Stool Glycoproteomics Signatures of Pre-Cancerous Lesions and Colorectal Cancer.

Colorectal cancer (CRC) screening relies primarily on stool analysis to identify occult blood. However, its sensitivity for detecting precancerous lesions is limited, requiring the development of new tools to improve CRC screening. Carcinogenesis involves significant alterations in mucosal epithelium glycocalyx that decisively contribute to disease progression. Building on this knowledge, we examined patient series comprehending premalignant lesions, colorectal tumors, and healthy controls for the T-antigen-a short-chain O-glycosylation of proteins considered a surrogate marker of malignancy in multiple solid cancers. We found the T-antigen in the secretions of dysplastic lesions as well as in cancer. In CRC, T-antigen expression was associated with the presence of distant metastases. In parallel, we analyzed a broad number of stools from individuals who underwent colonoscopy, which showed high T expressions in high-grade dysplasia and carcinomas. Employing mass spectrometry-based lectin-affinity enrichment, we identified a total of 262 proteins, 67% of which potentially exhibited altered glycosylation patterns associated with cancer and advanced pre-cancerous lesions. Also, we found that the stool (glyco)proteome of pre-cancerous lesions is enriched for protein species involved in key biological processes linked to humoral and innate immune responses. This study offers a thorough analysis of the stool glycoproteome, laying the groundwork for harnessing glycosylation alterations to improve non-invasive cancer detection.

Unmasking the Metabolite Signature of Bladder Cancer: A Systematic Review.

Bladder cancer (BCa) research relying on Omics approaches has increased over the last few decades, improving the understanding of BCa pathology and contributing to a better molecular classification of BCa subtypes. To gain further insight into the molecular profile underlying the development of BCa, a systematic literature search was performed in PubMed until November 2023, following the PRISMA guidelines. This search enabled the identification of 25 experimental studies using mass spectrometry or nuclear magnetic resonance-based approaches to characterize the metabolite signature associated with BCa. A total of 1562 metabolites were identified to be altered by BCa in different types of samples. Urine samples displayed a higher likelihood of containing metabolites that are also present in bladder tumor tissue and cell line cultures. The data from these comparisons suggest that increased concentrations of L-isoleucine, L-carnitine, oleamide, palmitamide, arachidonic acid and glycoursodeoxycholic acid and decreased content of deoxycytidine, 5-aminolevulinic acid and pantothenic acid should be considered components of a BCa metabolome signature. Overall, molecular profiling of biological samples by metabolomics is a promising approach to identifying potential biomarkers for early diagnosis of different BCa subtypes. However, future studies are needed to understand its biological significance in the context of BCa and to validate its clinical application.

The impact of chemotherapy on adipose tissue remodeling: The molecular players involved in this tissue wasting.

The depletion of visceral and subcutaneous adipose tissue (AT) during chemotherapy significantly correlates with diminished overall survival and progression-free survival. Despite its clinical significance, the intricate molecular mechanisms governing this AT loss and its chemotherapy-triggered initiation remain poorly understood. Notably, the evaluation of AT remodeling in most clinical trials has predominantly relied on computerized tomography scans or bioimpedance, with molecular studies often conducted using animal or in vitro models. To address this knowledge gap, a comprehensive narrative review was conducted. The findings underscore that chemotherapy serves as a key factor in inducing AT loss, exacerbating cachexia, a paraneoplastic syndrome that significantly compromises patient quality of life and survival. The mechanism driving AT loss appears intricately linked to alterations in AT metabolic remodeling, marked by heightened lipolysis and fatty acid oxidation, coupled with diminished lipogenesis. However, adipocyte stem cells' lost ability to divide due to chemotherapy also appears to be at the root of the loss of AT. Notably, chemotherapy seems to deactivate the mitochondrial antioxidant system by reducing key regulatory enzymes responsible for neutralizing reactive oxygen species (ROS), thereby impeding lipogenesis. Despite FDG-PET evidence of AT browning, no molecular evidence of thermogenesis was reported. Prospective investigations unraveling the molecular mechanisms modulated in AT by chemotherapy, along with therapeutic strategies aimed at preventing AT loss, promise to refine treatment paradigms and enhance patient outcomes.

