Abstract

We utilize the polarization and directionality of light emitted by fibrillar collagen via second harmonic generation to determine structural relationships between collagen in mouse mammary tumor models and the healthy mammary fat pad. In spite of the aberrations in collagen production and degradation that are the hallmarks of tumor stroma, we find that the characteristic angle of SHG scatterers within collagen fibrils, and the spatial extent over which they are appropriately ordered for SHG production, are the same in tumor and healthy collagen. This suggests that the SHG-producing subpopulation of collagen is unaffected by the altered collagen synthesis of the tumor stroma, and protected from its aberrant degradative environment.

© 2008 Optical Society of America

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  1. R. Kalluri and M. Zeisberg, "Fibroblasts in cancer," Nat. Rev. Cancer. 6,392-401 (2006).
    [CrossRef] [PubMed]
  2. M. P. Shekhar, R. Pauley, and G. Heppner, "Host microenvironment in breast cancer development: extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast," Breast Cancer Res. 5, 130-135 (2003).
    [CrossRef] [PubMed]
  3. S. Z. Haslam and T. L. Woodward, "Host microenvironment in breast cancer development: epithelial-cell-stromal-cell interactions and steroid hormone action in normal and cancerous mammary gland," Breast Cancer Res. 5, 208-215 (2003).
    [CrossRef] [PubMed]
  4. T. Hasebe,  et al., "Prognostic significance of fibrotic focus in invasive ductal carcinoma of the breast: a prospective observational study," Mod. Pathol. 15, 502-516 (2002).
    [CrossRef] [PubMed]
  5. A. Jukkola,  et al., "Postoperative PINP in serum reflects metastatic potential and poor survival in node-positive breast cancer," Anticancer Res. 21, 2873-2876 (2001).
    [PubMed]
  6. B. V. Jensen,  et al., "Extracellular matrix building marked by the N-terminal propeptide of procollagen type I reflect aggressiveness of recurrent breast cancer," Int. J. Cancer 98, 582-589 (2002).
    [CrossRef] [PubMed]
  7. R. Keskikuru,  et al., "Elevated preoperative serum ICTP is a prognostic factor for overall and disease-free survival in breast cancer," Oncol. Rep. 9, 1323-1327 (2002).
    [PubMed]
  8. Y. Guo,  et al., "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc Natl Acad Sci U. S. A. 96, 10854-6 (1999).
    [CrossRef] [PubMed]
  9. P. Wilder-Smith,  et al., "Noninvasive imaging of oral premalignancy and malignancy," J. Biomed. Opt. 10, 051601 (2005).
    [CrossRef] [PubMed]
  10. S.J. Lin,  et al., "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
    [CrossRef] [PubMed]
  11. P. P. Provenzano,  et al., "Collagen reorganization at the tumor-stromal interface facilitates local invasion". BMC Med. 4, 38 (2006).
    [CrossRef] [PubMed]
  12. E. Brown,  et al., "Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation," Nat. Med. 9, 796-800 (2003).
    [CrossRef] [PubMed]
  13. W. Wang,  et al., "Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling," Cancer Res. 62, 6278-6288 (2002).
    [PubMed]
  14. S. V. Plotnikov,  et al., "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90, 693-703 (2006).
    [CrossRef]
  15. H. F. Dvorak, "Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing," N. Engl. J. Med. 315, 1650-9 (1986).
    [PubMed]
  16. S. Kauppila,  et al., "Aberrant type I and type III collagen gene expression in human breast cancer in vivo". J. Pathol. 186, 262-8 (1998).
    [CrossRef]
  17. G. J. Cameron,  et al., "Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study," J. Struct. Biol. 137, 15-22 (2002).
    [CrossRef] [PubMed]
  18. R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-86 (2005).
    [CrossRef]
  19. J. Mertz and L. Moreaux, "Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Commun. 196, 325-330 (2001).
    [CrossRef]
  20. M. J. Duffy,  et al., "Metalloproteinases: role in breast carcinogenesis, invasion and metastasis," Breast Cancer Res. 2, 252-7 (2000).
    [CrossRef]
  21. Y. Chen,  et al., "DNA vaccines encoding full-length or truncated Neu induce protective immunity against Neu-expressing mammary tumors," Cancer Res. 58, 1965-71 (1998).
    [PubMed]
  22. C. J. Aslakson and F. R. Miller, "Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor," Cancer Res. 52, 1399-405 (1992).
    [PubMed]
  23. P. Stoller,  et al., "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-33342 (2002).
    [CrossRef] [PubMed]
  24. I. Freund, M. Deutsch, and A. Sprecher, "Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon". Biophys. J. 50, 693-712 (1986).
    [CrossRef] [PubMed]
  25. C. C. Reed and R. V. Iozzo, "The role of decorin in collagen fibrillogenesis and skin homeostasis," Glycoconj J. 19, 249-55 (2002).
    [CrossRef]
  26. A. S. Kamoun-Goldrat and M. F. Le Merrer, "Animal models of osteogenesis imperfecta and related syndromes," J. Bone Miner. Metab. 25, 211-8 (2007).
    [CrossRef] [PubMed]

