Extrinsic Mechanisms Research Articles

Overcoming Resistance Mechanisms to Melanoma Immunotherapy.

The advent of immune checkpoint inhibition has revolutionized treatment of advanced melanoma. While most patients derive survival benefit from established immunotherapies, notably monoclonal antibodies blocking cytotoxic T-lymphocyte antigen 4 and programmed cell death protein 1, a subset does not optimally respond due to the manifestation of innate or acquired resistance to these therapies. Combination regimens have proven efficacious relative to single-agent blockade, but also yield high-grade treatment toxicities that are often dose-limiting for patients. In this review, we discuss the significant strides made in the past half-decade toward expanding the melanoma immunotherapy treatment paradigm. These include newly approved therapies, adoption of neoadjuvant immunotherapy, and studies in the clinical trials pipeline targeting alternative immune checkpoints and key immunoregulatory molecules. We then review how developments in molecular and functional diagnostics have furthered our understanding of the tumor-intrinsic and -extrinsic mechanisms driving immunotherapy resistance, as well as highlight novel biomarkers for predicting treatment response. Throughout, we discuss potential approaches for targeting these resistance mechanisms in rational combination with established immunotherapies to improve outcomes for patients with melanoma.

Exploring giant dielectric constant in Dy2CoMnO6: evidence of non-overlapping small polaron hopping

Abstract Perovskites, both natural and synthetic, form a large class of materials are a vast class of materials with significant technological relevance due to their remarkable physical properties, such as superconductivity, magnetoresistance, ionic conductivity, and diverse dielectric behaviors. In this study, the dielectric relaxation and transport properties of the double perovskite Dy2CoMnO6(DCMO) synthesized via high-energy ball milling are investigated. DCMO exhibits a notably large dielectric constant, attributed to a combination of intrinsic and extrinsic mechanisms. Frequency-dependent dielectric studies reveal non-Debye-like behavior, validated by augmented Havriliak-Negami function fitting. Impedance spectroscopy confirms the semiconducting nature of DCMO, showing a negative temperature coefficient of resistance, and identifies two distinct relaxation processes corresponding to grain boundaries and grain interiors thereby highlighting the impact of microstructure and defects. The Cole-Cole plot further supports the non-Debye behavior, while thermally activated relaxation suggests damped charge carrier dynamics at grain boundaries. Conduction analysis using augmented Jonscher's power law reveals non-overlapping small polaron tunneling as the dominant mechanism driving both the dielectric response and transport properties, with DC conductivity suggesting a three-dimensional variable range hopping model. These results provide significant insights into the dielectric and transport properties of DCMO, highlighting its promising potential for advanced electronic applications.

Coexisting Ferromagnetic-Antiferromagnetic State and Giant Anomalous Hall Effect in Chemical Vapor Deposition-Grown 2D Cr5Te8.

Two-dimensional (2D) magnets, as an important member of the 2D material family, have emerged as a promising platform for spintronic devices. Herein, we report the chemical vapor deposition (CVD) growth of highly crystalline submillimeter-scale self-intercalated metallic 2D ferromagnetic (FM) trigonal chromium telluride (Cr5Te8) flakes on inert mica substrates. Through magneto-optical and magnetotransport measurements, we unveil the exceptional magnetic properties of these 2D flakes. The trigonal Cr5Te8 flakes exhibit a strong anisotropic FM order with a Curie temperature above 220 K. Notably, an emergent antiferromagnetic (AFM) state is observed in the MOKE signal from ultrathin Cr5Te8 flakes around the Curie temperature. The AFM state has a relatively weak interlayer exchange coupling, allowing a switching between the interlayer AFM and FM states by tuning the temperature. Meanwhile, the trigonal Cr5Te8 flakes exhibit a giant anomalous Hall effect (AHE), with an anomalous Hall conductivity of 710 Ω-1 cm-1 and an anomalous Hall angle of 3.5% at zero magnetic field, surpassing typical itinerant ferromagnets. Further analysis suggests that the AHE in trigonal Cr5Te8 is primarily driven by the skew-scattering mechanism rather than the intrinsic or extrinsic side-jump mechanism. These findings demonstrate the potential of CVD-grown ultrathin Cr5Te8 flakes as a promising 2D magnetic material with exceptional AHE properties for future spintronic applications.

