Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma.

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Silencing GMPPB Inhibits the Proliferation and Invasion of GBM via Hippo/MMP3 Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive malignancy and represents the most common brain tumor in adults. To better understand its biology for new and effective therapies, we examined the role of GDP-mannose pyrophosphorylase B (GMPPB), a key unit of the GDP-mannose pyrophosphorylase (GDP-MP) that catalyzes the formation of GDP-mannose. Impaired GMPPB function will reduce the amount of GDP-mannose available for O-mannosylation. Abnormal O-mannosylation of alpha dystroglycan (α-DG) has been reported to be involved in cancer metastasis and arenavirus entry. Here, we found that GMPPB is highly expressed in a panel of GBM cell lines and clinical samples and that expression of GMPPB is positively correlated with the WHO grade of gliomas. Additionally, expression of GMPPB was negatively correlated with the prognosis of GBM patients. We demonstrate that silencing GMPPB inhibits the proliferation, migration, and invasion of GBM cells both in vitro and in vivo and that overexpression of GMPPB exhibits the opposite effects. Consequently, targeting GMPPB in GBM cells results in impaired GBM tumor growth and invasion. Finally, we identify that the Hippo/MMP3 axis is essential for GMPPB-promoted GBM aggressiveness. These findings indicate that GMPPB represents a potential novel target for GBM treatment.

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies.

Background: Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations - demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency - enabling unlimited production of modified "off-the-shelf" hPSC-NKs. Methods: hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results: HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions: Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research - improving patient survival and quality of life.

Targeting GBM with an Oncolytic Picornavirus SVV-001 alone and in combination with fractionated Radiation in a Novel Panel of Orthotopic PDX models.

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.

Treatment of Hoffa fracture of femoral condyle with anchor combined with auxiliary fixation.

BACKGROUND: A Hoffa fracture is an unstable intra-articular break that is generally treated with surgery. OBJECTIVE: To evaluate the feasibility and clinical outcomes of using a suture anchor combined with auxiliary fixation for the treatment of a lateral femoral condyle Hoffa fracture. METHODS: The study retrospectively reviewed 8 patients (5 males and 3 females) with a lateral femoral condyle Hoffa fracture who had been treated by combining a suture anchor with auxiliary fixation between January 2016 and April 2020. The mean age of patients was 37.5 years (ranging from 23 to 45). According to Letenneur's classification, there were 4 cases of type I, 2 cases of type II, and 2 cases of type III fractures. The clinical outcomes were assessed using Letenneur's functional assessment. RESULTS: The follow-up duration ranged between 14-24 months. All patients achieved primary healing of the incision and fracture union, as well as normal flexion and extension of the knee joint, with 7 cases showing excellent outcomes and 1 case showing a good outcome. No postoperative complications, such as fracture displacement, anchor loosening, or fracture malunion, occurred in this series. CONCLUSION: Our results indicated that a suture anchor, combined with external fixation, was an effective treatment for a lateral femoral condyle Hoffa fracture. Accordingly, this procedure is worthy of wider clinical application.

Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Background: Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. Methods: We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. Results: We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. Conclusions: This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. Accession number: GSE197988.

Epigenetic Alterations of Repeated Relapses in Patient-matched Childhood Ependymomas.

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

A path to translation: How 3D patient tumor avatars enable next generation precision oncology.

3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.

Regulation of TORC1 by MAPK Signaling Determines Sensitivity and Acquired Resistance to Trametinib in Pediatric BRAFV600E Brain Tumor Models.

PURPOSE: We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes. EXPERIMENTAL DESIGN: Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized. RESULTS: In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance. CONCLUSIONS: In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

MiR-199a/b-3p inhibits colorectal cancer cell proliferation, migration and invasion through targeting PAK4 and BCAR3.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. P21 activated kinase 4 (PAK4) and Breast cancer anti-estrogen resistance 3 (BCAR3) have been reported to be involved in numerous aspects in tumorous progression. In this study, we propose to screen multi-targeted microRNAs. (miRNAs), which simultaneously inhibit neoplastic evolution through suppressing the transcription of target genes. METHODS: MTT and Colony formation assays measured cell's viability and proliferation. Scratch wound and Transwell assays detected the ability in migration and invasion for SW116 cells. The multi-targeted microRNAs of PAK4 and BCAR3 were predicted using bioinformatics analysis and verified by conducting dual luciferase reporter assay, western blot and qRT-PCR that could detect the expression levels of miR-199a/b-3p. RESULTS: The knockdown of PAK4 significantly impeded proliferation and colony formation of SW1116 cells when the knockdown of BCAR3 hindered migration and invasion of SW1116 cells. MiR-199a/b-3p directly targeted the 3'-UTR of PAK4 and BCAR3, further effected proliferation, colony formation, migration, and invasion of SW1116 cells. PAK4 or BCAR3 overexpression could partially reversed inhibitory effects of miR-199a/b-3p. CONCLUSIONS: These results provided a new multi-targeted cite for cancerous suppressant to improve the prognosis of CRC inpatients.

