Collagen density promotes mammary tumor initiation and progression
© Provenzano et al; licensee BioMed Central Ltd. 2008
Received: 30 October 2007
Accepted: 28 April 2008
Published: 28 April 2008
Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen. Thus, the influence of the extracellular matrix on breast carcinoma development and the underlying molecular mechanisms are not understood.
To study the effects of collagen density on mammary tumor formation and progression, we utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue. Imaging of the tumors and tumor-stromal interface in live tumor tissue was performed with multiphoton laser-scanning microscopy to generate multiphoton excitation and spectrally resolved fluorescent lifetimes of endogenous fluorophores. Second harmonic generation was utilized to image stromal collagen.
Herein we demonstrate that increased stromal collagen in mouse mammary tissue significantly increases tumor formation approximately three-fold (p < 0.00001) and results in a significantly more invasive phenotype with approximately three times more lung metastasis (p < 0.05). Furthermore, the increased invasive phenotype of tumor cells that arose within collagen-dense mammary tissues remains after tumor explants are cultured within reconstituted three-dimensional collagen gels. To better understand this behavior we imaged live tumors using nonlinear optical imaging approaches to demonstrate that local invasion is facilitated by stromal collagen re-organization and that this behavior is significantly increased in collagen-dense tissues. In addition, using multiphoton fluorescence and spectral lifetime imaging we identify a metabolic signature for flavin adenine dinucleotide, with increased fluorescent intensity and lifetime, in invading metastatic cells.
This study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. Furthermore, the imaging techniques and signature identified in this work may provide useful diagnostic tools to rapidly assess fresh tissue biopsies.
Mammographically dense breast tissue is linked to a greater than four-fold increased risk of breast carcinoma [1–3], and is one of the greatest independent risk factors for breast cancer [1, 2, 4]. For instance, breast density in more than 50% of the tissue may account for up to 30% of breast cancers, while BRCA1 and BRCA2 mutations, although conferring a greater relative risk, account for only 5% of breast cancers (see Boyd et al  and references therein). Breast tissue density is additionally increased with hormone replacement therapy , suggesting increased density may be part of the underlying mechanism by which hormone replacement therapy increases cancer risk. Furthermore, ductal carcinoma in situ (DCIS), a local precursor to some invasive breast cancers, arises overwhelmingly in dense regions of the breast ; and high breast tissue density is associated with a shift to more malignant tumors , an increased likelihood of DCIS [8, 9], invasive breast carcinoma [9, 10], lymphatic and vascular invasion , and an approximately three-fold greater risk of developing a second breast carcinoma . However, although there is considerable correlative data identifying breast density as a risk factor for developing carcinoma, a causal link between breast density and carcinoma has not been established. Moreover, the molecular mechanisms driving breast density-related tumor formation and progression remain largely unknown.
Importantly, areas of increased breast density are not only associated with increased epithelial and stromal cellularity [12–14], but also significantly increased fibrillar collagen deposition [8, 13, 14]. In addition, it has been reported that levels of total collagen increase as radiographic breast tissue density increases [8, 13]. This is significant since tissue microenvironments play an important role in maintaining normal cellular behavior [15, 16], and stroma surrounding breast epithelial cells is believed to be critically involved in epithelial transformation, carcinoma growth, and metastasis [17–21]. Consistent with this concept, adipose-derived type VI collagen promotes tumor growth , while disturbing the epithelial-stromal interaction by disrupting the β1-integrin in mammary epithelial cells inhibits tumorigenesis . A less considered aspect of the complexity of the epithelial-stromal interaction is the fact that the stroma is a dynamic mechanical microenvironment, with dense collagenous stroma transmitting multi-axial deformations to breast cells during tissue deformation and increasing resistance to cellular contractility. Such mechanical signals arising from increased density or rigidity of the microenvironment play a role in the transformed phenotype of breast epithelial cells [24, 25]. Hence, although tumor formation is a multistep process involving genetic alterations of the epithelial cell, it has become clear that the epithelial-stromal interaction plays a crucial role in tumor formation and progression. Therefore, due to the increased stroma associated with breast tissue density we hypothesized that increasing collagen density in the mammary gland would promote tumorigenesis. Although there is a strong correlative link between breast density and carcinoma, to date collagen density has not been causally linked to tumorigenesis, largely because studies utilizing animal models with different stromal density have not been performed previously. Here we demonstrate that mammary tumor formation, invasion, and metastasis are enhanced in collagen-dense stroma in a transgenic mouse model.
