Raman Spectroscopy Revealed Cell Passage-Dependent Distinct Biochemical Alterations in Radiation-Resistant Breast Cancer Cells.

Recapitulating radioresistant cell features in pertinent cell line models is essential for deciphering fundamental cellular mechanisms. The limited understanding of passage and cell cycle phases on radioresistant cells revived post-cryopreservation led us to investigate the effect of sub-culturing in parental and radioresistant MCF-7 cells. In this study, the radioresistant cells showed high-intensity nucleic acid and cytochrome bands, which are potentially a radiation-induced spectral marker. Raman spectroscopy data showed dynamic biochemical alterations in revived radioresistant G2/M synchronized cells at early cell passages 1 and 3 with stabilization at a latter cell passage, 5. The study highlights the importance of cell passaging and cell cycle phases in potentially changing the biochemical parameters during in vitro experiments after the revival of radioresistant cells post-cryopreservation.

SARS-CoV-2 severity classification from plasma sample using confocal Raman spectroscopy.

The world is on the brink of facing coronavirus's (COVID-19) fourth wave as the mutant forms of viruses are escaping neutralizing antibodies in spite of being vaccinated. As we have already witnessed that it has encumbered our health system, with hospitals swamped with infected patients observed during the viral outbreak. Rapid triage of patients infected with SARS-CoV-2 is required during hospitalization to prioritize and provide the best point of care. Traditional diagnostics techniques such as RT-PCR and clinical parameters such as symptoms, comorbidities, sex and age are not enough to identify the severity of patients. Here, we investigated the potential of confocal Raman microspectroscopy as a powerful tool to generate an expeditious blood-based test for the classification of COVID-19 disease severity using 65 patients plasma samples from cohorts infected with SARS-CoV-2. We designed an easy manageable blood test where we used a small volume (8 µl) of inactivated whole plasma samples from infected patients without any extra solvent usage in plasma processing. Raman spectra of plasma samples were acquired and multivariate exploratory analysis PC-LDA (principal component based linear discriminant analysis) was used to build a model, which segregated the severe from the non-severe COVID-19 group with a sensitivity of 83.87%, specificity of 70.60% and classification efficiency of 76.92%. Among the bands expressed in both the cohorts, the study led to the identification of Raman fingerprint regions corresponding to lipids (1661, 1742), proteins amide I and amide III (1555, 1247), proteins (Phe) (1006, 1034), and nucleic acids (760) to be differentially expressed in severe COVID-19 patient's samples. In summary, the current study exhibits the potential of confocal Raman to generate simple, rapid, and less expensive blood tests to triage the severity of patients infected with SARS-CoV-2.

Photothermal therapy (PTT) is an effective treatment measure against solid tumors which fails to respond conventional chemo/radiation therapies in clinic.

Photothermal therapy (PTT) has emerged as a fast, precisive, and cost-effective anticancer therapy protocol. Here we applied our previously designed nanomaterial (Tocophotoxil) for prospective PTT application to manage radiation- and chemo-resistant cancers in a preclinical model. A PTT dose vs. efficacy relationship was established for radioresistant breast (ZR-75-1 50Gy, 4T1 20Gy) and chemo-resistant ovarian (A2780LR) cancer cells and tumors in mice models. Compared to the sensitive cases, resistant cells treated with PTT for a shorter duration show higher endurance. However, preclinical tumor xenografts treated with optimal PTT dose show 2-3 fold higher longevity (P ≤ 0.05) of treated mice monitored by non-invasive imaging methods. Elevated ERK and AKT activation in radioresistant or only AKT activation in chemo-resistant cells were contributory to higher cell survival in sub-optimal PTT dose. A comprehensive single-cell Raman map of PTT treated ZR-75-1 cell reveals broad-spectrum macromolecular deformities, including protein damage features. Marked induction of pJNK, unfolded protein response (UPR) pathway, increased reactive oxygen species (ROS), and lipid peroxidation in PTT-treated cells disrupted the intracellular homeostasis. Analyzing cellular ultrastructure, the coexistence of swollen endoplasmic reticulum, and autophagic bodies after PTT indicate possible coordination between UPR and autophagy pathways. Therefore, this comprehensive study provides new evidence on the potential impact of PTT as a standalone therapy for ablation of failed conventional therapy-resistant cancers in vivo, the success of which is intricately linked to the PTT dose optimization. The study, for the first time, also illustrates that under PTT treatment, concerted action of novel molecular switches such as JNK activation and UPR activation plays a vital role in triggering autophagy and cancer cell death.

