The parasitic lifestyle of an archaeal symbiont.

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.

Phosphatidylserine regulates plasma membrane repair through tetraspanin-enriched macrodomains.

The integrity of the plasma membrane is critical to cell function and survival. Cells have developed multiple mechanisms to repair damaged plasma membranes. A key process during plasma membrane repair is to limit the size of the damage, which is facilitated by the presence of tetraspanin-enriched rings surrounding damage sites. Here, we identify phosphatidylserine-enriched rings surrounding damaged sites of the plasma membrane, resembling tetraspanin-enriched rings. Importantly, the formation of both the phosphatidylserine- and tetraspanin-enriched rings requires phosphatidylserine and its transfer proteins ORP5 and ORP9. Interestingly, ORP9, but not ORP5, is recruited to the damage sites, suggesting cells acquire phosphatidylserine from multiple sources upon plasma membrane damage. We further demonstrate that ORP9 contributes to efficient plasma membrane repair. Our results thus unveil a role for phosphatidylserine and its transfer proteins in facilitating the formation of tetraspanin-enriched macrodomains and plasma membrane repair.

ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

A novel method to quantify perivascular space enlargement near the syrinx in a rodent model of post-traumatic syringomyelia.

Posttraumatic syringomyelia (PTS) is an enigmatic condition characterized by the development of fluid-filled cysts (syrinxes) within the spinal cord. Perivascular spaces (PVS) are a critical component of fluid transport within the central nervous system (CNS), with dilated PVSs variably implicated in the pathogenesis of syringomyelia. The extent and spatial distribution of dilated PVSs in syringomyelia, however, remains unclear. This study aims to develop a method to assess PVS dimensions across multiple spinal cord segments in rats with PTS. Syrinxes were induced in two Sprague-Dawley rats at C6/7 with computer-controlled motorized spinal cord impaction; two control rats underwent sham laminectomies. Spinal cord segments were obtained at C4, C6 and C8, cleared via tissue clearing protocols, stained with immunofluorescent antibodies and imaged under confocal microscopy. Qualitative and quantitative analyses of PVS size were performed. Arteriolar PVSs were enlarged in the perisyringeal region of the spinal cord, compared to the control cord. No PVS enlargement was observed above or below the syrinx. These results confirm previous incidental findings of enlarged PVSs in the perisyringeal region, providing new insights into PVS dimensions across multiple spinal segments, and providing a novel method for quantifying spinal cord perivascular space size distributions.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-ß signaling pathways in cancer.

BACKGROUND: Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-ß levels and EMT signaling. Given that many drugs targeting TGF-ß have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-ß/EMT axis. RESULTS: Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-ß and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-ß levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-ß/SMAD2&3) and non-canonical (TGF-ß/PI3K/AKT, TGF-ß/RAS/RAF/MEK/ERK, and TGF-ß/WNT/ß-catenin) TGF-ß signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, ß-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. CONCLUSIONS: Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-ß and inhibiting EMT in a diverse range of cancers.

Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface.

Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host-pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis.

PI(3)P and DFCP1 regulate the biogenesis of lipid droplets.

The biogenesis of lipid droplets (LDs), key organelles for cellular lipid storage and homeostasis, remains poorly understood. Seipin is essential to normal LD biogenesis but exactly how it regulates LD initiation remains to be elucidated. Our previous results suggested that seipin may bind anionic phospholipids such as PI(3)P. Here, we investigate whether PI(3)P is functionally linked to seipin and whether PI(3)P can also impact LD biogenesis. In seipin-deficient cells, there were enlarged PI(3)P puncta where its effector, DFCP1, also appeared to congregate. Reducing cellular PI(3)P partially rescued the defective LD initiation caused by seipin deficiency. Increasing PI(3)P impeded the lipidation of nascent LDs. We further demonstrated that DFCP1 localized to LDs and facilitated the efficient lipidation of nascent LDs. However, the normal function and localization of DFCP1 were disrupted when cellular PI(3)P homeostasis was perturbed. Our results thus identify PI(3)P as a novel regulator of LD initiation and suggest that PI(3)P may impact the biogenesis of LDs through DFCP1.

