Bone Quality Heterogeneity in Mixed Femoral Metastases Revealed by Stimulated Raman Scattering Spectromicroscopy.

Skeletal metastases disrupt bone remodeling and compromise bone quality; however, existing analytical approaches have not provided spatially resolved characterization of chemical heterogeneity between regions of direct tumor involvement and areas distant to the tumor. Stimulated Raman scattering (SRS) spectromicroscopy is a label-free imaging technique that enables quantitative, spatially resolved assessment of tissue composition. This study characterized the organic and mineral phases of osteoblastic bone present in mixed femoral metastases using SRS microscopy, revealing whether tumor and bone interactions influence regions within the bone distant from the metastasis. Mixed metastases were induced in 5- to 6-week-old athymic male rats with ACE-1 canine prostate cancer cells via intracardiac injection. At 21-day post-inoculation, bilateral femora were harvested. Sagittal sections of the right femora were processed for histological analysis and SRS imaging of the organic phase, and undecalcified sections were used to assess the mineral phase. Regions of interest were selected within newly formed osteoblastic bone, bone distant from the tumor, and in distinct samples containing only healthy bone. Osteoblastic regions showed increases in amide I, amide III, and proline signal intensity, and a reduction in phosphate and phenylalanine signal intensities, reflecting enhanced collagen deposition with diminished mineral content and altered protein composition. Bone distant from the tumor displayed elevated proline and reduced phenylalanine signal intensity, supporting the hypothesis that tumor and bone interactions influence bone beyond areas of direct tumor involvement. These findings establish SRS microscopy as a non-destructive tool for detecting spatially resolved biochemical alterations in metastatic bone.