CRISPR editing to mimic porphyria combined with light: A new preclinical approach for prostate cancer.

Thanks to its very high genome-editing efficiency, CRISPR-Cas9 technology could be a promising anticancer weapon. Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy still has not been applied in clinical practice to directly target cancer cells. Targeting a canonical metabolic pathway essential to good functioning of cells without potential escape would represent an attractive strategy. We propose to mimic a genetic metabolic disorder in cancer cells to weaken cancer cells, independent of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to inactivate UROS, leading to porphyrin accumulation in a prostate cancer model. Prostate cancer is the leading cancer in men and has a high mortality rate despite therapeutic progress, with a primary tumor accessible to light. By combining light with gene therapy, we obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, we achieved the preclinical proof-of-principle of performing cancer CRISPR gene therapy.

Bioactive food components for colorectal cancer prevention and treatment: A good match.

Colorectal cancer (CRC) is the third most frequent cancer worldwide, accounts for about 10% of the total cancer cases, and ranks as the second cause of death by cancer. CRC is more prevalent in developed countries in close causal relation with occidental diets. Due to anatomy, the diet has a strong impact on CRC. High contents in meat are acknowledged risk factors whereas a diet rich in fruits and vegetables is an established CRC protective factor. Fruits and vegetables contain numerous Bioactive Food Components (BFCs), physiologically active food compounds, beneficial on health. Preventive and therapeutic benefits of BFCs in cancer have increasingly been reported over the past 20 years. BFCs show both chemopreventive and anti-tumor properties in CRC but more interestingly, abundant research describes BFCs as enhancers of conventional cancer treatments. Despite these promising results, their clinical transferability is slowed down by bioavailability interrogations and their poorly understood hormetic effect. In this review, we would like to reposition BFCs as well-fitted for applications in CRC. We provide a synthetic overview of trustworthy BFC applications in CRC, with a special highlight on combinatory approaches and conventional cancer treatment potentiation strategies.

Impact of Albumin Assays in the Diagnosis of Malnutrition in Hemodialysis Patients: A Cohort Study.

OBJECTIVE: In hemodialysis (HD) patients, malnutrition should be diagnosed by several assessment tools including a plasma albumin concentration of less than 3.8 g/dL or 3.5 g/dL using bromocresol green or immunonephelometry (IN), respectively. However, albumin measurement is not yet standardized and two alternative methods are also commonly used in laboratories: bromocresol purple (BCP) and immunoturbidimetry (IT). This study aimed to revisit the hypoalbuminemia thresholds for BCP and IT, in HD patients. METHODS: Plasma albumin was measured by the four analytical methods during the monthly HD nutritional assessment of 103 prospectively included patients. RESULTS: Significant differences in albumin levels were observed in HD patients depending on the method used. Using BCP or IT with the cut-off at 3.5 g/dL (determined for the general population) we obtained 33% and 9.7% of false hypoalbuminemia in comparison to IN (mean bias of -0.4 g/dL and -0.065 g/dL, respectively). The best hypoalbuminemia threshold for BCP was 3.05 g/dL and 3.4 g/dL for IT. Twenty percent of HD patients were classified as malnourished when albumin was determined by IN. Similar rates were obtained using the new hypoalbuminemia cut-offs for BCP (18.5%) and IT (19.5%). CONCLUSION: To avoid nutritional misclassification of HD patients, we should adjust hypoalbuminemia thresholds when BCP or IT methods are used in laboratories.

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid.

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy.

ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.

CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells.

CRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations.

Pigments test strips: A rapid companion test to exclude sub-arachnoid haemorrhage.

OBJECTIVES: Subarachnoid haemorrhage (SAH) is characterised by 25% of mortality or induces long-term care. It needs immediate diagnosis with computed tomography (CT) scan. For the inconclusive CT scans, the detection of haem pigments can be performed in the cerebrospinal fluid (CSF). The reference method is spectrophotometry but it requires a large volume of CSF, and specific equipment. Sometimes, urine test strips are used as an alternative method for haem pigments detection. However, this method needs validation in SAH context. The aim of the study was to compare the performance of Multistix® urine test strips for haem pigments detection to the reference spectrophotometry and the final clinical SAH diagnosis. METHODS: We collected 136 CSFs sampled for suspected SAH. We detected haem pigments with urine test strips and spectrophotometry and compared performances for 100 samples. RESULTS: Urine tests strips displayed a high sensitivity (0.97) as compared to the reference spectrophotometry for haem pigments detection. Interestingly, absence of haem pigments fully correlated with absence of SAH. CONCLUSIONS: Negative Multistix® urine test strips could help to exclude SAH diagnosis in combination with clinical data when a spectrophotometer is not available, or as a bedside diagnosis test.

Circulating Tumor Cell Clusters: United We Stand Divided We Fall.

The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that "united" CTCs, organized in clusters, resist better and carry stronger metastatic capacities than "divided" single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer.

Next-Generation Cancer Biomarkers: Extracellular Vesicle DNA as a Circulating Surrogate of Tumor DNA.

Extracellular vesicles (EVs) are produced by healthy tissues and tumor cells and are released in various bodily fluids, including blood. They are limited by bilayer phospholipidic membranes, and they carry a rich content in biomolecules. Their release cleanses the cells of their waste or serves as functional local and distant cell-cell communication and molecular exchange particles. This rich and heterogeneous content has been given intense attention in cancer physiopathology because EVs support cancer control and progression. Because of their specific active cargo, they are being evaluated as carriers of liquid biopsy biomarkers. Compared to soluble circulating biomarkers, their complexity might provide rich information on tumor and metastases status. Thanks to the acquired genomic changes commonly observed in oncogenic processes, double-stranded DNA (dsDNA) in EVs might be the latest most promising biomarker of tumor presence and complexity. This review will focus on the recent knowledge on the DNA inclusion in vesicles, the technical aspects of EV-DNA detection and quantification, and the use of EV-DNA as a clinical biomarker.

LC-MS/MS measurements of urinary guanidinoacetic acid and creatine: Method optimization by deleting derivatization step.

BACKGROUND: Cerebral Creatine deficiency syndromes (CCDS) include three hereditary diseases affecting the metabolism of creatine (Cr): arginine glycine amidinotransferase deficiency, guanidinoacetate methyltransferase deficiency and disorders of creatine transporter. These pathologies cause a brain creatine deficiency responsible of non-specific neurological impairments with mental retardation. LC-MS/MS measurements of guanidinoacetic acid (GAA) and creatine in urine and plasma are an important screening test to identify the deficit. Analysis of this polar and basic molecules not hold on standard column requires a derivatization step to butyl-esters. To overcome this long and fastidious derivatization, an ion pairing (IP) method was chosen in this study. METHOD: IP method was validated using Comité francais d'accréditation (COFRAC) recommendations. Then, urine GAA and creatine of 15 patients with a CDS deficiency suspected were tested y LC-MS/MS using IP technique, and performances were assessed with reference laboratory method (butylation method). Moreover, references values were suggested y the study of 100 urines samples of healthy patients. RESULTS: The method developed provided a good accuracy and precision with intra and inter-day coefficients of variation (CVs) <15%. The curve was linear for the biological and pathological concentrations. The comparison with the reference method did not reveal any significant difference for analytical performances but showed a simplification of the preparation of samples. CONCLUSION: The use of IP technique that we have developed demonstrated a good correlation with the butylation method. Moreover, this new method not only allows a simplification of the technique, but also decreases in run time.

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.