2007

A. S. Kamoun-Goldrat and M. F. Le Merrer, "Animal models of osteogenesis imperfecta and related syndromes," J. Bone Miner. Metab. 25, 211-8 (2007).
[CrossRef] [PubMed]

2006

R. Kalluri and M. Zeisberg, "Fibroblasts in cancer," Nat. Rev. Cancer. 6,392-401 (2006).
[CrossRef] [PubMed]

S.J. Lin,  et al., "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

P. P. Provenzano,  et al., "Collagen reorganization at the tumor-stromal interface facilitates local invasion". BMC Med. 4, 38 (2006).
[CrossRef] [PubMed]

S. V. Plotnikov,  et al., "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90, 693-703 (2006).
[CrossRef]

2005

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-86 (2005).
[CrossRef]

P. Wilder-Smith,  et al., "Noninvasive imaging of oral premalignancy and malignancy," J. Biomed. Opt. 10, 051601 (2005).
[CrossRef] [PubMed]

2003

E. Brown,  et al., "Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation," Nat. Med. 9, 796-800 (2003).
[CrossRef] [PubMed]

M. P. Shekhar, R. Pauley, and G. Heppner, "Host microenvironment in breast cancer development: extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast," Breast Cancer Res. 5, 130-135 (2003).
[CrossRef] [PubMed]

S. Z. Haslam and T. L. Woodward, "Host microenvironment in breast cancer development: epithelial-cell-stromal-cell interactions and steroid hormone action in normal and cancerous mammary gland," Breast Cancer Res. 5, 208-215 (2003).
[CrossRef] [PubMed]

2002

T. Hasebe,  et al., "Prognostic significance of fibrotic focus in invasive ductal carcinoma of the breast: a prospective observational study," Mod. Pathol. 15, 502-516 (2002).
[CrossRef] [PubMed]

W. Wang,  et al., "Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling," Cancer Res. 62, 6278-6288 (2002).
[PubMed]

B. V. Jensen,  et al., "Extracellular matrix building marked by the N-terminal propeptide of procollagen type I reflect aggressiveness of recurrent breast cancer," Int. J. Cancer 98, 582-589 (2002).
[CrossRef] [PubMed]

R. Keskikuru,  et al., "Elevated preoperative serum ICTP is a prognostic factor for overall and disease-free survival in breast cancer," Oncol. Rep. 9, 1323-1327 (2002).
[PubMed]

P. Stoller,  et al., "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-33342 (2002).
[CrossRef] [PubMed]

G. J. Cameron,  et al., "Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study," J. Struct. Biol. 137, 15-22 (2002).
[CrossRef] [PubMed]

C. C. Reed and R. V. Iozzo, "The role of decorin in collagen fibrillogenesis and skin homeostasis," Glycoconj J. 19, 249-55 (2002).
[CrossRef]

2001

J. Mertz and L. Moreaux, "Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Commun. 196, 325-330 (2001).
[CrossRef]

A. Jukkola,  et al., "Postoperative PINP in serum reflects metastatic potential and poor survival in node-positive breast cancer," Anticancer Res. 21, 2873-2876 (2001).
[PubMed]

2000

M. J. Duffy,  et al., "Metalloproteinases: role in breast carcinogenesis, invasion and metastasis," Breast Cancer Res. 2, 252-7 (2000).
[CrossRef]

1999

Y. Guo,  et al., "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc Natl Acad Sci U. S. A. 96, 10854-6 (1999).
[CrossRef] [PubMed]

1998

Y. Chen,  et al., "DNA vaccines encoding full-length or truncated Neu induce protective immunity against Neu-expressing mammary tumors," Cancer Res. 58, 1965-71 (1998).
[PubMed]

S. Kauppila,  et al., "Aberrant type I and type III collagen gene expression in human breast cancer in vivo". J. Pathol. 186, 262-8 (1998).
[CrossRef]

1992

C. J. Aslakson and F. R. Miller, "Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor," Cancer Res. 52, 1399-405 (1992).
[PubMed]

1986

H. F. Dvorak, "Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing," N. Engl. J. Med. 315, 1650-9 (1986).
[PubMed]

I. Freund, M. Deutsch, and A. Sprecher, "Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon". Biophys. J. 50, 693-712 (1986).
[CrossRef] [PubMed]

Anticancer Res.

