The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.

61 Biomarkers of favorable prognosis guides the identification of tumor reactive CD4+ and CD8+ TILs in endometrial cancer

BackgroundEndometrial cancer (EC) is the most prevalent gynecological cancer and it can be categorized into four molecular subtypes; ultramutated POLE, hypermutated MSI, CNL and CNH. To date, the prevalence, specificity and phenotype of tumor infiltrating lymphocytes (TILs), and their exact role in EC remain controversial. Thus, a thorough investigation deciphering the phenotypic landscape of EC-infiltrating T lymphocytes and their anti-tumor reactivity is still missing.MethodsIn order to study the implications of the T-cells infiltrating EC for prognosis or susceptibility to immune checkpoint inhibitors, we analyzed the phenotypic traits of CD8 and CD4 EC-resident T cells from single-cell suspensions (n=47) by multicolor flow cytometry (19 biomarkers). Correlation analyses between the phenotype of the TILs, EC molecular subgroups and the survival of the patients were performed to identify biomarkers that could predict prognosis or survival. This identification guided the isolation of specific CD8 and CD4 subpopulations based on the differential expression of the selected biomarkers to evaluate their ability to recognize tumor (figure 1).ResultsOur analysis evidenced the presence of CD8+ and CD4+ T cells with distinct levels of PD-1 expression: cells that did not express PD-1 (PD-1-), cells expressing intermediate (PD-1dim) or high (PD-1hi) levels of PD-1. We found that TIM-3+, CD39+, CXCL13+, BCL6+ or Ki67+ cells frequently co-expressed almost exclusively on the PD-1hi, but not on the PD-1- or dim CD8+ TILs. On the other hand, CD4+ TILs displayed co-expression of TIM-3, CD103, CD39, CD38, HLA-DR, CXCL13, BCL6, CXCR5 or Ki67 within the PD-1hi, but also PD-1dim cells. Importantly, our data shows that the frequency of PD-1hi or CD39+ CD8+ EC TILs, and of PD-1hi but not CD39+ CD4+ TILs was associated with improved survival. Of the 5 predominant CD8+ tumor-resident subpopulations observed (PD-1-CD39-, PD-1dimCD39-, PD-1dimCD39+, PD-1hiCD39- and PD-1hiCD39+) the PD-1hiCD39+, but not the PD-1hiCD39- lymphocytes, harbored the highest frequency of autologous tumor-reactive lymphocytes in all four EC tumors studied. However, both the CD4+ PD-1hiCD39+ and the PD-1hiCD39- contained autologous tumor-reactive lymphocytes in all four tumors screened; being the PD-1hiCD39+ cells the subpopulation containing the majority of tumor-reactive CD4+ cells.Abstract 61 Figure 1Schematic workflow of the study. A section of a primary EC tumor resection is digested and stained with specific antibodies to characterize the phenotype of CD8 and CD4 TILs by flow cytometry (upper panel). Targeted sequencing and IHC including typically altered genes in EC are performed in order to molecularly classify the patient‘s cohort into different disease subgroups. Combining the data generated by flow cytometry, targeted sequencing and IHC, specific biomarkers on CD8 and CD4 TILs are investigated for their potential role in predicting molecular subtypes or survival (middle panel). CD8 and CD4 TILs are isolated from the tumor digest based on the differential expression of PD-1 and CD39 and the specific isolated CD8 and CD4 subpopulations are cultured in vitro to expand them to large numbers. The expanded CD8 and CD4 subpopulations are subsequently screened for tumor recognition by co-culturing the isolated subpopulations with autologous tumor cell lines. Tumor recognition is assessed by measuring the up-regulation of the activation markers 4-1BB or OX40 on CD8+ or CD4+ T cells, respectively, by flow cytometry, and by measuring IFN-γ production by ELISPOT.ConclusionsOverall, our data suggest that CD39 expression on CD8+PD-1hi EC-resident T cells defines a tumor-reactive population that plays an important role in protecting patients from recurrence after surgery. However, PD-1 expression but not CD39 on CD4+ TILs, better guides the identification of lymphocyte subsets with enriched tumor-recognition potential that contribute to improved clinical benefit in EC.Ethics ApprovalThis study was approved by the ‘Comité de Ética de Investigación con Medicamentos del Hospital Universitario Vall d’Hebron’ institution’s Ethics Board; approval number PR(AG)537/2019.

Accurate Brain Tumor Recognition Using Double-Weighted Feature Extraction Labelling Model with Priority Weighted Feature Selection

In clinical practice and patient survival rates, early diagnosis of brain tumors plays a key role. Different forms of brain tumors and their properties and treatments are available. Therefore, tumor detection is complicated, time consuming and error-prone with manual brain tumor detection. Therefore, high-precision automated, computerized diagnostics are currently necessary. Feature extraction is a tumor prediction method for capturing the visual content of a picture. The extraction of features is the process through which the raw image is reduced and decisions like the pattern classification are facilitated. The MRI brain images are considered to be classified as a robust and more accurate classification that is able to serve as an expert assistant for healthcare practitioners. In this research, a new method for selecting and extracting features is introduced. The paper proposes to take into account the most important features for the classification of tumor and non-tumor cells using a Double-Weighted Feature Extraction Labelling Model with Priority Weighted Feature Selection (DWLM-PWFS). This approach combines the tumor's intensity, texture, shape and diagnostic properties. The selection of features with the technique proposed is most helpful for analyzing data according to grouping class variable and ensuring reduced feature setting with high classification accuracy. In contrast to the conventional model, the model proposed is shown to be highly efficient in comparison with traditional models.

