Age is a leading risk factor for developing breast cancer. This may be in part to the time required for acquiring sufficient cancer mutations; however, stromal cells that accumulate in tissues and undergo senescence eventually develop a senescence-associated secretory phenotype that alters the microenvironment to promote cancer. Our focus is on mesenchymal stem cells (MSCs) – stromal cells recruited to tumors due to their natural tropism for inflammatory tissues; MSCs have been shown to enhance the metastatic potential of tumor cells through direct interactions or paracrine signaling within the tumor. In the tumor, MSCs can differentiate into carcinoma-associated fibroblasts that play a central role in tumor growth and matrix remodeling. We recently investigated the molecular and mechanical differences in pre- and post- senescent MSCs and how their interactions with MDA-MB-231 breast cancer cells contribute to malignancy. Our data show post-senescent MSCs are larger and less motile, with more homogeneous mechanical properties than pre-senescent MSCs. In-depth omics analysis revealed differentially regulated genes and peptides including factors related to inflammatory cytokines, cell adhesion to the extracellular matrix, and cytoskeletal regulation. A 3D co-culture model was used to assess the effects of pre- and post- senescent MSCs on collagen matrix remodeling. Although post-senescent MSCs were far less motile than pre-senescent MSCs and less contractile with the matrix, they profoundly altered matrix protein deposition and crosslinking, which resulted in local matrix stiffening effects. Post-senescent MSCs also induced an invasive breast cancer cell phenotype, characterized by increased proliferation and invasion of breast cancer cells. This invasive breast cancer cell behavior was further amplified when MDA-MB-231 was co-cultured with a mixture of pre- and post- senescent MSCs; this result was attributed to matrix remodeling and soluble factor secretion effects of post-senescent MSCs, which enhanced the migration of pre-senescent MSCs allowing them to form tracks in the collagen network for cancer cells to follow. Finally, molecular inhibitors targeting actomyosin contractility and adhesion were used to alter MSC interactions with breast cancer cells. Actin depolymerizing agent and focal adhesion kinase inhibitor were most efficient and completely able to block the effects of post-senescent MSCs on MDA-MB-231 invasion in collagen gels. This comprehensive approach can be used to identify molecular pathways regulating heterotypic interactions of post-senescent MSCs with other cells in the tumor. Furthermore, the local matrix stiffening effect of post-senescent MSCs may play a critical role in breast cancer progression.
more »
« less
Macromolecular crowding tunes 3D collagen architecture and cell morphogenesis
Collagen I is the primary extracellular matrix component of most solid tumors and influences metastatic progression. Collagen matrix engineering techniques are useful for understanding how this complex biomaterial regulates cancer cell behavior and for improving in vitro cancer models. Here, we establish an approach to tune collagen fibril architecture using PEG as an inert molecular crowding agent during gelation and cell embedding. We find that crowding produces matrices with tighter fibril networks that are less susceptible to proteinase mediated degradation, but does not significantly alter matrix stiffness. The resulting matrices have the effect of preventing cell spreading, confining cells, and reducing cell contractility. Matrix degradability and fibril length are identified as strong predictors of cell confinement. Further, the degree of confinement predicts whether breast cancer cells will ultimately undergo individual or collective behaviors. Highly confined breast cancer cells undergo morphogenesis to form either invasive networks reminiscent of aggressive tumors or gland and lobule structures reminiscent of normal breast epithelia. This morphological transition is accompanied by expression of cell–cell adhesion genes, including PECAM1 and ICAM1. Our study suggests that cell confinement, mediated by matrix architecture, is a design feature that tunes the transcriptional and morphogenic state of breast cancer cells.
more »
« less
- Award ID(s):
- 1651855
- NSF-PAR ID:
- 10124612
- Date Published:
- Journal Name:
- Biomaterials Science
- Volume:
- 7
- Issue:
- 2
- ISSN:
- 2047-4830
- Page Range / eLocation ID:
- 618 to 633
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple “cryptic leading edges” within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as “cell polarization barriers,” decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.