The University of Wisconsin animal use and care committee approved this study. Breeding pairs of Col1a1tmJae mice  in the B6/129 background were obtained from Jackson Laboratory. Male FVB Polyomavirus middle-T mice under the control of the mammary specific MMTV promoter were originally obtained from Dr Amy Moser (University of Wisconsin) and are abbreviated PyVT following the Jackson Laboratory (from which they originated) nomenclature, but are also commonly abbreviated as PyMT or PyV MT. Col1a1tmJae homozygote males were crossed to C57BL/6 females to generate heterozygous females that were crossed to PyVT males to generate mice with normal and collagen-dense mammary tissues carrying the polyoma transgene. Mice were palpated every 2 to 3 days starting at 8 weeks of age to identify tumors. Genotyping by polymerase chain reaction (PCR) was performed on DNA extracted from tail biopsies (Wizard SV Genomic DNA Purification System, Promega, Madison, WI) using primers indicated in the strain information provided by The Jackson Laboratory. Mice were examined for palpable tumors starting at eight weeks of age and humanely killed at 15 weeks or when the tumor burden became excessive.
Histology and mammary gland whole mounts
Selected mammary tissues and tumors were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and then embedded in paraffin. In addition, all tissues imaged with multiphoton microscopy were subsequently fixed and processed for histology. Tissue sections were stained with hematoxylin and eosin (H&E) with adjacent sections stained with the selective collagen stain, picrosirius red. Mammary whole mounts were prepared by fixing tissues in Carnoy's solution (10% glacial acetic acid, 30% chloroform, and 60% absolute ethanol), followed by rehydration and staining with carmine alum. Tissues were then dehydrated, cleared with xylene, and mounted.
Immunofluorescent staining of mammary epithelial cells was performed in a manner similar to the methodology described by Wozniak and co-workers . Briefly, collagen gels were fixed in 4% paraformaldehyde for 20 minutes at room temperature. Following three washes in PBS, paraformaldehyde fluorescence was quenched with 0.15 M glycine in PBS then the gels were washed with PBS. Triton-X (0.2%) was added to permeabalize the cells, and gels blocked overnight with 2.5% fatty acid-free bovine serum albumin (BSA) + 1% donkey serum. Cell proliferation was then examined by staining with the anti-Ki-67 (mouse clone 7B11; Zymed) primary antibody in PBS containing 1% donkey serum for 30 minutes at room temperature. Following five 10-minute washes in PBS, gels were incubated with anti-mouse tetramethylrhodamine isothiocyanate (TRITC; Jackson ImmunoResearch Laboratories) secondary antibody, phalloidin-fluorescein isothiocyanate (FITC; Jackson ImmunoResearch Laboratories), and bisbenzimide (Sigma-Aldrich) in PBS containing 1% donkey serum for 30 minutes at room temperature. Gels were again washed with PBS and mounted with Prolong Antifade mounting media (Molecular Probes). Imaging was performed on a TE300 Nikon epifluorescence inverted microscope with acquired z-stacks deconvolved using Slidebook imaging software (Olympus).
Lungs from PyVT/wt (n = 4) and PyVT/Col1a1 (n = 4) mice (as well as wt/wt and wt/Col1a1 as negative controls) were harvested at 15 weeks, fixed in formalin, and processed for histology. Sections were cut every 50 μm through the entire tissue and sections stained with H&E. Total lung metastases over all sections were then counted.
Three-dimensional invasion assay
Uniform sized tumor explants were harvested from intact tumors using a tissue biopsy punch (3 mm diameter), rinsed with PBS (containing 100 units penicillin, 100 μg streptomycin, and 0.25 μg/ml amphotericin B), and placed into 2.0 mg/ml collagen gels (BD Biosciences, San Diego, CA) that were neutralized with 2× HEPES buffer. Tumors were maintained in collagen gels floated in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% fetal bovine serum (FBS), penicillin (100 units), streptomycin (100 μg), and amphotericin B (0.25 μg/ml) for 10 days over which time the number of distant multicellular colonies were counted.
Isolation of tumor cells and migration assay
Tumors from PyVT/wt and PyVT/Col1a1 backgrounds were minced and digested with 2 mg/ml collagenase and 10 μg/ml hyaluronidase in DMEM containing penicillin (100 units), streptomycin (100 μg), and amphotericin B (0.25 μg/ml). Following gentle shaking at 37°C for 3 hours, cells were pelleted, washed, and plated in DMEM containing 5% FBS. Thirty-six hours post-harvest the tumor cells were transferred to Transwell plates (Corning Inc, Corning, NY) using serum and soluble collagen containing media as the chemoattractant.