Anisotropy in fracture toughness and toughening mechanism of Ti-22Al-25Nb alloy related to texture and grain boundary damage

Combining the intrinsic (crack tip plasticity) and extrinsic (crack deflection) toughening mechanism, the anisotropy of fracture toughness for Ti-22Al-25Nb alloy obtain by B2 phase region isothermal forging process is discussed. The results show that the sequence of fracture toughness from high to low is RD (radial direction), OD (45° to RD) and AD (axial direction). Intrinsically, it is found that the damage of lamellar O phase ahead of the crack tip dominates crack propagation. Compared with grain interior, continuous lamellar O phase on grain boundary can more easily promote the nucleation and propagation of crack, reducing the intrinsic resistance of crack propagation. Consequently, AD sample with intergranular fracture has the smallest crack tip plastic zone than RD and OD samples with transgranular fracture. Due to the activation of prism <a> slip for O phase, the texture of RD sample has a higher Schmid factor than that of OD sample, resulting in better crack tip plasticity. Extrinsically, it can be concluded that grain boundaries have a significant effect on crack deflection due to the pancake shaped B2 morphology. For RD sample, the grain boundary is perpendicular to the initial crack plane, so the stress intensity factor (KI) required for cleavage fracture is less than that for intergranular fracture. The deflection of the cleavage facets results in the highest crack propagation tortuosity. Also, the crack propagation near grain boundary of OD sample leads to significant deflection because the grain boundary is located in the direction of maximum shear stress. Compared with RD and OD samples, the KI required for intergranular fracture of AD sample is minimum, results in a flat fracture path.

Dual inhibition of HERs and PD-1 counteract resistance in KRASG12C-mutant head and neck cancer

BackgroundBasket clinical trials targeting the KRASG12C-mutation in solid tumors have shown initial promise, including in orphan KRASG12C head and neck cancer (HNC). However, development of resistance to KRASG12C-mutant-specific inhibitors (KRASG12Ci) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRASG12Ci—MRTX849 and AMG510 in a unique syngenic murine KRASG12C-mutated HNC cell line.MethodsWestern-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRASG12Ci in KRASG12C-mutated HNC cell line, 4NQO-L. In vitro KRASG12C-acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8+ T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L– KRASG12Ci-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors.ResultsActivation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRASG12Ci in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8+ T-cells after treatment combination, and these CD8+ T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8+ T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8+ T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome.ConclusionsOur study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRASG12Ci-AcR tumors.

Paradox of Shorter Residual Fatigue Life due to Omission of Low‐Amplitude Cycles and Its Significance for Testing

ABSTRACTThe work investigates nonvalidity of the common presumption that the nondamaging cycles do not influence residual fatigue life. Paradoxically, application of the full loading spectrum (more cycles) resulted in approximately 2.3 times longer life of the fatigue crack growth specimens than application of the spectrum with 33% of the smallest amplitudes omitted. Unlike in humid air (controlled relative humidity of 50% at 23°C), the effect disappeared in a dry‐air chamber (relative humidity &lt;10% at 23°C), where both fatigue lives were short. The mechanism responsible for these effects was identified as the oxide‐induced crack closure, an extrinsic mechanism unrelated to material damage. Oxide debris developed on fracture surfaces was observed by light and scanning electron microscopy, whereas crack closure was measured during the experiments. The presented counterintuitive behavior in humid air may result in wrong assessment or prediction of components residual fatigue lives, which can be nonconservative in some scenarios.

