Abstract P6-01-18: Early detection of development of a pre-metastatic niche in lungs in response to primary breast tumor using Raman spectroscopy
Authors: Zheng C , Rizwan A , Paidi SK , Yu Z , Barman I , Glunde K 
Published: March 2017

Background: An alarmingly large proportion of cancer-related deaths result from metastatic cancers. Development of quick, reliable and non-invasive or minimally invasive approaches to objectively assess the secondary tissues (potentially in vivo) will be instrumental in substantially reducing the cancer burden due to metastasis, which accounts for majority of cancer related mortality. In our study we report the utilization of Raman spectroscopy and chemometric techniques in identifying formation of pre-metastatic niche in lungs prior to observing morphological changes.

Methods: Six-week-old female athymic nu/nu mice (NCI, MD) were implanted with 2x106 cells of human breast cancer cell lines - MDA-MB-231 (n=3), and MCF-7 (n=3) in their fourth right mammary fat pad orthotopically. And control mice (n=3) without tumor cell implantation were also employed. The primary tumor size was monitored and the mice were sacrificed within 8-12 weeks of cell implantation when the primary tumors volume grew to 500-600 mm3. Control mice were also sacrificed in this timeframe. The freshly excised lungs of the mice were cleaned in PBS and utilized for obtaining Raman spectra (830 nm, thermoelectrically cooled CCD). Each tissue was collected from multiple points. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) were employed as discriminating algorithm. Following spectral acquisition, the tissues were fixed in 10% formalin, embedded in paraffin, and then HE staining, and Masson's trichrome staining for collagen. Collagen quantification of Masson's trichrome stained slides was achieved using MATLAB (Mathworks, MA). The Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine approved the protocol of the study.

Results: 900 Raman spectra each acquired from the lungs of the control mice and mice bearing MCF-7 and MDA-MB-231 tumor xenografts were assigned class labels - 'Control', 'MCL' and 'MDL' respectively for further analysis. Select principal components from those obtained by subjecting all the chosen 900 spectra to PCA clearly evident that the differences in the Raman spectra belonging to tissues being primed by derivatives of different primary cells are quite pronounced. The average correct rates of PLS-DA prediction of 90.1%, 97.7% and 78.4% were obtained for the spectra belonging to the classes - Control, MCL and MDL respectively. The HE images are negative for any signs of cancerous lesions. Masson's trichrome staining results show that the metastatic potential of the cell lines responsible for the primary tumor is positively correlated with the collagen density in the pre-metastatic niche, the MDL shows the highest collagen density (P&lt;0.001). These differences clearly indicate the remodeling of extracellular matrix anticipating incoming tumor cells in response to primary tumor derived factors occurs very early in the metastatic cascade.

Conclusion: The current study introduces Raman spectroscopy in conjunction with chemometric techniques as are liable and minimally invasive tool for diagnosis of metastatic cancers significantly early in the metastatic cascade, and also opens a new route for early targeting of cancer metastasis and its associated burden.

Citation Format: Zheng C, Rizwan A, Paidi SK, Yu Z, Barman I, Glunde K. Early detection of development of a pre-metastatic niche in lungs in response to primary breast tumor using Raman spectroscopy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-18.