Unravelling the role of Silibinin in targeting CD44+ cancer stem cells: Therapeutic implications, effective strategies and approaches.

CD44+ cancer stem cells (CSCs) are believed to account for drug resistance and tumour recurrence due to their potential to self-renew and differentiate into heterogeneous lineages. Therefore, efficient treatment strategies targeting and eliminating these CSCs are required. The flavonolignan, Silibinin, has gained immense attention in targeting CD44+ CSCs as it alters functional properties like cell cycle arrest, apoptosis, inhibition of invasion and metastasis and also inhibits a range of molecular pathways. However, its limited bioavailability is a major hurdle in asserting Silibinin as a translational therapeutic agent. Combinatorial therapy of Silibinin with conventional chemotherapeutic drugs is an alternative approach in targeting CD44+ CSCs as it increases the efficacy and reduces the cytotoxicity of chemotherapeutic drugs, thus preventing drug resistance. Certain Silibinin-conjugated nano-formulations have also been successfully developed, through which there is improved absorptivity/bioavailability of Silibinin and a decrease in the concentration of therapeutic drugs leading to reduced cytotoxicity. In this review, we summarise the effectiveness of the synergistic therapeutic approach for Silibinin in targeting the molecular mechanisms of CD44+ CSCs and emphasise the potential role of Silibinin as a novel therapeutic agent.

CD44 targeted-chondroitin sulfate nanoparticles: Fine-tuning hydrophobic groups to enhance in vitro pH-responsiveness and in vivo efficacy for advanced breast cancer treatment.

The design of tumor-targeting nanoparticles with precisely controlled physical-biological properties may improve the delivery of chemotherapeutic agents. This study introduces pH-sensitive chondroitin sulfate-cholesterol (ChS-Chol) nano-assemblies for targeted intracellular doxorubicin (Dox) delivery in breast cancer treatment. Various ChS-Chol copolymers were synthesized, yielding self-assembling nanostructures with adjustable lipophilic content. In an aqueous environment, the ChS-Chol conjugates could form self-assembled nanostructures with a narrower size variation and a high negative potential. Moreover, the carriers would rapidly disassemble and release Dox in response to acidic pH. The in vitro cytotoxicity assay exhibited concentration-related anti-proliferation activity with Dox-loaded nanoparticles against 4T1, MCF-7, and MDA-MB-231 breast cancer cells. The nanoparticles demonstrated enhanced early apoptosis induction, efficient cellular uptake, and improved prevention of tumor cell proliferation compared to free Dox. In vivo results showcased significant tumor growth inhibition, underscoring the potential of these nanoparticle-based drug delivery systems for breast cancer therapy. The study emphasizes tailored nanocarrier design, leveraging pH-responsiveness and precise hydrophobic tuning to achieve targeted and potent therapeutic effects in the fight against breast cancer.

A highly potent small-molecule antagonist of exportin-1 selectively eliminates CD44+CD24- enriched breast cancer stem-like cells.

Breast cancer stem-like cells (BCSCs) have been suggested as the underlying cause of tumor recurrence, metastasis and drug resistance in triple-negative breast cancer (TNBC). Here, we report the discovery and biological evaluation of a highly potent small-molecule antagonist of exportin-1, LFS-1107. We ascertained that exportin-1 (also named as CRM1) is a main cellular target of LFS-1107 by nuclear export functional assay, bio-layer interferometry binding assay and C528S mutant cell line. We found that LFS-1107 significantly inhibited TNBC tumor cells at low-range nanomolar concentration and LFS-1107 can selectively eliminate CD44+CD24- enriched BCSCs. We demonstrated that LFS-1107 can induce the nuclear retention of Survivin and consequent strong suppression of STAT3 transactivation abilities and the expression of downstream stemness regulators. Administration of LFS-1107 can strongly inhibit tumor growth in mouse xenograft model and eradicate BCSCs in residual tumor tissues. Moreover, LFS-1107 can significantly ablate the patient-derived tumor organoids (PDTOs) of TNBC as compared to a few approved cancer drugs. Lastly, we revealed that LFS-1107 can enhance the killing effects of chemotherapy drugs and downregulate multidrug resistance related protein targets. These new findings provide preclinical evidence of defining LFS-1107 as a promising therapeutic agent to deplete BCSCs for the treatment of TNBC.