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma.

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.

Diagnosis and genetic analysis of a case with mandibuloacral dysplasia type B due to compound heterozygous mutations of the ZMPSTE24 gene.

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, mainly caused by pathogenic variants of the LMNA and ZMPSTE24 genes. In this study, we reported the first case of a patient with type B cranial and mandibular dysplasia in China. The patient presented with distinctive facial features, feeding difficulties, significant physical retardation, and overall developmental delay with abnormal tooth and bone development. Trio-whole exome sequencing analysis showed that the patient carried compound heterozygous mutations of c.743C>T (p.Pro248Leu) (dbSNP: rs121908095) and the loss of exons 1-10 of the ZMPSTE24 gene. Sanger sequencing and real-time quantitative PCR (RT-qPCR) showed that these two mutations were inherited from the patient's phenotypically normal mother and father, respectively. By summarizing and analyzing the characteristics of this case and the pedigree of the family, we suggested that trio-whole-exome sequencing could be performed to assist in the diagnosis of diseases that are difficult to be diagnosed definitively based on clinical phenotypes. The publication of this case has improved clinicians' understanding of MAD disease and provide new clinical information for the subsequent genetic study of this disease.

Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies.

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.

Spatial Dissection of Invasive Front from Tumor Mass Enables Discovery of Novel microRNA Drivers of Glioblastoma Invasion.

Diffuse invasion is the primary cause of treatment failure of glioblastoma (GBM). Previous studies on GBM invasion have long been forced to use the resected tumor mass cells. Here, a strategy to reliably isolate matching pairs of invasive (GBMINV ) and tumor core (GBMTC ) cells from the brains of 6 highly invasive patient-derived orthotopic models is described. Direct comparison of these GBMINV and GBMTC cells reveals a significantly elevated invasion capacity in GBMINV cells, detects 23/768 miRNAs over-expressed in the GBMINV cells (miRNAINV ) and 22/768 in the GBMTC cells (miRNATC ), respectively. Silencing the top 3 miRNAsINV (miR-126, miR-369-5p, miR-487b) successfully blocks invasion of GBMINV cells in vitro and in mouse brains. Integrated analysis with mRNA expression identifies miRNAINV target genes and discovers KCNA1 as the sole common computational target gene of which 3 inhibitors significantly suppress invasion in vitro. Furthermore, in vivo treatment with 4-aminopyridine (4-AP) effectively eliminates GBM invasion and significantly prolongs animal survival times (P = 0.035). The results highlight the power of spatial dissection of functionally accurate GBMINV and GBMTC cells in identifying novel drivers of GBM invasion and provide strong rationale to support the use of biologically accurate starting materials in understanding cancer invasion and metastasis.

Maximizing the potential of aggressive mouse tumor models in preclinical drug testing.

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT. First, genetically quiet, ATRT is a difficult tumor to target molecularly. Tumor cells need to divide to propagate tumor growth-intercepting the common crossroads in cell cycle progression is a feasible strategy. KIF11 is needed for bipolar spindle formation in metaphase. We identified KIF11 as a universal target of all ATRT-molecular-subtypes. Ispinesib, a KIF11-inhibitor, effectively inhibited tumor proliferation in all seven cell lines. A second challenge-a major challenge in preclinical drug testing in-vivo among aggressive tumor models, is the narrow therapeutic window to administer drugs within the limited murine lifespan. Our most aggressive ATRT tumor model was lethal in all mice within ~ 1 month of tumor implantation. Such short-surviving mouse models are difficult to employ for preclinical drug testing due to the narrow time window to administer drugs. To overcome this time restriction, we developed a clinical staging system which allowed physically-fit mice to continue treatment, in contrast to the conventional method of fixed drug-dose-duration regimen in preclinical testing which will not be feasible in such short-surviving mouse models. We validated this approach in a second embryonal brain tumor, medulloblastoma. This is a clinically relevant, cost-efficient approach in preclinical testing for cancer and non-cancer disease phenotypes. Widely used preclinical mouse models are not the most accurate and lack the aggressive tumor spectrum found within a single tumor type. Mice bearing the most aggressive tumor spectrum progress rapidly in the limited murine life-span, resulting in a narrow therapeutic window to administer drugs, and are thus difficult to employ in preclinical testing. Our approach overcomes this challenge. We discovered ispinesib is efficacious against two embryonal brain tumor types.