bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2024–05–26
fifty-four papers selected by
Kıvanç Görgülü, Technical University of Munich



  1. Cancer Res. 2024 May 22.
      Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), a deadly disease with limited preventive strategies. Lifestyle interventions to decrease obesity represent a potential approach to prevent obesity-associated PDAC. Here, we examined whether decreasing obesity through physical activity (PA) and/or dietary changes could decrease inflammation in humans and prevent obesity-associated PDAC in mice. Comparison of circulating inflammatory-associated cytokines in subjects (overweight and obese) before and after a PA intervention revealed PA lowered systemic inflammatory cytokines. Mice with pancreatic-specific inducible KrasG12D expression were exposed to PA and/or dietary interventions during and after obesity-associated cancer initiation. In mice with concurrent diet-induced obesity (DIO) and KrasG12D expression, the PA intervention led to lower weight gain, suppressed systemic inflammation, delayed tumor progression, and decreased pro-inflammatory signals in the adipose tissue. However, these benefits were not as evident when obesity preceded pancreatic KrasG12D expression. Combining PA with diet-induced weight loss (DI-WL) delayed obesity-associated PDAC progression in the genetically engineered mouse model, but neither PA alone nor combined with DI-WL or chemotherapy prevented PDAC tumor growth in orthotopic PDAC models regardless of obesity status. PA led to upregulation of IL-15ra in adipose tissue. Adipose-specific overexpression of IL-15 slowed PDAC growth but only in non-obese mice. Overall, our study suggests that PA alone or combined with DI-WL can reduce inflammation and delay obesity-associated PDAC development or progression. Lifestyle interventions that prevent or manage obesity or therapies that target weight loss-related molecular pathways could prevent progression of PDAC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-1045
  2. Acta Physiol (Oxf). 2024 May 23. e14167
       AIM: To investigate systemic regulators of the cancer-associated cachexia syndrome (CACS) in a pre-clinical model for lung cancer with the goal to identify therapeutic targets for tissue wasting.
    METHODS: Using the Kras/Lkb1 (KL) mouse model, we found that CACS is associated with white adipose tissue (WAT) dysfunction that directly affects skeletal muscle homeostasis. WAT transcriptomes showed evidence of reduced adipogenesis, and, in agreement, we found low levels of circulating adiponectin. To preserve adipogenesis and restore adiponectin levels, we treated mice with the PPAR-γ agonist, rosiglitazone.
    RESULTS: Rosiglitazone treatment increased serum adiponectin levels, delayed weight loss, and preserved skeletal muscle and adipose tissue mass, as compared to vehicle-treated mice. The preservation of muscle mass with rosiglitazone was associated with increases in AMPK and AKT activity. Similarly, activation of the adiponectin receptors in muscle cells increased AMPK activity, anabolic signaling, and protein synthesis.
    CONCLUSION: Our data suggest that PPAR-γ agonists may be a useful adjuvant therapy to preserve tissue mass in lung cancer.
    Keywords:  AMPK; adiponectin; cachexia; lung cancer; muscle wasting
    DOI:  https://doi.org/10.1111/apha.14167
  3. bioRxiv. 2024 May 13. pii: 2024.05.13.593939. [Epub ahead of print]
      Lipids are essential for tumours because of their structural, energetic, and signaling roles. While many cancer cells upregulate lipid synthesis, growing evidence suggests that tumours simultaneously intensify the uptake of circulating lipids carried by lipoproteins. Which mechanisms promote the uptake of extracellular lipids, and how this pool of lipids contributes to cancer progression, are poorly understood. Here, using functional genetic screens, we find that lipoprotein uptake confers resistance to lipid peroxidation and ferroptotic cell death. Lipoprotein supplementation robustly inhibits ferroptosis across numerous cancer types. Mechanistically, cancer cells take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. Tumour GAGs are a major determinant of the uptake of both low and high density lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of surface GAGs decreases the uptake of lipoproteins, sensitizes cells to ferroptosis and reduces tumour growth in mice. We also find that human clear cell renal cell carcinomas, a distinctively lipid-rich tumour type, display elevated levels of lipoprotein-derived antioxidants and the GAG chondroitin sulfate than non-malignant human kidney. Altogether, our work identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG biosynthesis as an unexpected mediator of this process.
    DOI:  https://doi.org/10.1101/2024.05.13.593939
  4. bioRxiv. 2024 May 09. pii: 2024.05.06.592780. [Epub ahead of print]
      Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.
    DOI:  https://doi.org/10.1101/2024.05.06.592780
  5. Cold Spring Harb Perspect Med. 2024 May 21. pii: a041349. [Epub ahead of print]
      A majority of cancer research is focused on defining the cellular and molecular basis of cancer cells and the signals that control oncogenic transformation; as a consequence, we know very little about the dynamic behavior of cancer cells in vivo. To begin to view and understand the mechanisms and interactions that control cancer initiation, growth, and metastatic progression and how these processes are influenced by the microenvironment and the signals derived from it, it is essential to develop strategies that allow imaging of the cancer cells in the context of the living microenvironment. Here, we discuss emerging work designed to visualize how cancer cells function within the microenvironment to discover how these interactions act coordinately to enable aberrant growth and to understand how they could be targeted to design new approaches to intercept the disease.
    DOI:  https://doi.org/10.1101/cshperspect.a041349
  6. Gastroenterology. 2024 May 18. pii: S0016-5085(24)04931-X. [Epub ahead of print]
      Present in all eukaryotic cells, the integrated stress response (ISR) is a highly coordinated signaling network that controls cellular behavior, metabolism and survival in response to diverse stresses. The ISR is initiated when any one of four stress sensing kinases (PERK, GCN2, PKR, HRI) becomes activated to phosphorylate the protein translation initiation factor eIF2α, shifting gene expression toward a comprehensive rewiring of cellular machinery to promote adaptation. While the ISR has been shown to play an important role in the homeostasis of multiple tissues, evidence suggests that it is particularly crucial for the development and ongoing health of the pancreas. Among the most synthetically dynamic tissues in the body, the exocrine and endocrine pancreas relies heavily on the ISR to rapidly adjust cell function to meet the metabolic demands of the organism. The hardwiring of the ISR into normal pancreatic functions and adaptation to stress may explain why it is a commonly utilized pro-oncogenic and therapy-resistance mechanism in both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (PanNETs). Here we review what is known about the key roles that the ISR plays in the development, homeostasis, and neoplasia of the pancreas.
    Keywords:  diabetes; pancreatic ductal adenocarcinoma; pancreatic neuroendocrine tumors; pancreatitis
    DOI:  https://doi.org/10.1053/j.gastro.2024.05.009
  7. Nat Commun. 2024 May 18. 15(1): 4244
      Cysteine metabolism occurs across cellular compartments to support diverse biological functions and prevent the induction of ferroptosis. Though the disruption of cytosolic cysteine metabolism is implicated in this form of cell death, it is unknown whether the substantial cysteine metabolism resident within the mitochondria is similarly pertinent to ferroptosis. Here, we show that despite the rapid depletion of intracellular cysteine upon loss of extracellular cystine, cysteine-dependent synthesis of Fe-S clusters persists in the mitochondria of lung cancer cells. This promotes a retention of respiratory function and a maintenance of the mitochondrial redox state. Under these limiting conditions, we find that glutathione catabolism by CHAC1 supports the mitochondrial cysteine pool to sustain the function of the Fe-S proteins critical to oxidative metabolism. We find that disrupting Fe-S cluster synthesis under cysteine restriction protects against the induction of ferroptosis, suggesting that the preservation of mitochondrial function is antagonistic to survival under starved conditions. Overall, our findings implicate mitochondrial cysteine metabolism in the induction of ferroptosis and reveal a mechanism of mitochondrial resilience in response to nutrient stress.
    DOI:  https://doi.org/10.1038/s41467-024-48695-2
  8. Cancer Res. 2024 May 22.
      Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide, primarily due to its rapid progression. The current treatment options for PDAC are limited, and a better understanding of the underlying mechanisms responsible for PDAC progression is required to identify improved therapeutic strategies. Here, we identified FBXO32 as an oncogenic driver in PDAC. FBXO32 was aberrantly upregulated in PDAC, and high FBXO32 expression was significantly associated with an unfavorable prognosis in PDAC patients. FRG1 deficiency promoted FBXO32 upregulation in PDAC. FBXO32 promoted cell migration and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, FBXO32 directly interacted with eEF1A1 and promoted its polyubiquitination at the K273 site, leading to enhanced activity of eEF1A1 and increased protein synthesis in PDAC cells. Moreover, FBXO32-catalyzed eEF1A1 ubiquitination boosted the translation of ITGB5 mRNA and activated FAK signaling, thereby facilitating focal adhesion assembly and driving PDAC progression. Importantly, interfering with the FBXO32-eEF1A1 axis or pharmaceutical inhibition of FAK by defactinib, an FDA-approved FAK inhibitor, substantially inhibited PDAC growth and metastasis driven by aberrantly activated FBXO32-eEF1A1 signaling. Overall, this study uncovers a mechanism by which PDAC cells rely on FBXO32-mediated eEF1A1 activation to drive progression and metastasis. FBXO32 may serve as a promising biomarker for selecting eligible PDAC patients for treatment with defactinib.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-3638
  9. Elife. 2024 May 24. pii: RP95652. [Epub ahead of print]13
      The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.
    Keywords:  cancer; cancer biology; human; metabolism; tumor microenvironment
    DOI:  https://doi.org/10.7554/eLife.95652
  10. Cell Mol Life Sci. 2024 May 22. 81(1): 227
      Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.
    Keywords:  Amyloid precursor protein; Autophagy; Bulk proteolysis; Cathepsins; Lysosome; Protease network; Proteolysis; Proteomics
    DOI:  https://doi.org/10.1007/s00018-024-05274-4
  11. Cold Spring Harb Perspect Med. 2024 May 21. pii: a041736. [Epub ahead of print]
      In the nearly 50 years since the original models of cancer first hit the stage, mouse models have become a major contributor to virtually all aspects of cancer research, and these have evolved well beyond simple transgenic or xenograft models to encompass a wide range of more complex models. As the sophistication of mouse models has increased, an explosion of new technologies has expanded the potential to both further develop and apply these models to address major challenges in cancer research. In the current era, cancer modeling has expanded to include nongermline genetically engineered mouse models (GEMMs), patient-derived models, organoids, and adaptations of the models better suited for cancer immunology research. New technologies that have transformed the field include the application of CRISPR-Cas9-mediated genome editing, in vivo imaging, and single-cell analysis to cancer modeling. Here, we provide a historical perspective on the evolution of mouse models of cancer, focusing on how far we have come in a relatively short time and how new technologies will shape the future development of mouse models of cancer.
    DOI:  https://doi.org/10.1101/cshperspect.a041736
  12. Sci Rep. 2024 05 18. 14(1): 11361
      Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. Tissue microarrays (TMA) are an established method of high throughput biomarker interrogation in tissues but may not capture histological features of cancer with potential biological relevance. Topographic TMAs (T-TMAs) representing pathophysiological hallmarks of cancer were constructed from representative, retrospective PDAC diagnostic material, including 72 individual core tissue samples. The T-TMA was interrogated with tissue hybridization-based experiments to confirm the accuracy of the topographic sampling, expression of pro-tumourigenic and immune mediators of cancer, totalling more than 750 individual biomarker analyses. A custom designed Next Generation Sequencing (NGS) panel and a spatial distribution-specific transcriptomic evaluation were also employed. The morphological choice of the pathophysiological hallmarks of cancer was confirmed by protein-specific expression. Quantitative analysis identified topography-specific patterns of expression in the IDO/TGF-β axis; with a heterogeneous relationship of inflammation and desmoplasia across hallmark areas and a general but variable protein and gene expression of c-MET. NGS results highlighted underlying genetic heterogeneity within samples, which may have a confounding influence on the expression of a particular biomarker. T-TMAs, integrated with quantitative biomarker digital scoring, are useful tools to identify hallmark specific expression of biomarkers in pancreatic cancer.
    Keywords:  Biomarkers; Digital spatial profiling; Image analysis; Pancreatic ductal adenocarcinoma; Topographic tissue microarrays; Tumour heterogeneity
    DOI:  https://doi.org/10.1038/s41598-024-62031-0
  13. Cell. 2024 May 23. pii: S0092-8674(24)00460-4. [Epub ahead of print]187(11): 2657-2681
      Turnover-constant component production and destruction-is ubiquitous in biology. Turnover occurs across organisms and scales, including for RNAs, proteins, membranes, macromolecular structures, organelles, cells, hair, feathers, nails, antlers, and teeth. For many systems, turnover might seem wasteful when degraded components are often fully functional. Some components turn over with shockingly high rates and others do not turn over at all, further making this process enigmatic. However, turnover can address fundamental problems by yielding powerful properties, including regeneration, rapid repair onset, clearance of unpredictable damage and errors, maintenance of low constitutive levels of disrepair, prevention of stable hazards, and transitions. I argue that trade-offs between turnover benefits and metabolic costs, combined with constraints on turnover, determine its presence and rates across distinct contexts. I suggest that the limits of turnover help explain aging and that turnover properties and the basis for its levels underlie this fundamental component of life.
    DOI:  https://doi.org/10.1016/j.cell.2024.04.034
  14. Trends Pharmacol Sci. 2024 May 17. pii: S0165-6147(24)00084-1. [Epub ahead of print]
      Cancer cells perturb lipid metabolic pathways for a variety of pro-tumorigenic functions, and deregulated cellular metabolism is a hallmark of cancer cells. Although alterations in lipid metabolism in cancer cells have been appreciated for over 20 years, there are no FDA-approved cancer treatments that target lipid-related pathways. Recent advances pertaining to cancer cell fatty acid synthesis (FAS), desaturation, and uptake, microenvironmental and dietary lipids, and lipid metabolism of tumor-infiltrating immune cells have illuminated promising clinical applications for targeting lipid metabolism. This review highlights emerging pathways and targets for tumor lipid metabolism that may soon impact clinical treatment.
    Keywords:  cancer metabolism; fatty acids; lipid metabolism
    DOI:  https://doi.org/10.1016/j.tips.2024.04.007
  15. Nat Metab. 2024 May 21.
      Lipid droplet tethering with mitochondria for fatty acid oxidation is critical for tumor cells to counteract energy stress. However, the underlying mechanism remains unclear. Here, we demonstrate that glucose deprivation induces phosphorylation of the glycolytic enzyme phosphofructokinase, liver type (PFKL), reducing its activity and favoring its interaction with perilipin 2 (PLIN2). On lipid droplets, PFKL acts as a protein kinase and phosphorylates PLIN2 to promote the binding of PLIN2 to carnitine palmitoyltransferase 1A (CPT1A). This results in the tethering of lipid droplets and mitochondria and the recruitment of adipose triglyceride lipase to the lipid droplet-mitochondria tethering regions to engage lipid mobilization. Interfering with this cascade inhibits tumor cell proliferation, promotes apoptosis and blunts liver tumor growth in male mice. These results reveal that energy stress confers a moonlight function to PFKL as a protein kinase to tether lipid droplets with mitochondria and highlight the crucial role of PFKL in the integrated regulation of glycolysis, lipid metabolism and mitochondrial oxidation.
    DOI:  https://doi.org/10.1038/s42255-024-01047-2
  16. J Cachexia Sarcopenia Muscle. 2024 May 23.
    Cancer Cachexia Endpoints Working Group
      Regulatory agencies require evidence that endpoints correlate with clinical benefit before they can be used to approve drugs. Biomarkers are often considered surrogate endpoints. In cancer cachexia trials, the measurement of biomarkers features frequently. The aim of this systematic review was to assess the frequency and diversity of biomarker endpoints in cancer cachexia trials. A comprehensive electronic literature search of MEDLINE, Embase and Cochrane (1990-2023) was completed. Eligible trials met the following criteria: adults (≥18 years), prospective design, more than 40 participants, use of a cachexia intervention for more than 14 days and use of a biomarker(s) as an endpoint. Biomarkers were defined as any objective measure that was assayed from a body fluid, including scoring systems based on these assays. Routine haematology and biochemistry to monitor intervention toxicity were not considered. Data extraction was performed using Covidence, and reporting followed PRISMA guidance (PROSPERO: CRD42022276710). A total of 5975 studies were assessed, of which 52 trials (total participants = 6522) included biomarkers as endpoints. Most studies (n = 29, 55.7%) included a variety of cancer types. Pharmacological interventions (n = 27, 51.9%) were most evaluated, followed by nutritional interventions (n = 20, 38.4%). Ninety-nine different biomarkers were used across the trials, and of these, 96 were assayed from blood. Albumin (n = 29, 55.8%) was assessed most often, followed by C-reactive protein (n = 22, 42.3%), interleukin-6 (n = 16, 30.8%) and tumour necrosis factor-α (n = 14, 26.9%), the latter being the only biomarker that was used to guide sample size calculations. Biomarkers were explicitly listed as a primary outcome in six trials. In total, 12 biomarkers (12.1% of 99) were used in six trials or more. Insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor 1 (IGF-1) levels both increased significantly in all three trials in which they were both used. This corresponded with a primary outcome, lean body mass, and was related to the pharmacological mechanism. Biomarkers were predominately used as exploratory rather than primary endpoints. The most commonly used biomarker, albumin, was limited by its lack of responsiveness to nutritional intervention. For a biomarker to be responsive to change, it must be related to the mechanism of action of the intervention and/or the underlying cachexia process that is modified by the intervention, as seen with IGFBP-3, IGF-1 and anamorelin. To reach regulatory approval as an endpoint, the relationship between the biomarker and clinical benefit must be clarified.
    Keywords:  biomarkers; cachexia; cancer; endpoints; trials
    DOI:  https://doi.org/10.1002/jcsm.13491
  17. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2315690121
      The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.
    Keywords:  Alzheimer’s disease; ESCRT; STORM; astrocyte; lysosome
    DOI:  https://doi.org/10.1073/pnas.2315690121
  18. Cancer Cell. 2024 May 18. pii: S1535-6108(24)00161-2. [Epub ahead of print]
      Most targeted cancer drugs inhibit the oncogenic signals to which cancer cells are addicted. We discuss here a counterintuitive approach to cancer therapy, which consists of deliberate overactivation of the oncogenic signals to overload the stress responses of cancer cells. We discuss why such overactivation of oncogenic signaling, combined with perturbation of the stress response pathways, can be potentially effective in killing cancer cells, aiming to inspire further discussion and consideration.
    DOI:  https://doi.org/10.1016/j.ccell.2024.04.014
  19. J Proteome Res. 2024 May 20.
      Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.
    Keywords:  Stroma; cancer; drug resistance; secretome
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00112
  20. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2318248121
      For eukaryotic cells to heal wounds, respond to immune signals, or metastasize, they must migrate, often by adhering to extracellular matrix (ECM). Cells may also deposit ECM components, leaving behind a footprint that influences their crawling. Recent experiments showed that some epithelial cell lines on micropatterned adhesive stripes move persistently in regions they have previously crawled over, where footprints have been formed, but barely advance into unexplored regions, creating an oscillatory migration of increasing amplitude. Here, we explore through mathematical modeling how footprint deposition and cell responses to footprint combine to allow cells to develop oscillation and other complex migratory motions. We simulate cell crawling with a phase field model coupled to a biochemical model of cell polarity, assuming local contact with the deposited footprint activates Rac1, a protein that establishes the cell's front. Depending on footprint deposition rate and response to the footprint, cells on micropatterned lines can display many types of motility, including confined, oscillatory, and persistent motion. On two-dimensional (2D) substrates, we predict a transition between cells undergoing circular motion and cells developing an exploratory phenotype. Small quantitative changes in a cell's interaction with its footprint can completely alter exploration, allowing cells to tightly regulate their motion, leading to different motility phenotypes (confined vs. exploratory) in different cells when deposition or sensing is variable from cell to cell. Consistent with our computational predictions, we find in earlier experimental data evidence of cells undergoing both circular and exploratory motion.
    Keywords:  Rho GTPases; cell motility; footprint; random walks
    DOI:  https://doi.org/10.1073/pnas.2318248121
  21. Cell. 2024 May 23. pii: S0092-8674(24)00463-X. [Epub ahead of print]187(11): 2601-2627
      Mitochondria reside at the crossroads of catabolic and anabolic metabolism-the essence of life. How their structure and function are dynamically tuned in response to tissue-specific needs for energy, growth repair, and renewal is being increasingly understood. Mitochondria respond to intrinsic and extrinsic stresses and can alter cell and organismal function by inducing metabolic signaling within cells and to distal cells and tissues. Here, we review how the centrality of mitochondrial functions manifests in health and a broad spectrum of diseases and aging.
    DOI:  https://doi.org/10.1016/j.cell.2024.04.037
  22. Curr Protoc. 2024 May;4(5): e1051
      Fluorescent imaging of cellular membranes is challenged by the size of lipid bilayers, which are smaller than the diffraction limit of light. Recently, expansion microscopy (ExM) has emerged as an approachable super-resolution method that requires only widely accessible confocal microscopes. In this method, biomolecules of interest are anchored to hydrogel-based, polymeric networks that are expanded through osmosis to physically separate and resolve features smaller than the diffraction limit of light. Whereas ExM has been employed for super-resolution imaging of proteins, DNA, RNA, and glycans, the application of this method to the study of lipids is challenged by the requirement of permeabilization procedures that remove lipids and compromise the integrity of the membrane. Here, we describe our recently developed protocols for lipid expansion microscopy (LExM), a method that enables ExM of membranes without permeabilization. These detailed protocols and accompanying commentary sections aim to make LExM accessible to any experimentalist interested in imaging membranes with super-resolution. © 2024 Wiley Periodicals LLC. Basic Protocol 1: LExM of alkyne-choline lipids Basic Protocol 2: LExM of IMPACT-labeled lipids Basic Protocol 3: LExM of clickable cholesterol Basic Protocol 4: Determining the expansion factor.
    Keywords:  bioorthogonal; click chemistry; lipid expansion microscopy; lipids; super‐resolution imaging
    DOI:  https://doi.org/10.1002/cpz1.1051
  23. IEEE J Transl Eng Health Med. 2024 ;12 413-434
      Mechanical force exerted on cancer cells by their microenvironment have been reported to drive cells toward invasive phenotypes by altering cells' motility, proliferation, and apoptosis. These mechanical forces include compressive, tensile, hydrostatic, and shear forces. The importance of forces is then hypothesized to be an alteration of cancer cells' and their microenvironment's biophysical properties as the indicator of a tumor's malignancy state. Our objective is to investigate and quantify the correlation between a tumor's malignancy state and forces experienced by the cancer cells and components of the microenvironment. In this study, we have developed a multicomponent, three-dimensional model of tumor tissue consisting of a cancer cell surrounded by fibroblasts and extracellular matrix (ECM). Our results on three different organs including breast, kidney, and pancreas show that: A) the stresses within tumor tissue are impacted by the organ specific ECM's biophysical properties, B) more invasive cancer cells experience higher stresses, C) in pancreas which has a softer ECM (Young modulus of 1.0 kPa) and stiffer cancer cells (Young modulus of 2.4 kPa and 1.7 kPa) than breast and kidney, cancer cells experienced significantly higher stresses, D) cancer cells in contact with ECM experienced higher stresses compared to cells surrounded by fibroblasts but the area of tumor stroma experiencing high stresses has a maximum length of 40 μm when the cancer cell is surrounded by fibroblasts and 12 μm for when the cancer cell is in vicinity of ECM. This study serves as an important first step in understanding of how the stresses experienced by cancer cells, fibroblasts, and ECM are associated with malignancy states of cancer cells in different organs. The quantification of forces exerted on cancer cells by different organ-specific ECM and at different stages of malignancy will help, first to develop theranostic strategies, second to predict accurately which tumors will become highly malignant, and third to establish accurate criteria controlling the progression of cancer cells malignancy. Furthermore, our in silico model of tumor tissue can yield critical, useful information for guiding ex vivo or in vitro experiments, narrowing down variables to be investigated, understanding what factors could be impacting cancer treatments or even biomarkers to be looking for.
    Keywords:  Cancer cells; ECM; cell mechanics; fibroblasts; forces on cells; malignancy; tumor microenvironment
    DOI:  https://doi.org/10.1109/JTEHM.2024.3388561
  24. Nat Cell Biol. 2024 May 23.
      Research into how the young and old differ, and which biomarkers reflect the diverse biological processes underlying ageing, is a current and fast-growing field. Biological clocks provide a means to evaluate whether a molecule, cell, tissue or even an entire organism is old or young. Here we summarize established and emerging molecular clocks as timepieces. We emphasize that intrinsically disordered proteins (IDPs) tend to transform into a β-sheet-rich aggregated state and accumulate in non-dividing or slowly dividing cells as they age. We hypothesize that understanding these protein-based molecular ageing mechanisms might provide a conceptual pathway to determining a cell's health age by probing the aggregation state of IDPs, which we term the IDP clock.
    DOI:  https://doi.org/10.1038/s41556-024-01423-w
  25. J Nucl Med. 2024 May 23. pii: jnumed.123.266262. [Epub ahead of print]
      Radiomics features can reveal hidden patterns in a tumor but usually lack an underlying biologic rationale. In this work, we aimed to investigate whether there is a correlation between radiomics features extracted from [18F]FDG PET images and histologic expression patterns of a glycolytic marker, monocarboxylate transporter-4 (MCT4), in pancreatic cancer. Methods: A cohort of pancreatic ductal adenocarcinoma patients (n = 29) for whom both tumor cross sections and [18F]FDG PET/CT scans were available was used to develop an [18F]FDG PET radiomics signature. By using immunohistochemistry for MCT4, we computed density maps of MCT4 expression and extracted pathomics features. Cluster analysis identified 2 subgroups with distinct MCT4 expression patterns. From corresponding [18F]FDG PET scans, radiomics features that associate with the predefined MCT4 subgroups were identified. Results: Complex heat map visualization showed that the MCT4-high/heterogeneous subgroup was correlating with a higher MCT4 expression level and local variation. This pattern linked to a specific [18F]FDG PET signature, characterized by a higher SUVmean and SUVmax and second-order radiomics features, correlating with local variation. This MCT4-based [18F]FDG PET signature of 7 radiomics features demonstrated prognostic value in an independent cohort of pancreatic cancer patients (n = 71) and identified patients with worse survival. Conclusion: Our cross-modal pipeline allows the development of PET scan signatures based on immunohistochemical analysis of markers of a particular biologic feature, here demonstrated on pancreatic cancer using intratumoral MCT4 expression levels to select [18F]FDG PET radiomics features. This study demonstrated the potential of radiomics scores to noninvasively capture intratumoral marker heterogeneity and identify a subset of pancreatic ductal adenocarcinoma patients with a poor prognosis.
    Keywords:  [18F]FDG PET; immunohistochemistry; machine learning; pancreatic cancer; tumor metabolism
    DOI:  https://doi.org/10.2967/jnumed.123.266262
  26. Mol Oncol. 2024 May 24.
      Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, characterized by late diagnosis and poor treatment response. Surgery is the only curative approach, only available to early-diagnosed patients. Current therapies have limited effects, cause severe toxicities, and minimally improve overall survival. Understanding of splicing machinery alterations in PDAC remains incomplete. Here, we comprehensively examined 59 splicing machinery components, uncovering dysregulation in pre-mRNA processing factor 8 (PRPF8) and RNA-binding motif protein X-linked (RBMX). Their downregulated expression was linked to poor prognosis and malignancy features, including tumor stage, invasion and metastasis, and associated with poorer survival and the mutation of key PDAC genes. Experimental modulation of these splicing factors in pancreatic cancer cell lines reverted their expression to non-tumor levels and resulted in decreased key tumor-related features. These results provide evidence that the splicing machinery is altered in PDAC, wherein PRPF8 and RBMX emerge as candidate actionable therapeutic targets.
    Keywords:  PRPF8; RBMX; pancreatic cancer; splicing; splicing factor
    DOI:  https://doi.org/10.1002/1878-0261.13658
  27. Autophagy. 2024 May 18. 1-8
      Segmenting autophagic bodies in yeast TEM images is a key technique for measuring changes in autophagosome size and number in order to better understand macroautophagy/autophagy. Manual segmentation of these images can be very time consuming, particularly because hundreds of images are needed for accurate measurements. Here we describe a validated Cellpose 2.0 model that can segment these images with accuracy comparable to that of human experts. This model can be used for fully automated segmentation, eliminating the need for manual body outlining, or for model-assisted segmentation, which allows human oversight but is still five times as fast as the current manual method. The model is specific to segmentation of autophagic bodies in yeast TEM images, but researchers working in other systems can use a similar process to generate their own Cellpose 2.0 models to attempt automated segmentations. Our model and instructions for its use are presented here for the autophagy community.Abbreviations: AB, autophagic body; AvP, average precision; GUI, graphical user interface; IoU, intersection over union; MVB, multivesicular body; ROI, region of interest; TEM, transmission electron microscopy; WT,wild type.
    Keywords:  Automated labeling; autophagy; computer vision; electron microscopy; image analysis; machine learning
    DOI:  https://doi.org/10.1080/15548627.2024.2353458
  28. Cancer Immunol Res. 2024 May 20.
      Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition (DDA) or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex-vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-23-0900
  29. Biochim Biophys Acta Mol Cell Biol Lipids. 2024 May 16. pii: S1388-1981(24)00062-3. [Epub ahead of print]1869(6): 159512
       OBJECTIVE: Excess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation.
    METHOD: Cholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits.
    RESULTS: Cholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH.
    CONCLUSION: SOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.
    Keywords:  Cholesterol crystal; Liver; MASH/NASH; Metabolism; SOAT/ACAT
    DOI:  https://doi.org/10.1016/j.bbalip.2024.159512
  30. Cold Spring Harb Perspect Med. 2024 May 21. pii: a041682. [Epub ahead of print]
      The recent rise in effective immuno-oncology therapies has increased demand for experimental approaches to model anticancer immunity. A variety of mouse models have been developed and used to study cancer immunology. These include mutagen-induced, genetically engineered, syngeneic, and other models of cancer immunology. These models each have the potential to define mechanistic aspects of anticancer immune responses, identify potential therapeutic targets, and serve as preclinical models for further therapeutic development. Specific benefits and liabilities are characteristic of particular cancer immunology modeling approaches. The optimal choice and utilization of models depends on the cancer immunology scientific question being addressed and can serve to increase mechanistic understanding and development of human immuno-oncology therapies.
    DOI:  https://doi.org/10.1101/cshperspect.a041682
  31. eGastroenterology. 2024 Apr;pii: e100057. [Epub ahead of print]2(2):
      Acute pancreatitis is a common inflammatory gastrointestinal disease without any successful treatment. Pancreatic exocrine acinar cells have high rates of protein synthesis to produce and secrete large amounts of digestive enzymes. When the regulation of organelle and protein homeostasis is disrupted, it can lead to endoplasmic reticulum (ER) stress, damage to the mitochondria and improper intracellular trypsinogen activation, ultimately resulting in acinar cell damage and the onset of pancreatitis. To balance the homeostasis of organelles and adapt to protect themselves from organelle stress, cells use protective mechanisms such as autophagy. In the mouse pancreas, defective basal autophagy disrupts ER homoeostasis, leading to ER stress and trypsinogen activation, resulting in spontaneous pancreatitis. In this review, we discuss the regulation of autophagy and its physiological role in maintaining acinar cell homeostasis and function. We also summarise the current understanding of the mechanisms and the role of defective autophagy at multiple stages in experimental pancreatitis induced by cerulein or alcohol.
    DOI:  https://doi.org/10.1136/egastro-2023-100057
  32. Mol Oncol. 2024 May 22.
      Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC-I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis-initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out.
    Keywords:  cancer; circulating tumor cells; glycoproteomics; immunopeptidomics; proteomics
    DOI:  https://doi.org/10.1002/1878-0261.13665
  33. J Biol Chem. 2024 May 21. pii: S0021-9258(24)01904-5. [Epub ahead of print] 107403
      Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Although there are functional impairments in both cases, the signaling consequences of primary mitochondrial dysfunction and lysosomal defects are dissimilar. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects to identify the global cellular consequences associated with mitochondrial or lysosomal dysfunction. We used these data to determine the pathways affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. We observed a transcriptional up-regulated of this pathway in cellular and murine models of lysosomal defects, while it is transcriptionally down-regulated in cellular and murine models of mitochondrial defects. We identified a role for the post-transcriptional regulation of transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, we found that retention of Ca2+ in lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo, using a model of mitochondria-associated disease in C. elegans, that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental delay induced by the respiratory chain deficiency.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107403
  34. Trends Cell Biol. 2024 May 23. pii: S0962-8924(24)00094-1. [Epub ahead of print]
      Over the past six decades, fluorescence-activated cell sorting (FACS) has become an essential technology for basic and clinical research by enabling the isolation of cells of interest in high throughput. Recent technological advancements have started a new era of flow cytometry. By combining the spatial resolution of microscopy with high-speed cell sorting, new instruments allow cell sorting based on simple image-derived parameters or sophisticated image analysis algorithms, thereby greatly expanding the scope of applications. In this review, we discuss the systems that are commercially available or have been described in enough methodological and engineering detail to allow their replication. We summarize their strengths and limitations and highlight applications that have the potential to transform various fields in basic life science research and clinical settings.
    Keywords:  cell sorting; flow cytometry; high-throughput imaging; image analysis
    DOI:  https://doi.org/10.1016/j.tcb.2024.04.007
  35. Biophys Rep. 2024 Apr 30. 10(2): 102-110
      The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein-protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.
    Keywords:  Co-immunoprecipitation; Lipid droplet; Protein interaction
    DOI:  https://doi.org/10.52601/bpr.2024.240007
  36. Am J Surg Pathol. 2024 May 20.
      Pancreatic ductal adenocarcinoma (PDAC) develops from 2 known precursor lesions: a majority (∼85%) develops from pancreatic intraepithelial neoplasia (PanIN), and a minority develops from intraductal papillary mucinous neoplasms (IPMNs). Clinical classification of PanIN and IPMN relies on a combination of low-resolution, 3-dimensional (D) imaging (computed tomography, CT), and high-resolution, 2D imaging (histology). The definitions of PanIN and IPMN currently rely heavily on size. IPMNs are defined as macroscopic: generally >1.0 cm and visible in CT, and PanINs are defined as microscopic: generally <0.5 cm and not identifiable in CT. As 2D evaluation fails to take into account 3D structures, we hypothesized that this classification would fail in evaluation of high-resolution, 3D images. To characterize the size and prevalence of PanINs in 3D, 47 thick slabs of pancreas were harvested from grossly normal areas of pancreatic resections, excluding samples from individuals with a diagnosis of an IPMN. All patients but one underwent preoperative CT scans. Through construction of cellular resolution 3D maps, we identified >1400 ductal precursor lesions that met the 2D histologic size criteria of PanINs. We show that, when 3D space is considered, 25 of these lesions can be digitally sectioned to meet the 2D histologic size criterion of IPMN. Re-evaluation of the preoperative CT images of individuals found to possess these large precursor lesions showed that nearly half are visible on imaging. These findings demonstrate that the clinical classification of PanIN and IPMN fails in evaluation of high-resolution, 3D images, emphasizing the need for re-evaluation of classification guidelines that place significant weight on 2D assessment of 3D structures.
    DOI:  https://doi.org/10.1097/PAS.0000000000002245
  37. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2317227121
      The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.
    Keywords:  cholesterol; giant unilamellar lipid vesicles; membrane biophysics; micropipette aspiration; polysaccharides
    DOI:  https://doi.org/10.1073/pnas.2317227121
  38. J Cell Sci. 2024 May 15. pii: jcs260943. [Epub ahead of print]137(10):
      Membrane trafficking, a fundamental cellular process encompassing the transport of molecules to specific organelles, endocytosis at the plasma membrane and protein secretion, is crucial for cellular homeostasis and signalling. Cancer cells adapt membrane trafficking to enhance their survival and metabolism, and understanding these adaptations is vital for improving patient responses to therapy and identifying therapeutic targets. In this Review, we provide a concise overview of major membrane trafficking pathways and detail adaptations in these pathways, including COPII-dependent endoplasmic reticulum (ER)-to-Golgi vesicle trafficking, COPI-dependent retrograde Golgi-to-ER trafficking and endocytosis, that have been found in cancer. We explore how these adaptations confer growth advantages or resistance to cell death and conclude by discussing the potential for utilising this knowledge in developing new treatment strategies and overcoming drug resistance for cancer patients.
    Keywords:  Cancer; Coatomer; Endocytosis; Imaging; Vesicle
    DOI:  https://doi.org/10.1242/jcs.260943
  39. Autophagy. 2024 May 18. 1-17
      Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects.Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.
    Keywords:  Autophagy; ROS; bleomycin; cellular senescence; lysosomal membrane permeabilization
    DOI:  https://doi.org/10.1080/15548627.2024.2353548
  40. J Pathol. 2024 May 23.
    Grand Challenge PRECISION Consortium
      Mutations are abundantly present in tissues of healthy individuals, including the breast epithelium. Yet it remains unknown whether mutant cells directly induce lesion formation or first spread, leading to a field of mutant cells that is predisposed towards lesion formation. To study the clonal and spatial relationships between morphologically normal breast epithelium adjacent to pre-cancerous lesions, we developed a three-dimensional (3D) imaging pipeline combined with spatially resolved genomics on archival, formalin-fixed breast tissue with the non-obligate breast cancer precursor ductal carcinoma in situ (DCIS). Using this 3D image-guided characterization method, we built high-resolution spatial maps of DNA copy number aberration (CNA) profiles within the DCIS lesion and the surrounding normal mammary ducts. We show that the local heterogeneity within a DCIS lesion is limited. However, by mapping the CNA profiles back onto the 3D reconstructed ductal subtree, we find that in eight out of 16 cases the healthy epithelium adjacent to the DCIS lesions has overlapping structural variations with the CNA profile of the DCIS. Together, our study indicates that pre-malignant breast transformations frequently develop within mutant clonal fields of morphologically normal-looking ducts. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
    Keywords:  3D pathology; copy number aberration; ductal carcinoma in situ; field cancerization; pre‐malignant transformation; spatial genomics
    DOI:  https://doi.org/10.1002/path.6289
  41. Curr Opin Cell Biol. 2024 May 23. pii: S0955-0674(24)00050-4. [Epub ahead of print]88 102371
      Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.
    DOI:  https://doi.org/10.1016/j.ceb.2024.102371
  42. Cell Death Differ. 2024 May 23.
      Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.
    DOI:  https://doi.org/10.1038/s41418-024-01313-6
  43. bioRxiv. 2024 May 10. pii: 2024.05.09.593302. [Epub ahead of print]
      Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.
    DOI:  https://doi.org/10.1101/2024.05.09.593302
  44. Mol Biol Cell. 2024 May 22. mbcE23090382
      Lipin 1 is an ER enzyme that produces diacylglycerol, the lipid intermediate that feeds into the synthesis of glycerophospholipids for membrane expansion or triacylglycerol for storage into lipid droplets. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but a role for CTDNEP1 in lipid storage in mammalian cells is not known. Here, we show that CTDNEP1 is reliant on its binding partner NEP1R1 for its stability and function in limiting ER expansion. CTDNEP1 contains an amphipathic helix at its N-terminus that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 and NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate that NEP1R1 binding to CTDNEP1 shields CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, we found that NEP1R1 is not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on NEP1R1 depends on cellular demands for membrane production versus lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis under different metabolic conditions.
    DOI:  https://doi.org/10.1091/mbc.E23-09-0382
  45. J Cell Biol. 2024 Aug 05. pii: e202309145. [Epub ahead of print]223(8):
      Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.
    DOI:  https://doi.org/10.1083/jcb.202309145
  46. J Proteome Res. 2024 May 22.
      Engineered macromolecules offer compelling means for the therapy of conventionally undruggable interactions in human disease. However, their efficacy is limited by barriers to tissue and intracellular delivery. Inspired by recent advances in molecular barcoding and evolution, we developed BarcodeBabel, a generalized method for the design of libraries of peptide barcodes suitable for high-throughput mass spectrometry proteomics. Combined with PeptideBabel, a Monte Carlo sampling algorithm for the design of peptides with evolvable physicochemical properties and sequence complexity, we developed a barcoded library of cell penetrating peptides (CPPs) with distinct physicochemical features. Using quantitative targeted mass spectrometry, we identified CPPS with improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell while maintaining minimal membrane disruption and negligible toxicity in vitro. These studies provide a proof of concept for peptide barcoding as a homogeneous high-throughput method for macromolecular screening and delivery. BarcodeBabel and PeptideBabel are available open-source from https://github.com/kentsisresearchgroup/.
    Keywords:  cell penetration; macromolecular drug delivery; mass spectrometry proteomics; molecular screening; peptide barcoding
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00068
  47. FEBS Lett. 2024 May;598(10): 1252-1273
      Over the past two decades, we have witnessed a growing appreciation for the importance of membrane contact sites (CS) in facilitating direct communication between organelles. CS are tiny regions where the membranes of two organelles meet but do not fuse and allow the transfer of metabolites between organelles, playing crucial roles in the coordination of cellular metabolic activities. The significant advancements in imaging techniques and molecular and cell biology research have revealed that CS are more complex than what originally thought, and as they are extremely dynamic, they can remodel their shape, composition, and functions in accordance with metabolic and environmental changes and can occur between more than two organelles. Here, we describe how recent studies led to the identification of a three-way mitochondria-ER-lipid droplet CS and discuss the emerging functions of these contacts in maintaining lipid storage, homeostasis, and balance. We also summarize the properties and functions of key protein components localized at the mitochondria-ER-lipid droplet interface, with a special focus on lipid transfer proteins. Understanding tripartite CS is essential for unraveling the complexities of inter-organelle communication and cooperation within cells.
