1.

Introduction

Breast cancer (BCa) is the second leading cause of cancer death in women in the United States, with $>\mathrm{230,000}$ invasive BCa diagnoses and 40,000 deaths estimated in 2014.¹ Despite recent advances in health awareness, patient screening, and cancer treatment, a significant proportion of women still develop advanced BCa, among which $\sim 70\%$ will develop bone metastases, causing chronic bone loss and destruction.^2–4 As a result, pathological bone fractures occur and represent one of the most serious problems associated with metastasis.^5–7 Therefore, it is important to predict patients’ fracture risk, thus providing guidance for personalized therapy. Current guidelines for the prediction of pathological fracture rely primarily on radiographs or computed tomography (CT). However, previous studies suggested that information from these examinations alone does not fully explain fracture risk.^8,9 Combining these current standards with other techniques could offer new opportunities to obtain complimentary information about bone quality and, therefore, improve the prediction of the risk for pathological fracture.

Raman spectroscopy (RS) is a vibrational spectroscopy technique that detects the inelastic scattering of photons upon their interactions with molecules. It provides molecular specific information regarding tissue’s compositional properties, which are key determinants for bone quality. In the past decades, RS has been successfully applied to characterize alterations in bone composition caused by aging and disease.^10–13 McCreadie et al. studied women proximal femurs and suggested bone compositions as risk factors for osteoporotic fracture.¹⁰ In another study, RS-derived bone material properties have exhibited significant correlation with the tissue-level mechanical function of bone, demonstrating promising potential in fracture risk prediction.¹⁴

Despite the promising results of RS in bone quality evaluation, the in vivo application of RS for orthopedic research has been limited by the shallow detection depth in the past decades. Both incident and Raman photons are highly scattered in biological tissues, hindering the technique’s ability to detect deeper layers in turbid media. Recent advances in spatially offset Raman spectroscopy (SORS) permitted the detection of Raman signals from depth up to several millimeters and, in some cases, a couple of centimeters by introducing a distance between the illumination and collection fibers.^15–18 Schulmerich et al. demonstrated the measurement of bone Raman spectra from live mice with optimized fiber configuration.¹⁵ Matousek et al. also successfully demonstrated that, using SORS, the spectrum of thumb distal phalanx could be obtained in vivo with a laser power below skin-safety level.¹⁸ Such a breakthrough in penetration depth has opened the door for noninvasive in vivo Raman interrogation of bone constituents. Furthermore, based on the concepts of spatial offsetting and multichannel collection, Raman tomography (RT) of tissue phantoms has recently been developed, indicating the potential to obtain three-dimensional compositional mapping of bone tissue.^19,20

Motivated by the need to improve fracture risk assessment in tumor-bearing bone and the promising advancement in SORS and RT, this study tries to determine the feasibility of assessing metastatic bone using RS. Tumor-bearing bones were investigated using Raman microspectroscopy to evaluate whether breast cancer metastases resulted in RS detectable local changes in bone tissue composition that may be linked to bone quality and fracture risk. The goal of the current study is to develop Raman spectral markers that are associated with tumor progression and resulting bone deterioration. These spectral markers may be used to evaluate the quality of metastatic bone and improve the estimation of the pathological fracture risk in the future in combination with noninvasive SORS and RT.

2.

Methods

3.

Results

3.1.

Bone Structural and Architectural Changes Induced by BCa Metastasis

BCa has been known to metastasize into bones and cause bone loss (osteolysis).^2,3,7 Representative radiographs in Fig. 1 exhibit the images from the control and the tumor-bearing tibiae. Most of the osteolytic lesions (indicated by white arrows) are in the metaphysis region. Further inspection of these lesions by μCT indicates that BCa metastases induced significant changes in the architectural properties of tibial metaphysis. A marked decrease in trabecular bone volume fraction and trabecular number [Figs. 2(a) and 2(b), both $p<0.001$], and a significant increase in trabecular space [Fig. 2(c), $p<0.001$] were observed in the metaphysis of tumor-bearing bone, confirming bone loss and structural disruption caused by BCa. No significant difference was observed in cortical TMD between tumor-bearing tibiae and the control group.