A. Jukkola,  et al., "Postoperative PINP in serum reflects metastatic potential and poor survival in node-positive breast cancer," Anticancer Res. 21, 2873-2876 (2001).
[PubMed]

Biophys J.

S. V. Plotnikov,  et al., "Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres," Biophys J. 90, 693-703 (2006).
[CrossRef]

Biophys. J.

R. M. Williams, W. R. Zipfel, and W. W. Webb, "Interpreting second-harmonic generation images of collagen I fibrils," Biophys. J. 88, 1377-86 (2005).
[CrossRef]

P. Stoller,  et al., "Polarization-modulated second harmonic generation in collagen," Biophys. J. 82, 3330-33342 (2002).
[CrossRef] [PubMed]

I. Freund, M. Deutsch, and A. Sprecher, "Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon". Biophys. J. 50, 693-712 (1986).
[CrossRef] [PubMed]

BMC Med.

P. P. Provenzano,  et al., "Collagen reorganization at the tumor-stromal interface facilitates local invasion". BMC Med. 4, 38 (2006).
[CrossRef] [PubMed]

Breast Cancer Res.

M. P. Shekhar, R. Pauley, and G. Heppner, "Host microenvironment in breast cancer development: extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast," Breast Cancer Res. 5, 130-135 (2003).
[CrossRef] [PubMed]

S. Z. Haslam and T. L. Woodward, "Host microenvironment in breast cancer development: epithelial-cell-stromal-cell interactions and steroid hormone action in normal and cancerous mammary gland," Breast Cancer Res. 5, 208-215 (2003).
[CrossRef] [PubMed]

M. J. Duffy,  et al., "Metalloproteinases: role in breast carcinogenesis, invasion and metastasis," Breast Cancer Res. 2, 252-7 (2000).
[CrossRef]

Cancer Res.

Y. Chen,  et al., "DNA vaccines encoding full-length or truncated Neu induce protective immunity against Neu-expressing mammary tumors," Cancer Res. 58, 1965-71 (1998).
[PubMed]

C. J. Aslakson and F. R. Miller, "Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor," Cancer Res. 52, 1399-405 (1992).
[PubMed]

W. Wang,  et al., "Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling," Cancer Res. 62, 6278-6288 (2002).
[PubMed]

Glycoconj J.

C. C. Reed and R. V. Iozzo, "The role of decorin in collagen fibrillogenesis and skin homeostasis," Glycoconj J. 19, 249-55 (2002).
[CrossRef]

Int. J. Cancer

B. V. Jensen,  et al., "Extracellular matrix building marked by the N-terminal propeptide of procollagen type I reflect aggressiveness of recurrent breast cancer," Int. J. Cancer 98, 582-589 (2002).
[CrossRef] [PubMed]

J. Biomed. Opt.

P. Wilder-Smith,  et al., "Noninvasive imaging of oral premalignancy and malignancy," J. Biomed. Opt. 10, 051601 (2005).
[CrossRef] [PubMed]

J. Bone Miner. Metab.

A. S. Kamoun-Goldrat and M. F. Le Merrer, "Animal models of osteogenesis imperfecta and related syndromes," J. Bone Miner. Metab. 25, 211-8 (2007).
[CrossRef] [PubMed]

J. Pathol.

S. Kauppila,  et al., "Aberrant type I and type III collagen gene expression in human breast cancer in vivo". J. Pathol. 186, 262-8 (1998).
[CrossRef]

J. Struct. Biol.

G. J. Cameron,  et al., "Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study," J. Struct. Biol. 137, 15-22 (2002).
[CrossRef] [PubMed]

Mod. Pathol.

T. Hasebe,  et al., "Prognostic significance of fibrotic focus in invasive ductal carcinoma of the breast: a prospective observational study," Mod. Pathol. 15, 502-516 (2002).
[CrossRef] [PubMed]

N. Engl. J. Med.

H. F. Dvorak, "Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing," N. Engl. J. Med. 315, 1650-9 (1986).
[PubMed]

Nat. Med.

E. Brown,  et al., "Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation," Nat. Med. 9, 796-800 (2003).
[CrossRef] [PubMed]

Nat. Rev. Cancer.

R. Kalluri and M. Zeisberg, "Fibroblasts in cancer," Nat. Rev. Cancer. 6,392-401 (2006).
[CrossRef] [PubMed]

Oncol. Rep.

