bims-cepepe 2024-02-25 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–02–25
27 papers selected by
Henry Lamb, Queensland University of Technology

Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.
Computational design of anti-cancer peptides tailored to target specific tumor markers.
Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides.
Deep learning for advancing peptide drug development: Tools and methods in structure prediction and design.
Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations.
Translational use of homing peptides: Tumor and placental targeting.
2023 FDA TIDES (Peptides and Oligonucleotides) Harvest.
Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-XL by engaging a single-residue discrepancy.
Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.
Enhanced Tumor Targeting and Antitumor Activity of Methylated β-Cyclodextrin-Threaded Polyrotaxanes by Conjugating Cyclic RGD Peptides.
A Tunable Glycosaminoglycan-Peptide Nanoparticle Platform for the Protection of Therapeutic Peptides.
Small molecule- and peptide-drug conjugates addressing integrins: A story of targeted cancer treatment.
Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.
[68Ga]Ga-FAP-2286, a novel promising theragnostic approach for PET/CT imaging in patients with various type of metastatic cancers.
Immunomodulatory Peptides as Vaccine Adjuvants and Antimicrobial Agents.
Selenium chemistry for spatio-selective peptide and protein functionalization.
Rigid-flexible nanocarriers loaded with active peptides for antioxidant and anti-inflammatory applications in skin.
Engineered exosomes with enhanced stability and delivery efficiency for glioblastoma therapy.
Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.
Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.
A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.
Dimeric Tubulin Modifies Mechanical Properties of Lipid Bilayer, as Probed Using Gramicidin A Channel.
Prediction, screening and characterization of novel bioactive tetra-peptide matrikines for skin rejuvenation.
Structural and Functional Insights into CRF Peptides and Their Receptors.
Exosomes in Glioma: Unraveling Their Roles in Progression, Diagnosis, and Therapy.
Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.
Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma, Quantitative Analysis, Optical Clearing, and Mathematical Modeling.

ACS Appl Mater Interfaces. 2024 Feb 22.

Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

Behnaz Ghaemi, Swati Tanwar, Aruna Singh, Dian R Arifin, Michael T McMahon, Ishan Barman, Jeff W M Bulte.

  For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Keywords:  arginine; biocompatibility; cell-penetrating peptide; enzyme-responsive peptide; legumain

DOI:  https://doi.org/10.1021/acsami.3c14908
BMC Chem. 2024 Feb 22. 18(1): 39

Computational design of anti-cancer peptides tailored to target specific tumor markers.

Aisha Naeem, Nighat Noureen, Shaikha Khalid Al-Naemi, Jawaher Ahmed Al-Emadi, Muhammad Jawad Khan.

  Anti-cancer peptides (ACPs) are short peptides known for their ability to inhibit tumor cell proliferation, migration, and the formation of tumor blood vessels. In this study, we designed ACPs to target receptors often overexpressed in cancer using a systematic in silico approach. Three target receptors (CXCR1, DcR3, and OPG) were selected for their significant roles in cancer pathogenesis and tumor cell proliferation. Our peptide design strategy involved identifying interacting residues (IR) of these receptors, with their natural ligands serving as a reference for designing peptides specific to each receptor. The natural ligands of these receptors, including IL8 for CXCR1, TL1A for DcR3, and RANKL for OPG, were identified from the literature. Using the identified interacting residues (IR), we generated a peptide library through simple permutation and predicted the structure of each peptide. All peptides were analyzed using the web-based prediction server for Anticancer peptides, AntiCP. Docking simulations were then conducted to analyze the binding efficiencies of peptides with their respective target receptors, using VEGA ZZ and Chimera for interaction analysis. Our analysis identified HPKFIKELR as the interacting residues (IR) of CXCR-IL8. For DcR3, we utilized three domains from TL1A (TDSYPEP, TKEDKTF, LGLAFTK) as templates, along with two regions (SIKIPSS and PDQDATYP) from RANKL, to generate a library of peptide analogs. Subsequently, peptides for each receptor were shortlisted based on their predicted anticancer properties as determined by AntiCP and were subjected to docking analysis. After docking, peptides that exhibited the least binding energy were further analyzed for their detailed interaction with their respective receptors. Among these, peptides C9 (HPKFELY) and C7 (HPKFEWL) for CXCR1, peptides D6 (ADSYPQP) and D18 (AFSYPFP) for DcR3, and peptides P19 (PDTYPQDP) and p16 (PDQDATYP) for OPG, demonstrated the highest affinity and stronger interactions compared to the other peptides. Although in silico predictions indicated a favorable binding affinity of the designed peptides with target receptors, further experimental validation is essential to confirm their binding affinity, stability and pharmacokinetic characteristics.

