on the right encountered CoA < 0.5 (randomly oriented fibers). To quantify cell migration, the distances from the origin to the left (r) and right (a) edges of the cell population were measured every 24 hours (n= 24, 48…..120) and characterized with Ω_n, defined as ∆r_n/∆a_n, was computed. Here, ∆r_n = r_n – r_n-24 and ∆a_n = a_n – a_n-24, with measurements taken up to 120 hours from seeding.

As shown in Figure 1E, Ω_n > 1 indicates biased movement of the cell front towards the left (increasing CoA). As shown in Figure 6, after 48 hours, Ω₄₈ = 1.1, indicating a slight leftward movement (towards increasing alignment). A day later, Ω₇₂ increased to 1.6, suggesting further leftward progression. The highest Ω₁₂₀ ratio of 3.2 was measured at 120 hours after seeding.

Thus, our results suggest that MB-231 cells are able to migrate directionally toward the increasing alignment and provides evidence to support the role of alignment gradients as possible taxis cue that cells can follow in the ECM. Future studies will concentrate on assessing the influence of different gradient slopes and exploring how these gradients impact cell migration.


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