Journal of Biomedical Science最新文献

Background: Triple-negative breast cancer (TNBC) has the highest mortality rate among all breast cancer subtypes. Although immunotherapy shows promise, its efficacy varies. CDK4/6 inhibitors can radiosensitize and modulate the immune system, and high-dose radiotherapy (RT) can enhance the effects of immunotherapy. This study explored the combination of RT with CDK4/6 inhibitors to improve TNBC immunotherapy by modulating the tumor microenvironment.

Methods: We assessed the radiosensitizing effects of abemaciclib (a CDK4/6 inhibitor) using clonogenic assays in three human TNBC cell lines (MDA-MB-231, MDA-MB-453, and MDA-MB-468) and two murine TNBC cell lines (4T1 and EMT6). The antitumor efficacy of the treatments (control, RT, abemaciclib, anti-PD-L1 antibody [aPD-L1], abemaciclib combined with aPD-L1, abemaciclib combined with RT, aPD-L1 combined with RT, and the triple combination of abemaciclib with aPD-L1 and RT) was evaluated in both 4T1 and EMT6 cell line-derived immunocompetent mouse models. Interferon-γ (IFN-γ) levels in mouse blood were monitored to gauge the immune response. Tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry and immunohistochemical staining.

Results: Clonogenic assays showed synergistic effects of RT and abemaciclib in all TNBC cell lines. RT increased PD-L1 expression, whereas abemaciclib did not alter PD-L1 expression. In the 4T1 and EMT6 mouse models, the triple combination treatment markedly inhibited tumor growth (P < 0.01). In the 4T1 mouse model, the triple combination group exhibited significantly greater circulating IFN-γ levels (P < 0.001) than the other groups. TIL analysis revealed a significant increase in CD4 + and CD8 + T cells and tumor-associated macrophages (P < 0.01) in the triple combination therapy group. Immunohistochemical staining confirmed increased infiltration of CD4 + T cells, CD8 + T cells, monocyte chemoattractant protein-1, CD80-, and iNOS- positive macrophages into the tumor microenvironment of this group, with a marked reduction in CD206-positive macrophages.

Conclusion: Combining CDK4/6 inhibitors with RT enhanced the antitumor effects of aPD-L1 immunotherapy against TNBC. This effect was correlated with increased IFN-γ secretion and recruitment of CD4 + and CD8 + T cells and M1 tumor-associated macrophages, leading to modulation of the tumor microenvironment.

Background: The pineal gland produces melatonin to control circadian rhythm via the final enzyme in the serotonin pathway, hydroxyindole O-methyltransferase (HIOMT). Interestingly, HIOMT is expressed by certain non-pineal cells. The main catalytically active of the three human HIOMT (hHIOMT) isoforms in pineal cells is hHIOMT345 (345 amino acids), while hHIOMT298 (298 amino acids) is the most active isoform in fibroblasts, where it converts 5-hydroxytryptophan to 5-methoxytryptophan (5-MTP). We previously demonstrated that exogenous 5-MTP protects the arteries. Nevertheless, whether vascular smooth muscle cells (VSMCs) per se synthesize 5-MTP is unknown.

Methods: We transfected primary wild-type VSMCs with different hHIOMT isoforms and treated them with inflammatory cytokines to examine hHIOMT's effects on p38 MAPK activation. Global and VSMC-specific hHIOMT transgenic mice were generated and subjected to an arterial injury model. Histological analysis was performed to evaluate intimal hyperplasia and expression of select tryptophan metabolites and their synthetic enzymes. We treated wild-type and transgenic VSMCs with interleukin-1 beta (IL-1β), with or without 5-MTP, to examine the levels of serotonin and aromatic L-amino acid decarboxylase (AADC). Serotonin's effects on VSMC functions were evaluated, and inhibitors of p38 MAPK and ERK1/2 were used to determine the signaling pathways. The effects of AADC on VSMCs were assessed by AADC knockdown or overexpression.

Results: Overexpression of the human full-length isoform of 373 amino acids (hHIOMT373) in VSMCs attenuated proinflammatory cytokine-induced p38 MAPK activation, similar to 5-MTP treatment. Global and VSMC-specific hHIOMT373 transgenic mice exhibited attenuated intimal hyperplasia following arterial injury. Intriguingly, the tryptophan metabolite serotonin and its synthetic enzyme AADC were reduced in transgenic arteries. In VSMCs, IL-1β increased AADC and serotonin levels that were mitigated by 5-MTP treatment or HIOMT overexpression via suppressing the p38 MAPK pathway. Interestingly, serotonin promoted VSMC proliferation and decreased VSMC marker levels through ERK1/2 activation. While AADC overexpression decreased VSMC contractile markers, AADC knockdown suppressed IL-1β-induced VSMC proliferation.

