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Abstract In our previous phase II T1219 trial for advanced biliary tract cancer (ABTC), the combination of nivolumab with modified gemcitabine and S-1 exhibited promising efficacy, while the programmed-death-ligand-1 (PD-L1) expression did not predict chemoimmunotherapy efficacy. Lymphocyte-activation-gene-3 (LAG-3), a negative immune checkpoint, is frequently co-expressed with PD-L1. This study assessed the predictive value of LAG-3 expression in ABTC patients who received chemoimmunotherapy. We analyzed 44 formalin-fixed ABTC samples using immunohistochemical staining for PD-L1 and LAG-3 and correlated them with the clinical efficacy of chemoimmunotherapy. Digital spatial profiling was conducted in selected regions of interest to examine immune cell infiltration and checkpoint expression in six cases. Three public BTC datasets were used for analysis: TCGA-CHOL, GSE32225, and GSE132305. LAG-3 positivity was observed in 38.6% of the ABTC samples and was significantly correlated with PD-L1 positivity (P < 0.001). The objective response rate (ORR) was significantly higher in LAG-3-positive tumors than in LAG-3-negative tumors (70.6% vs. 33.3%, P = 0.029). The LAG-3 expression level was associated with an increased ORR (33%, 58%, and 100% for LAG-3 < 1%, 1–9%, and ≥ 10%, respectively; P = 0.018) and a deeper therapeutic response (20.1%, 38.6%, and 57.6% for the same respective groups; P = 0.04). LAG-3 expression is positively correlated with the expression of numerous immune checkpoints. Enrichment of CD8+ T cells was observed in LAG-3-positive BTC, indicating that LAG-3 expression may serve as a biomarker for identifying immune-inflamed tumors and predicting the therapeutic response to chemoimmunotherapy in ABTC.

Abstract Transforming growth factor (TGF)-β signaling is critical for epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis. Disruption of Smad-depednent TGF-β signaling has been shown in CRC cells. However, TGF-β receptor remains expressed on CRC cells. Here, we investigated whether the cooperation between tumor-associated N-glycosylation and a glycan-binding protein modulated the TGF-β-driven signaling and metastasis of CRC. We showed that galectin-8, a galactose-binding lectin, hampered TGF-β-induced EMT by interacting with the type II TGF-β receptor and competing with TGF-β binding. Depletion of galectin-8 promoted the migration of CRC cells by increasing TGF-β-receptor-mediated RAS and Src signaling, which was attenuated after recombinant galectin-8 treatment. Treatment with recombinant galectin-8 also induces JNK-dependent apoptosis in CRC cells. The anti-migratory effect of galectin-8 depended on β4-galactosyltransferase-I (B4GALT1), an enzyme involved in N-glycan synthesis. Increased B4GALT1 expression was observed in clinical CRC samples. Depletion of B4GALT1 reduced the metastatic potential of CRC cells. Furthermore, inducible expression of galectin-8 attenuated tumor development and metastasis of CRC cells in an intra-splenic injection model. Our results thus demonstrate that galectin-8 alters non-canonical TGF-β response in CRC cells and suppresses CRC progression.

Background Cholangiocarcinoma is a challenging malignancy with limited responses to conventional therapies, particularly immune checkpoint inhibitor therapy. Tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) are key components of the tumor microenvironment (TME) and have been implicated in the immune response to cancer. However, the role and difference of TLSs and TILs in patients with cholangiocarcinoma remains unclear. This study elucidates their contributions to the TME.Methods We examined 16 tumor samples from a single-arm, phase II trial of nivolumab plus modified gemcitabine and S-1 and various datasets. Immunohistochemistry and RNA sequencing were employed to assess TLSs and TILs presence and activity. Differential gene expression and signature of immune cell composition were examined by GeoMx Digital Spatial Profiler and Cancer Transcriptome Altas analysis.Results TLS-positive (N=7) patients demonstrated significantly better immunotherapy outcomes compared with TLS-negative (N=9) patients, including higher objective response rates (71% vs 0%) and disease control rates (100% vs 67%). The presence of TLSs correlated with improved progression-free and overall survival (p=0.03). TLSs were associated with “inflamed” tumors characterized by substantial immune infiltration, particularly involving T and B cells. Gene expression analyses identified significant upregulation of B cell-related genes in TLSs. Additionally, TLSs exhibited higher properties of memory B cells and myeloid dendritic cells but lower levels of innate immune cells compared with TILs. T cells within TLSs showed elevated expression of precursor-exhausted-related genes and lower cytotoxicity signature. Furthermore, TILs in TLS-positive tumors had higher levels of exhaustion signatures compared with TILs in TLS-negative tumors. Clinical data corroborated these findings, with higher PD-L1 and LAG-3 expression in TLS-positive tumors.Conclusion Our findings revealed that TILs in TLS-positive tumors have more exhausted T cell signature and PD-1 and LAG-3 protein expression in CCA which support our clinical finding. TLSs can predict favorable immunotherapy responses in patients with cholangiocarcinoma, highlighting their potential as a biomarker and therapeutic target to enhance treatment efficacy.

