Real-time glioblastoma tumor microenvironment assessment by SpiderMass for improved patient management.

Glioblastoma is a highly heterogeneous and infiltrative form of brain cancer associated with a poor outcome and limited therapeutic effectiveness. The extent of the surgery is related to survival. Reaching an accurate diagnosis and prognosis assessment by the time of the initial surgery is therefore paramount in the management of glioblastoma. To this end, we are studying the performance of SpiderMass, an ambient ionization mass spectrometry technology that can be used in vivo without invasiveness, coupled to our recently established artificial intelligence pipeline. We demonstrate that we can both stratify isocitrate dehydrogenase (IDH)-wild-type glioblastoma patients into molecular sub-groups and achieve an accurate diagnosis with over 90% accuracy after cross-validation. Interestingly, the developed method offers the same accuracy for prognosis. In addition, we are testing the potential of an immunoscoring strategy based on SpiderMass fingerprints, showing the association between prognosis and immune cell infiltration, to predict patient outcome.

Large Vestibular Aqueduct-Associated Symptoms: Endolymphatic Duct Blockage as a Surgical Treatment.

OBJECTIVE: This study aimed to evaluate the effectiveness of endolymphatic duct blockage (EDB) on dizziness control in patients with a large vestibular aqueduct (LVA) and to evaluate its effect on hearing. STUDY DESIGN: This is a prospective nonrandomized study. SETTING: Five adults and one child with dizziness and five children with progressive hearing loss were referred to our tertiary centers. METHODS: The dizziness handicap inventory (DHI) and DHI-PC (dizziness handicap inventory-patient caregiver) questionnaires were used before and after surgery. All patients underwent a preoperative temporal bone HRCT scan and pure tone audiometry one day before surgery, then four and twelve months after surgery and at the last follow-up. The mean follow-up time was 5.6 years. Student's t-test was used to compare DHI/-PC results. RESULTS: The DHI scores were 44, 24, 84, 59 and 56 before surgery, respectively, for Patients 1 to 5. The DHI scores at four months was significantly different, i.e., 4, 6, 0, 7 and 18 (p = 0.001). No differences were found between 4 and 12 months. Patient 6 (child) had Trisomy 21; their DHI-PC score dropped from 38 (preoperative score) to 8 (postoperative score), showing no activity limitations; clinical evaluation showed the complete resolution of symptoms. We found no significant differences between hearing loss before the surgery and at 1 and 12 months post operation for four adult patients. Our fifth adult patient's hearing changed from severe to profound SNHL. For 5 out of 6 pediatric patients, preoperative PTA and mean ABG were 63 dB and 20 dB, respectively; postoperatively, they improved to 42 dB and 16 dB, respectively. The hearing loss level for the sixth pediatric patient dropped from moderate (PTA = 42 dB) to severe (PTA = 85 dB) due to an opening of the endolymphatic sac and a sudden leak of the endolymph. CONCLUSIONS: EDB, using two titanium clips, seems to be helpful for controlling vestibular symptoms and for stabilizing hearing or even to improve hearing in 82% of cases. Nevertheless, there is a risk of hearing worsening.

The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

OpenProt 2.0 builds a path to the functional characterization of alternative proteins.

The OpenProt proteogenomic resource (https://www.openprot.org/) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.

Comparison of Tympanostomy Tubes Under Local Anesthesia Versus General Anesthesia for Children.

OBJECTIVE: Tympanostomy tube insertion (TTI) is typically accomplished under general anesthesia (GA) in the operating room. We aimed to compare pain between GA and local anesthesia (LA) in surgically naïve children undergoing TTI. Secondary objectives examined patient's quality of life (QoL) and parent's satisfaction. STUDY DESIGN: Prospective single-center study. SETTING: Tertiary pediatric academic center. METHODS: Consecutive children who underwent TTI under GA were compared to patients under LA. Pain standardized observational pain scales (Face, Legs, Activity, Cry, Consolability Scale [FLACC], Children's hospital of Eastern Ontario Pain Scale [CHEOPS]) were completed pre-procedure, during the first tympanostomy and second tympanostomy, and post-procedure, as well as 1 week postoperatively. General health-related QoL (PedsQL) and QoL specific to otitis media (OM-6) were measured before insertion and 1 month postoperatively. Parental satisfaction was also evaluated using a qualitative scale. RESULTS: LA group had statistically significant higher pain levels at the beginning (7.3 vs. 0), during the first tympanostomy (7.8 vs. 0), during the second tympanostomy (7.7 vs. 0), and at end of the procedure (6.9 vs. 0) with the FLACC scale (all p < 0.01). Results were similar with the CHEOPS scale. No pain was noted 1 week after surgery in either group. Both groups had similar improvement in their QoL (p > 0.05). Minor complication occurred at a similar rate (p > 0.05). Parents were equally satisfied with their choice of anesthesia in both groups when initially questioned after the procedure (p > 0.05). CONCLUSIONS: Children experienced significantly less pain under GA than LA. If LA is to be used, pain and distress-reducing strategies are critical. Shared decision-making with families is essential. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:2422-2429, 2024.

