Invasive cancer cells soften collagen networks and disrupt stress-stiffening via volume exclusion, contractility and adhesion
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18392985.
The paper with the title "Invasive cancer cells soften collagen networks and disrupt stress-stiffening via volume exclusion, contractility and adhesion" written by Irène Nagle et al. addresses an important question at the interface of cancer biology and biophysics by investigating how the presence of invasive breast cancer cells influences the polymerization kinetics and mechanics of collagen networks. They perform bulk-shear rheology experiments on invasive and non-invasive cell lines in collagen networks as well as on soft hydrogel microparticles in collagen networks. Furthermore, they added a confocal microscopy setup to the rheometer to image the collagen polymerization process. They …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18392985.
The paper with the title "Invasive cancer cells soften collagen networks and disrupt stress-stiffening via volume exclusion, contractility and adhesion" written by Irène Nagle et al. addresses an important question at the interface of cancer biology and biophysics by investigating how the presence of invasive breast cancer cells influences the polymerization kinetics and mechanics of collagen networks. They perform bulk-shear rheology experiments on invasive and non-invasive cell lines in collagen networks as well as on soft hydrogel microparticles in collagen networks. Furthermore, they added a confocal microscopy setup to the rheometer to image the collagen polymerization process. They observed that a collagen network with embedded breast cancer cells is softer, compared to a pure collagen network, while embedded human dermal fibroblast cells stiffen the sample. To explain this effect, the authors hypothesize a threefold of causes: volume exclusion, cell-matrix adhesion, and active cell contractility. For each factor, an experimental condition is developed and performed, supporting the hypothesis.
Volume exclusion: passive soft hydrogel microparticles of cell-like size were able to reproduce a delay in polymerization and two-fold softening at high inclusion fraction, implicating volume exclusion. However, they do not show non-monotonic softening at 4 % volume fraction and did not impair stress-stiAening or rupture stress.
Cell-matrix adhesion: blocking β1-integrin mediated adhesion in the cancer cells removed the non-monotonic softening and restored stress stiAening, suggesting adhesion is required for active remodeling eAects.
Active cell contractility: inhibiting myosin II with blebbistatin prevented the non monotonic time-dependence of G'.
The manuscript is of high significance, as the suppression of stress-stiffening and reduced rupture strength by cancer cells point to a potentially important insight for future understanding and treatment of tumor cell activities. The manuscript is promising and clearly written but it lacks a more thorough theoretical background and requires stronger argumentation in several parts. I therefore recommend this manuscript to be accepted after major revisions.
Major Concerns:
First, though the manuscript seems experimentally thorough, the proposed mechanism of local fiber buckling and local remodeling of the collagen network by actin-mediated protrusions of the cancer cells is plausible but not quantified. Consider adding quantitative microstructure analysis as collagen fiber curvature or alignment around the cells or adding simulations to model the effects of local remodeling to global softening and the loss of stress-stiffening.
Second, this study uses collagen networks as a model system for the ECM to replicate natural tissues, following successful approaches from literature. However, this manuscript would benefit from further elaboration on why this is a suitable model for in vivo ECM. It would strengthen the argument to discuss the limitations of this model and to provide additional network characteristics such as mesh size or density.
Third, proteolytic degradation is introduced early as an alternative mechanism to alter the ECM. However, the treatment of this mechanism in the results section remains insuAiciently short. The discussion could be expanded to clarify why proteolysis is the main alternative explanation and how the current data rule it out. Without this, the inclusion of proteolysis as an alternative argument risks appearing half-hearted instead of strengthening the manuscript's main argument.
Minor Concerns:
Not all figures in the main part include number of samples (n) and number of independent experiments (N) for statistical analysis, even though this is stated in section 2.12.
Several figures show the storage modulus G' and differential modulus K as a function of polymerization time and stress. In some cases, the y-axis represents normalized moduli. Please either ensure consistency to (not) normalize in all f igures or clearly explain and highlight why different scales were chosen.
For the experiments with living cells, consider reporting cell viability and proliferation during and after polymerization and rheology. Especially at high volume fractions and during oscillatory shears, this may help to understand the full picture.
Some typographical and grammatical issues were noted that should be corrected for clarity and consistency:
Section 2.6: "a few tens of million of cells per mL" → should read "a few tens of millions of cells per mL."
Page 19, Paragraph 1: "between control networks versus networks with cells" → should read "between control networks and networks with cells."
Figure 3 Caption: "remodelling" → ensure consistency in spelling ("remodeling" or "remodelling") throughout the manuscript.
This manuscript addresses an important question at the interface of cancer biology and biophysics. The findings are potentially impactful, but the mechanical background and theoretical framework require further attention. Strengthening the quantification of remodeling effects and elaborating on the suitability of and limitations of collagen networks as a model system, potentially by using simulations, would considerably enhance the manuscript.
Competing interests
The author declares that they have no competing interests.
Use of Artificial Intelligence (AI)
The author declares that they did not use generative AI to come up with new ideas for their review.
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