Abstract Glioblastoma (GBM) is a type of brain cancer categorized as a high-grade glioma. GBM is characterized by limited treatment options, low patient survival rates, and abnormal serotonin metabolism. Previous studies have investigated the tumor suppressor function of aldolase C (ALDOC), a glycolytic enzyme in GBM. However, it is unclear how ALDOC regulates production of serotonin and its associated receptors, HTRs. In this study, we analyzed ALDOC mRNA levels and methylation status using sequencing data and in silico datasets. Furthermore, we investigated pathways, phenotypes, and drug effects using cell and mouse models. Our results suggest that loss of ALDOC function in GBM promotes tumor cell invasion and migration. We observed that hypermethylation, which results in loss of ALDOC expression, is associated with serotonin hypersecretion and the inhibition of PPAR-γ signaling. Using several omics datasets, we present evidence that ALDOC regulates serotonin levels and safeguards PPAR-γ against serotonin metabolism mediated by 5-HT, which leads to a reduction in PPAR-γ expression. PPAR-γ activation inhibits serotonin release by HTR and diminishes GBM tumor growth in our cellular and animal models. Importantly, research has demonstrated that PPAR-γ agonists prolong animal survival rates and increase the efficacy of temozolomide in an orthotopic brain model of GBM. The relationship and function of the ALDOC-PPAR-γ axis could serve as a potential prognostic indicator. Furthermore, PPAR-γ agonists offer a new treatment alternative for glioblastoma multiforme (GBM).

Halloysite nanotubes (HNTs) have a layered structure of clay silicate minerals and a tubular shape, which is suitable for the uniform loading of small substrates and drug molecules. The inner diameter of HNTs with an acidic solvent is selectively etched to increase the loading capacity of magnetic iron–platinum (FePt) nanoparticles. The FePt nanoparticles and etched HNTs (eHNT) are then composited by vacuum decompression. The resulting product is named FePt@eHNT and is used as a contrast agent for T2‐weighted magnetic resonance imaging. According to a comprehensive analysis of the material and its magnetic properties, by adding different proportions of HNTs before and after modification, the saturation magnetization can reach 23.769 emu g−1, which is higher than that of the composite materials studied in previous studies. This is because the tubular structure promotes the orderly displacement of the FePt nanoparticles under three‐dimensional space constraints and the uniform effect of the magnetic field. In addition, the magnetothermal effect of the composite material is observed and its potential as an imaging agent is investigated. In this study, the enhancement of its ferromagnetism and its potential to become a multifunctional composite material for applications in drug delivery, magnetic hyperthermia, and bioimaging is demonstrated.

Abstract Epithelial‐mesenchymal transition (EMT) is associated with tumorigenesis and drug resistance. The Rab superfamily of small G‐proteins plays a role in regulating cell cytoskeleton and vesicle transport. However, it is not yet clear how the Rab family contributes to cancer progression by participating in EMT. By analysing various in silico datasets, we identified a statistically significant increase in RAB31 expression in the oxaliplatin‐resistant group compared to that in the parental or other chemotherapy drug groups. Our findings highlight RAB31's powerful effect on colorectal cancer cell lines when compared with other family members. In a study that analysed multiple online meta‐databases, RAB31 RNA levels were continually detected in colorectal tissue arrays. Additionally, RAB31 protein levels were correlated with various clinical parameters in clinical databases and were associated with negative prognoses for patients. RAB31 expression levels in all three probes were calculated using a computer algorithm and were found to be positively correlated with EMT scores. The expression of the epithelial‐type marker CDH1 was suppressed in RAB31 overexpression models, whereas the expression of the mesenchymal‐type markers SNAI1 and SNAI2 increased. Notably, RAB31‐induced EMT and drug resistance are dependent on extracellular vesicle (EV) secretion. Interactome analysis confirmed that RAB31/AGR2 axis‐mediated exocytosis was responsible for maintaining colorectal cell resistance to oxaliplatin. Our study concluded that RAB31 alters the sensitivity of oxaliplatin, a supplementary chemotherapy approach, and is an independent prognostic factor that can be used in the treatment of colorectal cancer.