Keywords:  Anticancer peptides; CXCR1; DcR3; Homology modeling; OPG

DOI:  https://doi.org/10.1186/s13065-024-01143-0
Bioorg Chem. 2024 Feb 17. pii: S0045-2068(24)00125-1. [Epub ahead of print]145 107220

Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides.

Zhihan Huang, Mayumi Ishii, Eri Watanabe, Kayoko Kanamitsu, Kempei Tai, Hiroyuki Kusuhara, Tomohiko Ohwada, Yuko Otani.

  In this study, we explored the potential of the photoremovable o-nitrobenzyl (oNB) group as a tool to manipulate the membrane permeability and regulate the conformation of linear peptides by means of experimental and computational studies. We found that the introduction of one or more oNB groups markedly increased the permeability and altered the conformation, as compared to the corresponding unmodified peptides. We thoroughly investigated the impact of peptide length, number of oNB group, oNB insertion position, and introduction of N- and C-terminal protecting groups on the passive membrane permeability by means of parallel artificial membrane permeability assay (PAMPA). Photoreaction of peptides containing one or two oNB groups proceeded cleanly in moderate to high yields, releasing the unprotected parent linear peptide. The oNB-modified peptides showed a cis/trans conformational equilibrium, while after photolysis, the unprotected linear peptides showed only the trans-amide conformation. Furthermore, a comprehensive comparison of oNB-modified peptides and N-methylated peptides was conducted, encompassing conformational analysis and physicochemical properties. N-Substituted peptides favored a folded-like structure, which may contribute to the improvement in permeability.

Keywords:  PAMPA; Peptide membrane permeability; Photoreaction; Photoremovable o-nitrobenzyl group

DOI:  https://doi.org/10.1016/j.bioorg.2024.107220
Eur J Med Chem. 2024 Feb 19. pii: S0223-5234(24)00142-9. [Epub ahead of print]268 116262

Deep learning for advancing peptide drug development: Tools and methods in structure prediction and design.

Xinyi Wu, Huitian Lin, Renren Bai, Hongliang Duan.

  Peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, peptides' unique features, including smaller size, increased structural flexibility, and limited data availability, pose additional challenges to the design process compared to proteins. This review explores the dynamic field of peptide therapeutics, leveraging deep learning to enhance structure prediction and design. Our exploration encompasses various facets of peptide research, ranging from dataset curation handling to model development. As deep learning technologies become more refined, we channel our efforts into peptide structure prediction and design, aligning with the fundamental principles of structure-activity relationships in drug development. To guide researchers in harnessing the potential of deep learning to advance peptide drug development, our insights comprehensively explore current challenges and future directions of peptide therapeutics.

Keywords:  Artificial intelligence (AI); Deep learning (DL); Peptide design; Peptide structure prediction; Peptide-protein interaction (PepPI); Structure-based drug design (SBDD)

DOI:  https://doi.org/10.1016/j.ejmech.2024.116262
Nat Commun. 2024 Feb 21. 15(1): 1611

Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations.

Sijie Chen, Tong Lin, Ruchira Basu, Jeremy Ritchey, Shen Wang, Yichuan Luo, Xingcan Li, Dehua Pei, Levent Burak Kara, Xiaolin Cheng.

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target β-catenin and NF-κB essential modulator. Among the twelve β-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds β-catenin with an IC50 of 0.010 ± 0.06 μM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

DOI: https://doi.org/10.1038/s41467-024-45766-2
J Colloid Interface Sci. 2024 Feb 13. pii: S0021-9797(24)00347-3. [Epub ahead of print]662 1033-1043

Translational use of homing peptides: Tumor and placental targeting.

Abdulaziz A Alobaid, Maximilian W A Skoda, Lynda K Harris, Richard A Campbell.

   HYPOTHESIS: Tissue-specific homing peptides have been shown to improve chemotherapeutic efficacy due to their trophism for tumor cells. Other sequences that selectively home to the placenta are providing new and safer therapeutics to treat complications in pregnancy. Our hypothesis is that the placental homing peptide RSGVAKS (RSG) may have binding affinity to cancer cells, and that insight can be gained into the binding mechanisms of RSG and the tumor homing peptide CGKRK to model membranes that mimic the primary lipid compositions of the respective cells.
EXPERIMENTS: Following cell culture studies on the binding efficacy of the peptides on a breast cancer cell line, a systematic translational characterization is delivered using ellipsometry, Brewster angle microscopy and neutron reflectometry of the extents, structures, and dynamics of the interactions of the peptides with the model membranes on a Langmuir trough.
FINDINGS: We start by revealing that RSG does indeed have binding affinity to breast cancer cells. The peptide is then shown to exhibit stronger interactions and greater penetration than CGKRK into both model membranes, combined with greater disruption to the lipid component. RSG also forms aggregates bound to the model membranes, yet both peptides bind to a greater extent to the placental than cancer model membranes. The results demonstrate the potential for varying local reservoirs of peptide within cell membranes that may influence receptor binding. The innovative nature of our findings motivates the urgent need for more studies involving multifaceted experimental platforms to explore the use of specific peptide sequences to home to different cellular targets.