Multiphoton laser-scanning microscopy
For live tissue imaging by multiphoton excitation (MPE) and second harmonic generation (SHG), mammary tumors were harvested and live tissue maintained in buffered media at 37°C. All tissues were imaged immediately following tissue harvest using an Optical Workstation  that was constructed around a Nikon Eclipse TE300. A Tsunami Ti:sapphire laser driven by a Millenia 5 W pump laser (Spectra Physics, Mountain View, CA) excitation source producing around 100 fs pulse widths and tuned to 890 nm was utilized to generate both MPE and SHG. The beam was focused onto the sample with a Nikon 60X Plan Apo water-immersion lens (numerical aperture (NA) = 1.2). All SHG imaging was detected from the back-scattered SHG signal , and the presence of collagen confirmed in our tissues using fluorescence lifetime imaging microscopy (FLIM) on the same optical workstation, since the SHG from collagen has no lifetime. Furthermore, owing to the fundamentally different physical behavior of MPE and SHG, signals could be discriminated by filtering the emission signal. We used a 464 nm (cut-on) long pass filter to isolate the emission from autofluorescence from the conserved 445 nm SHG emission. A 445 nm (narrow-band pass) filter was therefore used to isolate the SHG emission. Acquisition was performed with WiscScan  a software acquisition package developed at LOCI (Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI) and image analysis for MPE/SHG was performed with ImageJ and VisBio  software. For TACS-1 image analysis, additional surface rendering plug-ins for ImageJ were utilized . For TACS-2 and -3, ImageJ was used to quantify the collagen fiber angle relative to the tumor. The tumor boundary was defined and the angle relative to the tangent of tumor boundary was measured every 10 μm as reported previously .
Fluorescence and spectral lifetime imaging microscopy
FLIM was performed on live tissue with the optical workstation described above and as described previously . Briefly, the Ti:sapphire laser (Millennium/Tsunami, Spectra-Physics, Mountain View, CA) was tuned to 890 nm with the beam focused onto the sample with a Nikon 60X Plan Apo water-immersion lens (NA = 1.2). Intensity and FLIM data were collected by a H7422 GaAsP photon-counting photomultiplier tube (PMT; Hamamatsu, Bridgewater, NJ) connected to a time-correlated single photon counting (TCSPC) system (SPC-730, Becker & Hickl, Berlin, Germany). Multiphoton spectral lifetime imaging microscopy (SLIM) was performed using a second-generation system that evolved from a previously described instrument  built around an inverted microscope (Eclipse TE2000, Nikon, Melville, NY). Briefly, a Mira Ti:sapphire mode-locking laser driven by a Verdi 8 W laser (Coherent Mira, Coherent, Santa Clara, CA) was used to generate pulse widths of approximately 120 fs at a repetition rate of 76 MHz. Intensity and fluorescence lifetime data were collected over 16 individual 10 nm spectral-width channels using a 16-anode photon counting linear PMT array (PML-16, Becker & Hickl) connected to a TCSPC system (SPC-830, Becker&Hickl). Fluorescent lifetime analysis from FLIM and SLIM was carried out with SPCImage (Becker & Hickl) as well as with a LOCI created computational tool, SlimPlotter , which allows visualization and analysis of the lifetimes by spectral channel.
For multi-group comparisons, one-way analysis of variance (ANOVA) with a post-hoc Tukey-Kramer test was used. We performed t-testing for two-group comparisons.