Abstract Cancer progression is influenced by junctional adhesion molecule (JAM) family members. The relationship between JAM family members and different types of cancer was examined using The Cancer Genome Atlas dataset. mRNA levels of the F11R (F11 receptor) in tumours were inversely correlated to the expression of JAM‐2 and JAM‐3. This relationship was unique to breast cancer (BCa) and was associated with poor prognosis (p = 0.024, hazard ratio = 1.44 [1.05–1.99]). A 50‐gene molecular signature (prediction analysis of microarray 50) was used to subtype BCa. F11R mRNA expression significantly increased in human epidermal growth factor receptor 2 (HER2)‐enriched (p = 0.0035) and basal‐like BCa tumours (p = 0.0005). We evaluated F11R protein levels in two different compositions of BCa subtype patient tissue array cohorts to determine the relationship between BCa subtype and prognosis. Immunohistochemistry staining revealed that a high F11R protein level was associated with poor overall survival (p

Rab GTPase 3C (RAB3C) is a peripheral membrane protein that is involved in membrane trafficking (vesicle formation) and cell movement. Recently, researchers have noted the exocytosis of RAB proteins, and their dysregulation is correlated with drug resistance and the altered tumor microenvironment in tumorigenesis. However, the molecular mechanisms of exocytotic RABs in the carcinogenicity of colorectal cancer (CRC) remain unknown. Researchers have used various in silico datasets to evaluate the expression profiles of RAB family members. We confirmed that RAB3C plays a key role in CRC progression. Its overexpression promotes exocytosis and is related to the resistance to several chemotherapeutic drugs. We established a proteomic dataset based on RAB3C, and found that dystrophin is one of the proteins that is upregulated with the overexpression of RAB3C. According to our results, RAB3C‐induced dystrophin expression promotes vesicle formation and packaging. A connectivity map predicted that the cannabinoid receptor 2 (CB2) agonists reverse RAB3C‐associated drug resistance, and that these agonists have synergistic effects when combined with standard chemotherapy regimens. Moreover, we found high dystrophin expression levels in CRC patients with poor survival outcomes. A combination of the dystrophin and RAB3C expression profiles can serve as an independent prognostic factor in CRC and is associated with several clinicopathological parameters. In addition, the RAB3C–dystrophin axis is positively correlated with the phosphatidylinositol 4,5‐bisphosphate 3‐kinase catalytic subunit alpha isoform (PIK3CA) genetic alterations in CRC patients. These findings can be used to provide novel combined therapeutic options for the treatment of CRC.

Abstract Cholangiocarcinoma (CCA) is a subtype of bile duct cancer usually diagnosed late with a low survival rate and no satisfactorily systemic treatment. Recently, regorafenib has been accepted as a second-line treatment for CCA patients. In this study, we investigated the potential signal transduction pathways mediated by regorafenib. We established a transcriptomic database for regorafenib-treated CCA cells using expression microarray chips. Our data indicate that regorafenib inhibits yes-associated protein 1 (YAP1) activity in various CCA cells. In addition, we demonstrated that YAP1 regulates epithelial-mesenchymal transition (EMT)-related genes, including E-cadherin and SNAI2. We further examined YAP1 activity, phosphorylation status, and expression levels of YAP1 downstream target genes in the regorafenib model. We found that regorafenib dramatically suppressed these events in CCA cells. Moreover, in vivo results revealed that regorafenib could significantly inhibit lung foci formation and tumorigenicity. Most importantly, regorafenib and amphiregulin (AREG) neutralize antibody exhibited synergistic effects against CCA cells. In a clinical setting, patients with high YAP1 and EMT expression had a worse survival rate than patients with low YAP1, and EMT expression did. In addition, we found that YAP1 upregulated the downstream target amphiregulin in CCA. Our findings suggest that AREG neutralizing antibody antibodies combined with regorafenib can reverse the CCA metastatic phenotype and EMT in vitro and in vivo. These findings provide novel therapeutic strategies to combat the metastasis of CCA.