Keywords:  CGKRK; Cancer; Cell cultures; Homing peptides; Neutron reflectivity; Placenta; RSG; Reflectometry; Tumor

DOI:  https://doi.org/10.1016/j.jcis.2024.02.103
Pharmaceuticals (Basel). 2024 Feb 13. pii: 243. [Epub ahead of print]17(2):

2023 FDA TIDES (Peptides and Oligonucleotides) Harvest.

Danah Al Shaer, Othman Al Musaimi, Fernando Albericio, Beatriz G de la Torre.

  A total of nine TIDES (pepTIDES and oligonucleoTIDES) were approved by the FDA during 2023. The four approved oligonucleotides are indicated for various types of disorders, including amyotrophic lateral sclerosis, geographic atrophy, primary hyperoxaluria type 1, and polyneuropathy of hereditary transthyretin-mediated amyloidosis. All oligonucleotides show chemically modified structures to enhance their stability and therapeutic effectiveness as antisense or aptamer oligomers. Some of them demonstrate various types of conjugation to driving ligands. The approved peptides comprise various structures, including linear, cyclic, and lipopeptides, and have diverse applications. Interestingly, the FDA has granted its first orphan drug designation for a peptide-based drug as a highly selective chemokine antagonist. Furthermore, Rett syndrome has found its first-ever core symptoms treatment, which is also peptide-based. Here, we analyze the TIDES approved in 2023 on the basis of their chemical structure, medical target, mode of action, administration route, and common adverse effects.

Keywords:  FDA approvals; avacincaptad pegol; drugs; eplontersen; flotufolastat F; motixafortide; nedosiran; oligonucleotides; peptides; rezafungin; tofersen; trofinetide; zilucoplan

DOI:  https://doi.org/10.3390/ph17020243
Nat Commun. 2024 Feb 17. 15(1): 1476

Cyclic peptides discriminate BCL-2 and its clinical mutants from BCL-XL by engaging a single-residue discrepancy.

Fengwei Li, Junjie Liu, Chao Liu, Ziyan Liu, Xiangda Peng, Yinyue Huang, Xiaoyu Chen, Xiangnan Sun, Sen Wang, Wei Chen, Dan Xiong, Xiaotong Diao, Sheng Wang, Jingjing Zhuang, Chuanliu Wu, Dalei Wu.

Overexpressed pro-survival B-cell lymphoma-2 (BCL-2) family proteins BCL-2 and BCL-XL can render tumor cells malignant. Leukemia drug venetoclax is currently the only approved selective BCL-2 inhibitor. However, its application has led to an emergence of resistant mutations, calling for drugs with an innovative mechanism of action. Herein we present cyclic peptides (CPs) with nanomolar-level binding affinities to BCL-2 or BCL-XL, and further reveal the structural and functional mechanisms of how these CPs target two proteins in a fashion that is remarkably different from traditional small-molecule inhibitors. In addition, these CPs can bind to the venetoclax-resistant clinical BCL-2 mutants with similar affinities as to the wild-type protein. Furthermore, we identify a single-residue discrepancy between BCL-2 D111 and BCL-XL A104 as a molecular "switch" that can differently engage CPs. Our study suggests that CPs may inhibit BCL-2 or BCL-XL by delicately modulating protein-protein interactions, potentially benefiting the development of next-generation therapeutics.

DOI: https://doi.org/10.1038/s41467-024-45848-1
Curr Protoc. 2024 Feb;4(2): e957

Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

Marwa Malhis, Susanne Aileen Funke.

  In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-β peptide (Aβ) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

Keywords:   D-enantiomer; mirror image phage display; peptide ligands; phage display

DOI:  https://doi.org/10.1002/cpz1.957
Biomolecules. 2024 Feb 15. pii: 223. [Epub ahead of print]14(2):

Enhanced Tumor Targeting and Antitumor Activity of Methylated β-Cyclodextrin-Threaded Polyrotaxanes by Conjugating Cyclic RGD Peptides.

Shunyao Zhang, Atsushi Tamura, Nobuhiko Yui.

  We previously reported that acid-degradable methylated β-cyclodextrins (Me-β-CDs)-threaded polyrotaxanes (Me-PRXs) can induce autophagic cell death through endoplasmic reticulum (ER) stress-related autophagy, even in apoptosis-resistant cells. Hence, Me-PRXs show great potential as anticancer therapeutics. In this study, peptide-supermolecule conjugates were designed to achieve the targeted delivery of Me-PRX to malignant tumors. Arg-Gly-Asp peptides are well-known binding motifs of integrin αvβ3, which is overexpressed on angiogenic sites and many malignant tumors. The tumor-targeted cyclic Arg-Gly-Asp (cRGD) peptide was orthogonally post-modified to Me-PRX via click chemistry. Surface plasmon resonance (SPR) results indicated that cRGD-Me-PRX strongly binds to integrin αvβ3, whereas non-targeted cyclic Arg-Ala-Glu (cRGE) peptide conjugated to Me-PRX (cRGE-Me-PRX) failed to interact with integrins αvβ3. In vitro, cRGD-Me-PRX demonstrated enhanced cellular internalization and antitumor activity in 4T1 cells than that of unmodified Me-PRX and non-targeted cRGE-Me-PRX, due to its ability to recognize integrin αvβ3. Furthermore, cRGD-Me-PRX accumulated effectively in tumors, leading to antitumor effects, and exhibited excellent biocompatibility and safety in vivo. Therefore, cRGD conjugation to enhance selectivity for integrin αvβ3-positive cancer cells is a promising design strategy for Me-PRXs in antitumor therapy.

Keywords:  antitumor activity; cyclic RGD peptide; methylated β-cyclodextrin; polyrotaxane; tumor targeting

DOI:  https://doi.org/10.3390/biom14020223
Pharmaceutics. 2024 Jan 25. pii: 173. [Epub ahead of print]16(2):

A Tunable Glycosaminoglycan-Peptide Nanoparticle Platform for the Protection of Therapeutic Peptides.

Harkanwalpreet Sodhi, Alyssa Panitch.

  The popularity of Glycosaminoglycans (GAGs) in drug delivery systems has grown as their innate ability to sequester and release charged molecules makes them adept in the controlled release of therapeutics. However, peptide therapeutics have been relegated to synthetic, polymeric systems, despite their high specificity and efficacy as therapeutics because they are rapidly degraded in vivo when not encapsulated. We present a GAG-based nanoparticle system for the easy encapsulation of cationic peptides, which offers control over particle diameter, peptide release behavior, and swelling behavior, as well as protection from proteolytic degradation, using a singular, organic polymer and no covalent linkages. These nanoparticles can encapsulate cargo with a particle diameter range spanning 130-220 nm and can be tuned to release cargo over a pH range of 4.5 to neutral through the modulation of the degree of sulfation and the molecular weight of the GAG. This particle system also confers better in vitro performance than the unencapsulated peptide via protection from enzymatic degradation. This method provides a facile way to protect therapeutic peptides via the inclusion of the presented binding sequence and can likely be expanded to larger, more diverse cargo as well, abrogating the complexity of previously demonstrated systems while offering broader tunability.

Keywords:  glycosaminoglycan; inflammation; nanoparticle; peptide; tunable

DOI:  https://doi.org/10.3390/pharmaceutics16020173
J Pept Sci. 2024 Feb 21. e3561

Small molecule- and peptide-drug conjugates addressing integrins: A story of targeted cancer treatment.

Jannik Paulus, Norbert Sewald.

  Targeted cancer treatment should avoid side effects and damage to healthy cells commonly encountered during traditional chemotherapy. By combining small molecule or peptidic ligands as homing devices with cytotoxic drugs connected by a cleavable or non-cleavable linker in peptide-drug conjugates (PDCs) or small molecule-drug conjugates (SMDCs), cancer cells and tumours can be selectively targeted. The development of highly affine, selective peptides and small molecules in recent years has allowed PDCs and SMDCs to increasingly compete with antibody-drug conjugates (ADCs). Integrins represent an excellent target for conjugates because they are overexpressed by most cancer cells and because of the broad knowledge about native binding partners as well as the multitude of small-molecule and peptidic ligands that have been developed over the last 30 years. In particular, integrin αV β3 has been addressed using a variety of different PDCs and SMDCs over the last two decades, following various strategies. This review summarises and describes integrin-addressing PDCs and SMDCs while highlighting points of great interest.

Keywords:  drug conjugates; integrins; peptides; peptidomimetics; tumor targeting

DOI:  https://doi.org/10.1002/psc.3561
Biochem J. 2024 Feb 21. pii: BCJ20240015. [Epub ahead of print]

Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.

Laura Weatherdon, Kate Stuart, Megan A Cassidy, Alberto Moreno de la Gándara, Hanneke Okkenhaug, Markus Muellener, Grahame Mckenzie, Simon J Cook, Rebecca Gilley.

  The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or Protein Interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

Keywords:  BRAF; ERK1/2; Gene reporters; KRAS; MEK1/2; small molecules

DOI:  https://doi.org/10.1042/BCJ20240015
Eur J Nucl Med Mol Imaging. 2024 Feb 20.