CSIG-24. BRAIN MICROENVIRONMENT-DEPENDENT NEURODEVELOPMENTAL LINEAGE PLASTICITY PROMOTES ADAPTATION TO ONCOGENE INHIBITION IN MALIGNANT GLIOMAS

Abstract Phenotypic plasticity drives non-genetic tumor adaptation in response to various microenvironmental and therapeutic pressures, consequently promoting tumor heterogeneity and progression. In malignant gliomas, the intrinsic and extrinsic mechanisms underlying phenotypic lineage plasticity and its contribution to drug resistance remain unclear. The epidermal growth factor receptor (EGFR) is frequently altered in glioma and serves as a critical regulator of normal radial glia (RG) cells – multipotent progenitors that give rise to cortical neurons and glia in the developing brain. Here, through interrogation of a large panel of diverse glioblastoma (GBM) patient-derived gliomaspheres and orthotopic xenografts, we find that enrichment of RG-like cells is significantly correlated with responses to pharmacological ablation of EGFR, highlighting EGFR as a crucial lineage survival factor for a population of malignant RG-like cells in GBM. Single-cell RNA sequencing and quantitative proteomics reveal that adaptation to EGFR inhibition is linked to a temporal contraction in RG-like cells and concomitant increase in lineage-restricted neuronal and oligodendrocyte progenitor (NPC/OPC)-like states exclusively in the orthotopic brain environment. This lineage transition is coupled to heightened RAS signaling gene expression programs and sustained activation of MAPK signaling, despite robust and durable inhibition of EGFR activation. Furthermore, constitutively active MEK – but not AKT – is sufficient to rescue tumor proliferation under EGFRi, suggesting that the brain microenvironment modulates RAS-MAPK oncogenic signaling to drive lineage transitions following EGFR blockade. Collectively, these results highlight a critical link between oncogenic signaling and neurodevelopmental lineage plasticity in malignant gliomas, presenting a compelling therapeutic opportunity to block tumor cell state transitions for more durable tumor responses in the native glioma tumor microenvironment.

Microglia induce an interferon-stimulated gene expression profile in glioblastoma and increase glioblastoma resistance to temozolomide.

Glioblastoma is the most malignant primary brain tumour. Even with standard treatment comprising surgery followed by radiation and concomitant temozolomide (TMZ) chemotherapy, glioblastoma remains incurable. Almost all patients with glioblastoma relapse owing to various intrinsic and extrinsic resistance mechanisms of the tumour cells. Glioblastomas are densely infiltrated with tumour-associated microglia and macrophages (TAMs). These immune cells affect the tumour cells in experimental studies and are associated with poor patient survival in clinical studies. The aim of the study was to investigate the impact of microglia on glioblastoma chemo-resistance. We co-cultured patient-derived glioblastoma spheroids with microglia at different TMZ concentrations and analysed cell death. In addition, we used RNA sequencing to explore differentially expressed genes after co-culture. Immunostaining was used for validation. Co-culture experiments showed that microglia significantly increased TMZ resistance in glioblastoma cells. RNA sequencing revealed upregulation of a clear interferon-stimulated gene (ISG) expression signature in the glioblastoma cells after co-culture with microglia, including genes such as IFI6, IFI27, BST2, MX1 and STAT1. This ISG expression signature is linked to STAT1 signalling, which was confirmed by immunostaining. The ISG expression profile observed in glioblastoma cells with enhanced TMZ resistance corresponded to the interferon-related DNA damage resistance signature (IRDS) described in different solid cancers. Here, we show that the IRDS signature, linked to chemo-resistance in other cancers, can be induced in glioblastoma by microglia. ISG genes and the microglia inducing the ISG expression could be promising novel therapeutic targets in glioblastoma.

Tunable Anomalous Hall Effect in Broken–Symmetry Integrated Perovskite/Brownmillerite Superlattices

AbstractThe anomalous Hall effect (AHE), a hallmark of broken time–reversal symmetry, serves as a powerful tool for probing magnetic states and elucidating complex spin–orbit interactions in transition metal oxides. It is reported here that the emergence of AHE signals and interfacial ferromagnetism is directly observed from broken–symmetry integrated perovskite PrCoO3 and brownmillerite SrCoO2.5 superlattices. Through the strategic combinations of these two complex oxide systems onto the substrates with tailored lattice parameters, squared AHE hysteresis loops with perpendicular magnetic anisotropy are found under compressive strain in the optimized superlattices, and the magnitude of the loops is more than three times greater compared to those under tensile strain. The manifestation of AHE in these superlattices is attributed to an extrinsic mechanism involving spin–polarized scattering of charge carriers, with the mismatched perovskite/brownmillerite interfaces acting as the primary scattering centers. These discoveries suggest a promising avenue for the development of artificial materials with controllable AHE properties.