Fallopian tube lesions as potential precursors of early ovarian cancer: a comprehensive proteomic analysis.

Ovarian cancer is the leading cause of death from gynecologic cancer worldwide. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype of ovarian cancer. While the origin of ovarian tumors is still debated, it has been suggested that HGSC originates from cells in the fallopian tube epithelium (FTE), specifically the epithelial cells in the region of the tubal-peritoneal junction. Three main lesions, p53 signatures, STILs, and STICs, have been defined based on the immunohistochemistry (IHC) pattern of p53 and Ki67 markers and the architectural alterations of the cells, using the Sectioning and Extensively Examining the Fimbriated End Protocol. In this study, we performed an in-depth proteomic analysis of these pre-neoplastic epithelial lesions guided by mass spectrometry imaging and IHC. We evaluated specific markers related to each preneoplastic lesion. The study identified specific lesion markers, such as CAVIN1, Emilin2, and FBLN5. We also used SpiderMass technology to perform a lipidomic analysis and identified the specific presence of specific lipids signature including dietary Fatty acids precursors in lesions. Our study provides new insights into the molecular mechanisms underlying the progression of ovarian cancer and confirms the fimbria origin of HGSC.

Comparing MS imaging of lipids by WALDI and MALDI: two technologies for evaluating a common ground truth in MS imaging.

In this study, we conducted a direct comparison of water-assisted laser desorption ionization (WALDI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging, with MALDI serving as the benchmark for label-free molecular tissue analysis in biomedical research. Specifically, we investigated the lipidomic profiles of several biological samples and calculated the similarity of detected peaks and Pearson's correlation of spectral profile intensities between the two techniques. We show that, overall, MALDI MS and WALDI MS present very close lipidomic analyses and that the highest similarity is obtained for the norharmane MALDI matrix. Indeed, for norharmane in negative ion mode, the lipidomic spectra revealed 100% similarity of detected peaks and over 0.90 intensity correlation between both technologies for five samples. The MALDI-MSI positive ion lipid spectra displayed more than 83% similarity of detected peaks compared to those of WALDI-MSI. However, we observed a lower percentage (77%) of detected peaks when comparing WALDI-MSI with MALDI-MSI due to the rich WALDI-MSI lipid spectra. Despite this difference, the global lipidomic spectra showed high consistency between the two technologies, indicating that they are governed by similar processes. Thanks to this similarity, we can increase datasets by including data from both modalities to either co-train classification models or obtain cross-interrogation.

Early Diagnosis: End-to-End CNN-LSTM Models for Mass Spectrometry Data Classification.

Liquid chromatography-mass spectrometry (LC-MS) is a powerful method for cell profiling. The use of LC-MS technology is a tool of choice for cancer research since it provides molecular fingerprints of analyzed tissues. However, the ubiquitous presence of noise, the peaks shift between acquisitions, and the huge amount of information owing to the high dimensionality of the data make rapid and accurate cancer diagnosis a challenging task. Deep learning (DL) models are not only effective classifiers but are also well suited to jointly learn feature representation and classification tasks. This is particularly relevant when applied to raw LC-MS data and hence avoid the need for costly preprocessing and complicated feature selection. In this study, we propose a new end-to-end DL methodology that addresses all of the above challenges at once, while preserving the high potential of LC-MS data. Our DL model is designed to early discriminate between tumoral and normal tissues. It is a combination of a convolutional neural network (CNN) and a long short-term memory (LSTM) Network. The CNN network allows for significantly reducing the high dimensionality of the data while learning spatially relevant features. The LSTM network enables our model to capture temporal patterns. We show that our model outperforms not only benchmark models but also state-of-the-art models developed on the same data. Our framework is a promising strategy for improving early cancer detection during a diagnostic process.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome.

The dogma "One gene, one protein" is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as "NOTCH1, EPHA3, IPO13" as well as membrane receptor protein including "CHRNA9; TGFBR, EPHB6, and TRAM". However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer.

Elevated levels of alkaline phosphatase (ALP) in the tumor microenvironment (TME) are a hallmark of cancer progression and thus inhibition of ALP could serve as an effective approach against cancer. Herein, we developed a novel prodrug approach to tackle cancer that bears self-inhibiting alkaline phosphatase-responsiveness properties that can enhance at the same time the solubility of the parent compound. To probe this novel concept, we selected apigenin as the cytotoxic agent since we first unveiled, that it directly interacts and inhibits ALP activity. Consequently, we rationally designed and synthesized, using a self-immolative linker, an ALP responsive apigenin-based phosphate prodrug, phospho-apigenin. Phospho-apigenin markedly increased the stability of the parent compound apigenin. Furthermore, the prodrug exhibited enhanced antiproliferative effect in malignant cells with elevated ALP levels, compared to apigenin. This recorded potency of the developed prodrug was further confirmed in vivo where phospho-apigenin significantly suppressed by 52.8% the growth of PC-3 xenograft tumors.Communicated by Ramaswamy H. Sarma.

