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Journal For Immunotherapy of Cancer May 2024Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer...
BACKGROUND
Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment.
METHODS
We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand.
RESULTS
We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4 and CD8 TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models.
CONCLUSIONS
Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation.
TRIAL REGISTRATION NUMBER
NCT00068003, NCT01174121, and NCT03412877.
Topics: Humans; Lymphocytes, Tumor-Infiltrating; Antigens, Neoplasm; Receptors, Antigen, T-Cell; Mice; Immunologic Memory; Animals; Female; Phenotype; Neoplasms
PubMed: 38816232
DOI: 10.1136/jitc-2023-008645 -
Life Science Alliance Aug 2024In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is...
In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.
Topics: Dynamins; Mitochondrial Dynamics; Humans; Single-Domain Antibodies; Mitochondria; Proteomics; Animals; Protein Binding; HeLa Cells; Mitochondrial Proteins
PubMed: 38816213
DOI: 10.26508/lsa.202402608 -
Cellular and Molecular Gastroenterology... May 2024Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known...
BACKGROUND & AIMS
Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.
METHODS
We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.
RESULTS
Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 upregulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.
CONCLUSIONS
IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration.
PubMed: 38815928
DOI: 10.1016/j.jcmgh.2024.101366 -
The Journal of Biological Chemistry May 2024ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice...
ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
PubMed: 38815867
DOI: 10.1016/j.jbc.2024.107418 -
The Journal of Biological Chemistry May 2024While the deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied....
While the deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied. Surprisingly, our findings demonstrate that ATXN3 exerts an anti-tumor effect in human colon cancers through potentiating Galectin-9-induced apoptosis. CRISPR-mediated ATXN3 deletion unexpectedly intensified colon cancer growth both in vitro and in xenograft colon cancers. At the molecular level, we identified ATXN3 as a bona fide deubiquitinase specifically targeting Galectin-9, as ATXN3 interacted with and inhibited Galectin-9 ubiquitination. Consequently, targeted ATXN3 ablation resulted in reduced Galectin-9 protein expression, thereby diminishing Galectin-9-induced colon cancer apoptosis and cell growth arrest. The ectopic expression of Galectin-9 fully reversed the growth of ATXN3-null colon cancer in mice. Furthermore, immunohistochemistry staining revealed a significant reduction in both ATXN3 and Galectin-9 protein expression, along with a positive correlation between them in human colon cancer. Our study identifies the first Galectin-9-specific deubiquitinase and unveils a tumor-suppressive role of ATXN3 in human colon cancer.
PubMed: 38815863
DOI: 10.1016/j.jbc.2024.107415 -
The Journal of Biological Chemistry May 2024The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel...
The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.
PubMed: 38815861
DOI: 10.1016/j.jbc.2024.107417 -
Molecular Metabolism May 2024p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of...
OBJECTIVE
p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of liver TAp63 in the progression of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis is unknown.
METHODS
We evaluated the hepatic p63 levels in different mouse models of steatohepatitis with fibrosis induced by diet. Next, we used virogenetic approaches to manipulate the expression of TAp63 in adult mice under diet-induced steatohepatitis with fibrosis and characterized the disease condition. Finally, we performed proteomics analysis in mice with overexpression and knockdown of hepatic TAp63.
RESULTS
Levels of TAp63, but not of ΔN isoform, are increased in the liver of mice with diet-induced steatohepatitis with fibrosis. Both preventive and interventional strategies for the knockdown of hepatic TAp63 significantly ameliorated diet-induced steatohepatitis with fibrosis in mice fed a methionine- and choline- deficient diet (MCDD) and choline deficient and high fat diet (CDHFD). The overexpression of hepatic TAp63 in mice aggravated the liver condition in mice fed a CDHFD. Proteomic analysis in the liver of these mice revealed alteration in multiple proteins and pathways, such as oxidative phosphorylation, antioxidant activity, peroxisome function and LDL clearance.
CONCLUSIONS
These results indicate that liver TAp63 plays a critical role in the progression of diet-induced steatohepatitis with fibrosis, and its inhibition ameliorates the disease.
