Among the reprogrammed metabolic pathways described in cancer stem cells, aberrant lipid metabolism has recently drawn increasing attention. Our study explored the contribution of fatty acids (FA) in the regulation of stem-like features in intrahepatic cholangiocarcinoma (iCCA). We previously identified a functional stem-like subset in human iCCA by using a three-dimensional sphere (SPH) model in comparison to parental cells grown as monolayers (MON). In this study, quantification of intracellular free FA and lipidomic analysis (triacylglycerol [TAG] composition, de novo synthesis products) was performed by Liquid chromatography–mass spectrometry (LC–MS); quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS), respectively, in both SPH and MON cultures. Stem-like SPH showed a superior content of free FA (citric, palmitic, stearic, and oleic acids) and unsaturated TAG. Molecularly, SPH showed upregulation of key metabolic enzymes involved in de novo FA biosynthesis (AceCS1, ACLY, ACAC, FASN, ACSL1) and the mTOR signalling pathway. In patients with iCCA (n = 68), tissue expression of FASN, a key gene involved in FA synthesis, correlated with 5-year overall survival. Interference with FASN activity in SPH cells through both specific gene silencing (siRNA) or pharmacological inhibition (orlistat) decreased sphere-forming ability and expression of stem-like markers. In a murine xenograft model obtained by injection of iCCA-SPH cells, FASN inhibition by orlistat or injection of FASN-silenced cells significantly reduced tumour growth and expression of stem-like genes. Altered FA metabolism contributes to the maintenance of a stem-like phenotype in iCCA. FASN inhibition may represent a new approach to interfere with the progression of this deadly disease. Recent evidence indicates that metabolic disorders correlate with an increased susceptibility to intrahepatic cholangiocarcinoma (iCCA). Our investigation emphasises the pivotal involvement of lipid metabolism in the tumour stem cell biology of iCCA, facilitated by the upregulation of crucial enzymes and the mTOR signalling pathway. From a clinical perspective, this underscores the dual role of FASN as both a prognostic indicator and a therapeutic target, suggesting that FASN inhibitors could enhance patient outcomes by diminishing stemness and tumour aggressiveness. These findings pave the way for novel therapeutic strategies for iCCA and shed light on its relationship with metabolic disorders such as diabetes, obesity, metabolic syndrome, and metabolic dysfunction-associated steatotic liver disease.

Pancreatic ductal adenocarcinoma (PDAC) represents an unmet medical need. Difficult/late diagnosis as well as the poor efficacy and high toxicity of chemotherapeutic drugs result in dismal prognosis. With the aim of improving the treatment outcome of PDAC, we tested the effect of combining Gemcitabine with a novel single chain bispecific antibody (scDb) targeting the cancer-specific hERG1/β1 integrin complex. First, using the scDb (scDb-hERG1-β1) in immunohistochemistry (IHC), Western blot (WB) analysis and immunofluorescence (IF), we confirmed the presence of the hERG1/β1 integrin complex in primary PDAC samples and PDAC cell lines. Combining Gemcitabine with scDb-hERG1-β1 improved its cytotoxicity on all PDAC cells tested in vitro. We also tested the combination treatment in vivo, using an orthotopic xenograft mouse model involving ultrasound-guided injection of PDAC cells. We first demonstrated good penetration of the scDb-hERG1-β1 conjugated with indocyanine green (ICG) into tumour masses by photoacoustic (PA) imaging. Next, we tested the effects of the combination at either therapeutic or sub-optimal doses of Gemcitabine (25 or 5 mg/kg, respectively). The combination of scDb-hERG1-β1 and sub-optimal doses of Gemcitabine reduced the tumour masses to the same extent as the therapeutic doses of Gemcitabine administrated alone; yielded increased survival; and was accompanied by minimised side effects (toxicity). These data pave the way for a novel therapeutic approach to PDAC, based on the combination of low doses of a chemotherapeutic drug (to minimize adverse side effects and the onset of resistance) and the novel scDb-hERG1-β1 targeting the hERG1/β1 integrin complex as neoantigen.

Targeted therapies and immunotherapies have largely improved cancer treatment in the last years. One of the most promising approaches is the induction of tumor apoptosis by TRAIL through its binding to apoptosis-inducing receptors DR4 and DR5 on the plasma membrane of target cells. However, some constraints (e.g., the short in vivo half-life and the poor activity on DR5 receptors) hinder the use of naked, soluble forms of TRAIL. Previous studies have shown that fusing TRAIL sequences with antibody-based moieties may represent a novel and efficacious strategy to overcome such hindrances. On these bases, novel TRAIL-related anticancer therapeutic strategies are being developed. In the present article, we describe a novel antibody represented by a single-chain diabody directed against a cancer-specific target, i.e., the hERG1/β1 integrin complex—scDb-hERG1-β1—fused with three TRAIL sequences. The scDb-hERG1-β1-TRAIL antibody combines the specific targeting and downregulation of cancer-specific signaling pathways by scDb-hERG1-β1 with the proapoptotic activity triggered by TRAIL. We provide substantial evidence of the efficacy of the scDb-hERG1-β1-TRAIL antibody to decrease tumor growth triggering apoptotic cell death in vitro in breast cancer cells as well as in vivo in a mouse model of triple-negative breast cancer. Being characterized by a favorable pharmacokinetic and toxicity profile, the scDb-hERG1-β1-TRAIL antibody can be proposed for the treatment of difficult-to-treat cancers, such as triple-negative breast cancer, which express the hERG1/β1 complex and TRAIL receptors.

Peptides used for ELISA assays.

A bioinformatic approach was applied to evaluate the effect of treatment with Bevacizumab on the gene expression pro-file of colorectal adenocarcinoma cells. The transcriptomic profile of Bevacizumab-adapted HCT-116 (Bev/A) colorectal adenocarcinoma cells was determined and compared with that of the corresponding control cell line by Agilent microar-ray analysis. Raw data were preprocessed, normalized, fil-tered, and subjected to a differential expression analysis us-ing standard R/Bioconductor packages (i.e., limma, RankProd). As consequence of Bevacizumab adaptation, 166 differentially expressed genes (DEGs) emerged, most of them (123) re-sulted downregulated and 43 overexpressed. The list of sta-tistically significant dysregulated genes was used as an in-put for functional overrepresentation analysis using Topp-Fun web tool. Such analysis pointed at cell adhesion, cell migration, extracellular matrix organization and angiogene-sis as the main dysregulated biological process involved in Bevacizumab-adaptation of HCT116 cells. In addition, gene set enrichment analysis was performed using GSEA, search-ing for enriched terms within the Hallmarks (H), Canonical Pathways (CP), and Gene Ontology (GO) gene sets. GO terms that showed significant enrichment included: transportome, vascularization, cell adhesion and cytoskeleton, extra cellu-lar matrix (ECM), differentiation and epithelial-mesenchymal transition (EMT), inflammation and immune response. Raw and normalized microarray data were deposited in the Gene Expression Omnibus (GEO) public repository with accession number GSE221948.(c) 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)