Ambient Mass Spectrometry Imaging by Water-Assisted Laser Desorption/Ionization for Ex Vivo and in Vivo Applications.

Water-assisted laser desorption/ionization mass spectrometry (WALDI-MS), also known as SpiderMass, is an emerging ambient ionization technique for in vivo and real-time analysis. It employs a remote infrared (IR) laser tuned to excite the most intense vibrational band (O-H) of water. The water molecules act as an endogenous matrix leading to the desorption/ionization of a variety of biomolecules from tissues, particularly metabolites and lipids. WALDI-MS was recently advanced into an imaging modality for ex vivo 2D sections and 3D in vivo real-time imaging. Here, we describe the methodological aspects for performing 2D and 3D imaging experiments with WALDI-MSI and the parameters for optimizing the image acquisition.

Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts. For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1.

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Employing non-targeted interactomics approach and subcellular fractionation to increase our understanding of the ghost proteome.

Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential new immunopeptide, and the interactions between HIST1H4F and several AltProts which can play a role in mRNA transcription. Thanks to the study of the interactome and the localization of AltProts, we can reveal more of the importance of the ghost proteome.

Prophylactic Radical Fimbriectomy with Delayed Oophorectomy in Women with a High Risk of Developing an Ovarian Carcinoma: Results of a Prospective National Pilot Study.

Risk-reducing salpingo-oophorectomy is the gold standard for the prophylaxis of ovarian cancer in high-risk women. Due to significant adverse effects, 20-30% of women delay or refuse early oophorectomy. This prospective pilot study (NCT01608074) aimed to assess the efficacy of radical fimbriectomy followed by a delayed oophorectomy in preventing ovarian and pelvic invasive cancer (the primary endpoint) and to evaluate the safety of both procedures. The key eligibility criteria were pre-menopausal women ≥35 years with a high risk of ovarian cancer who refused a risk-reducing salpingo-oophorectomy. All the surgical specimens were subjected to the SEE-FIM protocol. From January 2012 to October 2014, 121 patients underwent RF, with 51 in an ambulatory setting. Occult neoplasia was found in two cases, with one tubal high-grade serous ovarian carcinoma. Two patients experienced grade 1 intraoperative complications. No early or delayed grade ≥3 post-operative complications occurred. After 7.3 years of median follow-up, no cases of pelvic invasive cancer have been noted. Three of the fifty-two patients developed de novo breast cancer. One BRCA1-mutated woman delivered twins safely. Twenty-five patients underwent menopause, including fifteen who had received chemotherapy for breast cancer, and twenty-three underwent menopause before the delayed oophorectomy, while two did not undergo a delayed oophorectomy at all. Overall, 46 women underwent a delayed oophorectomy. No abnormalities were found in any delayed oophorectomy specimens. Radical fimbriectomy followed by delayed oophorectomy appears to be a safe and well-tolerated risk-reducing approach, which avoids early menopause for patients with a high risk of breast and ovarian cancer.

Establishment and characterization of canine mammary tumoroids for translational research.

BACKGROUND: Cancer heterogeneity is a main obstacle for the development of effective therapies, as its replication in in vitro preclinical models is challenging. Around 96% of developed drugs are estimated to fail from discovery to the clinical trial phase probably because of the unsuitability and unreliability of current preclinical models (Front Pharmacol 9:6, 2018; Nat Rev Cancer 8: 147-56, 2008) in replicating the overall biology of tumors, for instance the tumor microenvironment. Breast cancer is the most frequent cancer among women causing the greatest number of cancer-related deaths. Breast cancer can typically be modeled in vitro through the use of tumoroids; however, current approaches using mouse tumoroids fail to reproduce crucial aspect of human breast cancer, while access to human cells is limited and the focus of ethical concerns. New models of breast cancer, such as companion dogs, have emerged given the resemblance of developed spontaneous mammary tumors to human breast cancer in many clinical and molecular aspects; however, they have so far failed to replicate the tumor microenvironment. The present work aimed at developing a robust canine mammary tumor model in the form of tumoroids which recapitulate the tumor diversity and heterogeneity. RESULTS: We conducted a complete characterization of canine mammary tumoroids through histologic, molecular, and proteomic analysis, demonstrating their strong similarity to the primary tumor. We demonstrated that these tumoroids can be used as a drug screening model. In fact, we showed that paclitaxel, a human chemotherapeutic, could kill canine tumoroids with the same efficacy as human tumoroids with 0.1 to 1 µM of drug needed to kill 50% of the cells. Due to easy tissue availability, canine tumoroids can be produced at larger scale and cryopreserved to constitute a biobank. We have demonstrated that cryopreserved tumoroids keep the same histologic and molecular features (ER, PR, and HER2 expression) as fresh tumoroids. Furthermore, two cryopreservation techniques were compared from a proteomic point of view which showed that tumoroids made from frozen material allowed to maintain the same molecular diversity as from freshly dissociated tumor. CONCLUSIONS: These findings revealed that canine mammary tumoroids can be easily generated and may provide an adequate and more reliable preclinical model to investigate tumorigenesis mechanisms and develop new treatments for both veterinary and human medicine.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.