Spatially resolved proteomic profiling identifies tumor cell CD44 as a biomarker associated with sensitivity to PD-1 axis blockade in advanced non-small-cell lung cancer.

BACKGROUND: Most patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC. METHODS: We initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively. RESULTS: Using continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK-/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted p<0.05) upregulation of PD-L1, TIM-3, ICOS, and CD40 in two independent cohorts. CONCLUSIONS: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC.

PD1 CD44 antiviral peptide as an inhibitor of the protein-protein interaction in dengue virus invasion.

Dengue virus (DENV) infection is mediated by the interaction between the virus envelope protein and cellular receptors of the host cells. In this study, we designed peptides to inhibit protein-protein interaction between dengue virus and CD44 receptor, which is one of the receptors used by DENV for entry. In silico model complexes were designed between domain III of the viral envelope protein of dengue virus 2 and the domain of human CD44 receptor using ClusPro 2.0, (https://cluspro.bu.edu/login.php), and inhibition peptides were designed with Rosetta Online-Server(http://rosie.rosettacommons.org/peptiderive). We identified one linear antiviral peptide of 18 amino acids derived from the human CD44 receptor, PD1 CD44. It did not show hemolysis or toxicity in HepG2 or BHK cell lines, nor did it stimulate the release of IL-1ß, IL-6, TNF-α, and IFN-γ, below 100 µM. It had an IC50 of 13.8 µM and maximum effective dose of 54.9 µM evaluated in BHK cells. The decrease in plaque-forming units/mL for DENV1, DENV2, DENV3, and DENV4 was 99.60%, 99.40%, 97.80%, and 70.50%, respectively, and similar results were obtained by RT-qPCR. Non-structural protein 1 release was decreased in pre- and co-treatment but not in post-treatment. Competition assays between the DN59 peptide, envelope protein, and the fragment of domain III "MDKLQLKGMSYSMCTGKF" of the viral envelope of DENV2 and PD1 CD44 showed that our peptide lost its antiviral activity. We demonstrated that our peptide decreased endosome formation, and we propose that it binds to the envelope protein of DENV, inhibiting viral invasion/fusion.

Addition of HER2 and CD44 to 18F-FDG PET-based clinico-radiomic models enhances prediction of neoadjuvant chemoradiotherapy response in esophageal cancer.

OBJECTIVES: To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. METHODS: Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2-5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. RESULTS: Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance (R2s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. CONCLUSIONS: Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. KEY POINTS: • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an often highly invasive tumor, infiltrating functionally important tissue areas. Achieving complete tumor resection and preserving functionally relevant tissue structures depends on precise identification of tumor-free resection margins during surgery. Fluorescence-guided surgery (FGS), by intraoperative detection of tumor cells using a fluorescent tracer, may guide surgical excision and identify tumor-positive resection margins. Using a literature survey on potential surface molecules followed by immunohistochemical validation, we identified CD44 variant 6 (CD44v6) as a constitutively expressed antigen in the invasion zone of HNSCC lesions. The monoclonal anti-CD44v6 antibody BIWA was labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (Indium-111) and dual-modality imaging was applied in a locally invasive tumor mouse model. BIWA accurately detected human HNSCC xenografts in mice with a tumor uptake of 54 ± 11% ID/g and invasion regions with an accuracy of 94%. When dissected under clinical-like conditions, tumor remnants approximately 0.7 mm in diameter consisting of a few thousand cells were identified by fluorescence imaging, resulting in reliable dissection of invasive microregions. These data indicate that CD44v6 is a suitable target for reliable near-infrared detection and FGS of invasive HNSCC lesions in vivo.

The biology and role of CD44 in cancer progression: therapeutic implications.