R. Keskikuru,  et al., "Elevated preoperative serum ICTP is a prognostic factor for overall and disease-free survival in breast cancer," Oncol. Rep. 9, 1323-1327 (2002).
[PubMed]

Opt. Commun.

J. Mertz and L. Moreaux, "Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers," Opt. Commun. 196, 325-330 (2001).
[CrossRef]

Opt. Lett.

Proc Natl Acad Sci U. S. A.

Y. Guo,  et al., "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography," Proc Natl Acad Sci U. S. A. 96, 10854-6 (1999).
[CrossRef] [PubMed]

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Figures (8)

Fig. 1.
Fig. 1.

Enhanced stromal deposition characteristic of many tumors. Masson’s Trichrome staining of TG1-1 tumor cells grown in the mammary fat pad of FVB mice (left) as well as the healthy mammary fat pad of FVB mice (right). Abundant bands of ECM, primarily collagen, are evident throughout the tumor tissue as a blue staining (left), and are largely confined to isolated ducts in the healthy mammary fat pad (blue ring in center of right image). Images are 600 µm across.

Fig. 2.
Fig. 2.

Anti-Collagen I antibody staining of TG1-1 (left) and 4T1 (right) tumor sections, with DAB contrast. Both tumor types show the enhanced ECM deposition characteristic of tumor reactive stroma, evidenced by enhanced dark brown contrast around “islands” of lightly stained tumor cells. Left image is 600 µm across, right is 1.2 mm.

Fig. 3.
Fig. 3.

Experimental Apparatus.

Fig. 4.
Fig. 4.

Radar plot of detected intensity versus analyzer angle. This represents the intensity versus analyzer angle for each of five selected fibrils. In combination with the measured angle of the fibril relative to the laser polarization (vertical in the above graph), Ix, Iy, and hence θ can be extracted from this data for each fibril.

Fig. 5.
Fig. 5.

Measured values of θ in rat tail collagen as well as in different organ systems in the mouse. Error bars are standard deviations. All measurements in mouse organs are statistically significantly different from our measurements in rat (P<0.05), and are not statistically significantly different from each other (p>0.05).

Fig. 6.
Fig. 6.

Top Row: (a) Backward-, and (b) Forward-scattered image of collagen in an acute slice of a TG1-1 mammary adenocarcinoma. Bottom Row: (c) Backward-, and d) Forward-scattered image of collagen in an acute slice of a healthy FVB mammary fat pad. The small dots in the images are calibration beads. Images are 680 microns across.

Fig. 7.
Fig. 7.

The F/B ratio does not vary with apparent diameter of collagen fibrils. A fit straight line slope of zero is within the 95% confidence interval. Note that the PSF has an e-2 radius of 0.67 um. Consequently the stated apparent diameter is a convolution of the Gaussian PSF with the true fibril diameter. However, a deconvolution of the Gaussian PSF with the unknown true distribution (i.e. solid rod, hollow tube, etc.) will rescale the independent variable but will not impart a trend in the data which is not already apparent in the above plots.

Fig. 8.
Fig. 8.

TEM images of collagen in TG1-1 tumor (a), 4T1 tumor (b), and the corresponding healthy FVB and BALB mammary fat pads (c and d, respectively) consists of bundles of rods with a relatively constant characteristic diameter on the order of the ~70 nm predicted by SHG F/B ratio. Collagen fibrils intersecting the image plane transversely appear as streaks (a, b, d) and fibrils intersecting the plane perpendicularly appear as discs (c and d).

Equations (14)

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P = χ ( 1 ) * E + χ ( 2 ) * E * E + χ ( 3 ) * E * E * E
P = a s ̂ ( s ̂ · E ) 2 + b s ̂ ( E · E ) + c E ( s ̂ · E )
I e ( P · e ̂ ) 2 [ a cos α cos 2 φ + b cos α + c cos ( α φ ) cos φ ] 2
I y [ ( a + c ) cos 2 φ + b ] 2
I x [ c 2 sin 2 φ ] 2
I y I x = [ ( a + c ) cos 2 φ + b ] 2 [ c 2 sin ( 2 φ ) ] 2
a = n 3 m
c = 2 b = 2 m
n = χ zzz ( 2 ) = N cos 3 θ β
m = χ zxx ( 2 ) = χ xxz ( 2 ) = N 2 cos θ sin 2 θ β
tan 2 θ = 2 cos 2 φ I y I x sin ( 2 φ ) sin 2 ( φ )
I y ( φ ) = I p [ ρ cos 2 φ + sin 2 φ ] 2
I x ( φ ) = I p [ sin 2 φ ] 2
tan 2 θ = 2 ρ

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