Discontinuous phase diagram of amorphous carbons.

The short-range order and medium-range order of amorphous carbons demonstrated in experiments allow us to rethink whether there exist intrinsic properties hidden by atomic disordering. Here we presented six representative phases of amorphous carbons (0.1-3.4 g/cm3), namely, disordered graphene network (DGN), high-density amorphous carbon (HDAC), amorphous diaphite (a-DG), amorphous diamond (a-D), paracrystalline diamond (p-D), and nano-polycrystalline diamond (NPD), respectively, classified by their topological features and microstructural characterizations that are comparable with experiments. To achieve a comprehensive physical landscape for amorphous carbons, a phase diagram was plotted in the sp3/sp2 versus density plane, in which the counterintuitive discontinuity originates from the inherent difference in topological microstructures, further guiding us to discover a variety of phase transitions among different amorphous carbons. Intriguingly, the power law, log(sp3/sp2) ∝ ρn, hints at intrinsic topology and hidden order in amorphous carbons, providing an insightful perspective to reacquaint atomic disorder in non-crystalline carbons.

Temperature-Dependent Paracrystalline Nucleation in Atomically Disordered Diamonds.

Atomically disordered diamonds with medium-range order realized in recent experiments extend our knowledge of atomic disorder in materials. However, the current understanding of amorphous carbons cannot answer why paracrystalline diamond (p-D) can be formed inherently different from other tetrahedral amorphous carbons (ta-Cs), and the emergence of p-D seems to be easily hindered by inappropriate temperatures. Herein, we performed atomistic-based simulations to shed light on temperature-dependent paracrystalline nucleation in atomically disordered diamonds. Using metadynamics and two carefully designed collective variables, reversible phase transitions among different ta-Cs can be presented under different temperatures, evidenced by corresponding local minima on the free energy surface and reaction path along the free energy gradient. We found that p-D is preferred in a narrow range of temperatures, which is comparable to real experimental temperatures under the Arrhenius framework. The insights and related methods should open up a perspective for investigating other amorphous carbons.

Trastuzumab Deruxtecan, Antibody-Drug Conjugate Targeting HER2, Is Effective in Pediatric Malignancies: A Report by the Pediatric Preclinical Testing Consortium.

HER2 is expressed in many pediatric solid tumors and is a target for innovative immune therapies including CAR-T cells and antibody-drug conjugates (ADC). We evaluated the preclinical efficacy of trastuzumab deruxtecan (T-DXd, DS-8201a), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient- and cell line-derived xenograft (PDX/CDX) models. HER2 mRNA expression was determined using RNA-seq and protein expression via IHC across multiple pediatric tumor PDX models. Osteosarcoma (OS), malignant rhabdoid tumor (MRT), and Wilms tumor (WT) models with varying HER2 expression were tested using 10 mice per group. Additional histologies such as Ewing sarcoma (EWS), rhabdomyosarcoma (RMS), neuroblastoma (NB), and brain tumors were evaluated using single mouse testing (SMT) experiments. T-DXd or vehicle control was administered intravenously to mice harboring established flank tumors at a dose of 5 mg/kg on day 1. Event-free survival (EFS) and objective response were compared between treatment and control groups. HER2 mRNA expression was observed across histologies, with the highest expression in WT (median = 22 FPKM), followed by MRT, OS, and EWS. The relationship between HER2 protein and mRNA expression was inconsistent. T-DXd significantly prolonged EFS in 6/7 OS, 2/2 MRT, and 3/3 WT PDX models. Complete response (CR) or maintained CR (MCR) were observed for 4/5 WT and MRT models, whereas stable disease was the best response among OS models. SMT experiments also demonstrated activity across multiple solid tumors. Clinical trials assessing the efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered.

Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma.

Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP-targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors.

BACKGROUND: Adoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. METHODS: We generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells, chimeric antigen receptor-T cells, and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells. RESULTS: attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells targeted tumor cells expressing cell-surface vimentin, enriching effector T cell and interferon γ production in tumors, which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects. CONCLUSIONS: This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors.

Dose-response effect of eribulin in preclinical models of osteosarcoma by the pediatric preclinical testing consortium.

The pediatric preclinical testing program previously demonstrated activity of eribulin in osteosarcoma patient-derived xenograft (PDX) models. The phase 2 trial in patients with relapsed osteosarcoma failed to meet response endpoints. Eribulin was evaluated in the original and an expanded set of PDX models and tested at multiple dose levels and schedules to evaluate dose-response. Maximal response was observed at the highest dose, consistent with prior results. The alternative schedule generated similar responses. We demonstrate steep dose-response for eribulin in osteosarcoma PDX models, implying that any deviation from achievement of effective concentrations may have a significant impact on activity.

Tumor-targeted IL-12 combined with tumor resection yields a survival-favorable immune profile.