Improving Vibrational Spectroscopy Prospects in Frontline Clinical Diagnosis: Fourier Transform Infrared on Buccal Mucosa Cancer.

We report a novel method with higher than 90% accuracy in diagnosing buccal mucosa cancer. We use Fourier transform infrared spectroscopic analysis of human serum by suppressing confounding high molecular weight signals, thus relatively enhancing the biomarkers' signals. A narrower range molecular weight window of the serum was also investigated that yielded even higher accuracy on diagnosis. The most accurate results were produced in the serum's 10-30 kDa molecular weight region to distinguish between the two hardest to discern classes, i.e., premalignant and cancer patients. This work promises an avenue for earlier diagnosis with high accuracy as well as greater insight into the molecular origins of these signals by identifying a key molecular weight region to focus on.

Raman micro-spectroscopic map estimating in vivo precision of tumor ablative effect achieved by photothermal therapy procedure.

Photothermal-therapy (PTT) inculcates near-infrared laser guided local heating effect, where high degree of precision is expected, but not well proven to-date. An ex vivo tissue biochemical map with molecular/biochemical response showing the coverage area out of an optimized PTT procedure can reveal precision information. In this work, Raman-microscopic mapping and linear discriminant analysis of spectra of PTT treated and surrounding tissue areas ex vivo are done, revealing three distinct spectral clusters/zones, with minimal overlap between the core treated and adjacent untreated zone. The core treated zone showed intense nucleic-acid, cytochrome/mitochondria and protein damage, an adjacent zone showed lesser degree of damages and far zone showed minimal/no damage. Immunohistochemistry for γH2AX (DNA damage marker protein) in PTT exposed tissue also revealed similar results. Altogether, this study reveals the utility of Raman-microspectroscopy for fine-tuning safety parameters and precision that can be achieved from PTT mediated tumor ablation in preclinical/clinical application.

Exploring the effect of vitamin E in cancer chemotherapy-A biochemical and biophysical insight.

Many oncologists contend that patient undergoing chemotherapy must avoid antioxidant supplementation as it may interfere with the activity of the drug. In the present investigation, we have explored the influence of vitamin E, a well-known antioxidant on Camptothecin (CPT), a potent anti-cancer drug induced cell apoptosis and death of cervical cancer cells. HeLa cells were treated with different concentrations of CPT in presence and absence of 100 µm vitamin E. Treated cells were subjected to cytotoxicity studies, catalase assay, DNA fragmentation assay, clonogenic assay and flow cytometry based apoptosis detection. Also, Raman spectroscopy a label free technique which provides global information, in conjunction with multivariate tools like PCA, PCLDA and FDA, was investigated to explore vitamin E supplementation induced alterations. Our data based on biochemical and biophysical experimental analysis reveals that CPT causes DNA damage along with protein and lipid alteration culminating in cell death. Importantly, Raman spectroscopic analysis could uniquely differentiate the cluster of control and vitamin E control from CPT and CPT + Vit E treated cells. We conclusively prove that presence of vitamin E at 100 µM concentration shows promising antioxidant activity and displays no modulatory role on CPT induced effect, thereby causing no possible hindrance with the efficacy of the drug. Vitamin E may prove beneficial to alleviate chemotherapy associated side effects in patients during clinical settings which may open the doors further for subsequent exploration in in vivo preclinical studies.

Raman spectroscopic study of radioresistant oral cancer sublines established by fractionated ionizing radiation.

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer.