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.

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide, with the 5 year survival rate in metastatic cases limited to 12%. The design of targeted and effective therapeutics remains a major unmet clinical need in CRC treatment. Carcinoembryonic antigen (CEA), a glycoprotein overexpressed in most colorectal tumors, may constitute a promising molecule for generating novel CEA-targeted therapeutic strategies for CRC treatment. Here, we developed a smart nanoplatform based on chemical conjugation of an anti-CEA single-chain variable fragment (scFv), MFE-23, with PLGA-PEG polymers to deliver the standard 5-Fluorouracil (5-FU) chemotherapy to CRC cells. We confirmed the specificity of the developed CEA-targeted NPs on the internalization by CEA-expressing CRC cells, with an enhance of threefold in the cell uptake. Additionally, CEA-targeted NPs loaded with 5-FU induced higher cytotoxicity in CEA-expressing cells, after 24 h and 48 h of treatment, reinforcing the specificity of the targeted NPs. Lastly, the safety of CEA-targeted NPs loaded with 5-FU was evaluated in donor-isolated macrophages, with no relevant impact on their metabolic activity nor polarization. Altogether, this proof of concept supports the CEA-mediated internalization of targeted NPs as a promising chemotherapeutic strategy for further investigation in different CEA-associated cancers and respective metastatic sites.Authors: Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [Maria José] Last name [Silveira]. Author 7 Given name: [Maria José] Last name [Oliveira]. Also, kindly confirm the details in the metadata are correctokAffiliations: Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.ok.

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages.

Introduction: Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3'UTR-APA and IPA in primary human macrophages was unclear. Methods: In this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3'RNA-Seq was performed to quantify gene expression and characterize new 3'UTR-APA and IPA mRNA isoforms. Results: Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3'UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3'UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Discussion: Our results reveal new 3'UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for SRSF12 in pro-inflammatory macrophages, key cells in the tumor response.

Special Issue: Nanotherapeutics in Women's Health Emerging Nanotechnologies for Triple-Negative Breast Cancer Treatment.

Breast cancer appears as the major cause of cancer-related deaths in women, with more than 2 260 000 cases reported worldwide in 2020, resulting in 684 996 deaths. Triple-negative breast cancer (TNBC), characterized by the absence of estrogen, progesterone, and human epidermal growth factor type 2 receptors, represents ≈20% of all breast cancers. TNBC has a highly aggressive clinical course and is more prevalent in younger women. The standard therapy for advanced TNBC is chemotherapy, but responses are often short-lived, with high rate of relapse. The lack of therapeutic targets and the limited therapeutic options confer to individuals suffering from TNBC the poorest prognosis among breast cancer patients, remaining a major clinical challenge. In recent years, advances in cancer nanomedicine provided innovative therapeutic options, as nanoformulations play an important role in overcoming the shortcomings left by conventional therapies: payload degradation and its low solubility, stability, and circulating half-life, and difficulties regarding biodistribution due to physiological and biological barriers. In this integrative review, the recent advances in the nanomedicine field for TNBC treatment, including the novel nanoparticle-, exosome-, and hybrid-based therapeutic formulations are summarized and their drawbacks and challenges are discussed for future clinical applications.

Incidência de neoplasias malignas em Porto Alegre e Salvador em 2020 de acordo com o DATASUS / Incidence of malignant neoplasms in Porto Alegre and Salvador in 2020 according to DATASUS / Incidencia de neoplasias malignas en Porto Alegre y Salvador en 2020 según DATASUS

Objetivo: analisar a incidência de neoplasias malignas em 2020. Métodos: estudo ecológico com análise comparativa entre as populações de Porto Alegre e Salvador. Foram extraídos dados do DATASUS, analisados em tabelas e apresentados em gráficos. Resultados: A incidência de neoplasias malignas em mulheres entre 30 a 34 anos é maior em Porto Alegre que em Salvador, sendo quase o dobro de casos de mulheres em relação aos homens. Entre 65 a 69 anos, mulheres representaram 20 casos a mais em Porto Alegre, e, em Salvador, o sexo masculino apresentou 28 casos a mais. As mulheres realizaram mais quimioterapias e os homens mais cirurgias. Conclusão: Houve diferença entre a incidência de neoplasias nas cidades podendo associar variáveis determinantes como sexo biológico feminino ao tipo de câncer e idade avançada. A maior incidência de casos na região sul pode estar associada aos hábitos de vida como alimentação e cultura desta região.

Objective: to analyze the incidence of malignant neoplasms in 2020 in two Brazilian cities. Methods: this is an ecological study with comparative analysis between the populations of the cities of Porto Alegre, and Salvador. Data were extracted from the DATASUS, analyzed in tables and presented in descriptive. Results: The incidence of malignant neoplasms in women aged 30 to 34 years is higher of Porto Alegre than in Salvador, with almost double the number of cases in women compared to men in both cities. In the age 65 to 69, women accounted for 20 more cases in Porto Alegre, and in Salvador, males had 28 more cases. Women underwent more chemotherapy and men more surgical in both cities. Conclusion: Differences were observed between the incidence of neoplasms for the cities compared, which could associate determinant variables such as female biological sex with the type of cancer and advanced age. In addition, there is evidence that the southern region of Brazil has a higher incidence than the northeast region, which may be associated with lifestyle habits such as food and culture in the region.

Objetivo: analizar la incidencia de neoplasias malignas en 2020 en dos ciudades brasileñas. Métodos: se trata de un estudio ecológico con análisis comparativo entre las poblaciones de Porto Alegre y Salvador. Los datos fueron extraídos del DATASUS, analizados en tablas y presentados en gráficos. Resultados: La incidencia de neoplasias malignas en mujeres de 30 a 34 años es mayor en Porto Alegre que en Salvador, con casi el doble de casos en mujeres que en hombres. Entre 65 a 69 años, las mujeres representaron 20 casos más en Porto Alegre, y en Salvador, los hombres tuvieron 28 casos más. Las mujeres se sometieron más a quimioterapia y los hombres más a quirúrgias. Conclusión: Se observaron diferencias entre la incidencia de neoplasias, que podrían asociar variables determinantes como el sexo biológico femenino con el tipo de cáncer y la edad avanzada. Existe evidencia de que la región sur de Brasil tiene una mayor incidencia que la región noreste, lo que puede estar asociado con hábitos de estilo de vida como la alimentación y la cultura en la región.

Incidência de neoplasias malignas em Porto Alegre e Salvador em 2020 de acordo com o DATASUS / Incidence of malignant neoplasms in Porto Alegre and Salvador in 2020 according to DATASUS / Incidencia de neoplasias malignas en Porto Alegre y Salvador en 2020 según DATASUS

Objetivo: analisar a incidência de neoplasias malignas em 2020. Métodos: estudo ecológico com análise comparativa entre as populações de Porto Alegre e Salvador. Foram extraídos dados do DATASUS, analisados em tabelas e apresentados em gráficos. Resultados: A incidência de neoplasias malignas em mulheres entre 30 a 34 anos é maior em Porto Alegre que em Salvador, sendo quase o dobro de casos de mulheres em relação aos homens. Entre 65 a 69 anos, mulheres representaram 20 casos a mais em Porto Alegre, e, em Salvador, o sexo masculino apresentou 28 casos a mais. As mulheres realizaram mais quimioterapias e os homens mais cirurgias. Conclusão: Houve diferença entre a incidência de neoplasias nas cidades podendo associar variáveis determinantes como sexo biológico feminino ao tipo de câncer e idade avançada. A maior incidência de casos na região sul pode estar associada aos hábitos de vida como alimentação e cultura desta região.

Objective: to analyze the incidence of malignant neoplasms in 2020 in two Brazilian cities. Methods: this is an ecological study with comparative analysis between the populations of the cities of Porto Alegre, and Salvador. Data were extracted from the DATASUS, analyzed in tables and presented in descriptive. Results: The incidence of malignant neoplasms in women aged 30 to 34 years is higher of Porto Alegre than in Salvador, with almost double the number of cases in women compared to men in both cities. In the age 65 to 69, women accounted for 20 more cases in Porto Alegre, and in Salvador, males had 28 more cases. Women underwent more chemotherapy and men more surgical in both cities. Conclusion: Differences were observed between the incidence of neoplasms for the cities compared, which could associate determinant variables such as female biological sex with the type of cancer and advanced age. In addition, there is evidence that the southern region of Brazil has a higher incidence than the northeast region, which may be associated with lifestyle habits such as food and culture in the region.

Objetivo: analizar la incidencia de neoplasias malignas en 2020 en dos ciudades brasileñas. Métodos: se trata de un estudio ecológico con análisis comparativo entre las poblaciones de Porto Alegre y Salvador. Los datos fueron extraídos del DATASUS, analizados en tablas y presentados en gráficos. Resultados: La incidencia de neoplasias malignas en mujeres de 30 a 34 años es mayor en Porto Alegre que en Salvador, con casi el doble de casos en mujeres que en hombres. Entre 65 a 69 años, las mujeres representaron 20 casos más en Porto Alegre, y en Salvador, los hombres tuvieron 28 casos más. Las mujeres se sometieron más a quimioterapia y los hombres más a quirúrgias. Conclusión: Se observaron diferencias entre la incidencia de neoplasias, que podrían asociar variables determinantes como el sexo biológico femenino con el tipo de cáncer y la edad avanzada. Existe evidencia de que la región sur de Brasil tiene una mayor incidencia que la región noreste, lo que puede estar asociado con hábitos de estilo de vida como la alimentación y la cultura en la región.

Mechanical Properties of Colorectal Cancer Cells Determined by Dynamic Atomic Force Microscopy: A Novel Biomarker.

Colorectal cancer (CRC) has been addressed in the framework of molecular, cellular biology, and biochemical traits. A new approach to studying CRC is focused on the relationship between biochemical pathways and biophysical cues, which may contribute to disease understanding and therapy development. Herein, we investigated the mechanical properties of CRC cells, namely, HCT116, HCT15, and SW620, using static and dynamic methodologies by atomic force microscopy (AFM). The static method quantifies Young's modulus; the dynamic method allows the determination of elasticity, viscosity, and fluidity. AFM results were correlated with confocal laser scanning microscopy and cell migration assay data. The SW620 metastatic cells presented the highest Young's and storage moduli, with a defined cortical actin ring with distributed F-actin filaments, scarce vinculin expression, abundant total focal adhesions (FAK), and no filopodia formation, which could explain the lessened migratory behavior. In contrast, HCT15 cells presented lower Young's and storage moduli, high cortical tubulin, less cortical F-actin and less FAK, and more filopodia formation, probably explaining the higher migratory behavior. HCT116 cells presented Young's and storage moduli values in between the other cell lines, high cortical F-actin expression, intermediate levels of total FAK, and abundant filopodia formation, possibly explaining the highest migratory behavior.

Modulation of Fibroblast Phenotype by Colorectal Cancer Cell-Secreted Factors Is Mostly Independent of Oncogenic KRAS.

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment. Herein, we studied the impact of mutant KRAS on the modulation of the pro-tumorigenic properties of cancer-associated fibroblasts (CAFs), including α-SMA expression, TGFß1 and HGF production, extracellular matrix components and metalloproteinases expression as well as collagen contraction and migration capacities. To do so, CCD-18Co normal-like colon fibroblasts were challenged with conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Our results showed that the mutant KRAS CRC cell-secreted factors were capable of turning normal-like fibroblasts into CAF-like by modulating the α-SMA expression, TGFß1 and HGF production and migration capacity. Oncogenic KRAS played a secondary role as its silencing did not completely impair the capacity of CRC cells to modulate most of the fibroblast properties analyzed. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell-secreted factors that modulate the behavior of fibroblasts. The fact that CRC cells retain the capacity to modulate the pro-tumorigenic features of fibroblasts independently of KRAS silencing is likely to negatively impact their response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS inhibition with potential therapeutical relevance.

Mutant KRAS-Associated Proteome Is Mainly Controlled by Exogenous Factors.

Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFß1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFß1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.

Mutant KRAS modulates colorectal cancer cells invasive response to fibroblast-secreted factors through the HGF/C-MET axis.

Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblast-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and nonautonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent nonautonomous effect, resulting from the cooperation between fibroblast-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promote invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells.

Crucial Role of Oncogenic KRAS Mutations in Apoptosis and Autophagy Regulation: Therapeutic Implications.

KRAS, one of the RAS protein family members, plays an important role in autophagy and apoptosis, through the regulation of several downstream effectors. In cancer cells, KRAS mutations confer the constitutive activation of this oncogene, stimulating cell proliferation, inducing autophagy, suppressing apoptosis, altering cell metabolism, changing cell motility and invasion and modulating the tumor microenvironment. In order to inhibit apoptosis, these oncogenic mutations were reported to upregulate anti-apoptotic proteins, including Bcl-xL and survivin, and to downregulate proteins related to apoptosis induction, including thymine-DNA glycosylase (TDG) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). In addition, KRAS mutations are known to induce autophagy in order to promote cell survival and tumor progression through MAPK and PI3K regulation. Thus, these mutations confer resistance to anti-cancer drug treatment and, consequently, result in poor prognosis. Several therapies have been developed in order to overcome KRAS-induced cell death resistance and the downstream signaling pathways blockade, especially by combining MAPK and PI3K inhibitors, which demonstrated promising results. Understanding the involvement of KRAS mutations in apoptosis and autophagy regulation, might bring new avenues to the discovery of therapeutic approaches for CRCs harboring KRAS mutations.

C-Reactive Protein as Predictive Biomarker for Response to Chemoradiotherapy in Patients with Locally Advanced Rectal Cancer: A Retrospective Study.

The standard of care for the treatment of locally advanced rectal cancer is neoadjuvant chemoradiotherapy (nCRT) followed by surgery, but complete response rates are reduced. To find predictive biomarkers of response to therapy, we conducted a retrospective study evaluating blood biomarkers before nCRT. Hemoglobin (Hg), C-reactive protein (CRP), platelets, carcinoembryonic antigen, carbohydrate antigen 19.9 levels, and neutrophil/lymphocyte ratio were obtained from 171 rectal cancer patients before nCRT. Patients were classified as responders (Ryan 0-1; ycT0N0), 59.6% (n = 102), or nonresponders (Ryan 2-3), 40.3% (n = 69), in accordance with the Ryan classification. A logistic regression using prognostic pretreatment factors identified CRP ≤ 3.5 (OR = 0.05; 95%CI: 0.01-0.21) as a strong independent predictor of response to treatment. Multivariate analysis showed that CRP was an independent predictor of disease-free survival (DFS) (HR = 5.48; 95%CI: 1.54-19.48) and overall survival (HR = 6.10; 95%CI 1.27-29.33) in patients treated with nCRT. Platelets were an independent predictor of DFS (HR = 3.068; 95%CI: 1.29-7.30) and OS (HR= 4.65; 95%CI: 1.66-13.05) and Hg was revealed to be an independent predictor of DFS (HR = 0.37; 95%CI: 0.15-0.90) in rectal cancer patients treated with nCRT. The lower expression of CRP is independently associated with an improved response to nCRT, DFS, and OS.

KRAS as a Modulator of the Inflammatory Tumor Microenvironment: Therapeutic Implications.

KRAS mutations are one of the most frequent oncogenic mutations of all human cancers, being more prevalent in pancreatic, colorectal, and lung cancers. Intensive efforts have been encouraged in order to understand the effect of KRAS mutations, not only on tumor cells but also on the dynamic network composed by the tumor microenvironment (TME). The relevance of the TME in cancer biology has been increasing due to its impact on the modulation of cancer cell activities, which can dictate the success of tumor progression. Here, we aimed to clarify the pro- and anti-inflammatory role of KRAS mutations over the TME, detailing the context and the signaling pathways involved. In this review, we expect to open new avenues for investigating the potential of KRAS mutations on inflammatory TME modulation, opening a different vision of therapeutic combined approaches to overcome KRAS-associated therapy inefficacy and resistance in cancer.

Decellularized Colorectal Cancer Matrices as Bioactive Scaffolds for Studying Tumor-Stroma Interactions.

More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.

Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression.

PURPOSE: To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. METHODS: Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient's radiotherapy. Protein expression profile by proteomics was performed. RESULTS: A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. CONCLUSIONS: Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

O idoso, a desospitalização e a família: os desafios para pratica do cuidado domiciliar / Personas mayores, deshospitalización y familia: los desafíos para la práctica de cuidado en casa / The elderly, de-hospitalization and the family: challenges for the practice of home care

O envelhecimento da população tem exigido uma maior sobrecarga de cuidados por parte dos sistemas de saúde e familiares dos idosos, principalmente após a hospitalização. Os profissionais de saúde, em especial os enfermeiros, são responsáveis pela educação em saúde, para que a reabilitação seja segura e o cuidado seja exitoso. O objetivo deste estudo é relatar a experiência de educação em saúde realizada com familiares (cuidadores) e idosos após a alta para prevenir quedas no domicílio. Trata-se de um relato de experiência oriundo de visitas domiciliares realizadas três meses após a alta hospitalar de idosos que receberam tratamento cirúrgico de fratura de membros inferiores em um hospital público de Porto Alegre-RS. Realizamos a pesquisa de campo em três etapas, que são descritas a seguir: contato telefônico prévio para agendamento da visita, durante a visita iniciamos a abordagem baseada no diálogo informal, resgatando as vivências relacionadas ao ato de cuidar do idoso por conta própria. despesas, retorno ao domicílio e, por fim, orientações sobre cuidados domiciliares para prevenção de novas quedas. Foram realizadas 55 visitas domiciliares em Porto Alegre e região metropolitana. A educação em saúde utilizou o calendário como recurso para a discussão das melhores práticas na atenção domiciliar para prevenir o risco de quedas e promover a autonomia e independência dos idosos. Esta intervenção conseguiu reabilitar a autoconfiança, autonomia e independência do idoso, promovendo qualidade de vida e reinserção social, além de tornar o familiar solidariamente responsável por um cuidado seguro.

El envejecimiento de la población ha exigido una mayor carga asistencial por parte de los sistemas de salud de las personas mayores y de los familiares, especialmente después de la hospitalización. Los profesionales de la salud, especialmente las enfermeras, son responsables de la educación sanitaria, para que la rehabilitación sea segura y la atención sea exitosa. El objetivo de este estudio es relatar la experiencia de educación sanitaria realizada con familiares (cuidadores) y personas mayores tras el alta para prevenir caídas en el domicilio. Se trata de un relato de experiencia derivada de las visitas domiciliarias realizadas tres meses después del alta hospitalaria de ancianos que recibieron tratamiento quirúrgico por fracturas de miembros inferiores en un hospital público de Porto Alegre-RS. Realizamos la investigación de campo en tres etapas, las cuales se describen a continuación: contacto telefónico previo a programar la visita, durante la visita iniciamos el abordaje basado en el diálogo informal, rescatando las experiencias relacionadas con el acto de cuidar al adulto mayor por su cuenta. gastos, regreso a casa y, fi nalmente, orientación sobre cuidados domiciliarios para evitar nuevas caídas. Se realizaron 55 visitas domiciliarias en Porto Alegre y su región metropolitana. La educación para la salud utilizó el calendario como un recurso para discutir las mejores prácticas en la atención domiciliaria para prevenir el riesgo de caídas y promover la autonomía e independencia de las personas mayores. Esta intervención logró rehabilitar la autoconfianza, autonomía e independencia de las personas mayores, promoviendo la calidad de vida y la reinserción social, además de responsabilizar solidariamente al familiar de un cuidado seguro.

The aging of the population has demanded a greater burden of care on the part of the elderly's health systems and family members, especially after hospitalization. Health professionals, especially nurses, are responsible for health education, so that rehabilitation is safe and care is successful. The aim of this study is to report the experience of health education carried out with family members (caregivers) and elderly people after discharge to prevent falls at home. This is an experience report arising from home visits carried out three months after hospital discharge of elderly people who received surgical treatment for fractures of the lower limbs in a public hospital in Porto Alegre-RS. We carried out the field research in three stages, which are described below telephone contact prior to scheduling the visit, during the visit we started the approach based on informal dialogue, rescuing the experiences related to the act of caring for the elderly on their own. Expenses, return home and, finally, guidance on home care to prevent further falls. 55 home visits were carried out in Porto Alegre and its metropolitan region. Health education used the calendar as a resource for discussing best practices in home care to prevent the risk of falls and promote the autonomy and independence of the elderly. The intervention managed to rehabilitate the elderly's self-confidence, autonomy and independence, promoting quality of life and social reintegration, in addition to making the family member jointly responsible for safe care.

Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer.

BACKGROUND: Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. METHODS: A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. RESULTS: Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. CONCLUSION: HOMER3-glycoforms allow the identification of patients' subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established.

Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells.

Transcription termination in eukaryotic cells involves the recognition of polyadenylation signals (PAS) that signal the site of pre-mRNA cleavage and polyadenylation. Most eukaryotic genes contain multiple PAS that are used by alternative polyadenylation (APA), a co-transcriptional process that increases transcriptomic diversity and modulates the fate of the mRNA and protein produced. However, current tools to pinpoint the relationship between mRNAs in different subcellular fractions and the gene expression outcome are lacking, particularly in primary human immune cells, which, due to their nature, are challenging to study. Here, we describe an integrative approach using subcellular fractionation and RNA isolation, chromatin-bound and nucleoplasmic RNA-Sequencing, 3' RNA-Sequencing and bioinformatics, to identify accurate APA mRNA isoforms and to quantify gene expression in primary human macrophages. Our protocol includes macrophage differentiation and polarization, co-culture with cancer cells, and gene silencing by siRNA. This method allows the simultaneous identification of macrophage APA mRNA isoforms integrated with the characterization of nuclear APA events, the identification of the molecular mechanisms involved, as well as the gene expression alterations caused by the cancer-macrophage crosstalk. With this methodology we identified macrophage APA mRNA signatures driven by the cancer cells that alter the macrophage inflammatory and transcriptomic profiles, with consequences for macrophage physiology and tumor evasion.