Theranostics最新文献

[This corrects the article DOI: 10.7150/thno.20942.].

[This corrects the article DOI: 10.7150/thno.43427.].

Background: Stem cell-like properties are known to promote the recurrence and metastasis of hepatocellular carcinoma (HCC), contributing to a poor prognosis for HCC patients. Betaine, an important phytochemical and a methyl-donor related substance, has shown protective effects against liver diseases. However, its effect on HCC stem cell-like properties and the underlying mechanisms remains uninvestigated. Methods: We measured the effects of betaine on the stem cell-like properties and malignant progression of HCC using patient-derived xenografts, cell-derived xenografts, tail vein-lung metastasis models, in vitro limiting dilution, tumor sphere formation, colony formation, and transwell assays. Mechanistic exploration was conducted using western blots, dot blots, methylated RNA immunoprecipitation-qPCR, RNA stability assays, RNA immunoprecipitation-qPCR, RNA pull-down, and gene mutation assays. Results: A cohort study of HCC found that a higher serum concentration of betaine was associated with decreased levels of stemness-related markers. Furthermore, in HCC cells and xenograft mice, betaine suppressed the stem cell-like properties of HCC by activating autophagy. Mechanistically, betaine increased the m⁶A modification in HCC by producing S-adenosylmethionine (SAM) via betaine-homocysteine S-methyltransferase (BHMT). This increase in SAM subsequently triggered autophagy by enhancing the stability of autophagy-related protein 3 (ATG3) via YTHDF1 in an m⁶A-dependent manner, thereby inhibiting the stem cell-like properties of HCC cells. Conclusions: These findings indicate that betaine inhibits the stem cell-like properties of HCC via the SAM/m⁶A/YTHDF1/ATG3 pathway. This study underscores the potential anti-tumor effects of betaine on HCC and offers novel therapeutic prospects for HCC patients.

Rationale: Mesenchymal stem cells (MSCs) possess potent immunomodulatory capability, but occasionally, clinical application of MSCs is hindered by compromised cell functionality and insufficient therapeutic efficacy. Methods: Here, well-established mouse models of dextran sulfate sodium (DSS)-induced colitis and streptozotocin (STZ)-induced type 1 diabetes (T1D) were used to evaluate therapeutic immunomodulatory effects of human umbilical cord-derived MSCs. MSCs were examined at the fifth (P5) and the fifteenth (P15) passages, and three-dimensional (3D) culture was conducted by Matrigel incorporation. A series of biochemical, histopathological and cellular assays were performed to investigate the MSC function and therapeutic performance, and immunoregulation was evaluated by in vitro co-culture with T cells and in vivo analyses of T-cell infiltration into target tissues. RNA sequencing (RNA-seq) analysis followed by immunofluorescence staining, gene expression analyses and chemical regulation were used to investigate the molecular targets. Results: MSCs lose therapeutic immunomodulatory effects after extensive expansion to P15 when cell senescence occurs. Intriguingly, 3D preconditioning of MSCs in Matrigel promotes diminished immunoregulatory capability despite extensive passages, which benefits function of P15-MSCs to modulate T-cell subsets in co-culture, suppress infiltration of pro-inflammatory T cells in the colon and pancreas tissues after infusion, ameliorate systemic inflammation, and alleviate colitis and T1D in mice. Mechanistically, 3D culture provokes transcriptomic reprogramming of MSCs toward a Yes-associated protein 1 (YAP1)-marked, Hippo signaling pathway-upregulated state with promoted release of the anti-inflammatory cytokine, transforming growth factor-beta1 (TGF-β1). Moreover, chemical regulation of YAP1 by clinically relevant drugs, verteporfin (VP) and prostaglandin E2 (PGE2), affects TGF-β1 expression and the immunomodulatory capability of MSCs during dimensional culture. Conclusions: Taken together, these findings unravel YAP1-based dimensional and chemical coordination of expanded MSC immunoregulation, which will shed light on precisely controlled translational application.