CD44, a non-kinase transmembrane glycoprotein, is overexpressed in several cell types including cancer stem cells and frequently shows alternative spliced variants that are thought to play a role in cancer development and progression. Hyaluronan, the main ligand for CD44, binds to and activates CD44 resulting in activation of cell signaling pathways that induces cell proliferation, increases cell survival, modulates cytoskeletal changes, and enhances cellular motility. The different functional roles of CD44 standard (CD44s) and specific CD44 variant (CD44v) isoforms are not fully understood. CD44v contain additional peptide motifs that can interact with and sequester growth factors and cytokines at the cell surface thereby functioning as coreceptors to facilitate cell signaling. Moreover, CD44v were expressed in metastasized tumors, whereas switching between CD44v and CD44s may play a role in regulating epithelial to mesenchymal transition (EMT) and in the adaptive plasticity of cancer cells. Here, we review current data on the structural and functional properties of CD44, the known roles for CD44 in tumorigencity, the regulation of CD44 expression, and the potential for targeting CD44 for cancer therapy.

Ascaris lumbricoides: Capacidad de unión a hialuronato / Ascaris lumbricoides: Hyaluronan binding capacity

El ácido hialurónico tiene importantes funciones en los procesos inflamatorios y de reparación tisular. Sus principales receptores son CD44, RHAMM e ICAM-I. Debido a la variedad de estrategias utilizadas por los parásitos para evadir la respuesta inmune del hospedador y considerando las múltiples funciones e importancia fisiológica del ácido hialurónico, el objetivo de este trabajo fue determinar si Ascaris lumbricoides tiene capacidad de unión a hialuronato. Se trabajó con 36 extractos de A. lumbricoides obtenidos por remoción quirúrgica de la cutícula y ruptura mecánica refrigerada. Se modificó la técnica de detección de CD44 soluble en suero por Inhibición de la Agregación por adhesión. Los resultados demostraron que 23 de los 36 extractos estudiados tenían capacidad de unión a hialuronato. Este hecho podría deberse a la existencia de algún receptor en el parásito que une hialuronato y que eventualmente competiría con los receptores habituales del hospedador. A. lumbricoides podría utilizar este mecanismo para evadir la respuesta inmune.

Hyaluronan acid has important functions in inflammatory and tissue reparation processes. Its main receptors are CD44, RHAMM and ICAM-I. Owing to the varied strategies of the parasites to evade the host´s immune response, and also considering the multiple functions and physiological importance of hyaluronan acid, the aim was to study if Ascaris lumbricoides has hyaluronan binding capacity. Extracts of A. lumbricoides were prepared by surgical remotion of the cuticle and refrigerated mechanical rupture. The study was done on 36 parasite extracts. The test of serum soluble CD44 Detection by Aggregation Inhibition was modified. Of the 36 extracts studied, 23 presented hyaluronan binding capacity. This fact can possibly be due to the existence of a receptor with hyaluronan acid binding capacity in the parasite, which eventually might compete with the usual receptors of the host. A. lumbricoides might use this mechanism to evade the immune response.

Antitumor effects of vaccination with dendritic cells transfected with modified receptor for hyaluronan-mediated motility mRNA in a mouse glioma model.

OBJECT: The receptor for hyaluronan-mediated motility (RHAMM) is frequently overexpressed in brain tumors and was recently identified as an immunogenic antigen by using serological screening of cDNA expression libraries. In this study, which was conducted using a mouse glioma model, the authors tested the hypothesis that vaccination with dendritic cells transfected with RHAMM mRNA induces strong immunological antitumor effects. METHODS: The authors constructed a plasmid for transduction of the mRNAs transcribed in vitro into dendritic cells, which were then used to transport the intracellular protein RHAMM efficiently into major histocompatibility complex class II compartments by adding a late endosomal-lysosomal sorting signal to the RHAMM gene. The dendritic cells transfected with this RHAMM mRNA were injected intraperitoneally into the mouse glioma model 3 and 10 days after tumor cell implantation. The antitumor effects of the vaccine were estimated by the survival rate, histological analysis, and immunohistochemical findings for immune cells. Mice in the group treated by vaccination therapy with dendritic cells transfected with RHAMM mRNA survived significantly longer than those in the control groups. Immunohistochemical analysis revealed that greater numbers of T lymphocytes containing T cells activated by CD4+, CD8+, and CD25+ were found in the group vaccinated with dendritic cells transfected with RHAMM mRNA. CONCLUSIONS: These results demonstrate the therapeutic potential of vaccination with dendritic cells transfected with RHAMM mRNA for the treatment of malignant glioma.