Distinct spectral signatures unfold ECM stiffness-triggered biochemical changes in breast cancer cells.

Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening.

Thearubigins/Polymeric black tea polyphenols (PBPs) do not prevent benzo[a]pyrene (B[a]P) induced lung tumors in A/J mice.

OBJECTIVES: Previously we have demonstrated the chemopreventive effect of Thearubigins/Polymeric Black-tea Polyphenols (PBPs) upon pre-treatment to a combination of carcinogens, that is, Benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which are present in Tobacco smoke (TS). However, the chemopreventive effect in response to B[a]P as a single carcinogen remains unexplored. B[a]P is a universal carcinogen and an important constituent of particulate matter 2.5 (PM2.5) found in the environment and in TS. METHODS: We investigated the pre-treatment of Thearubigins/PBPs as a chemopreventive agent at three doses (1.5, 5, 10%) against B[a]P-induced lung carcinogenesis at early & late time points. We also investigated the effect of PBPs at early time points to understand molecular changes by employing western blotting in xenobiotic metabolism pathways, DNA damage, inflammation, apoptosis, and proliferation as they are modulated in response to carcinogens. We used 6-8 weeks male A/J mice for tumorigenicty and western blotting to probe the molecular biomarkers. RESULTS: We report no decrease in tumor incidence and multiplicity upon pre and concurrent treatment of Thearubigins/PBPs. Further, we also report no changes in molecular markers at early time points, in agreement with former observations. CONCLUSION: Our results suggest that chemopreventive agents need to be tested with different combinations of carcinogens and regimens to fully understand the complex interplay between carcinogenesis and the efficacy of chemopreventive agents. Studies like these will provide meaningful data before initiating large-scale clinical trials.