Abstract Pulmonary metastasis occurring via the colonization of circulating cancer stem cells is a major cause of oral squamous cell carcinoma (OSCC)-related death. Thus, understanding the mechanism of OSCC pulmonary metastasis may provide a new opportunity for OSCC treatment. FAS, a well-known apoptosis-inducing death receptor, has multiple nonapoptotic, protumorigenic functions. Previously, we found that SAS OSCC cells with FAS receptor knockout did not affect orthotopic tumor growth or cervical lymph node metastasis. However, FAS knockout cells could not colonize in distant organs to form metastases upon intravenous injection, which hinted at the cancer stemness function of the FAS receptor. Immunohistochemistry staining indicated that the FAS receptor serves as a poor prognosis marker in OSCC patients. FAS knockout inhibited in vitro cancer spheroid formation, migration and invasion, and prevented mesenchymal transition in OSCC cells and inhibited OSCC pulmonary metastasis in vivo. To determine the regulatory mechanism by which the FAS receptor exerts its oncogenic function, we utilized cDNA microarrays and phosphoprotein arrays to discover key candidate genes and signaling pathway regulators. JAG1 expression and NOTCH pathway activation were controlled by the FAS receptor through ERK phosphorylation. Both JAG1 and NOTCH1 silencing decreased in vitro cancer spheroid formation. In OSCC cells, FAS ligand or JAG1 protein treatment increased NOTCH pathway activity, which could be abolished by FAS receptor knockout. In FAS knockout cells, restoring the NOTCH1 intracellular domain stimulated cancer spheroid formation. Both JAG1 and NOTCH1 silencing decreased in vivo OSCC growth. In conclusion, we found a novel FAS-ERK-JAG1-NOTCH1 axis that may contribute to OSCC stemness and pulmonary metastasis.

Purpose: Immune checkpoint inhibitors (ICIs) alone or in combination with chemotherapy can improve the limited efficacy of colorectal cancer (CRC) immunotherapy. CX-5461 causes substantial DNA damage and genomic instability and can increase ICIs’ therapeutic efficacies through tumor microenvironment alteration. Results: We analyzed whether CX-5461 enhances ICIs’ effects in CRC and discovered that CX-5461 causes severe DNA damage, including cytosolic dsDNA appearance, in various human and mouse CRC cells. Our bioinformatics analysis predicted CX-5461-based interferon (IFN) signaling pathway activation in these cells, which was verified by the finding that CX-5461 induces IFN-α and IFN-β secretion in these cells. Next, cGAMP, phospho-IRF3, CCL5, and CXCL10 levels exhibited significant posttreatment increases in CRC cells, indicating that CX-5461 activates the cGAS-STING-IFN pathway. CX-5461 also enhanced PD-L1 expression through STAT1 activation. CX-5461 alone inhibited tumor growth and prolonged survival in mice. CX-5461+anti-PD-1 or anti-PD-L1 alone exhibited synergistic growth-suppressive effects against CRC and breast cancer. CX-5461 alone or CX-5461+anti-PD-1 increased cytotoxic T-cell numbers and reduced myeloid-derived suppressor cell numbers in mouse spleens. Conclusions: Therefore, clinically, CX-5461 combined with ICIs for CRC therapy warrants consideration because CX-5461 can turn cold tumors into hot ones.