L2pB1 Cells Contribute to Tumor Growth Inhibition

Natural IgM (nIgM) antibodies play critical roles in cancer immunosurveillance. However, the role of B-1 B cells, the lymphocytes that produce nIgM, remains to be elucidated. L2pB1 cells, a subpopulation of B-1 B cells, have a unique poly-self-reactive nIgM repertoire and are capable of phagocytosis, potent antigen presentation, and immunomodulation. Using an inducible knock-in and knockout mouse model, we investigated the effect of the loss of L2pB1 cells in a B16F10 melanoma model. Our results show active tumor infiltration of L2pB1 cells in wild type mice, and conversely, depletion of L2pB1 cells results in larger tumor mass and increased angiogenesis. In vitro analysis revealed that L2pB1 cells contribute to the growth inhibition of melanoma cells in both 2D cell culture and 3D tumor spheroids. Similar effects were observed in an MC38 murine colon cancer model. Moreover, our data suggest that one of the ways that L2pB1 cells can induce tumor cell death is via lipoptosis. Lastly, we tested whether L2pB1 cell-derived monoclonal nIgM antibodies can specifically recognize tumor spheroids. Nine of the 28 nIgM-secreting L2pB1 clones demonstrated specific binding to tumor spheroids but did not bind control murine embryonic fibroblasts. Our study provides evidence that L2pB1 cells contribute to cancer immunity through their unique nIgM repertoire, tumor recognition, and lipoptosis. Taken together, because of their ability to recognize common features of tumors that are independent of genetic mutations, L2pB1 cells and their nIgM could be potential candidates for cancer treatment that can overcome tumor heterogeneity-associated drug resistance.

An Efficient Magnetic Nanocatalyst Induced Chemo- and Ferroptosis Synergistic Cancer Therapy in Combination with T1-T2 Dual-Mode Magnetic Resonance Imaging through Doxorubicin Delivery

Excessive iron ions in cancer cells can catalyze H2O2 into highly toxic ·OH and then promote the generation of reactive oxygen species (ROS), inducing cancer ferroptosis. However, the efficacy of ferroptosis catalyst is still insufficient because of low Fe(II) release, which severely limited its application in clinics. Herein, we developed a novel magnetic nanocatalyst for MRI-guided chemo- and ferroptosis synergistic cancer therapies through iRGD-PEG-ss-PEG modified gadolinium engineering magnetic iron oxide loaded Dox (ipGdIO-Dox). The introduction of gadolinium compound disturbed the structure of ipGdIO-Dox, making magnetic nanocatalyst be more sensitive to weak acid. When the ipGdIO-Dox entered into cancer cells, abundance of Fe(II) ions were released and then catalyzed H2O2 into highly toxic OH·, which would elevate cellular oxidative-stress to damage mitochondria and cell membranes and induced cancer ferroptosis. In addition, the iRGD-PEG-ss-PEG chain coated onto nanoplatform were also broken by high expression of GSH, and then the Dox was released. This process not only effectively inhibited DNA replication, but further activated cellular ROS, making nanoplatform achieve stronger anticancer ability. Besides, the systemic delivery ipGdIO-Dox significantly enhanced T1- and T2-weighted MRI signal of tumor, endowing accurate diagnostic capability for tumor recognition. Therefore, the ipGdIO-Dox might be a promising candidate for developing MRI guided chemo- and chemdynamic synergistic theranostic system.

PTEN Hamartoma Tumor Syndrome/Cowden Syndrome: Genomics, Oncogenesis, and Imaging Review for Associated Lesions and Malignancy

PTEN hamartoma tumor syndrome/Cowden syndrome (CS) is a rare autosomal dominant syndrome containing a germline PTEN mutation that leads to the development of multisystem hamartomas and oncogenesis. Benign tumors such as Lhermitte–Duclos disease and malignant tumors involving the breast, thyroid, kidneys, and uterus are seen in CS. Radiologists have an integral role in the comanagement of CS patients. We present the associated imaging findings and imaging screening recommendations. Knowledge of the types of cancers commonly seen in CS and their imaging findings can aid in early tumor recognition during cancer screening to help ensure near-normal life spans in CS patients.

Paclitaxel-Loaded Magnetic Nanoparticles Based on Biotinylated N-Palmitoyl Chitosan: Synthesis, Characterization and Preliminary In Vitro Studies

A considerable interest in cancer research is represented by the development of magnetic nanoparticles based on biofunctionalized polymers for controlled-release systems of hydrophobic chemotherapeutic drugs targeted only to the tumor sites, without affecting normal cells. The objective of the paper is to present the synthesis and in vitro evaluation of the nanocomposites that include a magnetic core able to direct the systems to the target, a polymeric surface shell that provides stabilization and multi-functionality, a chemotherapeutic agent, Paclitaxel (PTX), and a biotin tumor recognition layer. To our best knowledge, there are no studies concerning development of magnetic nanoparticles obtained by partial oxidation, based on biotinylated N-palmitoyl chitosan loaded with PTX. The structure, external morphology, size distribution, colloidal and magnetic properties analyses confirmed the formation of well-defined crystalline magnetite conjugates, with broad distribution, relatively high saturation magnetization and irregular shape. Even if the ability of the nanoparticles to release the drug in 72 h was demonstrated, further complex in vitro and in vivo studies will be performed in order to validate the magnetic nanoparticles as PTX delivery system.