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect.

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Application of Rapid Fluorescence Lifetime Imaging Microscopy (RapidFLIM) to Examine Dynamics of Nanoparticle Uptake in Live Cells.

A key challenge in nanomedicine stems from the continued need for a systematic understanding of the delivery of nanoparticles in live cells. Complexities in delivery are often influenced by the biophysical characteristics of nanoparticles, where even subtle changes to nanoparticle designs can alter cellular uptake, transport and activity. Close examination of these processes, especially with imaging, offers important insights that can aid in future nanoparticle design or translation. Rapid fluorescence lifetime imaging microscopy (RapidFLIM) is a potentially valuable technology for examining intracellular mechanisms of nanoparticle delivery by directly correlating visual data with changes in the biological environment. To date, applications for this technology in nanoparticle research have not been explored. A PicoQuant RapidFLIM system was used together with commercial silica nanoparticles to follow particle uptake in glioblastoma cells. Importantly, RapidFLIM imaging showed significantly improved image acquisition speeds over traditional FLIM, which enabled the tracking of nanoparticle uptake into subcellular compartments. We determined mean lifetime changes and used this to delineate significant changes in nanoparticle lifetimes (>0.39 ns), which showed clustering of these tracks proximal to both extracellular and nuclear membrane boundaries. These findings demonstrate the ability of RapidFLIM to track, localize and quantify changes in single nanoparticle fluorescence lifetimes and highlight RapidFLIM as a valuable tool for multiparameter visualization and analysis of nanoparticle molecular dynamics in live cells.

Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity.

Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.

Rapid FLIM Measurement of Membrane Tension Probe Flipper-TR.

In the following protocol, we describe the application of rapid fluorescence lifetime imaging to the measurement of membrane tension. The recent developments in tension sensing probes have resulted in probes which allow for quantification of membrane tension through measurement of fluorescence lifetime change with increasing or decreasing tension. In this protocol, we describe the acquisition and analysis steps required for these types of experiments and demonstrate how the fluorescence lifetime reports on change in membrane tension as a result of osmotic shock in live HeLa cells.

Spatio-temporal analysis of nanoparticles in live tumor spheroids impacted by cell origin and density.

Nanoparticles hold great preclinical promise in cancer therapy but continue to suffer attrition through clinical trials. Advanced, three dimensional (3D) cellular models such as tumor spheroids can recapitulate elements of the tumor environment and are considered the superior model to evaluate nanoparticle designs. However, there is an important need to better understand nanoparticle penetration kinetics and determine how different cell characteristics may influence this nanoparticle uptake. A key challenge with current approaches for measuring nanoparticle accumulation in spheroids is that they are often static, losing spatial and temporal information which may be necessary for effective nanoparticle evaluation in 3D cell models. To overcome this challenge, we developed an analysis platform, termed the Determination of Nanoparticle Uptake in Tumor Spheroids (DONUTS), which retains spatial and temporal information during quantification, enabling evaluation of nanoparticle uptake in 3D tumor spheroids. Outperforming linear profiling methods, DONUTS was able to measure silica nanoparticle uptake to 10 µm accuracy in both isotropic and irregularly shaped cancer cell spheroids. This was then extended to determine penetration kinetics, first by a forward-in-time, center-in-space model, and then by mathematical modelling, which enabled the direct evaluation of nanoparticle penetration kinetics in different spheroid models. Nanoparticle uptake was shown to inversely relate to particle size and varied depending on the cell type, cell stiffness and density of the spheroid model. The automated analysis method we have developed can be applied to live spheroids in situ, for the advanced evaluation of nanoparticles as delivery agents in cancer therapy.

Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ macrophages as a predictor of improved survival in triple negative breast cancer.

Triple negative breast cancer (TNBC) comprises 10-15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33-0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25-0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine.

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.

Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.

BACKGROUND: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation. METHODS: Nasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition" (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult™-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADiALI under basal and CFTR corrector treated (VX-809) conditions. RESULTS: No significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. CONCLUSION: Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.