Aberrant metabolism has been proposed as one of the emerging hallmarks of cancer. However, the interplay between metabolic disorders and cancer metastasis remains to be defined. To explore the sophisticated metabolic processes during metastatic progression, we analyzed differentially expressed metabolic genes during the epithelial-mesenchymal transition (EMT) of lung cancer cells and defined the EMT-associated metabolic gene signature in lung adenocarcinoma patients. We found that the glycosaminoglycan (GAG)-chondroitin sulfate (CS) biosynthesis pathway was upregulated in the mesenchymal state of lung cancer and associated with poor prognosis. Notably, carbohydrate sulfotransferase 11 (CHST11), a crucial CS biosynthetic enzyme, was confirmed as a poor prognosis marker in non-small cell lung cancer (NSCLC) by immunohistochemical analysis. Moreover, forced CHST11 expression promoted invasion and metastasis, which was abolished by depleting the final product of CS biosynthesis by chondroitinase ABC treatment or active-domain negative CHST11. In vivo metastasis mouse models showed that CHST11 increased lung colonies number and sulfated mucosubstance expression. Furthermore, microarray analysis revealed ceruloplasmin (CP), which facilitated iron metabolism, was the downstream effector of CHST11. CP was upregulated by CHST11 through interferon-γ signaling pathway stimulation and related to unfavorable prognosis. Both forced CP expression and long-term iron treatment increased invasion and lung colony formation. Furthermore, we found 3-AP, an iron chelator, hampered the CHST11-induced metastasis. Our findings implicate that the novel CHST11-CP-iron axis enhances EMT and may serve as a new therapeutic target to treat NSCLC patients.

Molecular biology applications based on gold nanotechnology have revolutionary impacts, especially in diagnosing and treating molecular and cellular levels. The combination of plasmonic resonance, biochemistry, and optoelectronic engineering has increased the detection of molecules and the possibility of atoms. These advantages have brought medical research to the cellular level for application potential. Many research groups are working towards this. The superior analytical properties of gold nanoparticles can not only be used as an effective drug screening instrument for gene sequencing in new drug development but also as an essential tool for detecting physiological functions, such as blood glucose, antigen-antibody analysis, etc. The review introduces the principles of biomedical sensing systems, the principles of nanomaterial analysis applied to biomedicine at home and abroad, and the chemical surface modification of various gold nanoparticles.

Because of the unique physicochemical properties of magnetic iron-based nanoparticles, such as superparamagnetism, high saturation magnetization, and high effective surface area, they have been applied in biomedical fields such as diagnostic imaging, disease treatment, and biochemical separation. Iron-based nanoparticles have been used in magnetic resonance imaging (MRI) to produce clearer and more detailed images, and they have therapeutic applications in magnetic fluid hyperthermia (MFH). In recent years, researchers have used clay minerals, such as ceramic materials with iron-based nanoparticles, to construct nanocomposite materials with enhanced saturation, magnetization, and thermal effects. Owing to their unique structure and large specific surface area, iron-based nanoparticles can be homogenized by adding different proportions of ceramic minerals before and after modification to enhance saturation magnetization. In this review, we assess the potential to improve the magnetic properties of iron-based nanoparticles and in the preparation of multifunctional composite materials through their combination with ceramic materials. We demonstrate the potential of ferromagnetic enhancement and multifunctional composite materials for MRI diagnosis, drug delivery, MFH therapy, and cellular imaging applications.

Cancer is a disease characterized by abnormal cell growth. According to a report published by the World Health Organization (WHO), cancer is the second leading cause of death globally, responsible for an estimated 9.6 million deaths in 2018. It should be noted that ultrasound is already widely used as a diagnostic procedure for detecting tumorigenesis. In addition, ultrasound energy can also be utilized effectively for treating cancer. By filling the interior of lipospheres with gas molecules, these particles can serve both as contrast agents for ultrasonic imaging and as delivery systems for drugs such as microbubbles and nanobubbles. Therefore, this review aims to describe the nanoparticle-assisted drug delivery system and how it can enhance image analysis and biomedicine. The formation characteristics of nanoparticles indicate that they will accumulate at the tumor site upon ultrasonic imaging, in accordance with their modification characteristics. As a result of changing the accumulation of materials, it is possible to examine the results by comparing images of other tumor cell lines. It is also possible to investigate ultrasound images for evidence of cellular effects. In combination with a precision ultrasound imaging system, drug-carrying lipospheres can precisely track tumor tissue and deliver drugs to tumor cells to enhance the ability of this nanocomposite to treat cancer.