Conclusions: Our results unveiled a unique function of HIOMT in vascular disease. In VSMCs, hHIOMT373 reprogrammed tryptophan metabolism to increase 5-MTP and decrease serotonin levels, thereby protecting against injury-induced intimal hyperplasia. Mechanistically, HIOMT-5-MTP suppressed AADC-serotonin induction through inhibiting p38 MAPK activation.

Background: Postnatal skeletal muscle development leads to increased muscle mass, strength, and mitochondrial function, but the role of mitochondrial remodeling during this period is unclear. This study investigates mitochondrial remodeling during postnatal muscle development and examines how constitutive autophagy deficiency impacts these processes.

Methods: We initially performed a broad RNA-Seq analysis using a publicly available GEO database of skeletal muscle from postnatal day 7 (P7) to postnatal day 112 (P112) to identify differentially expressed genes. This was followed by investigation of postnatal skeletal muscle development using the mitophagy report mouse line (mt-Kiema mice), as well as conditional skeletal muscle knockout (Atg7^{f/f:Acta1-Cre}) mice.

Results: Our study observed rapid growth of body and skeletal muscle mass, along with increased fiber cross-sectional area and grip strength. Mitochondrial maturation was indicated by enhanced maximal respiration, reduced electron leak, and elevated mitophagic flux, as well as increased mitochondrial localization of autophagy and mitophagy proteins. Anabolic signaling was also upregulated, coinciding with increased mitophagy and fusion signaling, and decreased biogenesis signaling. Despite the loss of mitophagic flux in skeletal muscle-specific Atg7 knockout mice, there were no changes in body or skeletal muscle mass; however, hypertrophy was observed in type IIX fibers. This lack of Atg7 and loss of mitophagy was associated with the activation of mitochondrial apoptotic signaling as well as ubiquitin-proteasome signaling, suggesting a shift in degradation mechanisms. Inhibition of the ubiquitin-proteasome system (UPS) in autophagy-deficient skeletal muscle led to significant atrophy, increased reactive oxygen species production, and mitochondrial apoptotic signaling.

Conclusion: These results highlight the role of mitophagy in postnatal skeletal muscle development and suggest that autophagy-deficiency triggers compensatory degradative pathways (i.e., UPS) to prevent mitochondrial apoptotic signaling and thus preserve skeletal muscle integrity in developing mice.

Background: Rickettsiosis is among the deadliest vector-borne infectious diseases worldwide, in part because rickettsiae replicate within human cells, where antibodies and most drugs cannot effectively reach this obligatory intracellular pathogen. Ehrlichia chaffeensis, an emerging rickettsia, is the causative agent of human monocytic ehrlichiosis. We therefore aim to generate intrabodies (IBs), the variable domain of heavy chain of heavy-chain-only antibodies (VHHs) that bind intracellular bacterial proteins to inhibit E. chaffeensis infection.

Methods: E. chaffeensis replicates in membrane-bound vacuoles resembling early endosomes in human monocytes/macrophages. The type IV secretion system effector Ehrlichia translocated factor-2 (Etf-2) directly binds to RAB5-GTP on E. chaffeensis-containing vacuoles. Consequently, Etf-2 hinders the engagement of RAB5 GTPase-activating protein with RAB5-GTP, delays maturation of Ehrlichia vacuoles to late endosomes, thus facilitates infection. As C-terminal half of Etf-2 (Etf-2C) binds RAB5-GTP, a random synthetic library of VHHs was screened for binding to Etf-2C, and for inhibition of Etf-2 binding to RAB5 in human cells when expressed intracellularly (IBs). Positive IBs were tested for inhibition of Etf-2 functions and E. chaffeensis infection, and lipid nanoparticles-encapsulated mRNAs (mRNAs-LNP) platform was used to deliver IBs in vitro and in mice.

Results: We have identified two distinct IBs that inhibit Etf-2 binding to RAB5 and Etf-2 functions in vitro. Synthesized mRNA-LNP encoding anti-Etf-2 IBs significantly inhibited E. chaffeensis infection in cell cultures and in a mouse model.

Conclusions: The results demonstrate the feasibility of mRNA-LNP encoding IBs as intracellular probes and a precision therapy addressing underlying cause of obligatory intracellular infection.

Background: Therapeutic options against metallo-β-lactamase producing P. aeruginosa (MBL-PA) are limited due to multi-drug resistance. A jumbo phage isolated from wastewater in Greece was characterized microbiologically and genetically and evaluated for its potential as a therapeutic agent alone or in combination with antibiotics in an experimental thigh infection mouse model.

Methods: The host range of the jumbo phage vB_PaerM_AttikonH10 (AttikonH10) against 20 MBL-PA clinical isolates and 10 susceptible strains, one-step phage growth and growth curves of mid-exponential phase bacteria upon addition of the phage were analyzed. Whole-genome sequencing was performed and the de novo assembled complete phage genome was compared with other jumbo phages. In vivo pharmacokinetics in different tissues as well as the efficacy of two dosing regimens 10⁹ and 10⁶ PFU/mouse administered intraperitoneally alone and in combination with amikacin (384 mg/kg/day) was tested against an MBL-PA clinical isolate in murine thigh infection model.

Results: The phage formed small plaques in double-layer agar and demonstrated clear or semi-clear lysis in 83.3% (25/30) of P. aeruginosa clinical isolates. Growth curves showed a > 94% growth inhibition in the presence of phage even at the lowest multiplicity of infection ratio tested (10^-5). Whole genome analysis indicated a jumbo dsDNA phage with 278,406 bp (36.92% GC) belonging to Phikzvirus that is predicted to host up to 413 putative ORFs and 6 tRNA genes. No known lysogeny-associated genes, virulence factors, or antimicrobial resistance genes were identified within the genome. Phage titres 10⁴-10⁶ PFU/tissue were detected in all mouse tissues with elimination half-life of 3.4 h except in bronchoalveolar lavage where no phages were found. Only the high phage dose (10⁹ PFU/mouse) reduced bacterial load in thigh by 1.09 log₁₀ cfu/thigh compared to placebo, similar to amikacin monotherapy (1.19 log₁₀ cfu/thigh), while their combination achieved a greater reduction of 2.07 log₁₀ cfu/thigh compared to each monotherapy (p = 0.0044-0.0048).

Conclusions: The newly reported Phikzvirus jumbo phage AttikonH10 demonstrated a broad host range, strong lytic activity and synergistic effects with amikacin against MBL-PA isolates making it a candidate for phage therapy.

Pyruvate kinase M2 (PKM2) is a key enzyme involved in glycolysis, yet its role in cancer extends far beyond metabolic flux. Unlike its isoform PKM1, PKM2 exhibits unique regulatory properties due to alternative splicing and dynamic structural plasticity, enabling it to translocate into the nucleus. Once nuclear, PKM2 functions as a signal receiver, gene programmer, and metabolic modulator by acting as a co-transcriptional activator and protein kinase. In this capacity, nPKM2 (nuclear PKM2) orchestrates the transcription of genes involved in glycolysis, lipogenesis, redox homeostasis, and cell cycle progression, thereby reinforcing the Warburg effect and promoting tumor growth, metastasis, and resistance to stress. In this regard, nPKM2 can be considered as the oncogenic component of PKM2. This review consolidates current knowledge on the structural basis of PKM2 assembly and the post-translational modifications that govern its oligomeric state and nuclear import. We also explore emerging therapeutic strategies aimed at targeting nPKM2, including small-molecule modulators that stabilize its cytosolic tetrameric form or disrupt its nuclear functions. Ultimately, the multifaceted roles of nuclear PKM2 underscore its significance as a critical oncoprotein and a promising target for precision cancer therapy.

Background: CD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However, its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here, we investigated the role of CD24a, the murine CD24 gene, in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.

Methods: Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations, including myeloid-derived suppressor cells (MDSCs), natural killer cells, T cells, and macrophages, within tumors, spleens, and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.

Results: Knocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8⁺ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage-mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity, effects that were partially reversed by re-expression of CD24a. Moreover, in vivo depletion of macrophages and CD8⁺ T cells reverted the delayed tumor growth caused by CD24a knockout, underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore, in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1, CXCL5, and CXCL10, chemokines known to recruit gMDSCs, further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.

Conclusions: Our findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state, thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.

Background: Taiwan has the highest prevalence of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) globally, making them major public health concerns with significant morbidity, mortality, and healthcare burden. While genetic risk factors for kidney disease have been identified in previous studies, the contribution of rare genetic variants remains unclear.