    Keywords:  MAM; contact sites; fatty acids; lipid metabolism; lipid transfer proteins
    DOI:  https://doi.org/10.1002/1873-3468.14893
  48. Front Nutr. 2024 ;11 1388986
      The importance of meal distribution of dietary protein to optimize muscle mass and body remains unclear, and the findings are intertwined with age, physical activity, and the total quantity and quality of protein consumed. The concept of meal distribution evolved from multiple discoveries about regulating protein synthesis in skeletal muscle. The most significant was the discovery of the role of the branched-chain amino acid leucine as a metabolic signal to initiate a post-meal anabolic period of muscle protein synthesis (MPS) in older adults. Aging is often characterized by loss of muscle mass and function associated with a decline in protein synthesis. The age-related changes in protein synthesis and subsequent muscle atrophy were generally considered inevitable until the discovery of the unique role of leucine for the activation of the mTOR signal complex for the initiation of MPS. Clinical studies demonstrated that older adults (>60 years) require meals with at least 2.8 g of leucine (~30 g of protein) to stimulate MPS. This meal requirement for leucine is not observed in younger adults (<30 years), who produce a nearly linear response of MPS in proportion to the protein content of a meal. These findings suggest that while the efficiency of dietary protein to stimulate MPS declines with aging, the capacity for MPS to respond is maintained if a meal provides adequate protein. While the meal response of MPS to total protein and leucine is established, the long-term impact on muscle mass and body composition remains less clear, at least in part, because the rate of change in muscle mass with aging is small. Because direct diet studies for meal distribution during aging are impractical, research groups have applied meal distribution and the leucine threshold to protein-sparing concepts during acute catabolic conditions such as weight loss. These studies demonstrate enhanced MPS at the first meal after an overnight fast and net sparing of lean body mass during weight loss. While the anabolic benefits of increased protein at the first meal to stimulate MPS are clear, the benefits to long-term changes in muscle mass and body composition in aging adults remain speculative.
    Keywords:  leucine; muscle mass; muscle protein synthesis; protein requirements; sarcopenia
    DOI:  https://doi.org/10.3389/fnut.2024.1388986
  49. Theranostics. 2024 ;14(7): 2675-2686
      Cyanine dyes are widely used organic probes for in vivo imaging due to their tunable fluorescence. They can form complexes with endogenous albumin, resulting in enhanced brightness and photostability. However, this binding is uncontrollable and irreversible, leading to considerable nonspecific background signals and unregulated circulation time. Methods: Here, we connect varying numbers of 4-(4-iodophenyl) butanoic acid (IP) as albumin-binding moieties (ABM) to the cyanine dye, enabling dynamic and controllable binding with albumin. Meanwhile, we provide a blocking method to completely release the dye from covalent capture with albumin, resulting in specific targeting fluorescence. Furthermore, we evaluate the pharmacokinetics and tumor targeting of the developed dyes. Results: The engineered dyes can dynamically and selectively bind with multiple albumins to change the in situ size of assemblies and circulation time, providing programmable regulation over the imaging time window. The nucleophilic substitution of meso-Cl with water-soluble amino acids or targeting peptides for IP-engineered dye further addresses the nonspecific signals caused by albumin, allowing for adjustable angiography time and efficient tumor targeting. Conclusion: This study rationalizes the binding modes of dyes and proteins, applicable to a wide range of near-infrared (NIR) dyes for improving their in vivo molecular imaging.
    Keywords:  Dye@albumin nanoparticles; Dynamic albumin binder; Programmable imaging windows; Protein pocket-escaping design; Tumor-targeting dyes
    DOI:  https://doi.org/10.7150/thno.92991
  50. EMBO J. 2024 May 22.
      Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.
    Keywords:  Circadian; Proteostasis; Ribosome; SILAC
    DOI:  https://doi.org/10.1038/s44318-024-00121-5
  51. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2321600121
      Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.
    Keywords:  T cell receptor; antigen processing; peptide-loading complex; transporter associated with antigen processing TAP; tumor-associated antigen
    DOI:  https://doi.org/10.1073/pnas.2321600121
  52. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2322479121
      The significance of biochemical cues in the tumor immune microenvironment in affecting cancer metastasis is well established, but the role of physical factors in the microenvironment remains largely unexplored. In this article, we investigated how the mechanical interaction between cancer cells and immune cells, mediated by extracellular matrix (ECM), influences immune escape of cancer cells. We focus on the mechanical regulation of macrophages' targeting ability on two distinct types of colorectal carcinoma (CRC) cells with different metastatic potentials. Our results show that macrophages can effectively target CRC cells with low metastatic potential, due to the strong contraction exhibited by the cancer cells on the ECM, and that cancer cells with high metastatic potential demonstrated weakened contractions on the ECM and can thus evade macrophage attack to achieve immune escape. Our findings regarding the intricate mechanical interactions between immune cells and cancer cells can serve as a crucial reference for further exploration of cancer immunotherapy strategies.
    Keywords:  extracellular matrix; immune escape; mechanical signals
    DOI:  https://doi.org/10.1073/pnas.2322479121
  53. bioRxiv. 2024 May 07. pii: 2024.04.20.590392. [Epub ahead of print]
      Most common cytometry methods, including flow cytometry, observe suspended or fixed cells and cannot evaluate their structural roles in 3D tissues. However, cellular physical interactions are critical in physiological, developmental, and pathological processes. Here, we present a novel optical visco-elastography that characterizes single-cellular physical interactions by applying in-situ micro-mechanical perturbation to live microtissues under 3D lightsheet microscopy. The 4D digital image correlation (DIC) analysis of ∼20,000 nodes tracked the compressive deformation of 3D tissues containing ∼500 cells. The computational 3D image segmentation allowed cell-by-cell qualitative observation and statistical analysis, directly correlating multi-channel fluorescence and viscoelasticity. To represent epithelia-stroma interactions, we used a 3D organoid model of maternal-fetal interface and visualized solid-like, well-aligned displacement and liquid-like random motion between individual cells. The statistical analysis through our unique cytometry confirmed that endometrial stromal fibroblasts stiffen in response to decidualization. Moreover, we demonstrated in the 3D model that interaction with placental extravillous trophoblasts partially reverses the attained stiffness, which was supported by the gene expression analysis. Placentation shares critical cellular and molecular significance with various fundamental biological events such as cancer metastasis, wound healing, and gastrulation. Our analysis confirmed existing beliefs and discovered new insights, proving the broad applicability of our method.
    DOI:  https://doi.org/10.1101/2024.04.20.590392