Measure Of Cellular Metabolism Research Articles

Determination of CAR T cell metabolism in an optimized protocol.

Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

Assessment of bacterial viability: a comprehensive review on recent advances and challenges.

Assessing bacterial contamination in environmental samples is critical in determining threats to public health. The classical methods are time-consuming and only recognize species that grow easily on culture media. Viable but non-culturable (VBNC) bacteria are a possible threat that may resuscitate and cause infections. Recent dye-based screening techniques employ nucleic acid dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), along with many fluorescent dyes, which are an effective alternative for viability assessment. The measurement of cellular metabolism, heat flow and ATP production has also been widely applied in detection approaches. In addition, RNA-based detection methods, including nucleic acid sequence-based amplification (NASBA), have been applied for bacterial pathogen determination. Stable isotope probing using 13C, 15 N and 18O, which are mobilized by microbes, can also be used for effective viability assessment. Future detection tools, such as microarrays, BioNEMS and BioMEMS, which are currently being validated, might offer better microbial viability detection.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence.

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

Monitoring the metabolic response to nanoencapsulated silibinin treatment in DMBA-induced oral carcinogenesis using endogenous fluorescence

Autofluorescence spectroscopy is a very sensitive tool for detecting early metabolic response after cancer treatment. The present work aims to investigate the chemopreventive effect of the prepared silibinin-loaded nanoparticles (SILNPs) relative to the efficacy of free silibinin (SIL) for monitoring the changes in the endogenous fluorophore emission and to quantify the metabolic changes in the redox state during 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch (HBP) carcinogenesis using autofluorescence spectroscopy. Significant differences in the autofluorescence spectral signatures between the control and the experimental animals have been noticed under the excitation wavelength at 320 nm with emission ranging from 350–550 nm. The tumor tissues are characterized by a decrease in the emission of collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) compared to the control tissues. Further, the optical oxidation–reduction (redox) ratio is a measure of cellular metabolism and can be determined by the relative change in the fluorescence emission intensity of NADH and FAD. The optical redox ratio will provide sufficient information on metabolic changes associated with tumor transformation. The results revealed that a significant decrease in the optical redox ratio is observed in DMBA-induced tumor tissues, which indicates increased metabolic activity compared to the control tissues. Moreover, an oral administration of SIL and its nanoparticulates restored the status of endogenous fluorophore emission and led to a higher redox ratio in the buccal mucosa of DMBA-painted animals. On a comparative basis, treatment with nanoparticulate silibinin was found to be more effective than free silibinin for reducing the formation of squamous cell carcinoma and improving the status of endogenous fluorophore emission to a near normal range in DMBA-induced hamster buccal pouch carcinogenesis. Furthermore, the diagnostic algorithms based on principal component analysis followed by linear discriminant analysis (PC–LDA) achieved an overall diagnostic sensitivity of 96.46% and specificity of 93.64% for separating the control from the experimental groups. The results of this study further suggest that the fluorescence spectroscopic technique in conjunction with PC–LDA has a potential for rapid and sensitive detection of specific metabolic alteration and changes in the endogenous fluorophores in response to anti-cancer drug treatments.

Optical Redox Ratio Differentiates Breast Cancer Cell Lines Based on Estrogen Receptor Status

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearson's correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.