Increased collagen density promotes mammary epithelial cell proliferation in reconstituted three-dimensional matrices
Increased tumor incidence in collagen-dense mammary tissues
With a defined model for breast tissue density in place, we set out to test the hypothesis that increased mammary collagen density increases tumor formation. Mammary tumors were initiated with the PyVT transgene. This breast tumor model correlates well with many features of human cancer, progresses from hyperplasia to adenoma to early and late carcinoma , and is reliably invasive and metastatic . When mice carrying the PyVT transgene under the control of the mammary epithelial-specific MMTV promoter were crossed with heterozygous Col1a1tmJae mice, we observed an approximately three-fold increase in early tumor formation in collagen-dense tissues (that is, a three-fold greater number of tumors per mouse; see Figure 2b). This trend of increased tumor incidence in collagen-dense glands continued through week 15 (Figure 2b), and two additional PyVT/Col1a1 mice needed to be euthanized by week 13 due to excessive tumor burden (not shown). Consistent with these observations, quantitative analysis of whole mounts of the fourth mammary gland (n = 3 pairs) show significantly increased areas of hyperplasia (Figure 2c) with collagen-dense tissues showing increased cell growth out from the gland (Figure 2c arrowhead and Figure 2d). Furthermore, tumors progressing in collagen-dense tissues at 10 weeks had a more invasive morphology (Figure 2e). Of note is the fact that tumors arising in collagen-dense mammary tissue retain increased collagen density (Figure 2e and confirmed with collagen selective picrosirius red staining (not shown)). In fact, collagen levels in PyVT/Col1a1 tumor-bearing glands appear to be increased relative to non-tumor bearing collagen-dense glands (Figure 2e). This observation possibly indicates an amplified shift in the imbalance between collagen synthesis and degradation in the Col1a1 mice following tumor initiation, and may represent an increased desmoplastic response.
Increased invasion and metastasis associated with dense stromal collagen
Data showing increased invasion in tumors that arose in collagen-dense tissue was obtained by quantifying invasion from tumor explants into three-dimensional collagen gels. To determine whether local invasion was a simple reflection of increased local collagen that facilitates invasion or also due to an intrinsic property of tumor cells arising in a collagen-dense stroma, tumor explants of defined size were placed into three-dimensional collagen gels and the number of distant colonies was counted. After 10 days in culture, explants from collagen-dense tissues resulted in significantly more colonies (Figure 3b). Furthermore, tumor cells isolated from collagen-dense tissues were in fact more migratory (Figure 3c), indicating that the earlier onset of invasiveness is likely not the sole cause for increased metastasis but that the tumor cells themselves are more invasive (Figure 3b) and migratory (Figure 3c).
Changes in the tumor-stromal interaction associated with increased stromal collagen
Spectral-lifetime imaging of the tumor-stromal interaction suggests a metabolic signature associated with invasion
Exploiting cellular FAD as an endogenous biomarker to visualize cells, we further explored the difference in FAD signal between stromal and tumor cells, using FLIM. Differences in the fluorescence lifetime of FAD between primary tumor cells and stromal cells were color mapped (Figure 6b) and quantified (Figure 6c). Stromal cells possessed a higher second component (τ2) and weighted mean (τm) of the fluorescent lifetime, allowing stromal cells to be easily differentiated from epithelial tumor cells (Figure 6b and 6c).
In addition to identifying key differences in measurable fluorescent intensity and lifetime associated with invading cells, FLIM analysis confirmed results shown in Figure 4 demonstrating a shift towards TACS-3 and increased local invasion with higher collagen density (see Figure 7b). Invading cells associated with TACS-3 could be clearly differentiated in collagen-dense tissues (the right panel in Figure 7b) while PyVT/wt tumors (the left panel in Figure 7b) were not highly invasive at this stage (week 10).
Collagen density and tumor formation and progression
Although the increased risk for breast carcinoma associated with collagen-dense breast tissue has been described [1–3], a causal link between increased stromal collagen and increased breast carcinoma has not been previously established. Moreover, little is known about the molecular mechanisms underlying increased collagen deposition and its influence on the interactions between stromal collagen, fibroblasts, and epithelial cells, or how increased collagen affects tumorigenesis and tumor cell phenotype. This is due in large part to the fact that no animal model system had previously existed to study these phenomena in vivo. Herein we demonstrate that mice with increased stromal collagen have increased mammary tumors that are more invasive and metastatic, and thus provide a causal link between stromal density and carcinoma progression, consistent with reports of human breast carcinoma risk.
However, the possibility that altered matrix remodeling associated with the Col1a1 model is playing a role also warrants consideration. However, in theory, a significant defect in matrix remodeling should inhibit tumor progression, and the fraction of collagen that is collagenase-resistant can be degraded/remodeled at a second site by the rodent collagenase and other proteases that are expressed by tumor and tumor-associated cells. Hence, while limitations of the model must be taken into account when considering the presented data, it appears unlikely that a defect in matrix remodeling associated with the use of the Col1a1 model is causal for the increases in tumor formation and progression observed in this study.