Abstract The sense of agency refers to the feeling of initiating and controlling one’s actions and their resulting effects on the external environment. Previous studies have uncovered behavioral evidence of excessive self-attribution and, conversely, a reduction in the sense of agency in patients with schizophrenia. We hypothesize that this apparent paradox is likely to result from impairment in lower-level processes underlying the sense of agency, combined with a higher-level compensational bias. The present study employed three behavioral tasks utilizing the same stimuli and experimental design to systematically evaluate multiple factors that influence the sense of agency, including motor control, sensorimotor processing, and self-attribution. Participants’ real-time mouse movements were combined with prerecorded motions of others in ratios of 30/70, 55/45, or 80/20, with an additional angular bias of either 0° or 90°. Twenty-six patients with schizophrenia and 27 health control volunteers participated in the three tasks. Patients with schizophrenia performed significantly worse in the reaching and control detection tasks than healthy controls. However, their self-attribution in the control judgment task was comparable to that of the healthy controls. Patients with schizophrenia were impaired in motor control components and in the detection of control using sensorimotor information, but their evaluation of agency remained relatively less affected. This underscores the importance of distinguishing between different subcomponents when addressing the abnormal sense of agency in patients with schizophrenia. Subsequent cluster analysis revealed that the combined task performance accurately distinguished between the patients and healthy control participants.

Background: Brain oscillations in the delta frequency band have been linked with deep sleep and consolidation of declarative memory during sleep. However, the causal relationship of these associations remains not competely clarified, primarily due to constraints by technical limitations of brain stimulation approaches suited to induce and stabilize respective oscillatory activity in the human brain. The objective of this study was to establish a non-invasive brain stimulation protocol capable of reliably inducing, and stabilizing respective oscillatory activity in the delta frequency range. Hypothesis: We aimed to develop an efficient non-invasive brain stimulation (NIBS) protocol for delta frequency induction and stabilization via concurrent, phase-locked repetitive transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS). We hypothesized that rTMS induces oscillatory resting-state activity in the delta frequency and that tACS stabilizes this effect, as has been shown before for alpha and theta frequencies. Methods: 19 healthy participants took part in a repeated-measures experimental protocol. We applied rTMS pulses synchronized with the peak or trough phase of 0.75Hz tACS over the bilateral prefrontal cortex. Resting state EEG in eyes-open (EO) and eyes-closed (EC) conditions was recorded before, immediately after and every 10 min for up to 1 h after intervention. Results: rTMS phase-synchronized to the trough of the tACS waveform significantly increased delta frequency activity for up to 60 min in both EO and EC conditions after stimulation. The effects extended from frontal to temporal regions and this enhancement of oscillatory activity was shown to be specific for the delta frequency range. Conclusion: Concurrent, trough-synchronized 0.75 Hz rTMS combined with tACS may be a reliable protocol to induce long-lasting oscillatory activity in the delta frequency range. The results of the current study might perspectively be relevant for clinical treatment of sleep disturbances which are accompanied by pathologically altered brain oscillations, and enhancement of memory consolidation.

Abstract The face serves as a crucial cue for self-identification, while the sense of agency plays a significant role in determining our influence through actions in the environment. The current study investigates how self-identification through facial recognition may influence the perception of control via motion. We propose that self-identification might engender a belief in having control over one’s own face, leading to a more acute detection and greater emphasis on discrepancies between their actions and the sensory feedback in control judgments. We refer to the condition governed by the belief in having control as the exploitation mode. Conversely, when manipulating another individual’s face, the belief in personal control is absent. In such cases, individuals are likely to rely on the regularity between actions and sensory input for control judgments, exhibiting behaviors that are exploratory in nature to glean such information. This condition is termed the explorative mode. The study utilized a face-motion mixing paradigm, employing a deep generative model to enable participants to interact with either their own or another person’s face through facial and head movements. During the experiment, participants observed either their own face or someone else’s face (self-face vs. other-face) on the screen. The motion of the face was driven either purely by their own facial and head motion or by an average of the participant’s and the experimenter’s motion (full control vs. partial control). The results showed that participants reported a higher sense of agency over the other-face than the self-face, while their self-identification rating was significantly higher for the self-face. More importantly, controlling someone else’s face resulted in more movement diversity than controlling one’s own face. These findings support our exploration–exploitation theory: When participants had a strong belief in control triggered by the self-face, they became highly sensitive to any sensorimotor prediction errors, leading to a lower sense of agency. In contrast, when the belief of control was absent, the exploration mode triggered more explorative behaviors, allowing participants to efficiently gather information to establish a sense of agency.