Fig. 1

Representative radiographs from the control (a) and the metastatic tibia (b). Osteolytic bone lesions are indicated by the white arrows.

Fig. 2

μCT analysis of metastatic tibiae. Significant bone loss was observed in the trabecular bone of tumor-bearing tibia (a). Significantly reduced bone volume fraction (b), trabecular number (c), and enhanced trabecular space (d) indicate bone loss and architectural disruption in metastatic tibiae (***$p<0.001$).

3.2.

RS Reveals Tumor-Associated Compositional Changes

RS was used to measure mineral and matrix constituents of tibial cortical bones, with special interest at the metaphysis region where metastasis generally starts. Representative Raman spectra from tumor-bearing and control tibiae were compared in Fig. 3. Selective Raman peaks, including phosphate, proline, hydroxyproline, phenylalanine, carbonate, and protein amide, are labeled in Fig. 3 with their corresponding biochemical assignments. The variations in the intensities of these Raman signature bands reveal the changes in molecular content and organization induced by BCa metastasis.

Fig. 3

Representative Raman spectra from tumor-bearing (dashed line) and control (solid line) tibiae. Selective Raman peaks are labeled on the figure.

Quantitative analysis of cortical composition was performed by calculating the intensity of Raman features that were associated with tissue-level bone material properties in previous reports.¹⁴ These spectral features include the degree of collagen mineralization, which is calculated by the peak ratio of phosphate $\nu 1$ and amide 1 (or proline); type B carbonate substitution level, which is determined by the peak ratio of carbonate and phosphate $\nu 1$; and hydroxyapatite crystallinity, which is calculated as the reciprocal of full width half maximum of phosphate $\nu 1$ peak. Figure 4 shows the comparison of the aforementioned bone material properties between tumor-bearing and control tibiae when the averaged value from each bone was compared ($n=30$ for tumor and $n=14$ for control). The tumor-bearing group showed significant increases in mineralization (phosphate $\nu 1/\text{amide}\mathrm{I}$) [Fig. 4(a)] and carbonate substitution [Fig. 4(c)], but no significant difference in mineral crystallinity [Fig. 4(d)] and phosphate $\nu 1/\text{proline}$ [Fig. 4(b)].

Fig. 4

Compositional parameters derived from Raman analysis. Significantly higher level of mineralization and carbonation was observed in tumor-bearing tibiae (*$p<0.05$; **$p<0.01$; ***$p<0.001$).

Fig. 5

Effect of tissue heterogeneity and proximity to tumor on Raman spectral parameters. Collagen mineralization by phosphate $\nu 1/\text{amide}\mathrm{I}$ (a) and phosphate $\nu 1/\text{proline}$ (b), and mineral crystallinity (d) showed significant differences only at the proximal positions to metaphysis in the tumor-bearing tibiae, while carbonation level (c) was enhanced at all measurement locations. The measurement locations on tibiae are shown in (e). (*$p<0.05$; **$p<0.01$; ***$p<0.001$).

To further evaluate the effect of tissue heterogeneity and proximity to tumor on the acquired spectral parameters, Raman spectra from the same relative location on tibiae were grouped and compared to four other sampling locations. Figure 5 shows tumor-associated compositional changes as a function of the distance to metaphysis. When sorted by locations, the elevation in mineralization is significant only at the proximal positions to metaphysis [Figs. 5(a) and 5(b)], where osteolytic lesions started and were most severe in all the animals (Fig. 1). Similarly, mineral crystallinity is significantly higher only at the metaphysis [Fig. 5(d)] in the tumor-bearing group. In contrast, carbonation level [Fig. 5(c)] was enhanced at all five measurement locations on the tibiae, indicating a possible alteration in bone material properties even in the absence of direct interaction with the tumor. Table 1 lists the COV in all the above compositional parameters from control and tumor groups. The variations in carbonation and mineral crystallinity are less pronounced than the mineralization parameters.

Table 1

The coefficient of variation in compositional parameters from control and tumor groups.