-
Background: Cell migration and invasion are essential processes for metastatic dissemination of cancer cells. Significant progress has been made in developing new therapies against oncogenic signaling to eliminate cancer cells and shrink tumors. However, inherent heterogeneity and treatment-induced adaptation to drugs commonly enable subsets of cancer cells to survive therapy. In addition to local recurrence, these cells escape a primary tumor and migrate through the stroma to access the circulation and metastasize to different organs, leading to an incurable disease. As such, therapeutics that block migration and invasion of cancer cells may inhibit or reduce metastasis and significantly improve cancer therapy. This is particularly more important for cancers, such as triple negative breast cancer, that currently lack targeted drugs. Methods: We used cell migration, 3D invasion, zebrafish metastasis model, and phosphorylation analysis of 43 protein kinases in nine triple negative breast cancer (TNBC) cell lines to study effects of fisetin and quercetin on inhibition of TNBC cell migration, invasion, and metastasis. Results: Fisetin and quercetin were highly effective against migration of all nine TNBC cell lines with up to 76 and 74% inhibitory effects, respectively. In addition, treatments significantly reduced 3D invasion of highly motile TNBC cells from spheroids into a collagen matrix and their metastasis in vivo. Fisetin and quercetin commonly targeted different components and substrates of the oncogenic PI3K/AKT pathway and significantly reduced their activities. Additionally, both compounds disrupted activities of several protein kinases in MAPK and STAT pathways. We used molecular inhibitors specific to these signaling proteins to establish the migration-inhibitory role of the two phytochemicals against TNBC cells. Conclusions: We established that fisetin and quercetin potently inhibit migration of metastatic TNBC cells by interfering with activities of oncogenic protein kinases in multiple pathways.more » « less
-
Mesenchymal stem cells (MSCs) that accumulate in the primary tumor due to their natural tropism for inflammatory tissues enhance the metastatic potential of tumor cells through direct interactions with tumor cells or paracrine signaling within the tumor microenvironment. MSCs also undergo senescence, which leads to increased production of pro-inflammatory cytokines and matrix-degrading enzymes. Senescence is a critical mechanism of limiting abnormal growth and cancer development through tumor suppression; however, senescent cells that accumulate in tissues eventually develop a senescence-associated secretory phenotype that alters the microenvironment to promote cancer. Increased understanding of the biophysical properties of senescent MSCs and how they mediate cell-cell interactions in the tumor may be useful in identifying novel biomarkers for senescent stromal cells in tissues or aggressive cancer cells that form in an aging stroma. A high-content single cell biophysical approach was used to define the mechanical properties of pre- and post- senescent MSCs. Our data shows post-senescent MSCs are larger and less motile, with more homogeneous mechanical properties than their pre-senescent counterparts. A robust molecular screening approach combining genome-wide microarray analysis with mass spec-based proteomics was used to establish the molecular differences in pre- and post- senescent MSCs. Our data show a consistent correlation of up and down regulated gene and peptide expression. A 3D co-culture model was used to assess the effects of pre- and post- senescent MSCs on breast cancer cell motility and invasion in 3D collagen gels. Post-senescent MSCs induced an invasive breast cancer cell phenotype, characterized by increased spreading of breast cancer cells in collagen, increased numbers of invading cells, and morphological elongation of breast cancer cells. Surprisingly, this invasive breast cancer cell behavior was further amplified when breast cancer cells were co-cultured with both pre- and post- senescent cells.more » « less
-
more » « less
Abstract Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A‐derived tumor cell lines of varying malignant potential and lean or obese ASCs are embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell‐cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time‐resolved optical coherence tomography imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)‐dependent proteolytic activity significantly contributes to ASC‐mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co‐inhibition of both MMPs and contractility is necessary to completely abrogate ASC‐mediated tumor cell migration. These findings imply that obesity‐mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c8bm01188e
