Here we employed an in situ
AFM-based approach to quantify and spatially characterize the biomechanical properties of freshly excised, intact mammary gland tissue as it transformed from normal to frank malignancy. The influence of biomechanics on tumor epithelial cell behavior is recognized; however analysis of the relationship between cell and tissue stiffening to cancer progression at the molecular scale and in living tissues has been technically challenging. Sonoelastography, MRI elastography, TDI analysis and physical palpation leave no doubt that the pathogenesis of invasive mammary cancer is associated with tissue stiffening.22–28
Multiple factors clearly contribute to tissue stiffening in tumors including elevated interstitial pressure and compression, enhanced cell contractility and elevated tumor cell mass as well as increased vascular density and ECM deposition, cross-linking and remodeling.2,12,19–21
Yet, the limited resolution of imaging modalities has compromised the use of these approaches to clarify the molecular basis for the biomechanical changes in cancerous tissue.
Using a combination of genetic and molecular fluorescent tagging with AFM and a novel tissue vitrification approach, we physically quantified and characterized the nature of the biomechanical stiffening associated with tumor progression in an experimental model of mammary gland cancer. We also found there was a coordinate stiffening of the ECM and the epithelium as the mammary gland transitioned from normal tissue to frank malignancy. Tumorigenesis is associated with an increase in microvascular density;42,43
and we determined that the vasculature associated with the developing tumors was stiffer than the vasculature of normal tissue. Our data also demonstrated that there were inherent differences in the mechanical properties of the vasculature within the tumor core versus
the vessels at the invasive front. Indeed, we found that these different mechanical phenotypes in the vascular beds were associated with quantifiable changes in the morphology and functional behavior of the vessels. By exploiting high-resolution AFM we also uncovered inherent biomechanical heterogeneities within the epithelium and the ECM of cancerous tissue. Thus, not only do our data clarify the anatomical components contributing to tumor stiffening but our findings also raise the possibility that mechanical heterogeneity within tumors could contribute to the heterogeneous behavior of some cancers.46–48
Therefore our approach offers a high-resolution strategy with which to clarify and study the role of biomechanical heterogeneity in cancer progression.
Our in situ
AFM force mapping studies using double transgenic MMTV-PyMT/ACTB-ECFP mice showed that mammary epithelial cell stiffness increased at least three-fold as the epithelium transformed from normal to frankly invasive lesions (). The findings demonstrate that the tumor epithelium is a significant biomechanical contributor to the overall stiffening of the transformed tissue. The results agree with prior bulk property analyses of transformed mouse and human tumor tissue and are consistent with the idea that cells “tune” their mechanical properties in response to their mechanical microenvironment.49,50
In marked contradiction to our findings however, prior work suggested that the mechanical properties of isolated cancer cells are, paradoxically, softer than non-transformed cells and imply that this ‘softer phenotype’ is a prerequisite for tumor metastasis.29,30
Such findings are at odds with in vivo
imaging studies that clearly demonstrate that solid tumors are significantly stiffer than normal tissue, and that tissues incrementally stiffen with cancer progression.22–28
Indeed, intravital microscopy imaging of metastasizing mammary gland tumor epithelium illustrate how tumor cells disseminate from primary tumors and migrate along thick and presumably “stiff” collagen fibers; through a process that culture manipulations suggest is favored by high traction forces and elevated tumor cell mechano-responsiveness (Lopez et al
., unpublished observations).18,32,34
One major difference between these prior in vitro
cellular rheology findings and our current study is that our measurements were conducted in situ
under conditions in which heterotypic and homotypic cellular interactions and ECM associations were strictly maintained. These conditions impart critical biochemical and biomechanical constraints on cellular behavior. For instance, epithelial cells sense and respond to the mechanical compliance of the microenvironment by pulling on their surroundings in an actomyosin-dependent fashion that alters the cells intrinsic stiffness.49,50
Our AFM indentation experiments demonstrated that although isolated mammary epithelial tumor cells were stiffer than normal mammary epithelial cells they were substantially softer when measured ex vivo
than in situ
(). Previous work in which the rheology of metastatic tumor cells was characterized examined isolated pleural effusion or tumor cells which by definition lack biochemical and physical connections to surrounding tissue elements.29,30
By contrast, the current study examined the mechanical properties of tumor cells in situ
, under conditions that preserve the native cellular associations between the tumor cells and the surrounding microenvironment.