Study on the fracture behavior and toughening mechanisms of continuous fiber reinforced Wf/Y2O3/W composites fabricated via powder metallurgy

Tungsten (W) is a promising candidate material for the plasma facing components in fusion reactors. However, it has issues regarding the intrinsic brittleness. Tungsten fiber reinforced tungsten composites (Wf/W) have been developed based on the concept of extrinsic toughening mechanisms and they show a pseudo-ductile behavior during the fracture process. In the present work, continuous fiber reinforced Wf/Y2O3/W composites were fabricated via a powder metallurgy (PM) process, and the microstructure and mechanical properties were characterized. The fracture behavior and toughening mechanisms were analyzed in detail combining the results of experiments and numerical simulation. The Wf/Y2O3/W composites is toughened by multiple mechanisms such as fiber bridging, crack bending and deflection, interface de-bonding and plastic deformation of fiber. The energy dissipation by interface de-bonding can be neglected. However, it is a necessary factor to ensure any extrinsic toughening mechanisms. The main contribution of the energy dissipation while composite failure is the plastic deformation of fibers.

Optimizing heterostructure parameters towards enhanced toughening in micro/nano-reinforced bimodal-grained Al alloy composites

We manipulated soft coarse-grained (CG) domains to design optimized and toughened B4Cp/6061Al composites, featuring bimodal domains with in-situ MgO nanoparticles (n-MgO) and ex-situ carbon nanotubes (CNTs). Manipulating CG fractions influenced heterostructure parameters such as CG band width and domain distribution. A systematic optimization strategy integrating intrinsic/extrinsic toughening and strengthening mechanisms identified optimal conditions for maximizing strength-ductility-toughness. The optimal 20:80 CG-to-ultrafine-grain (UFG) weight ratio offered an ultimate strength of 550 MPa and ∼7 % elongation. Intrinsic toughening mechanisms enhanced UFG domain dislocation storage capacity. Multiscale analysis of crack behavior revealed pronounced crack-blunting in well-dispersed CG domains. Extrinsic toughening mechanisms included nanobridge formation, crack-deflection, micro-crack proliferation, and crack-branching. Micromechanical behavior was examined using the strain gradient model. Designing strong and ductile micro/nano-reinforced bimodal grained composites requires selecting a smaller amount of CG domains, a CG band width larger than double the interface affected zone (IAZ), and larger grain sizes in the CG domains.

Recent advancements in self-healing materials and their application in coating industry

Self-healing polymers (SHP), inspired by nature, are materials that have the ability to recover themselves from various physical damages in the presence of different inducing environments. The self-recovering property indicates the capability to heal the cracks in their very nascent stage at their micro or nano-level size and provides a door to prevent them from any major catastrophic failure. In this article, a comprehensive review with updated reported work on various types of self-healing mechanisms and their advantages and limitations has been discussed, along with their applications. The main focus of the review has been aligned with the challenges and future scopes associated with the exploration of self-healing concepts for industrial coating applications. The different extrinsic, intrinsic, and combined healing mechanisms have been explained, along with their application in coating. Finally, the recent status of healing technology and future research trends has been discussed in this review article.

Microstructure and fracture behaviour of biomimetic Al2O3-ZrO2 composite with 2-levels of structural hierarchy

We report new class of natural nacre-like alumina toughened by zirconia fabricated by the simple two-step approach of unidirectional freeze-casting and spark plasma sintering. The biomimetic alumina-zirconia with a relative density of more than 98 % was achieved by consolidating at temperature of 1300–1425 °C with 50 MPa pressure. The resultant microstructure consists of two levels of structural hierarchy, i.e. level-1: bulk lamellar brick phase (alumina platelets) and level-2: mortar phase (yttria stabilized tetragonal zirconia polycrystals). The increase in volume fraction of zirconia (in tetragonal form) from 0 to 15 vol% was effective in progressively reducing the size of alumina platelets from 12 to 9 μm, and significantly improved the flexural strength of the composite by 60 % (from 172 to 270 MPa). By optimizing the hierarchical architecture from micro to macroscopic level, the structural performance of our synthetic nacre, where strength and toughness of 270 MPa and 13.5 MPa√m respectively are superior to that of natural nacre and many other engineering materials (illustrated in the form of Ashby map). The exceptional damage resistance is rationalized by both intrinsic (stress induced tetragonal to monoclinic phase transformation of zirconia) and extrinsic (crack deflection, crack branching and bridging, multiple cracking, platelets interlocking) toughening mechanisms. For 15 vol% ZrO2 composition, the contribution of intrinsic and extrinsic toughness was quantitatively evaluated as 0.9 ± 0.03 and 4.3 ± 0.07 MPa√m respectively (with experimentally measured toughness at crack initiation as 5.2 ± 0.2 MPa√m).

Disentangling cell-intrinsic and extrinsic factors underlying evolution.

A key goal of developmental biology is to determine the extent to which cells and organs develop autonomously, as opposed to requiring interactions with other cells or environmental factors. Chimeras have played a foundational role in this by enabling qualitative classification of cell-intrinsically vs. extrinsically driven processes. Here, we extend this framework to precisely decompose evolutionary divergence in any quantitative trait into cell-intrinsic, extrinsic, and intrinsic-extrinsic interaction components. Applying this framework to thousands of gene expression levels in reciprocal rat-mouse chimeras, we found that the majority of their divergence is attributable to cell-intrinsic factors, though extrinsic factors also play an integral role. For example, a rat-like extracellular environment extrinsically up-regulates the expression of a key transcriptional regulator of the endoplasmic reticulum (ER) stress response in some but not all cell types, which in turn strongly predicts extrinsic up-regulation of its target genes and of the ER stress response pathway as a whole. This effect is also seen at the protein level, suggesting propagation through multiple regulatory levels. Applying our framework to a cellular trait, neuronal differentiation, revealed a complex interaction of intrinsic and extrinsic factors. Finally, we show that imprinted genes are dramatically mis-expressed in species-mismatched environments, suggesting that mismatch between rapidly evolving intrinsic and extrinsic mechanisms controlling gene imprinting may contribute to barriers to interspecies chimerism. Overall, our conceptual framework opens new avenues to investigate the mechanistic basis of developmental processes and evolutionary divergence across myriad quantitative traits in any multicellular organism.

Enhanced strength–ductility synergy of SiC particles reinforced aluminum matrix composite via dual configuration design of reinforcement and matrix

To overcome the strength-ductility conflict in particle reinforced aluminum matrix composites (PRAMCs), a novel dual configuration strategy of microstructure was proposed in this work. The dual configuration including both heterogeneous grain structure and hybrid reinforcements was obtained by powder metallurgy, which was designed as submicron-sized SiC particles (SiCsm)/Al and micron-sized SiC particles (SiCm)/2024Al components. Representative alternating coarse grain (CG) bands containing SiCm and ultra fine grain (UFG) bands with dispersed SiCsm were revealed in microstructural characterizations. Compared to corresponding homogeneous composites with the same fraction particles, the dual configuration composite achieved simultaneous enhancement in strength and ductility and remained a comparable Young’s modulus of 98GPa, which represented 442 MPa in yield strength, 590 MPa in ultimate strength and 8.7 % in fracture elongation. The extra strength provided by hetero-deformation induced (HDI) strengthening is a potential dominant strengthening mechanism. The intrinsic toughening mechanisms primarily contribute to HDI strain hardening and enhanced dislocation accumulation capacity, while the extrinsic toughening mechanisms presumably attribute to more uniform microcrack distribution and blunting effect of CG zones on cracks. Our work provides a feasible route including ball milling and powder assembly to preparate high-modulus aluminum matrix composites for strength-ductility balance design.