Aberrantly Glycosylated GLUT1 as a Poor Prognosis Marker in Aggressive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

A multivalent CD44 glycoconjugate vaccine candidate for cancer immunotherapy.

Cancer presents a high mortality rate due to ineffective treatments and tumour relapse with progression. Cancer vaccines hold tremendous potential due to their capability to eradicate tumour and prevent relapse. In this study, we present a novel glycovaccine for precise targeting and immunotherapy of aggressive solid tumours that overexpress CD44 standard isoform (CD44s) carrying immature Tn and sialyl-Tn (sTn) O-glycans. We describe an enzymatic method and an enrichment strategy to generate libraries of well-characterized cancer-specific CD44s-Tn and/or sTn glycoproteoforms, which mimic the heterogeneity found in tumours. We conjugated CD44-Tn-derived glycopeptides with carrier proteins making them more immunogenic, with further demonstration of the importance of this conjugation to overcome the glycopeptides' intrinsic toxicity. We have optimized the glycopeptide-protein maleimide-thiol conjugation chemistry to avoid undesirable cross-linking between carrier proteins and CD44s glycopeptides. The resulting glycovaccines candidates were well-tolerated in vivo, inducing both humoral and cellular immunity, including immunological memory. The generated antibodies exhibited specific reactivity against synthetic CD44s-Tn glycopeptides, CD44s-Tn glycoengineered cells, and human tumours. In summary, we present a promising prototype of a cancer glycovaccine for future therapeutical pre-clinical efficacy validation.

Unexpected pancreatic mixed neuroendocrine-nonneuroendocrine neoplasms (MiNEN)-reflection on a case report.

The authors present a case involving a 51-year-old male who was diagnosed with a 4-cm mass in the body of the pancreas, initially suspected to be a ductal adenocarcinoma due to an elevated Ca 19.9 during routine analysis. Subsequent imaging studies confirmed a resectable disease without suspicious lymph nodes or distant metastasis, leading to the proposal of surgery. The patient underwent a laparoscopic distal splenopancreatectomy, which was uneventful. The histopathological examination revealed a 3.7-cm pancreatic mixed neuroendocrine neoplasia (MiNEN) with a predominant high-grade ductal adenocarcinoma component and a concurrent high-grade neuroendocrine carcinoma, with negative margins. Two lymph node metastases were identified, each representing metastasis of one of the components. The tumor was classified as pT2N1M0. Currently, the patient is undergoing chemotherapy with FOLFIRINOX. This case prompts reflection on the optimal treatment strategy for pancreatic MiNEN and raises the question of how the preoperative diagnosis could influence the patient's outcome.

Minimizing false positives for CTC identification.

BACKGROUND: Cancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. RESULTS: This study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. SIGNIFICANCE: This study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.

Fellowship in surgical oncology: The results of an experience in Portuguese-speaking African countries.

Cancer incidence rates are increasing worldwide including in Portuguese speaking African countries. We present the results of the fellowship in surgical oncology promoted by the Portuguese Institute of Oncology in Porto (IPO), Fernando Pessoa University, Portugal, and the Calouste Gulbenkian Foundation, which involved the training of residents and surgeons from Portuguese-speaking African countries in Portugal. The program's structure and content was the same of UMES/ESSO. The hands-on activity took place at the IPO. After the fellowship, a theoretical and practical assessment was held, and all fellows scored above the threshold to pass. The surgical performance of the fellows was assessed in their workplaces in Africa. The impact of the training was evaluated by a questionnaire completed by all the fellows. It was reported an increase in knowledge, skills and organizational skills that fellows are committed to implement. As the fellowship is certified by a Portuguese University, it may be recognized by the Local Health and University Authorities, impacting on the professional progression of fellows.

Textbook Oncological Outcome in European GASTRODATA.