[68Ga]Ga-FAP-2286, a novel promising theragnostic approach for PET/CT imaging in patients with various type of metastatic cancers.

Seyedeh Somayyeh Banihashemian, Ghasemali Divband, Elahe Pirayesh, Babak Nikkholgh, Hamidreza Amini, Abdolghafar Abolhosseini Shahrnoy, Reza Nami, Mohammad Esmaeil Akbari.

   BACKGROUND: Fibroblast activation protein (FAP) has emerged as a promising target for diagnosis and therapeutic intervention due to high expression and accumulation in the stromal compartments of a variety of malignant tumors. FAP-2286 utilizes cyclic peptides with FAP-binding characteristics to enhance the retention of the imaging agent within tumors, in contrast to the small-molecule FAP inhibitors (FAPI) like FAPI-04/46. The aim of this study was to quantify the tumor uptake of [68Ga] Gallium-FAP-2286 within primary solid tumors, adjacent excised tissues, and metastatic lesions.
METHODS: In this prospective study, 21 patients (average age 51.9) with various diagnoses of remaining and metastatic cancers participated. Among them, six had metastatic sarcoma, and 14 had adenocarcinoma, including eight breast, two rectum, two lung, two pancreas, and one thyroid cases. The patients underwent a [68Ga]Ga-FAP-2286 PET/CT scan. An hour post-administration of [68Ga]Ga-FAP-2286, a visual assessment of whole body scans and semi-quantification of the PET/CT results were carried out. The standardized uptake values (SUV)max of [68Ga]Ga-FAP-2286 in tumor lesions and the tumor-to-background ratio (TBR) were then calculated.
RESULTS: The vital signs of the patients, such as heart rate, blood pressure, and temperature, were observed before, during, and after the diagnostic procedure during the 4-h follow-up. All individuals underwent the [68Ga]Ga-FAP-2286 PET/CT scans without any signs of drug-associated pharmacological effects. The PET/CT scans displayed substantial absorption of [68Ga]Ga-FAP-2286 in tumor lesions in all patients (100% (21/21)). Irrespective of the tumors' origins (epithelial or mesothelium) and whether they exhibited local recurrence, distant recurrence, or metastatic lesions, the PET/CT scans revealed the uptake of [68Ga]Ga-FAP-2286 in these lesions.
CONCLUSION: Overall, these data suggest that [68Ga]Ga-FAP-2286 is a promising FAP derivative for efficient metastatic cancer diagnosis and being considered as a potential compound for therapeutic application in patients with advanced metastatic cancers.

Keywords:  Fibroblast activation protein; Metastatic cancers; Theragnostic radionuclides; [68Ga]Ga-PET/CT imaging

DOI:  https://doi.org/10.1007/s00259-024-06635-8
Pharmaceuticals (Basel). 2024 Feb 02. pii: 201. [Epub ahead of print]17(2):

Immunomodulatory Peptides as Vaccine Adjuvants and Antimicrobial Agents.

Shiva Hemmati, Zahra Saeidikia, Hassan Seradj, Abdolali Mohagheghzadeh.

  The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides.

Keywords:  adjuvant; antibiotics; anticancer peptide; antigen-presenting cell; antimicrobial peptide; artificial intelligence; cell-penetrating peptide; cytokine; immunotherapy

DOI:  https://doi.org/10.3390/ph17020201
Nat Rev Chem. 2024 Feb 22.

Selenium chemistry for spatio-selective peptide and protein functionalization.

Zhenguang Zhao, Shay Laps, Jacob S Gichtin, Norman Metanis.

The ability to construct a peptide or protein in a spatio-specific manner is of great interest for therapeutic and biochemical research. However, the various functional groups present in peptide sequences and the need to perform chemistry under mild and aqueous conditions make selective protein functionalization one of the greatest synthetic challenges. The fascinating paradox of selenium (Se) - being found in both toxic compounds and also harnessed by nature for essential biochemical processes - has inspired the recent exploration of selenium chemistry for site-selective functionalization of peptides and proteins. In this Review, we discuss such approaches, including metal-free and metal-catalysed transformations, as well as traceless chemical modifications. We report their advantages, limitations and applications, as well as future research avenues.

DOI: https://doi.org/10.1038/s41570-024-00579-1
Colloids Surf B Biointerfaces. 2024 Jan 30. pii: S0927-7765(24)00030-4. [Epub ahead of print]236 113772

Rigid-flexible nanocarriers loaded with active peptides for antioxidant and anti-inflammatory applications in skin.

Yan Wang, Jialiang Lin, Zihao Yu, Jinbo Cheng, Jianhua Cheng, Weikang Cui.

  Peptides are recognized as highly effective and safe bioactive ingredients. However, t their practical application is limited and hampered by harsh conditions for practical drug delivery. Hence, a novel peptide nanocarrier of copper peptide (GHK-Cu) encapsulation developed by liposome technology combined with the classical Chinese concept of rigidity and flexibility. Different polyols were selected as modification ligands for phospholipid bilayers to construct a nano drug-carrying system with high loading rate, good stability and biocompatibility. In vitro, this complex not only significantly retarded the release ability of copper peptides, but also enabled copper peptides to be effectively resistant to enzymatic degradation. Furthermore, cellular experiments showed that this system mainly regulates Nrf2, SIRT1, and PEG2/COX-2-related signaling pathways, thus effectively counteracting cellular inflammation, senescence, and apoptosis from oxidative damage. Interestingly, a green, non-toxic, efficient and convenient antioxidant system was developed for the prevention and deceleration of skin aging.

Keywords:  Anti-inflammatory; Antioxidant; Bioactive peptides; Nanoliposomes

DOI:  https://doi.org/10.1016/j.colsurfb.2024.113772
J Control Release. 2024 Feb 19. pii: S0168-3659(24)00102-0. [Epub ahead of print]

Engineered exosomes with enhanced stability and delivery efficiency for glioblastoma therapy.

Yutong Wang, Yiming Huo, Chunyuan Zhao, Heng Liu, Yurou Shao, Chenqi Zhu, Lan An, Xiao Chen, Zhipeng Chen.

  Due to the blood-brain barrier (BBB), the application of chemical drugs for glioblastoma treatment is severely limited. Recently, exosomes have been widely applied for drug delivery to the brain. However, the differences in brain targeting efficiency among exosomes derived from different cell sources, as well as the premature drug leakage during circulation, still limit the therapeutic efficacy. Here, we designed a functional oligopeptide-modified exosome loaded with doxorubicin (Pep2-Exos-DOX) for glioblastoma treatment. BV2 mouse microglial cell line was selected as the exosome source due to the favorable BBB penetration. To avoid drug release in the circulation, a redox-response oligopeptide was designed for incorporation into the membranes of exosomes to lock the drug during circulation. The enrichment of the drug in glioblastoma was confirmed. Pharmacodynamic evaluation showed Pep2-Exos-DOX possessed significant anti-cancer activity against glioblastoma as well as relative biosafety. This exosome-based drug delivery system modified with redox-response oligopeptides provides us a novel strategy for brain diseases treatment.

Keywords:  BBB penetration; Exosome; Functional oligopeptide; Glioma treatment; Redox-response

DOI:  https://doi.org/10.1016/j.jconrel.2024.02.015
Mol Pharm. 2024 Feb 23.

Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.

Biao Hu, Xiaopan Ma, Linqing Shi, Tianyu Liu, Liqiang Li, Meinan Yao, Chenzhen Li, Bing Jia.

  The expression level of PD-L1 in tumor tissue is considered one of the effective biomarkers to guide PD-1/PD-L1 therapy. Quantifying whole-body PD-L1 expression by SPECT imaging may help in selecting patients that potentially respond to PD-1/PD-L1 therapy. Nanobody is the smallest antibody fragment with antigen-binding ability that is well suited for radionuclide imaging. Nevertheless, high retention of radioactivity in the kidney may limit its clinical translation. The present study aimed to screen, design, and prepare a nanobody-based SPECT probe with rapid renal clearance to evaluate the PD-L1 expression level in vivo noninvasively. A phage library was constructed by immunizing alpaca with recombinant human PD-L1 protein, and 17 anti-PD-L1 nanobodies were screened by the phage display technique. After sequence alignment and flow cytometry analysis, APN09 was selected as the candidate nanobody, and a GGGC chelator was attached to its C-terminus for 99mTc labeling to prepare a SPECT imaging probe. The affinity and specificity of 99mTc-APN09 were evaluated by protein and cell-binding experiments, and SPECT imaging and biodistribution were performed in a mouse model with bilateral transplantation of A549 and A549PD-L1 tumors. The ability of 99mTc-APN09 to quantify the PD-L1 expression level in vivo was validated in tumor models with different PD-L1 expression levels. 99mTc-APN09 had a radiochemical purity higher than 99% and a binding equilibrium dissociation constant of 21.44 ± 1.65 nM with hPD-L1, showing high affinity. SPECT imaging results showed that 99mTc-APN09 could efficiently detect PD-L1-positive tumors within 0.5 h, and the quantitative results of SPECT were well correlated with the expression level of PD-L1 in cell lines. SPECT imaging and biodistribution results also showed that 99mTc-APN09 was rapidly cleared from the kidney in 2 h postinjection. 99mTc-APN09 was a simple and stable tool for visualizing PD-L1 expression in the whole body. In addition, due to its significant reduction in renal retention, it has better prospects for clinical translation.