Collagen signatures and local invasion
In a previous study we described the use of collagen alignment to quantify local invasion with the level of TACS-2 (alignment tangential to the tumor boundary at a 0° angle) and TACS-3 (alignment radial to the tumor boundary at an angle of 90°) providing a novel quantitative assessment of tumor progression . In this study, the analysis of collagen radial alignment in samples from 8- and 10-week tumors demonstrates a transition from TACS-2 to TACS-3. We observe a broad distribution of fiber angles away from zero but not yet tightly grouped as late-stage tumor at the radial alignment (90°) associated with the high degree of local invasion previously reported for 15-week tumors . This result suggests that the move toward invasive behavior is a transitional process increasing with time. We find that tumor cells in collagen-dense tumors are not only more invasive and metastatic in vivo, but were also more invasive and migratory in vitro (Figure 3b and 3c), indicating that the increased invasiveness is not only the result of earlier tumorigenesis that had more time to progress, but also due to tumor cells that are fundamentally more invasive because they arose within collagen-dense tissues. This finding suggests that cellular behavior is altered by epigenetic changes signaled from the collagen-dense stroma, consistent with findings that increased collagen density alters epithelial cell signaling and behavior in vitro .
FAD and local invasion
Interestingly, we measured an increase in the fluorescence lifetime for the metabolite FAD associated with invading cells. While this information provides a valuable biomarker for use with an optical biopsy, the biological relevance of this finding is not well understood. It is known that transformed cells often undergo increased glycolysis in the cytosol, a phenomena known as the Warburg effect , and that the fluorescent lifetimes of NADH and FAD, and in particular the redox ratio of these two metabolites, is altered in transformed cells . Of interest, Skala and co-workers  recently reported an increase in the τ1 component of the FAD lifetime in precancerous cells when compared with normal epithelium. In the current study we compare non-invasive transformed cells with invading cells, and as such we speculate that the alteration in FAD state seen in transformation may become increasingly mis-regulated in the more metastatic population of transformed cells. Furthermore, the biological implications of the increased FAD intensity and fluorescent lifetime may be found in the possibility that increased glycolysis is increasing levels of NADH, a known regulator of transcription , and resulting in more lactic acid production  with less pyruvate entering into the citric acid cycle and, as a consequence, less FAD being reduced to FADH2. Moreover, it has also been reported that the fluorescence lifetime of FAD can decrease due to quenching from NAD+, other molecular interactions, or environmental conditions [62, 63], and thus the increased fluorescence lifetime of FAD could also be indicative of less available NAD+, particularly in the cytosol, and other unknown changes in FAD binding and localization. Hence, the biological implications of altered FAD intensity and fluorescence lifetime remain elusive. However, our results provide evidence that changes in FAD signals can be found within a more invasive subpopulation of carcinoma cells and as such understanding the regulatory mechanisms associated with these observations may provide great insight into tumor cell metastasis.
In summary, increased collagen density increases tumorigenesis, local invasion, and metastasis, causally linking increased stromal collagen to tumor formation and progression. Imaging with combined MPE and SHG in tumors allows visualization of cellular autofluorescence and defined collagen structures that identify key differences associated with high collagen density and may provide useful diagnostic tools to rapidly assess fresh tissue biopsies. Furthermore, imaging live tissues with FLIM and SLIM confirms results obtained with MPE/SHG and identifies significant differences in fluorescence lifetimes that are indicative of invasive cells. Thus, FLIM and SLIM serve as powerful tools to evaluate the invasiveness of tumor cells in mammary tissues. Given the significant findings associated with high breast tissue density and the now available utility of a mouse model for breast tissue density, fundamental questions regarding the molecular mechanisms associated with breast tissue density-related carcinoma can now be further addressed in vivo.
List of abbreviations
analysis of variance
bovine serum albumin
ductal carcinoma in situ
Dulbecco/Vogt modified Eagle's minimal essential medium
hematoxylin and eosin
flavin adenine dinucleotide
fetal bovine serum
fluorescence lifetime imaging microscopy
multiphoton laser-scanning microscopy
nicotinamide adenine dinucleotide
phosphate buffered saline
polymerase chain reaction
second harmonic generation
spectral-lifetime imaging microscopy
tumor-associated collagen signature
time-correlated single photon counting
The authors thank Dr. Caroline Alexander helpful discussions regarding mice. This work was supported by grants from the DOD-CDMRP/BCRP (W81XWH-04-1-042 to PPP, and W81XWH-06-1-0397 to LY), the Susan G Komen Foundation (BCTR02-1841), the American Cancer Society (RSG-00-339CSM), NIH-NCI (R01-CA076537 to PJK) and NIH NIBIB (R01-EB000184 to JGW and KWE).
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