OBJECTIVE: To assess the rate of textbook outcome (TO) and textbook oncological outcome (TOO) in the European population based on the GASTRODATA registry. BACKGROUND: TO is a composite parameter assessing surgical quality and strongly correlates with improved overall survival. Following the standard of treatment for locally advanced gastric cancer, TOO was proposed as a quality and optimal multimodal treatment parameter. METHODS: TO was achieved when all the following criteria were met: no intraoperative complications, radical resection according to the surgeon, pR0 resection, retrieval of at least 15 lymph nodes, no severe postoperative complications, no reintervention, no admission to the intensive care unit, no prolonged length of stay, no postoperative mortality and no hospital readmission. TOO was defined as TO with the addition of perioperative chemotherapy compliance. RESULTS: Of the 2558 patients, 1700 were included in the analysis. TO was achieved in 1164 (68.5%) patients. The use of neoadjuvant chemotherapy [odds ratio (OR) = 1.33, 95% CI: 1.04-1.70] and D2 or D2+ lymphadenectomy (OR = 1.55, 95% CI: 1.15-2.10) had a positive impact on TO achievement. Older age (OR = 0.73, 95% CI: 0.54-0.94), pT3/4 (OR = 0.79, 95% CI: 0.63-0.99), ASA 3/4 (OR = 0.68, 95% CI: 0.54-0.86) and total gastrectomy (OR = 0.56, 95% CI: 0.45-0.70), had a negative impact on TO achievement. TOO was achieved in 388 (22.8%) patients. Older age (OR = 0.37, 95% CI: 0.27-0.53), pT3 or pT4 (OR = 0.52, 95% CI: 0.39-0.69), and ASA 3 or 4 (OR = 0.58, 95% CI: 0.43-0.79) had a negative impact on TOO achievement. CONCLUSIONS: Despite successively improved surgical outcomes, stage-appropriate chemotherapy in adherence to the current guidelines for multimodal treatment of gastric cancer remains poor. Further implementation of oncologic quality metrics should include greater emphasis on perioperative chemotherapy and adequate lymphadenectomy.

XPERT® breast cancer STRAT4 as an alternative method of identifying breast cancer phenotype in Cape Verde (preliminary results).

Introduction: Breast cancer (BC) is a public health problem in developing countries, including Cape Verde. Immunohistochemistry (IHC) is the gold standard technique used for BC phenotypic characterisation to support efficient therapeutic decisions. However, IHC is a demanding technique that requires knowledge, trained technicians, expensive antibodies and reagents, controls, and results validation. The low number of cases in Cape Verde increases the risk of expiring the validity of the antibodies, and manual procedures often jeopardise the quality of the results. Thus, IHC is limited in Cape Verde, and an alternative technically easy solution is needed. A point-of-care messenger RNA (mRNA) STRAT4 BC assay to assess estrogen (ER), progesterone (PR), hormone growth factor 2 receptor (HER2), and Ki67, using the GeneXpert platform, has been recently validated on tissues from internationally accredited laboratories, showing excellent concordance with IHC results.To assess whether this technology can be implemented in Cape Verde to guide BC treatment we decided to study the level of agreement between the findings yielded by BC STRAT4 and the results are the same cases obtained by IHC. Methods: Formalin-fixed and paraffin-embedded (FFPE) tissue samples from 29 Cabo Verdean BC patients diagnosed in Agostinho Neto University Hospital were analysed by applying IHC and BC STRAT4 assay. The time between sample collection and pre-analytic procedures is unknown. All the samples were pre-processed in Cabo Verde (fixed in formalin and embedded in paraffin). IHC studies were performed in referenced laboratories in Portugal. STRAT4 and IHC result concordance was assessed by calculating the percentage of results agreement and Cohen's Kappa (K) statistics. Results: STRAT4 assay failed in 2 out of the 29 analysed samples. Of the 27 successfully analysed samples, STRAT4/IHC results for ER, PR, HER2, and Ki67 were concordant in 25, 24, 25, and 18 cases, respectively. Ki67 was indeterminate in three cases, and PR was indeterminate once.The percentage of agreement between STRAT4 and IHC results for ER, PR, HER2, and Ki67 was 92.59%, 92.31%, 92.59% and 81.82%, respectively. The Cohen's K statistic coefficients for each biomarker were 0.809, 0.845, 0.757 and 0.506, respectively. Conclusions: According to our preliminary results, a point-of-care mRNA STRAT4 BC assay may be an alternative in laboratories unable to provide quality and/or cost-efficient IHC services. However, more data and improvement on sample pre-analytic processes are required to implement this BC STRAT4 Assay in Cape Verde.

Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

Neoadjuvant Gastric Cancer Treatment and Associated Nutritional Critical Domains for the Optimization of Care Pathways: A Systematic Review.

(1) Background: Gastric cancer patients are known to be at a high risk of malnutrition, sarcopenia, and cachexia, and the latter impairs the patient's nutritional status during their clinical course and also treatment response. A clearer identification of nutrition-related critical points during neoadjuvant treatment for gastric cancer is relevant to managing patient care and predicting clinical outcomes. The aim of this systematic review was to identify and describe nutrition-related critical domains associated with clinical outcomes. (2) Methods: We performed a systematic review (PROSPERO ID:CRD42021266760); (3) Results: This review included 14 studies compiled into three critical domains: patient-related, clinical-related (disease and treatment), and healthcare-related. Body composition changes during neoadjuvant chemotherapy (NAC) accounted for the early termination of chemotherapy and reduced overall survival. Sarcopenia was confirmed to have an independent prognostic value. The role of nutritional interventions during NAC has not been fully explored. (4) Conclusions: Understanding critical domain exposures affecting nutritional status will enable better clinical approaches to optimize care plans. It may also provide an opportunity for the mitigation of poor nutritional status and sarcopenia and their deleterious clinical consequences.

Immunomodulatory glycomedicine: Introducing next generation cancer glycovaccines.

Cancer remains a leading cause of death worldwide due to the lack of safer and more effective therapies. Cancer vaccines developed from neoantigens are an emerging strategy to promote protective and therapeutic anti-cancer immune responses. Advances in glycomics and glycoproteomics have unveiled several cancer-specific glycosignatures, holding tremendous potential to foster effective cancer glycovaccines. However, the immunosuppressive nature of tumours poses a major obstacle to vaccine-based immunotherapy. Chemical modification of tumour associated glycans, conjugation with immunogenic carriers and administration in combination with potent immune adjuvants constitute emerging strategies to address this bottleneck. Moreover, novel vaccine vehicles have been optimized to enhance immune responses against otherwise poorly immunogenic cancer epitopes. Nanovehicles have shown increased affinity for antigen presenting cells (APCs) in lymph nodes and tumours, while reducing treatment toxicity. Designs exploiting glycans recognized by APCs have further enhanced the delivery of antigenic payloads, improving glycovaccine's capacity to elicit innate and acquired immune responses. These solutions show potential to reduce tumour burden, while generating immunological memory. Building on this rationale, we provide a comprehensive overview on emerging cancer glycovaccines, emphasizing the potential of nanotechnology in this context. A roadmap towards clinical implementation is also delivered foreseeing advances in glycan-based immunomodulatory cancer medicine.

Chemotherapy-Induced Molecular Changes in Skeletal Muscle.

Paraneoplastic conditions such as cancer cachexia are often exacerbated by chemotherapy, which affects the patient's quality of life as well as the response to therapy. The aim of this narrative review was to overview the body-composition-related changes and molecular effects of different chemotherapy agents used in cancer treatment on skeletal-muscle remodeling. A literature search was performed using the Web of Science, Scopus, and Science Direct databases and a total of 77 papers was retrieved. In general, the literature survey showed that the molecular changes induced by chemotherapy in skeletal muscle have been studied mainly in animal models and mostly in non-tumor-bearing rodents, whereas clinical studies have essentially assessed changes in body composition by computerized tomography. Data from preclinical studies showed that chemotherapy modulates several molecular pathways in skeletal muscle, including the ubiquitin-proteasome pathway, autophagy, IGF-1/PI3K/Akt/mTOR, IL-6/JAK/STAT, and NF-κB pathway; however, the newest chemotherapy agents are underexplored. In conclusion, chemotherapy exacerbates skeletal-muscle wasting in cancer patients; however, the incomplete characterization of the chemotherapy-related molecular effects on skeletal muscle makes the development of new preventive anti-wasting strategies difficult. Therefore, further investigation on molecular mechanisms and clinical studies are necessary.