Keywords:  99mTc; APN09; PD-L1; SPECT imaging; nanobody

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c01219
Pharmaceuticals (Basel). 2024 Feb 08. pii: 225. [Epub ahead of print]17(2):

Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Xinxin Rao, Zhibin Qiao, Yang Yang, Yun Deng, Zhen Zhang, Xiaoli Yu, Xiaomao Guo.

  Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

Keywords:  drug screening; epigenetic inhibitors; patient-derived organoids; treatment; triple-negative breast cancer

DOI:  https://doi.org/10.3390/ph17020225
J Nat Prod. 2024 Feb 22.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.

Tim Berger, Judith Alenfelder, Sophie Steinmüller, Dominik Heimann, Namrata Gohain, Daniel Petras, Mingxun Wang, Robert Berger, Evi Kostenis, Raphael Reher.

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily.

DOI: https://doi.org/10.1021/acs.jnatprod.3c00750
Int J Mol Sci. 2024 Feb 12. pii: 2204. [Epub ahead of print]25(4):

Dimeric Tubulin Modifies Mechanical Properties of Lipid Bilayer, as Probed Using Gramicidin A Channel.

Tatiana K Rostovtseva, Michael Weinrich, Daniel Jacobs, William M Rosencrans, Sergey M Bezrukov.

  Using the gramicidin A channel as a molecular probe, we show that tubulin binding to planar lipid membranes changes the channel kinetics-seen as an increase in the lifetime of the channel dimer-and thus points towards modification of the membrane's mechanical properties. The effect is more pronounced in the presence of non-lamellar lipids in the lipid mixture used for membrane formation. To interpret these findings, we propose that tubulin binding redistributes the lateral pressure of lipid packing along the membrane depth, making it closer to the profile expected for lamellar lipids. This redistribution happens because tubulin perturbs the lipid headgroup spacing to reach the membrane's hydrophobic core via its amphiphilic α-helical domain. Specifically, it increases the forces of repulsion between the lipid headgroups and reduces such forces in the hydrophobic region. We suggest that the effect is reciprocal, meaning that alterations in lipid bilayer mechanics caused by membrane remodeling during cell proliferation in disease and development may also modulate tubulin membrane binding, thus exerting regulatory functions. One of those functions includes the regulation of protein-protein interactions at the membrane surface, as exemplified by VDAC complexation with tubulin.

Keywords:  amphitropic proteins; lipid packing stress; non-lamellar lipids; planar lipid membranes; protein–lipid interactions

DOI:  https://doi.org/10.3390/ijms25042204
Br J Dermatol. 2024 Feb 20. pii: ljae061. [Epub ahead of print]

Prediction, screening and characterization of novel bioactive tetra-peptide matrikines for skin rejuvenation.

Nathan Jariwala, Matiss Ozols, Alexander Eckersley, Bezaleel Mambwe, Rachel E B Watson, Leo Zeef, Andrew Gilmore, Laurent Debelle, Mike Bell, Eleanor J Bradley, Yegor Doush, Amy Keenan, Carole Courage, Richard Leroux, Olivier Peschard, Philippe Mondon, Caroline Ringenbach, Laure Bernard, Aurelien Pitois, Michael J Sherratt.

BACKGROUND: Extracellular matrices play a critical role in tissue structure and function and aberrant remodelling of these matrices is a hallmark of many age-related diseases. In skin, loss of dermal collagens and disorganisation elastic fibre components are key features of photo-ageing. Although application of some small matrix-derived peptides to aged skin has been shown to beneficially affect in vitro cell behaviour and, in vivo, molecular architecture and clinical appearance, the discovery of new peptides has lacked a guiding hypothesis.
OBJECTIVES: As endogenous matrix-derived peptides can act as cell-signalling molecules (matrikines), we hypothesised that protease cleavage site prediction could identify novel putative matrikines with beneficial activities for skin composition and structure.
METHODS: Here, we present an in silico (peptide cleavage prediction) to in vitro (proteomic and transcriptomic activity testing in cultured human dermal fibroblasts) to in vivo (short term patch test and longer term split-face clinical study) discovery pipeline, which enables the identification and characterisation of peptides with differential activities.
RESULTS: Using this pipeline we show that cultured fibroblasts are responsive to all applied peptides but their associated bioactivity is sequence-dependent. Based on bioactivity, toxicity and protein source we further characterised a combination of two novel peptides, GPKG and LSVD, that act in vitro to enhance the transcription of matrix organisation and cell proliferation genes and in vivo, in a short-term patch test, to promote processes associated with epithelial and dermal maintenance and remodelling. Prolonged use of a formulation containing these peptides in a split-face clinical study led to significantly improved measures of crow's feet and firmness in a mixed-ethnicity population.
CONCLUSIONS: We conclude that this approach to peptide discovery and testing can identify new synthetic matrikines, providing insights into biological mechanisms of tissue homeostasis and repair and new pathways to clinical intervention.