BACKGROUND: Although accumulated evidence provides a strong scientific premise for using immune profiles to predict survival in patients with cancer, a universal immune profile across tumor types is still lacking, and how to achieve a survival-associated immune profile remains to be evaluated. METHODS: We analyzed datasets from The Cancer Genome Atlas to identify an immune profile associated with prolonged overall survival in multiple tumor types and tested the efficacy of tumor cell-surface vimentin-targeted interleukin 12 (ttIL-12) in inducing that immune profile and prolonging survival in both mouse and patient-derived xenograft tumor models. RESULTS: We identified an immune profile (IFNγHiCD8HiFOXP3LowCD33Low) associated with prolonged overall survival across several human tumor types. ttIL-12 in combination with surgical resection of the primary tumor transformed tumors to this immune profile. Intriguingly, this immune profile transformation led to inhibition of metastasis and to prolonged survival in both mouse and patient-derived xenograft malignant models. Wild-type IL-12 combined with surgery was significantly less effective. In the IL-12-sensitive C3H mouse strain, in fact, wild-type IL-12 and surgery resulted in shorter overall survival than in mice treated with control pDNA; this surprising result is believed to be attributable to IL-12 toxicity, which was absent in the mice treated with ttIL-12. The ttIL-12-induced immune profile associated with longer overall survival was also associated with a greater accumulation of CD8+ T cells and reduced infiltration of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The underlying mechanism for this transformation by ttIL-12 treatment was induction of expression of CXCL9 and reduction of expression of CXCL2 and CCL22 in tumors. CONCLUSIONS: ttIL-12 when combined with surgery led to conversion to the IFNγHiCD8HiFOXP3LowCD33Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.

p53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis.

Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis.

Renal tubule-specific expression and urinary secretion of human growth hormone: a kidney-based transgenic bioreactor growth.

Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.

Overexpression of epidermal growth factor receptor in urothelium elicits urothelial hyperplasia and promotes bladder tumor growth.

Although urothelium is constantly bathed in high concentrations of epidermal growth factor (EGF) and most urothelial carcinomas overexpress EGF receptor (EGFr), relatively little is known about the role of EGFr signaling pathway in urothelial growth and transformation. In the present study, we used the uroplakin II gene promoter to drive the urothelial overexpression of EGFr in transgenic mice. Three transgenic lines were established, all expressing a higher level of the EGFr mRNA and protein in the urothelium than the nontransgenic controls. The overexpressed EGFr was functionally active because it was autophosphorylated, and its downstream mitogen-activated protein kinases were highly activated. Phenotypically, the urinary bladders of all transgenic lines developed simple urothelial hyperplasia that was strongly positive for proliferative cell nuclear antigen and weakly positive for bromodeoxyuridine incorporation. When coexpressed with the activated Ha-ras oncogene in double transgenic mice, EGFr had no apparent tumor-enhancing effects over the urothelial hyperplastic phenotype induced by Ha-ras oncogene. However, when coexpressed with the SV40 large T antigen, EGFr accelerated tumor growth and converted the carcinoma in situ of the SV40T mice into high-grade bladder carcinomas, without triggering tumor invasion. Our studies indicate that urothelial overexpression of EGFr can induce urothelial proliferation but not frank carcinoma formation. Our results also suggest that, whereas EGFr and Ha-ras, both of which act in the same signal transduction cascade, stimulated urothelial hyperplasia, they were not synergistic in urothelial tumorigenesis, and EGFr overexpression can cooperate with p53 and pRB dysfunction (as occurring in SV40T transgenic mice) to promote bladder tumor growth.

Isolation of mouse THP gene promoter and demonstration of its kidney-specific activity in transgenic mice.

Tamm-Horsfall protein (THP), the most abundant urinary protein synthesized by the kidney epithelial cells, is believed to play important and diverse roles in the urinary system, including renal water balance, immunosuppression, urinary stone formation, and inhibition of bacterial adhesion. In the present study, we describe the isolation of a 9.3-kb, 5'-region of the mouse THP gene and show the highly conserved nature of its proximal 589-bp, 5'-flanking sequence with that in rats, cattle, and humans. We also demonstrate using the transgenic mouse approach that a 3.0-kb, proximal 5'-flanking sequence is sufficient to drive the kidney-specific expression of a heterologous reporter gene. Within the kidney, transgene expression was confined to the renal tubules that endogenously expressed the THP protein, which suggests specific transgene activity in the thick ascending limb of the loop of Henle and early distal convoluted tubules. Our results establish the kidney- and nephron-segment-specific expression of the mouse THP gene. The availability of the mouse THP gene promoter that functions in vivo should facilitate additional studies of the molecular mechanisms of kidney-specific gene regulation and should provide new molecular tools for better understanding renal physiology and disease through nephron-specific gene targeting.