PubMed: 38815625
DOI: 10.1016/j.molmet.2024.101962 -
Clinics (Sao Paulo, Brazil) May 2024To investigate the influence of aerobic exercise on myocardial injury, NF-B expression, glucolipid metabolism and inflammatory factors in rats with Coronary Heart...
OBJECTIVE
To investigate the influence of aerobic exercise on myocardial injury, NF-B expression, glucolipid metabolism and inflammatory factors in rats with Coronary Heart Disease (CHD) and explore the possible causative role.
METHODS
45 Sprague Dawley® rats were randomized into model, control and experimental groups. A high-fat diet was adopted for generating a rat CHD model, and the experimental group was given a 4-week aerobic exercise intervention. ECG was utilized to evaluate the cardiac function of the rats; HE staining to evaluate the damage of myocardial tissue; TUNEL staining to evaluate cardiomyocyte apoptosis level; ELISA to assay the contents of inflammatory factors and glucolipid metabolism in cardiomyocytes; qPCR to assay IB- and NF-B mRNA expression; Western-blot to assay the apoptosis-related proteins and NF-B signaling pathway-related proteins expressions in myocardial tissue.
RESULTS
In contrast to the model group, aerobic exercise strongly improved the rat's cardiac function and glucolipid metabolism (p < 0.01), enhanced IL-10 content, Bcl-2/Bax level as well as IB- protein and mRNA expression (p < 0.01), and reduced myocardial injury and cardiomyocyte apoptosis, the contents of IL-6, IL-1 and TNF-, Caspase 3 level, NF-B mRNA and protein expression and p-p38 and p-STAT3 expressions (p < 0.01).
CONCLUSION
Aerobic exercise can not only effectively reduce myocardial injury, the release of inflammatory factors and NF-B expression in CHD rats, but also improve cardiac function and glucolipid metabolism. Its mechanism is likely to be related to the inhibition of the NF-B signaling pathway.
PubMed: 38815541
DOI: 10.1016/j.clinsp.2024.100386 -
PLoS Neglected Tropical Diseases May 2024Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are...
BACKGROUND
Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated.
METHODOLOGY/PRINCIPAL FINDINGS
C2C12 myoblast cells were exposed to BjsuV (12.5 μg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кβ, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-β, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-β, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP.
CONCLUSION/SIGNIFICANCE
These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.
PubMed: 38814992
DOI: 10.1371/journal.pntd.0012227 -
PLoS Pathogens May 2024The eggs of the blood fluke Schistosoma mansoni are the main cause of the clinical manifestations of chronic schistosomiasis. After laying, the egg "winners" attach to...
The eggs of the blood fluke Schistosoma mansoni are the main cause of the clinical manifestations of chronic schistosomiasis. After laying, the egg "winners" attach to the endothelium of the mesenteric vein and, after a period of development, induce the growth of a small granuloma, which facilitates their passage to the intestinal lumen. Egg "losers" carried by the bloodstream to non-specific tissues also undergo full development and induce large granuloma formation, but their life ends there. Although these trapped eggs represent a dead end in the parasite life cycle, the vast majority of studies attempting to describe the biology of the S. mansoni eggs have studied these liver-trapped "losers" instead of migrating intestinal "winners". This raises the fundamental question of how these eggs differ. With robust comparative transcriptomic analysis performed on S. mansoni eggs isolated 7 weeks post infection, we show that gene expression is critically dependent on tissue localization, both in the early and late stages of development. While mitochondrial genes and venom allergen-like proteins are significantly upregulated in mature intestinal eggs, well-described egg immunomodulators IPSE/alpha-1 and omega-1, together with micro-exon genes, are predominantly expressed in liver eggs. In addition, several proteases and protease inhibitors previously implicated in egg-host interactions display clear tissue-specific gene expression patterns. These major differences in gene expression could be then reflected in the observed different ability of liver and intestinal soluble egg antigens to elicit host immune responses and in the shorter viability of miracidia hatched from liver eggs. Our comparative analysis provides a new perspective on the biology of parasite's eggs in the context of their development and tissue localization. These findings could contribute to a broader and more accurate understanding of parasite eggs interactions with the host, which have historically been often restricted to liver eggs and sometimes inaccurately generalized.
PubMed: 38814989
DOI: 10.1371/journal.ppat.1012268