Background: Identifying biomarkers that predict immunotherapy efficacy and discovering new targets for combination therapies are critical elements for improving the prognosis of bladder cancer (BLCA) patients. Methods: Firstly, we explored the expression patterns of TBX3 in normal and pan-cancer tissues and the correlation between TBX3 and the immune microenvironment using data from multiple public databases. Then, we combined various techniques, including bulk RNA sequencing, single-cell RNA sequencing, high-throughput cytokine arrays, functional experiments, ProcartaPlex multiplex immunoassays and TissueFAXS panoramic tissue quantification assays, to demonstrate that TBX3 shapes an immunosuppressive tumor microenvironment (TME) in BLCA. Results: We identified TBX3 as a key factor associated with the immunosuppressive microenvironment in BLCA through a systematic multi-omics analysis. We found that TBX3 is primarily expressed in malignant cells, where TBX3^high tumor cells increase the secretion of TGFβ1, which promotes the infiltration of cancer-associated fibroblasts (CAFs), thereby forming an immunosuppressive microenvironment. We further demonstrated that TBX3 enhances TGFβ1 expression by binding to the TGFβ1 promoter, and blocking TGFβ1 counteracts the immunosuppressive effects of TBX3. Moreover, TBX3 reduced the cancer-killing efficiency of CD8⁺ T cells by decreasing the proportion of GZMB⁺ CD8⁺ T cells, and knocking down TBX3 combined with anti-PD-1 treatment increased CD8⁺ T cell infiltration and reduced CAFs in vivo. We also validated the inverse relationship between TBX3⁺ malignant cells and CD8⁺ T cells and the positive relationship with CAFs in tissue microarrays. Lastly, we found that TBX3 predicted immunotherapy efficacy in our real-world immunotherapy cohort and multiple public cohorts. Conclusion: In summary, TBX3 promotes BLCA progression and immunotherapy resistance by inducing an immunosuppressive microenvironment, and targeting TBX3 could enhance the efficacy of immunotherapy for BLCA.

The Ser/Thr kinase RAF, particularly BRAF isoform is a dominant target of oncogenic mutations and many mutations have been identified in various cancers. However, how these mutations except V600E evade the regulatory machinery of RAF protein and hence trigger its oncogenicity remains unclear. Methods: In this study, we used mutagenesis, peptide affinity assay, immunoprecipitation, immunoblot, and complementary split luciferase assay as well as mouse xenograft tumour model to investigate how the function of RAF is cooperatively regulated by Cdc37/Hsp90 chaperones and 14-3-3 scaffolds and how this regulatory machinery is evaded by prevalent non-V600 mutations. Results: We found that Cdc37/Hsp90 chaperones engaged with mature BRAF proteins promoted together with 14-3-3 scaffolds a switch of BRAF proteins from active open dimers into inactive close monomers. Most non-V600 mutations were enriched on or around the Cdc37/Hsp90-binding segments of BRAF, which impair association of CDc37/Hsp90 chaperones with BRAF and hence trap BRAF in active open conformation favouring dimerization. These BRAF mutants with high dimer propensity sustained a prolonged ERK signaling, and were effectively targeted by RAF dimer breaker plx8394 in vitro and in vivo. In contrast, CRAF and ARAF existed as immature monomers highly packaged with Cdc37/Hsp90 chaperones, which will be released upon dimerization driven by RAS-GTP binding with their N-terminus as well as 14-3-3 scaffold association with their C-terminus. Mature CRAF and ARAF dimers also sustained a prolonged ERK signaling as non-V600 BRAF mutants by virtue of absence of the C-terminal Cdc37/Hsp90-binding segment. Conclusions: Cdc37/Hsp90 chaperones and 14-3-3 scaffolds cooperatively facilitate the switch of RAF proteins from open active dimers to close inactive monomers. Non-V600 mutations disrupt this regulatory machinery, and trap RAF in dimers, which could be targeted by RAF dimer breakers.

Rationale: Stress granules (SGs) are membraneless organelles that are formed in response to various stresses. Multiple cellular processes have been reported to be involved in SG formation. However, the signaling cascades that coordinate SG formation remain to be elucidated. Methods: By performing two high-content imaging-based phenomic screens, we identified multiple signaling components that form a possible signal transduction pathway that regulates SG formation. Results: We found that Sch9 and Ypk1 function in an early step of SG formation, leading to a decrease in intermediate long-chain base sphingolipids (LCBs). This further downregulates the polyubiquitin precursor protein Ubi4 through upregulating the deubiquitinase Ubp3. Decreased levels of cellular free ubiquitin may subsequently facilitate Lsm7 phase separation and thus trigger SG formation. Conclusion: The signaling pathway identified in this work, together with its conserved components, provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.

