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Abstract Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

Partial prostatectomy has been described as an alternative to focal ablation therapy for the management of localized low- to intermediate-risk prostate cancer. This report aims to describe the long-term outcomes in a series of 28 men (2000–2022) who underwent robotic-assisted anterior partial prostatectomy (APP) for anteriorly located tumors entirely or partially within the anterior fibromuscular stroma. The median follow-up is 7 yr (interquartile range [IQR]: 4.2–8). The median prostate-specific antigen (PSA) before APP was 9.6 (6–11). Continence remained uninterrupted in 92% of patients. Erectile function without drug remained uninterrupted in 69%. The median nadir PSA after APP was 0.36 ng/ml (IQR: 0.25–0.60). Cancer recurrence at biopsies at the margins of the primary cancer resected area in case of a PSA elevation was observed in eight patients and led to salvage completion robotic radical prostatectomy at a median time of 3.25 yr (IQR: 2.4–6). Freedom from post-APP cancer recurrence at 7 yr was 62.7% (35.0–81.3%). Pre-APP tumor volume at magnetic resonance imaging (MRI) and volume of grade 4/5 were predictive of recurrence. Freedom from biochemical recurrence after completion radical prostatectomy at 7 yr was 94.7% (68.1–99.3%). All 28 patients are alive. No one had systemic treatment or metastases. These results confirm our initial report of robotic APP with good functional results and acceptable oncological results. The use of the inclusion criteria of pre-APP tumor volume at MRI

Background: The risk of prostate cancer metastatic is correlated with its volume and grade. These parameters are now best estimated preoperatively with magnetic resonance imaging (MRI) and MRI-guided biopsy. Objective: To estimate the risk of metastatic recurrence after radical prostatectomy (RP) in our model versus conventional clinical European Association of Urology (EAU) classification. The secondary objective is biochemical recurrence (BCR). Design, setting, and participants: A retrospective study was conducted of a cohort of 713 patients having undergone MRI-guided biopsies and RP between 2009 and 2018. The preoperative variables included prostate-specific antigen, cT stage, tumor volume (TV) based on the lesion’s largest diameter at MRI, percentage of Gleason pattern 4/5 (%GP4/5) at MRI-guided biopsy, and volume of GP4/5 (VolGP4/5) calculated as TV × %GP4/5. Outcome measurements and statistical analysis: The variables’ ability to predict recurrence was determined in univariable and multivariable Fine-and-Gray models, according to the Akaike information criterion (AIC) and Harrell’s C-index. Results and limitations: Overall, 176 (25%), 430 (60%), and 107 (15%) patients had low, intermediate, and high-risk disease, respectively, according to the EAU classification. During a median follow-up period of 57 mo, metastatic recurrence was observed in 48 patients with a 5-yr probability of 5.6% (95% confidence interval [CI] 3.9–7.7). VolGP4/5 (categories: 3.2 ml) was the parameter with the lowest AIC and the highest C-index for metastatic recurrence of 0.82 (95% CI 0.76–0.88), and for BCR it was 0.73 (95% CI 0.68–0.78). In a multivariable model that included %GP4/5 and TV, C-index values were 0.86 (95% CI 0.79–0.91) for metastatic recurrence and 0.77 (0.72–0.82) for BCR. The same results for EAU classification were 0.74 (0.67–0.80) and 0.67 (0.63–0.72), respectively. Limitations are related to short follow-up and expertise of radiologists and urologists. Conclusions: We developed a preoperative risk tool integrating the VolGP4/5 based on MRI and MRI-guided biopsies to predict metastatic recurrence after RP. Our model showed higher accuracy than conventional clinical risk models. These findings might enable physicians to provide more personalized patient care. Patient summary: Aggressiveness of prostate cancer evaluated before treatment by incorporating magnetic resonance imaging (MRI) and MRI-guided biopsy results gives a better estimate of the risk of metastatic recurrence than previous parameters not based on MRI.