Measurement locations (μm)	Carbonation		Mineralization		Mineral/Collagen		Mineral crystallinity
	Control (%)	Tumor (%)	Control (%)	Tumor (%)	Control (%)	Tumor (%)	Control (%)	Tumor (%)
0	7.56	6.58	36.22	23.86	19.96	16.37	2.68	1.60
500	6.86	6.65	26.15	26.41	14.48	14.43	1.42	1.10
1000	5.59	4.81	35.16	30.22	14.32	16.74	1.04	1.51
1500	6.70	4.60	36.59	23.98	13.39	11.99	1.22	0.98
2000	4.86	4.56	35.91	18.99	16.30	10.62	1.35	0.97

3.3.

Carbonation Level Is Correlated to the Size of Bone Lesions

Pearson’s correlation analysis was performed to interrogate the association between Raman-derived compositional properties and the size of bone lesions. The carbonation level showed significant positive correlation with the size of osteolytic lesions (Fig. 6), indicating that carbonate/phosphate $\nu 1$ could potentially be used as a spectral marker to evaluate tumor-induced bone degeneration.

Fig. 6

Significant positive correlation was shown between carbonation level and the size of osteolytic lesions ($p<0.05$).

4.

Discussion

Bone is a common site for BCa metastasis.²⁷ In breast cancer patients, these bone metastases are typically osteolytic, or bone destructive.^2,3 Indeed, both digital radiograph and μCT results in our study showed osteolytic lesions in the tibiae of all tumor-bearing mice, which is in agreement with the bone structural changes observed in previous studies.^2–4

While numerous studies have been focused on the structural and architectural changes of bone in BCa metastasis, other aspects of bone quality, particularly the compositional or material properties, have largely been neglected, likely due to a lack of effective detection methods. Bone TMD calculated from μCT has been suggested as an indicator for bone mineralization. However, μCT cannot detect the organic matrix in bone. Furthermore, the TMD value from the tumor-bearing tibiae in our study did not differ from that of the control group, indicating that μCT alone might not be enough to capture tumor-associated alterations in biochemical compositions. Therefore, techniques that can provide complementary information in bone composition can potentially expand current capability for bone quality evaluation. In the present study, the tumor-bearing tibiae exhibited significant differences in Raman-derived compositional properties, suggesting the feasibility to use RS for the detection of changes in bone quality related to breast cancer metastases.

Raman-derived compositional properties, including mineralization, mineral crystallinity, and carbonation, are important predictors of bone mechanical functions.^12,14,28 Studies on animal and human bones revealed that these Raman parameters are indicative of bone quality alterations associated with age and disease at tissue level.^29–31 Collagen mineralization indicates the amount of bone minerals, mainly consisting of calcium phosphate in an apatite crystalline structure, deposited in collagen matrix.^32,33 Hydroxyapatite crystallinity is an indicator for the size and stoichiometric perfection of the deposited mineral crystal.³⁴ Both properties have been found to increase with tissue age and demonstrated reduced heterogeneity within aged tissue, along with deteriorating mechanical properties.^12,29,31 Since bone is a birefringent material, the polarization of incident light and molecular orientation in the tissue can also affect the intensities of Raman bands.^35–38 The major vibration of phosphate $\nu 1$ is along the direction of collagen fibrils, while the carbonyl groups (amide I) of collagen mainly stretch perpendicular to the fibrils. Therefore, both bands are very sensitive to sample orientation and the instrument polarization. The phase mismatch between phosphate $\nu 1$ and amide I could introduce uncertainty when interpreting the difference of phosphate $\nu 1/\text{amide}\mathrm{I}$ between groups. As a result, other Raman bands, such as phosphate $\nu 2$ and $\nu 4$, amide III, and proline, could be essential quality control parameters for compositional analysis.^35,37 The area ratio of phosphate $\nu 2$ to amide III has been reported as insensitive to sample orientation, indicating phosphate $\nu 2/\text{amide}\mathrm{III}$ is more relevant to the mineralization than phosphate $\nu 1/\text{amide}\mathrm{I}$.^36,37 Since the intensities of phosphate $\nu 1$ and proline change with phase matching under polarized light, their ratio (phosphate $\nu 1/\text{proline}$) has also been recommended for the calculation of mineralization.³⁵ In the current study, both phosphate $\nu 1/\text{amide}\mathrm{I}$ and phosphate $\nu 1/\text{proline}$ have been investigated and demonstrated consistent outcomes at the metaphysis region (positions at 0 and 500 μm). The significance of differences ($p$ value) between the two groups in phosphate $\nu 1/\text{proline}$ is not as dramatic as those in phosphate $\nu 1/\text{amide}\mathrm{I}$ at the same position, indicating that there might be variations in the orientation of collagen fibrils besides mineralization in the tumor-bearing bones.