Rationale: Fecal microbiota transplantation (FMT) is advantageous for treating intractable diseases via the microbiota-gut-organ axis. However, invasive administration of gut microbiota via nasal feeding tubes limits the widespread application of FMT. Here, we attempted to develop a novel strategy to deliver gut microbiota using nanocapsules. Methods: Single-cell nanocapsules were fabricated within 1 h by layer-by-layer assembly of silk fibroin and phosphatidylcholine to generate a protective nanoshell on the cell surface of complicated microbiota. The physical properties of the microbiota nanocapsules were analyzed. The protective effects of nanocapsules on the gastrointestinal tract were analyzed both in vitro and in vivo. The efficacy of FMT assisted by single-cell nanocapsules (NanoFMT) was evaluated using the inflammatory response, gut microbiota balance, and histopathological analysis in animal model. Results: The nanocapsules achieved a good coating ratio for a single type of microbe and complex microbiota, resulting in a remarkable increase in the survival rate of microbes in the gastrointestinal tract. NanoFMT improved the diversity and abundance of the gut microbiota better than common FMT in germ-free mice. Moreover, NanoFMT alleviated intestinal inflammation and positively reversed the microbiota balance in a mouse model of colitis compared with common FMT, assisted by the inherent anti-inflammatory effects of silk fibroin and phosphatidylcholine. Conclusions: Considering its rapid preparation, convenient delivery, and perfect therapeutic effect, we anticipate that NanoFMT may be a promising clinical candidate for next-generation FMT treatment.

Solid tissue biopsy is fundamental in guiding surgeons during intraoperative and peri-operative management of cancer patients. However, conventional histopathologic methods depend heavily on the expertise of trained pathologists, facing challenges in accuracy and efficiency. Methods: Here, we show that unbiased labeling of proteins within tissue sections using tumor-selective dyes enhances tumor-specific signals, enabling robust and accurate differentiation of tumors from normal tissues in less than 45 min. This diagnostic approach combines a tumor-selective dye labeling strategy and a three-dimensional (3D) histological electrophoresis separation strategy to visualize protein differences between tissues and exclude off-target interference. Results: We successfully diagnose and delineate malignant tissue from frozen and fresh surgical specimens from 34 patients across six types of cancer (mean AUC = 0.93). Furthermore, we apply this method to distinguish different histological characteristics in liver cancer surgical specimens, as well as identify and quantify the degree of inflammation in tumor-surrounding tissues. Conclusion: This rapid, accurate, unbiased, and marker-free approach may enhance intraoperative detection of multiple types of tumor specimens.

Rationale: Silybum marianum is used to protect against degenerative liver damage. The molecular mechanisms of its bioactive component, silybin, remained enigmatic, although membrane-stabilizing properties, modulation of membrane protein function, and metabolic regulation have been discussed for decades. Methods: Experiments were performed with hepatocyte cell lines and primary monocytes in vitro under both basal and stressed conditions, and in mice in vivo. Quantitative lipidomics was used to detect changes in phospholipids and triglycerides. Key findings were confirmed by Western blotting, quantitative PCR, microscopy, enzyme activity assays, metabolic flux studies, and functional relationships were investigated using selective inhibitors. Results: We show that specifically the stereoisomer silybin A decreases triglyceride levels and lipid droplet content, while enriching major phospholipid classes and maintaining a homeostatic phospholipid composition in human hepatocytes in vitro and in mouse liver in vivo under normal and pre-disease conditions. Conversely, in cell-based disease models of lipid overload and lipotoxic stress, silybin treatment primarily depletes triglycerides. Mechanistically, silymarin/silybin suppresses phospholipid-degrading enzymes, induces phospholipid biosynthesis to varying degrees depending on the conditions, and down-regulates triglyceride remodeling/biosynthesis, while inducing complex changes in sterol and fatty acid metabolism. Structure-activity relationship studies highlight the importance of the 1,4-benzodioxane ring configuration of silybin A in triglyceride reduction and the saturated 2,3-bond of the flavanonol moiety in phospholipid accumulation. Enrichment of hepatic phospholipids and intracellular membrane expansion are associated with a heightened biotransformation capacity. Conclusion: Our study deciphers the structural features of silybin contributing to hepatic lipid remodeling and suggests that silymarin/silybin protects the liver in individuals with mild metabolic dysregulation, involving a lipid class switch from triglycerides to phospholipids, whereas it may be less effective in disease states associated with severe metabolic dysregulation.