Study of synergistic and competitive mechanisms on toughening CFRP composites with intrinsic and extrinsic multiscale interleaves

Delamination damage is a common failure mode of carbon fiber reinforced polymer (CFRP) laminates, which severely limits their application. This paper proposes a novel interlaminar toughening structure based on intrinsic and extrinsic multiscale toughening mechanisms using nanoscale multi-walled carbon nanotubes (MWCNTs) and microscale core-shell rubber (CSR). The resin sample M-0.5/C-8, containing 0.5 wt% MWCNTs and 8 wt% CSR, exhibited significant improvements in fracture toughness, with increases of 183.3 % in the mode-I critical stress intensity factor (KIC, Resin) and 412.0 % in the critical energy release rate of resin (GIC, Resin). However, its flexural strength and modulus decreased by 3.1 % and 3.3 %, respectively. Introducing MWCNTs and CSR into the interlayers of the CFRP, the interleaved toughened CFRP laminate exhibited a 123.3 % improvement in the mode-I critical energy release rate (GIC, Comp) and a 79.6 % increase in the mode-II critical energy release rate (GIIC, Comp) during the crack propagation stage, without sacrificing flexural performance. This remarkable enhancement in interlaminar fracture toughness results from the simultaneous triggering of nanoscale extrinsic toughening and microscale intrinsic toughening during crack growth. The flexural properties and fracture toughness of Epoxy/MWCNTs/CSR composites were also investigated to understand the properties transformation from resin matrix to CFRP. Morphological characterization and numerical analysis were used to analyze the synergies and constraints between two energy dissipation behaviors.

Intrinsic and extrinsic relaxation mechanisms for controlling spin current intensity in Fe 100−x Co x /Ta bilayers

Controlling the damping parameter in metallic ferromagnetic thin films is a key step for spintronic applications in which spin currents are generated by spin pumping. The coexistence of two states with low and high damping constant values would allow to obtain states of high and low spin current intensity, respectively. We have fabricated Fe 100−x Co x /Ta (with nominal x = 0, 15, 20, 25, 30 and 35) bilayers in which the Fe 100−x Co x layers grow epitaxially and the Ta layer is polycrystalline. We have found the coexistence of Gilbert damping and two magnon scattering mechanisms linked to a sign change in the magnetocrystalline anisotropy constant that allows the manipulation of low and high intensity states of the measured inverse spin Hall effect voltage. Bilayers with lower Co concentrations ( x⩽ 25%) present different relaxation mechanisms (isotropic Gilbert damping and two magnon scattering) and an extra ferromagnetic resonance linewidth broadening produced due to mosaicity. Bilayers with Co concentration x> 25% present a dominating Gilbert damping for all directions in the film plane. However, in this concentration range the damping constant is anisotropic and when the magnetic field is applied along the hard magnetization direction α increases ∼420% with respect to the value obtained for the easy magnetization direction. Coexistence of isotropic Gilbert damping and two magnon scattering generated spin currents 2.5 times larger when the field is applied along the hard magnetization axis compared to the value observed in the easy magnetization axis. Thesefindings make the Fe 100−x Co x /Ta system an excellent candidate for spintronic device applications.

Chronic TNF in the aging microenvironment exacerbates Tet2 loss-of-function myeloid expansion

AbstractSomatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cells (HSCs) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing tumor necrosis factor (TNF) favors TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2 mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF–/– genotypes reconstituted with WT CD45.1+ and Tet2–/– CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2–/– cells in old WT recipient mice, with strong skewing toward the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF–/– recipient mice, suggesting that TNF signaling is essential for the expansion Tet2-mutant myeloid clones. Examination of human patients with rheumatoid arthritis with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