Importantly, while there was a consistent overall increase in tumor cell stiffness, we also noted that there was considerable biomechanical heterogeneity within the epithelium of each tumor (). The heterogeneous mechanical properties of the cancer cells within each tumor mass could reflect the non-uniform state of the local cellular and extracellular microenvironment including whether or not the tumor cells were adjacent to a necrotic region or were proximal to a blood vessel or collagen fibril.49,50
Alternatively, this heterogeneity could be due to cell intrinsic properties linked to either an inherent genetic heterogeneity that enhances cellular contractility or differential mechanical properties of cells from distinct origins. It is tempting to speculate that the observed biomechanical tumor cell heterogeneity could reflect the unique mechanical behavior of a subpopulation of tumor stem or progenitor cells, especially given recent evidence that stem cells are softer and exhibit differential biomechanical behaviors than their differentiated progeny.51–53
Tumor progression is intimately linked to angiogenesis and we observed changes in vascular mechanical properties as mammary tumors developed.42,43
AFM force mapping revealed the two distinct types of tumor-associated vasculature. We noted that the vasculature associated with the tumor core was quite stiff, stiffer even than the surrounding transformed epithelium and that the vasculature at the tumor front was by contrast relatively soft (). The rigidity of the vasculature at the tumor core might reflect either differences in the maturity of the vasculature or physical changes in the intra-tumoral vessels that have collapsed due to high solid stresses exerted by the proliferating tumor mass or interstitial tumor pressure.11,42,54
CD31 immunofluorescent staining of Rh-lectin perfused mammary tumors revealed non-patent vessels within the tumor core and leaky vessels at the invasive front of the tumors () that are typical of the different types of vessels found in aggressive tumors.42,43
It is feasible that differences in the biomechanical properties of these vessels reflect the maturity and perfusion of these vessels. Regardless, immature vessels and collapsed vessels compromise the transport of cytotoxic cancer therapeutic drugs, and increased tumor stiffness is a primary impediment to efficient drug delivery.54
Interestingly, the soft neovasculature at the invasive front of tumors was located within a region of the ECM that was quite stiff. Previous work has demonstrated that a very stiff ECM compromises vascular integrity possibly by inhibiting vascular endothelial pseudopodial branch initiation and disrupting vascular network assembly, implicating ECM rigidity as a key regulator of vasculature heterogeneity.55–57
Whether ECM stiffness could actively promote disease progression and induce drug resistance by regulating the phenotype of the tumor-associated vasculature awaits further “mechanistic” analysis.
To establish the relationship between ECM biomechanical property changes and tumor progression necessitated the development of new in situ
approaches in which the ECM could be accurately identified and the biochemical and morphological makeup of the spatially-probed region comprehensively analyzed. Consequently, a rapid freeze/rapid thaw tissue vitrification approach was developed to preserve the biomechanical integrity of the tissue so that tissues were amenable for sectioning, and thus precise positioning of the AFM probe to the ECM could be achieved (). Although freezing and vitrification have been previously used to preserve the integrity of both stiff and soft tissues such as bone and cartilage, as well as skin and adipose, it had not been coupled to AFM force mapping.58–62
Similarly, AFM has been used previously to visualize the characteristics of heart, cornea, liver and bone tissues at a micro and nano-scale and in rare instances to obtain low-resolution mechanical information.63–67
However, until our study AFM was never coupled with traditional histopathologic methods to register the molecular and cellular composition and behavior of the tissue that was being force mapped. Through the application of tissue vitrification we were able to examine the morphological and mechanical behavior of the ECM in the mammary gland as it transitioned from the normal to the cancerous state. Using this approach we confirmed that the ECM progressively stiffened during tumor progression (). Although collagen-dense breast tissue has been associated with increased risk to breast malignancy, a casual link between altered collagen deposition and tissue mechanical properties in breast cancer progression has not been explored.22–28,68–70
This is primarily due to the fact that until our study no in situ
approach existed to accurately mechanically and histopathogically examine tissues at high resolution. Using AFM histopathologic examination we were able to demonstrate that while the fibrillar collagen stiffened during tumor progression, collagen deposition alone was not able to account for the observed biomechanical stiffening. Instead our findings suggest that ECM remodeling, altered crosslinking, fibril linearization and orientation also must contribute to increased ECM mechanical properties.12
Our findings are consistent with and extend prior work from our group and others strongly implicating collagen remodeling and stiffening in mammary gland tumor transition to invasion and metastasis.2,12,13,71
Indeed, the fact that we noted consistent stiffening during tumor progression suggests that the oriented, thickened collagen fibers along which mammary gland tumor cells have been seen to migrate are indeed a source of the ECM stiffening and suggest that these biomaterials properties facilitate tumor cell invasion and metastasis.13,72,73
Interestingly, protein degradation mediated by matrix metalloproteinases (MMPs) has been implicated in tumor progression and metastasis and an overwhelming body of evidence support the contention that MMPs are absolutely critical for fostering the transition of oncogenically-transformed epithelia to an invasive phenotype.74,75
Our observation that invasive mammary gland tumors are characterized by a stiffened ECM suggests that perhaps MMPs play a more nuanced role in fomenting tumor progression by “remodeling” and creating rigid collagen fibrils that foster tumor cell migration and invasion into the interstitial matrix; this possibility must be rigorously tested.
This study provides important new insight into the interplay between tumor evolution and the biomechanics of cancer. Nevertheless many questions remain, not the least of which is whether or not different subtypes of mammary gland tumors (basal, luminal, ER/PR positive, Her2 positive) evolve within unique biomechanical microenvironments that foster their histophenotype and behavior. Moreover, it is not obvious whether there exist temporal- and cell-type specific biomechanical hierarchies within a tissue and if so whether and how these physical parameters influence tumor evolution. What is clear is that at present we lack the ability to predict which tumors will progress to malignancy and which will be most lethal.76,77
Perhaps by clarifying the role of biomechanics in tumorigenesis at the micro-scale we will obtain the critical information required to clarify this dilemma. Our findings suggest that tumor and ECM stiffness may be novel prognostic metrics that could indicate whether or not a noninvasive lesion will progress. The next challenge will be to develop tractable approaches to accurately image and quantify these physical changes so that they can be translated to the clinic.