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. 2017 Jan 15;77(2):247-256.
doi: 10.1158/0008-5472.CAN-16-1862. Epub 2016 Nov 15.

Label-Free Raman Spectroscopy Detects Stromal Adaptations in Premetastatic Lungs Primed by Breast Cancer

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Label-Free Raman Spectroscopy Detects Stromal Adaptations in Premetastatic Lungs Primed by Breast Cancer

Santosh Kumar Paidi et al. Cancer Res. .
Free PMC article


Recent advances in animal modeling, imaging technology, and functional genomics have permitted precise molecular observations of the metastatic process. However, a comprehensive understanding of the premetastatic niche remains elusive, owing to the limited tools that can map subtle differences in molecular mediators in organ-specific microenvironments. Here, we report the ability to detect premetastatic changes in the lung microenvironment, in response to primary breast tumors, using a combination of metastatic mouse models, Raman spectroscopy, and multivariate analysis of consistent patterns in molecular expression. We used tdTomato fluorescent protein expressing MDA-MB-231 and MCF-7 cells of high and low metastatic potential, respectively, to grow orthotopic xenografts in athymic nude mice and allow spontaneous dissemination from the primary mammary fat pad tumor. Label-free Raman spectroscopic mapping was used to record the molecular content of premetastatic lungs. These measurements show reliable distinctions in vibrational features, characteristic of the collageneous stroma and its cross-linkers as well as proteoglycans, which uniquely identify the metastatic potential of the primary tumor by recapitulating the compositional changes in the lungs. Consistent with histological assessment and gene expression analysis, our study suggests that remodeling of the extracellular matrix components may present promising markers for objective recognition of the premetastatic niche, independent of conventional clinical information. Cancer Res; 77(2); 247-56. ©2016 AACR.


Figure 1
Figure 1. Raman spectroscopic profiling of pre -metastatic lungs
(A) Mouse models, orthotopically xenografted with human breast cancer cells of different metastatic potential (MCF-7 and MDA-MB-231), were used to study stromal adaptations in the lung, prior to seeding of tumor cells. (B) Representative in vivo brightfield (left) and fluorescence (right) images of mouse growing a tdTomato-expressing breast tumor xenograft. (C) Mean Raman spectra (with the shadow representing ±1 standard deviation) acquired from lungs of normal mice, and pre-metastatic lungs of MCF-7 and MDA-MB-231 xenografted mice are shown.
Figure 2
Figure 2. Principal component analysis of the acquired Raman spectra
(A) PC loadings derived from spectra of lungs from control mice, i.e. bearing no tumor xenograft. (B) PC loadings derived from spectra of lungs belonging to mice bearing MCF-7 xenografts (labeled as MCL in the text). (C) PC loadings derived from spectra of lungs belonging to mice with MDA-MB-231 xenografts (labeled as MDL in the text). Dotted and dot-dashed lines highlight collagen and proteoglycan features, respectively.
Figure 3
Figure 3. Visualization of spectroscopic differences due to pre-metastatic adaptations
Radial visualization plot showing clusters formed by spectra recorded from lung samples of sacrificed mice bearing MDA-MB-231 and MCF-7 breast cancer xenografts as well as controls without xenografts.
Figure 4
Figure 4. Histological assessment of pre-metastatic lungs shows stromal changes
Top (A-C) and middle (D-F) panels display representative microscopic images of H&E and Masson's trichrome stained slides at 5x and 10x magnifications, respectively. The H&E stained sections confirm the absence of tumor cell seeding in the lungs of controls. Masson's trichrome stain delineates collagen fibers in the extracellular matrix and is quantified through image processing, as shown in the bottom panel (G-I). The left (A, D, G) panel shows lung sections derived from control mice whereas the middle (B, E, H) and right (C, F, I) panels represent lung sections from mice bearing MCF-7 (non-metastatic) and MDA-MB-231 (metastatic) tumor xenografts, respectively. The scale bars in the top and middle panels represent 1,000 and 500 μm, respectively.
Figure 5
Figure 5. Quantification of collagen fiber density in pre-metastatic lungs
(A) Bar plot showing mean and standard deviation of collagen density across the three classes (with all mice included) along with pairwise Student's t-test p-values. (B) Bar plot showing mean and standard deviation of collagen content across the three classes (after exclusion of MDA-MB-231 xenograft bearing mouse displaying atypical Raman data) along with pairwise Student's t-test p-values.
Figure 6
Figure 6. Gene expression changes in pre-metastatic lungs as a function of metastatic potential of primary tumor
Microarray gene expression data heat map was obtained by analyzing the publicly available dataset GSE62816 on the Gene-e data visualization and analysis platform. The sample cohort includes lungs of mice bearing breast tumor xenografts of different metastatic potential. Total RNA was isolated from the pre-metastatic lungs and hybridized on an Affymetrix Mouse Genome 430 2.0 Array. Genes that are relevant to spectral markers identified in the current study and overexpressed in response to the metastatic potential of the primary tumor were analyzed. Moderated F-value of 2.53 was set as the criterion for inclusion.

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