The development of bone metastases significantly alters the bone remodeling process and, thus causes pathologic compositional changes. Generally, prostate cancer bone metastasis results in mixed but osteoblastic (more bone formation) dominant lesions, while breast cancer leads to osteolytic (more bone destruction) lesions.⁷ Decreased mineralization and crystallinity have been reported in animal models of prostate cancer metastasis,^25,39,40 which is in agreement with the formation of less mineralized woven bones at the lesion. In the breast cancer metastasis model presented here, both properties increased at the metaphysis region in the tumor-bearing tibiae where the most severe lesions exist, possibly due to the tissue’s compensatory response in cortex to the trabecular bone loss caused by osteolysis lesions. Therefore, mineralization and crystallinity in metastatic bones can vary markedly with different primary tumors. These two properties should be examined cautiously when applied as spectral markers to estimate fracture risk in metastasis.

Typically, bone minerals contain $\sim 5$ to 8 wt.% carbonate, predominantly in the form of type B carbonate substitution in which a $\mathrm{P}{\mathrm{O}}_4^{3-}$ is replaced by a $\mathrm{C}{\mathrm{O}}_3^{2-}$.^32,33 The concentration of carbonate varies with the tissue age, mineral crystallinity, and bone-remodeling rate,^12,13 and, thus provides important information regarding bone quality. The peak at $\sim 1070\mathrm{c}{\mathrm{m}}^{-1}$ in the Raman spectrum of bone is generally assigned to the carbonate $\nu 1$ mode. Although the neighboring bands from phosphate $\nu 3$ ($1076\mathrm{c}{\mathrm{m}}^{-1}$) and lipid ($1060\mathrm{c}{\mathrm{m}}^{-1}$) could potentially interfere with the precision in peak height and area calculation, the ratio of carbonate (at $1070\mathrm{c}{\mathrm{m}}^{-1}$) and phosphate $\nu 1$ ($960\mathrm{c}{\mathrm{m}}^{-1}$) have been calibrated with various weight% of carbonated apatite and validated as an efficient measure for carbonation quantification in bone.³² Therefore, the Raman spectral parameter carbonate/phosphate $\nu 1$ reflects the carbonation level and has been suggested to be predictive for osteoporotic fractures.¹⁰ With aging, the carbonation level increases possibly due to the accumulation of bicarbonate ions produced by osteoclasts in the tissue.²⁵ This is a particularly attractive hypothesis here, since metastasis-bearing bone has extremely elevated osteoclast activity. Previous RS studies on metastatic bones from the prostate cancer model showed significantly elevated carbonation level than controls. In the current study, breast cancer bone metastasis also caused significant and consistent enhancement in carbonate substitution across the entire measurement region (2 cm). In addition, such enhancement is positively correlated with the size of bone metastatic lesions. These results suggest that Raman-derived carbonate substitution parameter, i.e., carbonate/phosphate $\nu 1$, can potentially be used as a unique spectral marker to evaluate bone quality alteration with tumor progression and may provide important information for the prediction of pathological fractures.

In summary, the present paper investigated the potential of RS in assessing pathological fracture risk. An RS spectral marker has been discovered, reflective of bone degeneration and metastatic lesion development. Although this ex vivo study exploited conventional RS, it paved the road to noninvasive assessment of metastatic bone using SORS or RT.