DOI: https://doi.org/10.1093/bjd/ljae061
Biology (Basel). 2024 Feb 13. pii: 120. [Epub ahead of print]13(2):

Structural and Functional Insights into CRF Peptides and Their Receptors.

Minos-Timotheos Matsoukas, Vasilis Panagiotopoulos, Vlasios Karageorgos, George P Chrousos, Maria Venihaki, George Liapakis.

  Corticotropin-releasing factor or hormone (CRF or CRH) and the urocortins regulate a plethora of physiological functions and are involved in many pathophysiological processes. CRF and urocortins belong to the family of CRF peptides (CRF family), which includes sauvagine, urotensin, and many synthetic peptide and non-peptide CRF analogs. Several of the CRF analogs have shown considerable therapeutic potential in the treatment of various diseases. The CRF peptide family act by interacting with two types of plasma membrane proteins, type 1 (CRF1R) and type 2 (CRF2R), which belong to subfamily B1 of the family B G-protein-coupled receptors (GPCRs). This work describes the structure of CRF peptides and their receptors and the activation mechanism of the latter, which is compared with that of other GPCRs. It also discusses recent structural information that rationalizes the selective binding of various ligands to the two CRF receptor types and the activation of receptors by different agonists.

Keywords:  CRF-peptides; CRF-receptors; activation; agonists; antagonists; binding; structure

DOI:  https://doi.org/10.3390/biology13020120
Cancers (Basel). 2024 Feb 18. pii: 823. [Epub ahead of print]16(4):

Exosomes in Glioma: Unraveling Their Roles in Progression, Diagnosis, and Therapy.

Song Yang, Yumeng Sun, Wei Liu, Yi Zhang, Guozhu Sun, Bai Xiang, Jiankai Yang.

  Gliomas, the most prevalent primary malignant brain tumors, present a challenging prognosis even after undergoing surgery, radiation, and chemotherapy. Exosomes, nano-sized extracellular vesicles secreted by various cells, play a pivotal role in glioma progression and contribute to resistance against chemotherapy and radiotherapy by facilitating the transportation of biological molecules and promoting intercellular communication within the tumor microenvironment. Moreover, exosomes exhibit the remarkable ability to traverse the blood-brain barrier, positioning them as potent carriers for therapeutic delivery. These attributes hold promise for enhancing glioma diagnosis, prognosis, and treatment. Recent years have witnessed significant advancements in exosome research within the realm of tumors. In this article, we primarily focus on elucidating the role of exosomes in glioma development, highlighting the latest breakthroughs in therapeutic and diagnostic approaches, and outlining prospective directions for future research.

Keywords:  diagnosis; drug delivery system; exosomes; gliomas; treatment

DOI:  https://doi.org/10.3390/cancers16040823
Biosensors (Basel). 2024 Feb 11. pii: 96. [Epub ahead of print]14(2):

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Santhosh Kannan, Chien-Chung Peng, Hsiao-Mei Wu, Yi-Chung Tung.

  Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Keywords:  frequency domain fluorescence lifetime imaging microscopy (FD-FLIM); microfluidics; oxygen consumption; spheroid

DOI:  https://doi.org/10.3390/bios14020096
Methods Mol Biol. 2024 ;2764 291-310

Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma, Quantitative Analysis, Optical Clearing, and Mathematical Modeling.

Loredana Spoerri, Kimberley A Beaumont, Andrea Anfosso, Ryan J Murphy, Alexander P Browning, Gency Gunasingh, Nikolas K Haass.

  Aberrant cell cycle progression is a hallmark of solid tumors. Therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information. In this chapter, we describe a technique that allows spatiotemporal real-time tracking of cell cycle progression of individual cells in a multicellular context. The power of this system lies in the use of 3D melanoma spheroids generated from melanoma cells engineered with the fluorescent ubiquitination-based cell cycle indicator (FUCCI). This technique, combined with mathematical modeling, allows us to gain further and more detailed insight into several relevant aspects of solid cancer cell biology, such as tumor growth, proliferation, invasion, and drug sensitivity.

Keywords:  3D spheroid; Cancer drug resistance; Fluorescent ubiquitination-based cell cycle indicator (FUCCI); Invasion; Mathematical modeling; Migration; Real-time imaging; Tumor heterogeneity; Tumor microenvironment

DOI:  https://doi.org/10.1007/978-1-0716-3674-9_19