Abstract Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm −1 , which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.
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Prostate Cancer Phenotype Influences Bone Mineralization at Metastasis: A Study Using an In Vitro Prostate Cancer Metastasis Testbed
In this study, two types of prostate cancer cell lines, highly metastatic PC‐3 and low metastatic MDA PCa 2b (PCa) were cultured on bone mimetic scaffolds to recapitulate metastasis to bone. A unique in vitro 3D tumor model that uses a sequential culture (SC) of human mesenchymal stem cells followed by seeding with cancer cells after bone formation was initiated to study the phenotype‐specific interaction between prostate cancer cells and bone microenvironment. The PCa cells were observed to be less prolific and less metastatic, and to form multicellular tumoroids in the bone microenvironment, whereas PC‐3 cells were more prolific and were highly metastatic, and did not form multicellular tumoroids in the bone microenvironment. The metastatic process exhibited by these two prostate cancer cell lines showed a significant and different effect on bone mineralization and extracellular matrix formation. Excessive bone formation in the presence of PC‐3 and significant osteolysis in the presence of PCa were observed, which was also indicated by osteocalcin and MMP‐9 expression as measured by ELISA and qRT‐PCR. The field emission scanning electron microscopy images revealed that the structure of mineralized collagen in the presence of PC‐3 is different than the one observed in healthy bone. All experimental results indicated that both osteolytic and osteoblastic bone lesions can be recapitulated in our tumor testbed model and that different cancer phenotypes have a very different influence on bone at metastasis. The 3D in vitro model presented in this study provides an improved, reproducible, and controllable system that is a useful tool to elucidate osteotropism of prostate cancer cells. © 2019 The Authors.
- NSF-PAR ID:
- 10458320
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- JBMR Plus
- Volume:
- 4
- Issue:
- 2
- ISSN:
- 2473-4039
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Current preclinical models of metastatic prostate cancer (PCa) require sophisticated technologies and/or genetically engineered cells for the noninvasive monitoring of tumors in remote sites, such as bone. Recent developments in circulating tumor DNA (ctDNA) analysis provide an alternative method for noninvasive tumor monitoring at a low cost. Here, we sought to evaluate human Alu and LINE‐1 ctDNA for the longitudinal measurement of subcutaneous and intratibial human PCa xenograft growth and response to ionizing radiation (IR) through comparison with standard slide caliper and bioluminescence measurements.
Material and Methods Intratibial and subcutaneous xenografts were established in male athymic nude mice using LNCaP cells that stably express firefly luciferase. A subset of tumors was treated with a single dose of IR (CT‐guided focal IR, 6 Gy). Tumor measurements were simultaneously taken by slide caliper (subcutaneous only), in vivo bioluminescence imaging, and quantitative real‐time PCR (qPCR) of human‐specific Alu and LINE‐1 ctDNA for several weeks.
Results Levels of ctDNA and bioluminescence increased concordantly with subcutaneous and intratibial tumor growth. A statistically significant correlation (Spearman) was observed between ctDNA and subcutaneous tumor volume (LINE‐1,
r = .94 and Alu,r = .95,p < .0001), ctDNA and bioluminescence (LINE‐1,r = .66 and Alu,r = .60,p < .002), and bioluminescence and tumor volume (r = .66,p = .0003). Bioluminescence and ctDNA were also significantly correlated in intratibial tumors (LINE‐1,r = .82 and Alu,r = .81,p < .0001). Following external beam IR, the tumor responses varied briefly by method of measurement, but followed a similar trend. Statistically significant correlations were maintained between ctDNA and slide caliper measurement in irradiated subcutaneous tumors (LINE‐1,r = .64 and Alu,r = .44,p < .02), and ctDNA and bioluminescence in intratibial tumors (LINE‐1,r = .55,p = .018).Conclusions Real‐time qPCR of circulating human Alu and LINE‐1 DNA provides an accurate measurement of subcutaneous and intratibial xenograft burden that is comparable with conventional bioluminescence imaging and slide caliper measurement. Transient differences in measurements were observed following tumor‐targeted IR, but overall all measurements mirrored tumor growth and response.