Methods: This study utilized whole-exome sequencing (WES) to investigate the role of missense rare variants in CKD and ESKD susceptibility. Genomic data from 500 Taiwanese individuals at Taipei Medical University Hospital were included based on strict clinical diagnostic criteria, comprising 200 CKD cases, 200 ESKD cases, and 100 healthy controls. Independent validation was performed using ESKD Asian cohorts from the All of Us Research Program (AoU) (N = 222) and the Tohoku Medical Megabank Organization (ToMMo) (N = 140).

Results: We identified rare pathogenic variants in known monogenic kidney disease genes, including PKD1 and COL4A4, confirming their role in disease susceptibility. We replicated GWAS-reported genes such as SPI1, RIN3, FTO, SIPA1L3, and EEF1E1, highlighting their contribution through both common and rare variants. Beyond previously reported genes, we identified novel rare pathogenic variants in PEX1, GANAB, DYNC2H1, and PROKR2. Pathway enrichment analysis suggested that ciliopathies, inflammation, and metabolic dysfunction may contribute to kidney disease progression. Furthermore, the polygenic score (PGS) for ESKD demonstrated strong predictive utility for kidney function, with high genetic risk having a greater influence than comorbidities such as diabetes and overweight. The prediction power of ESKD PGS was further confirmed in the AoU Asian population.

Conclusions: This study provides novel insights into the genetic architecture of CKD and ESKD in the Taiwanese population, utilizing a hospital-based cohort with strict clinical diagnostic criteria to ensure precise phenotype classification. We propose that individuals with high genetic risk may benefit from earlier interventions, while those with lower PGS may be better managed through lifestyle modifications targeting comorbidities. The findings highlight the importance of preventive strategies and precision medicine in kidney disease management.

Glucose metabolism is a pivotal hub for cellular energy production and the generation of building blocks that support cell growth, survival, and differentiation. Cancer cells undergo metabolic reprogramming to sustain rapid proliferation, survive in harsh microenvironments, and resist therapies. Beyond producing energy and building blocks to meet cancer cell demands, glucose metabolism generates numerous metabolites that serve as signaling molecules, orchestrating signaling pathways and epigenetic modifications that regulate cancer cell phenotypes and immunity. In this review, we discuss how glucose, through its metabolism and direct actions, influences diverse biological processes driving cancer progression and therapeutic resistance, while also exploring metabolic vulnerabilities in cancer for therapeutic strategies.

Background: Immunomodulatory agents benefit a small percentage of patients with oral cancer (OC), a subset of head and neck cancer. Cathepsin S (CTSS), a lysosomal protease, has been frequently associated with tumor immunity. This study aimed to investigate the mechanism by which tumor CTSS affects anti-tumor immunity through the regulation of interleukin-7 (IL-7) to overcome this obstacle.

Methods: OC patients' samples were used to disclose the correlation among CTSS and CD8⁺ T cell infiltration levels. The cytokine array was used to investigate the effect of CTSS on the secretion of cytokine/chemokines. We utilized various cell biology experiments to investigate the molecular mechanism of CTSS that mediates IL-7 secretion in OC cell lines, including fluorescence resonance energy transfer, immunogold-labeled transmission electron microscopy, IL-7-enzyme-linked immunosorbent assay, immunofluorescence staining, and pull-down assay. Two syngeneic OC mice models were utilized to investigate the anti-cancer effects and the tumor immunity modulation effects of RJW-58, a CTSS activity inhibitor, and the combination with the anti-PD-1 antibody.

Results: CTSS expression was inversely correlated with CD8⁺ T-cell infiltration in clinical samples. In vivo and in vitro studies using a mouse OC tumor model showed that CTSS-knockdown inhibited tumor growth and enhanced CD8⁺ T cell proliferation. These results were counteracted by co-treatment with anti-CD8 or anti-IL-7 antibodies. CTSS inhibition also remodeled the memory CD8⁺ T cell subsets within tumor tissues in vivo. Mechanistically, CTSS inhibited IL-7 secretion by disrupting its intracellular transport route. This was achieved by recognizing the intracellular domain of the IL-7 receptor (IL-7R), which bound IL-7 in granular vesicles. RJW-58 enhanced IL-7 secretion and exerted an anti-tumor effect. RJW-58 enhanced the therapeutic effect of the anti-PD-1 antibody in syngeneic mouse models.

Conclusion: The findings indicate that CTSS negatively regulates IL-7 secretion by interacting with IL-7R. The CTSS-targeting strategy has the potential to reinvigorate IL-7-directed anti-tumor T cell immunity and enhance the therapeutic effect of the anti-PD-1 antibody.