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Abstract Glycogen storage disease type Ia (GSD Ia) is a rare metabolic disease due to glucose‐6‐phosphatase deficiency. Chronic kidney disease is a frequent complication that may manifest itself by glomerular lesions and tubular dysfunction from the second decade of life. We report two young GSDIa patients with malignant renal tumor. The first patient was a 25‐year‐old man. He had chronic metabolic imbalance without kidney involvement. The tumor, a type 2 papillary renal carcinoma, was accidentally discovered during follow‐up. The second patient was a 27‐year‐old woman with chronic metabolic imbalance and chronic kidney involvement. The tumor, a grade 2 papillary carcinoma, was accidentally discovered during follow‐up. These two observations are, to date, the first to be reported. We suggest that annual monitoring of kidney imaging in GSDI patients should be systematic to detect renal cancer, from the second decade of life.

Objective: Recently, we observed that the specialized proresolving mediator (SPM) entity resolvin D1 activates lipoxin A4/formyl peptide receptor 2 (ALX/FPR2), which facilitates cardiac healing and persistent inflammation is a hallmark of impaired cardiac repair in aging. Splenic leukocyte-directed SPMs are essential for the safe clearance of inflammation and cardiac repair after injury; however, the target of SPMs remains undefined in cardiac healing and repair. Methods: To define the mechanistic basis of ALX/FPR2 as a resolvin D1 target, ALX/FPR2-null mice were examined extensively. The systolic-diastolic heart function was assessed using echocardiography, leukocytes were phenotyped using flow cytometry, and SPMs were quantitated using mass spectrometry. The presence of cardiorenal syndrome was validated using histology and renal markers. Results: Lack of ALX/FPR2 led to the development of spontaneous obesity and diastolic dysfunction with reduced survival with aging. After cardiac injury, ALX/FPR2−/− mice showed lower expression of lipoxygenases (−5, −12, −15) and a reduction in SPMs in the infarcted left ventricle and spleen, indicating nonresolving inflammation. Reduced SPM levels in the infarcted heart and spleen are suggestive of impaired cross-talk between the injured heart and splenic leukocytes, which are required for the resolution of inflammation. In contrast, cyclooxygenases (−1 and −2) were over amplified in the infarcted heart. Together, these results suggest interorgan signaling in which the spleen acts as both an SPM biosynthesizer and supplier in acute heart failure. ALX/FPR2 dysfunction magnified obesogenic cardiomyopathy and renal inflammation (↑NGAL, ↑TNF-α, ↑CCL2, ↑IL-1β) with elevated plasma creatinine levels in aging mice. At the cellular level, ALX/FPR2−/− mice showed impairment of macrophage phagocytic function ex-vivo with expansion of neutrophils after myocardial infarction. Conclusions: Lack of ALX/FPR2 induced obesity, reduced the life span, amplified leukocyte dysfunction, and facilitated profound interorgan nonresolving inflammation. Our study shows the integrative and indispensable role of ALX/FPR2 in lipid metabolism, cardiac inflammation–resolution processes, obesogenic aging, and renal homeostasis. Keywords: Inflammation, Leukocytes, Myocardial infarction, Nonresolving inflammation, Obesogenic aging, Resolution of inflammation

Summary: Esthesioneuroblastoma (ENB) is a rare cancer of the olfactory mucosa, with no established molecular stratification to date. We report similarities of ENB with tumors arising in the neural crest and perform integrative analysis of these tumors. We propose a molecular-based subtype classification of ENB as basal or neural, both of which have distinct pathological, transcriptomic, proteomic, and immune features. Among the basal subtype, we uncovered an IDH2 R172 mutant-enriched subgroup (∼35%) harboring a CpG island methylator phenotype reminiscent of IDH2 mutant gliomas. Compared with the basal ENB methylome, the neural ENB methylome shows genome-wide reprogramming with loss of DNA methylation at the enhancers of axonal guidance genes. Our study reveals insights into the molecular pathogenesis of ENB and provides classification information of potential therapeutic relevance. : Classe et al. report an integrative multi-omics analysis of esthesioneuroblastomas (ENBs) and identify two subgroups of ENBs: neural-like and basal-like. These subgroups are linked to cell ontogeny and are associated with distinct clinicopathological features and patient outcomes. Notably, one-third of basal ENBs harbor an IDH2 R172 mutation with a CpG island methylator phenotype. Keywords: esthesioneuroblastomas, IDH2, neural, basal, DNA methylation, sequencing, genetics, epigenetics, survival, ki67

Renal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome.

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.