Novel Antioxidant Therapy with the Immediate Precursor to Glutathione, γ-Glutamylcysteine (GGC), Ameliorates LPS-Induced Cellular Stress in In Vitro 3D-Differentiated Airway Model from Primary Cystic Fibrosis Human Bronchial Cells.

Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.

TILs Immunophenotype in Breast Cancer Predicts Local Failure and Overall Survival: Analysis in a Large Radiotherapy Trial with Long-Term Follow-Up.

AIM: To determine the prognostic significance of the immunophenotype of tumour-infiltrating lymphocytes (TILs) within a cohort of breast cancer patients with long-term follow-up. METHODS: Multiplexed immunofluorescence and automated image analysis were used to assess the expression of CD3, CD8, CD20, CD68, Fox P3, PD-1 and PD-L1 in a clinical trial of local excision and radiotherapy randomised to a cavity boost or not (n = 485, median follow-up 16 years). Kaplan-Meier and Cox multivariate analysis (MVA) methodology were used to ascertain relationships with local recurrence (LR), overall survival (OS) and disease-free survival (DFS). NanoString BC360 gene expression panel was applied to a subset of luminal patients to identify pathways associated with LR. RESULTS: LR was predicted by low CD8 in MVA in the whole cohort (HR 2.34, CI 1.4-4.02, p = 0.002) and luminal tumours (HR 2.19, CI 1.23-3.92, p = 0.008) with associations with increased stromal components, decreased Tregs (FoxP3), inflammatory chemokines and SOX2. Poor OS was associated with low CD20 in the whole cohort (HR 1.73, CI 1.2-2.4, p = 0.002) and luminal tumours on MVA and low PD-L1 in triple-negative cancer (HR 3.44, CI 1.5-7, p = 0.003). CONCLUSIONS: Immunophenotype adds further prognostic data to help further stratify risk of LR and OS even in TILs low-luminal tumours.

Nanomolar ATP binding to single myosin cross-bridges in rigor: a molecular approach to studying myosin ATP kinetics using single human cardiomyocytes.

Our knowledge in the field of cardiac muscle and associated cardiomyopathies has been evolving incrementally over the past 60 years and all was possible due to the parallel progress in techniques and methods allowing to take a fresh glimpse at an old problem. Here, we describe an exciting tool used to examine the various states of the human cardiac myosin at the single molecule level. By imaging single Alexa647-ATP binding to permeabilised cardiomyocytes using total internal reflection fluorescence (TIRF) microscopy, we are able to acquire large populations of events in a short timeframe (~ 5000 sites in ~ 10 min) and measure each binding event with high spatio-temporal resolution. The applied algorithm decomposes the point pattern of single molecule binding events into individually distinct binding sites that enables us to recover kinetic parameters, such as bound or free time per site. This single molecule binding approach is a useful tool used to examine muscle contractility. Of particular importance is its application to probing the dynamic lifetimes and proportion of myosins in the super-relaxed state in human cardiomyopathies.

Identification of Novel Medulloblastoma Cell-Targeting Peptides for Use in Selective Chemotherapy Drug Delivery.

Medulloblastoma is a malignant brain tumor diagnosed in children. Chemotherapy has improved survival rates to approximately 70%; however, children are often left with long-term treatment side effects. New therapies that maintain a high cure rate while reducing off-target toxicity are required. We describe for the first time the use of a bacteriophage-peptide display library to identify heptapeptides that bind to medulloblastoma cells. Two heptapeptides that demonstrated high [E1-3 (1)] or low [E1-7 (2)] medulloblastoma cell binding affinity were synthesized. The potential of the peptides to deliver a therapeutic drug to medulloblastoma cells with specificity was investigated by conjugating E1-3 (1) or E1-7 (2) to doxorubicin (5). Both peptide-drug conjugates were cytotoxic to medulloblastoma cells. E1-3 doxorubicin (3) could permeabilize an in vitro blood-brain barrier and showed a marked reduction in cytotoxicity compared to free doxorubicin (5) in nontumor cells. This study provides proof-of-concept for developing peptide-drug conjugates to inhibit medulloblastoma cell growth while minimizing off-target toxicity.