Risk factors for oesophageal squamous cell carcinoma in Mozambique.

Studies evaluating risk factors for the occurrence of oesophageal squamous cell carcinoma (ESCC) in high-risk regions might contribute to a better understanding of the oesophageal cancer aetiology and incidence variation worldwide. We aimed to quantify the association between alcohol, tobacco and dietary history, and the occurrence of ESCC in Mozambique. A case-control study was conducted at Maputo Central Hospital. Cases (n = 143) were patients with newly diagnosed oesophageal cancer recruited in the Gastroenterology Service. Controls (n = 212) were selected in the Orthopaedic Ward among subjects with pathologies related to trauma. Crude and adjusted odds ratios (ORs), and the corresponding 95% confidence intervals (CI) were computed using non-conditional logistic regression. The risk of ESCC was higher in older participants and lower in those with higher household income. Alcohol drinking (lifetime consumption ≥ 55.1 versus 0 kg ethanol: OR = 5.56; 95% CI: 2.43-12.73) and tobacco smoking (lifetime consumption ≥ 20 versus 0 pack/years: OR=7.26; 95% CI: 1.42-37.17) were associated with increased risk of ESCC. Tea (at least twice daily versus less than daily: OR = 5.09; 95% CI: 2.45-10.58) was also associated with the occurrence of ESCC. No significant differences were observed for fruit and vegetable and for smoked meat or fish consumption. Findings from this study show that in our sample, the occurrence of ESCC is strongly influenced by lifetime consumption of tobacco and alcohol, and with tea drinking. This highlights the importance of preventive measures based on the promotion of healthier lifestyles.

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion.

Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods:CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O-glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O-glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics allowed, for the first time, the comprehensive characterization of CD44 splicing code at the protein level. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification as well as associated glycoforms. Finally, we confirmed the link between CD44 and invasion in CD44s-enriched cells in vitro by small interfering RNA (siRNA) knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Conclusions: Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/ Sialyl-Tn (STn) as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.

A roadmap for translational cancer glycoimmunology at single cell resolution.

Cancer cells can evade immune responses by exploiting inhibitory immune checkpoints. Immune checkpoint inhibitor (ICI) therapies based on anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have been extensively explored over the recent years to unleash otherwise compromised anti-cancer immune responses. However, it is also well established that immune suppression is a multifactorial process involving an intricate crosstalk between cancer cells and the immune systems. The cancer glycome is emerging as a relevant source of immune checkpoints governing immunosuppressive behaviour in immune cells, paving an avenue for novel immunotherapeutic options. This review addresses the current state-of-the-art concerning the role played by glycans controlling innate and adaptive immune responses, while shedding light on available experimental models for glycoimmunology. We also emphasize the tremendous progress observed in the development of humanized models for immunology, the paramount contribution of advances in high-throughput single-cell analysis in this context, and the importance of including predictive machine learning algorithms in translational research. This may constitute an important roadmap for glycoimmunology, supporting careful adoption of models foreseeing clinical translation of fundamental glycobiology knowledge towards next generation immunotherapies.

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients.

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

The Tumour Microenvironment and Circulating Tumour Cells: A Partnership Driving Metastasis and Glycan-Based Opportunities for Cancer Control.

Circulating tumour cells (CTC) are rare cells that actively detach or are shed from primary tumours into the lymph and blood. Some CTC subpopulations gain the capacity to survive, home and colonize distant locations, forming metastasis. This results from a multifactorial process in which cancer cells optimize motility, invasion, immune escape and cooperative relationships with microenvironmental cues. Here we present evidences of a self-fuelling molecular crosstalk between cancer cells and the tumour stroma supporting the main milestones leading to metastasis. We discuss how the tumour microenvironment supports pre-metastatic niches and CTC development and ultimately dictates CTC fate in targeted organs. Finally, we highlight the key role played by protein glycosylation in metastasis development, its prompt response to microenvironmental stimuli and the tremendous potential of glycan-based molecular signatures for liquid biopsies and targeted therapeutics.