STEM-05. CREATING PATIENT-DERIVED TUMOROIDS TO INVESTIGATE RADIORESISTANCE IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Brain cancers are the most frequently diagnosed tumor types in pediatric and young adult populations. Unfortunately, they often carry a poor prognosis and face common challenges such as treatment resistances due to intra- and intertumoral heterogeneity. To fully understand the intrinsic and extrinsic mechanisms which might hinder proper care, there is an emerging need to develop precise models of brain tumors to understand resistances and to predict therapeutic responses. While tumor-derived primary cell lines are relatively easy to produce, fully replicate the complexity of tumors as they grow without interactions with and from the microenvironment. Therefore, the advent of organoid cultures represents a significant advancement in modelling development. Derived from this technology, tumoroids offer a promising solution for modeling 3D tumors and their complex microenvironment. METHODS We develop tumoroids from fresh biopsies and patient-derived tumor xenografts. These models were rigorously validated against to initial tumor using confocal microscopy, methylome analyses, metabolomics and single-cell RNAseq. To assess the intrinsic hypoxic environment and its spatial heterogeneity, characteristic of aggressive cancers, we employed fluorescent oxygen-sensitive dye loaded polymeric nanorods. RESULTS We successfully generated tumoroids using PDX of ependymomas (BT39) and it paired relapse (BT42), two H3.3 mutated high-grade gliomas (BT35 and BT69), one ganglioglioma (BT63) and directly from fresh biopsies of DIPG sample (BT140). Several passages were obtained after primary derivation, and cryopreservation demonstrating the stability of the models over time. Tumoroids displayed similar protein and omics markers as their parental tumors and harbored specific stem cell and neural progenitor markers. The methylation patterns were entirely preserved comparing 3D models and their parental tumors, and other multiomics and metabolic approaches confirmed the accurate reconstitution of tumor complexity. The study of oxygen-sensitive nanorods highlighted the heterogeneous oxygen gradient within tumoroids. CONCLUSIONS All these initial characterized tumoroids helped us to develop radioresistant paired models.

STEM-04. BIOBANKING PATIENT-DERIVED MODELS OFFERS A PRECISE SPATIAL AND EPIGENETIC APPROACH TO UNRAVEL THE INITIATION, PROGRESSION, AND THERAPEUTIC RESISTANCE OF PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Aggressive brain cancers represent the most lethal forms of tumors in pediatric population. Their molecular drivers and co-alterations are currently guiding therapeutic strategies. Due to heterogeneous nature of these tumors, they are quickly developing resistance to treatment. Recognizing intrinsic and extrinsic mechanisms underlying therapeutic resistance, international scientific community has recognized an urgent need to create precise models of brain tumors capable of predicting therapeutic responses. So far, patient-derived cell lines (PDCL) remain the most reliable model to estimate and mimic evolving scenarios. The primary challenges lie in derivation methods and in cellular and spatial validation. METHODS In the context of the PEDIAMODECAN program, we collected 105 fresh brain tumor samples, including 34 LGG, 24 HGG, 19 ependymomas, 19 medulloblastomas, and 9 other rare entities, to concurrently generate PDCLs and patient-derived xenografts (PDX). RESULTS Regarding the derivation process itself, we successfully maintained 80% of samples in 2D and 54% in PDX models. As anticipated, the LGG group showed the higher percentage of PDX failure (90%). Nevertheless, for high-grade subtypes, PDX generation rate increased to 85,7%. No correlation was found between derivation success and a shorter interval between surgical removal and dissociation processes. Histological analysis of PDXs models confirmed the similar microscopic features to those of primary tumors. Next-Generation Sequencing (NGS), methylome assays and spatial transcriptomic analyses of derived models matched those of the original patient tumors. Epigenetic approaches were proved to be the most effective methods for validating models, in addition to spatial transcriptomic analyses that highlighted similar inter-tumor heterogeneity between PDX models and primary tumor counterparts. CONCLUSIONS Establishing a systematic PDCL/PDX biobank for all pediatric brain tumors represents one of the most effective approaches to develop models capable of accurately reproducing tumoral heterogeneity, histological and molecular diversity, and facilitating the design predictive screenings for targeted therapies combined with irradiation.