Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

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.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with CAR T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons (CSE) present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify CSE targets, we analyzed 1,532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We found 2,933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n=148) or the alternatively spliced (AS) isoform (n=9) level. Expression of selected AS targets, including the EDB domain of FN1 (EDB), and gene targets, such as COL11A1, were validated in pediatric PDX tumors. We generated CAR T cells specific to EDB or COL11A1 and demonstrated that COL11A1-CAR T-cells have potent antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Impact of environment on pediatric and adult brain tumors: The 2023 Brain Tumor Epidemiology Consortium meeting report.

The Brain Tumor Epidemiology Consortium (BTEC) is an international organization with membership of individuals from the scientific community with interests related to brain tumor epidemiology including surveillance, classification, methodology, etiology, and factors associated with morbidity and mortality. The 2023 annual BTEC meeting entitled "Impact of Environment on Pediatric and Adult Brain Tumors" was held in Lexington, KY, USA on May 22 - 24, 2023. The meeting gathered scientists from the United States, Canada, Australia, and Europe and included four keynote sessions covering genomic, epigenomic, and metabolomic considerations in brain tumor epidemiology, cancer clusters, environmental risk factors, and new approaches to cancer investigation. The meeting also included three abstract sessions and a brainstorming session. A summary of the meeting content is included in this report.

Adjuvant activities of immunostimulating natural products: Astragalus membranaceus (Fisch.) Bge. and Coriolus versicolor in BNT162b2 vaccination against COVID-19 infection.

The global pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been developing all over the world for more than 3 years. In late 2020, several variants of concern of SARS-CoV-2 virus emerged, with increased viral fitness and transmissibility by mutations of the spike proteins of the viral particle, denting hopes of the use of early-generation vaccines for a widespread protective immunity against viral infection. The use of adjuvants may enhance the immune responses of the conventional application of the COVID-19 vaccine. We have shown that the water extract of 2 ß-glucan-enriched immunostimulating natural products, Astragalus membranaceus (Fisch.) Bge. (AM) and Coriolus versicolor (CV), could induce innate immunity-related cytokines from human monocytes (CCL5, interleukin [IL]-6, IL-10, and tumor necrosis factor α) and monocyte-derived dendritic cells (IL-1ß, IL-10, IL-12, and tumor necrosis factor α). Using BALB/c mice, orally administrated AM and CV (1,384 and 742 mg/kg/d) for 4 d after vaccination, respectively, could enhance (1) the immunoglobulin G binding activities of BNT162b2 vaccination against ancestral and Delta SARS-CoV-2 spike proteins by 5.8- and 4.3-fold, respectively; (2) the immunoglobulin G3 subclass production of BNT162b2 vaccination against ancestral and variant SARS-CoV-2 spike proteins; and (3) the in vitro antibody-neutralizing activities of BNT162b2 vaccinated mice. In conclusion, combining AM and CV was effective in acting as an oral adjuvant with the messenger RNA vaccine BNT162b2 to improve the antigen binding activities against SARS-CoV-2 ancestral and variant SARS-CoV-2 spike proteins, probably via trained immunity of macrophages and dendritic cells.

Cryopreservation of mouse thymus depletes thymocytes but supports immune reconstitution on transplantation.

Cryopreservation of mouse thymus depletes donor thymocytes but preserves thymus function when transplanted after thawing into athymic mice. No differences in immune reconstitution were observed between fresh and frozen/thawed transplants suggesting that donor thymocyte depletion does not affect outcome. Thus, cryopreservation of thymus may improve outcomes in thymus transplant patients.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Creating a Vaccine-like Supplement against Respiratory Infection Using Recombinant Bacillus subtilis Spores Expressing SARS-CoV-2 Spike Protein with Natural Products.

Vaccination is the most effective method of combating COVID-19 infection, but people with a psychological fear of needles and side effects are hesitant to receive the current vaccination, and alternative delivery methods may help. Bacillus subtilis, a harmless intestinal commensal, has recently earned a strong reputation as a vaccine production host and delivery vector, with advantages such as low cost, safety for human consumption, and straightforward oral administration. In this study, we have succeeded generating "S spores" by engineering B. subtilis with spore coat proteins resembling the spike (S) protein of the ancestral SARS-CoV-2 coronavirus. With the addition of two immunostimulating natural products as adjuvants, namely Astragalus membranaceus (Fisch.) Bge (AM) and Coriolus versicolor (CV), oral administration of S spores could elicit mild immune responses against COVID-19 infection without toxicity. Mucosal IgA against the S protein was enhanced by co-feeding with AM and CV in an S spores-inoculated mouse model. Faster and stronger IgG responses against the S protein were observed when the mice were fed with S spores prior to vaccination with the commercial COVID-19 vaccine CoronaVac. In vitro studies demonstrated that AM, CV, and B. subtilis spores could dose-dependently activate both macrophages and dendritic cells by secreting innate immunity-related IL-1ß, IL-6, and TNF-α, and some other proinflammatory chemokines and cytokines. In conclusion, the combination of S spores with AM and CV may be helpful in developing a vaccine-like supplement against respiratory infection.

Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

Allograft vasculopathy (AV) leads to chronic rejection of organ transplants, but its causes are obscure. New research from the Jane-Wit laboratory showed that Sonic Hedgehog (SHH) signalling from damaged graft endothelium drives vasculopathy by promoting proinflammatory cytokine production and NLRP3-inflammasome activation in alloreactive CD4+PTCH1hiPD-1hiT memory cells, offering new diagnostic and therapeutic strategies.

Iron Deficiency among School-Aged Adolescents in Hong Kong: Prevalence, Predictors, and Effects on Health-Related Quality of Life.

Iron deficiency (ID) is a prevalent nutritional deficiency affecting children/adolescents worldwide. We reported (1) the prevalence of ID and ID with anemia (IDA) among Chinese school-aged adolescents, (2) clinical and dietary predictors of iron status, and (3) its impact on health-related qualities of life (HRQoL). This cross-sectional study recruited 183 boys and 340 girls (mean age = 17.55) from 16 schools in Hong Kong. ID is defined as serum ferritin <15 µg/L. The participants reported their dietary habits, menstrual patterns (girls), and HRQoL using structured questionnaires. The overall prevalence of ID was 11.1%. None of the boys had ID or IDA. Among girls, the rate of ID was 17.1% and IDA was 10.9%. One-third (36.3%) reported a regular habit of skipping ≥1 meal/day. Lower ferritin was found in adolescents who skipped meals (Est = -35.1, p = 0.017). Lower ferritin is correlated with poorer school functioning (Est = 0.81, p = 0.045) and fatigue (Est = 0.92, p = 0.016). Skipping meals is associated with poorer physical (p = 0.0017) and school functioning (p = 0.027). To conclude, 1 in 10 school-aged adolescents in Hong Kong are iron-deficient. The ID rate in girls (17.1%) is similar to that in other industrialized countries (5.2-16.6%). Future work should promote awareness on the potential health consequences of poor dietary habits on ID and the well-being of adolescents.

Pediatric brain tumors: Origins, epidemiology, and classification - The 2022 Brain Tumor Epidemiology Consortium meeting report.

The Brain Tumor Epidemiology Consortium (BTEC) is an international organization that fosters collaboration among scientists focused on understanding the epidemiology of brain tumors with interests ranging from the etiology of brain tumor development and outcomes to the control of morbidity and mortality. The 2022 annual BTEC meeting with the theme "Pediatric Brain Tumors: Origins, Epidemiology, and Classification" was held in Lyon, France on June 20 - 22, 2022. Scientists from North America and Europe presented recent research and progress in the field. The meeting content is summarized in this report.

Early experience with targeted therapy as a first-line adjuvant treatment for pediatric low-grade glioma.

OBJECTIVE: Pediatric low-grade gliomas (pLGGs) frequently exhibit dysregulation of the mitogen-activated protein kinase (MAPK) pathway. Targeted therapies, including mutant BRAF inhibitors (dabrafenib) and MEK inhibitors (trametinib), have shown promise in patients in whom conventional chemotherapy has failed. However, few studies have investigated the use of targeted therapy as a first-line treatment for pLGG. Here, the authors reviewed their institutional experience with using a personalized medicine approach to patients with newly diagnosed pLGGs. METHODS: All pediatric patients at the authors' institution who had been treated with dabrafenib or trametinib for pLGG without first receiving conventional chemotherapy or radiation were retrospectively reviewed. Demographic, clinical, and radiological data were collected. RESULTS: Eight patients underwent targeted therapy as a first-line treatment for pLGG. Five patients had a BRAF alteration (1 with a BRAFV600E mutation, 4 with a KIAA1549:BRAF fusion), and 3 patients had an NF1 mutation. One of the 8 patients was initially treated with dabrafenib, and trametinib was added later. Seven patients were initially treated with trametinib; of these, 2 later transitioned to dual therapy, whereas 5 continued with trametinib monotherapy. Six patients (75%) demonstrated a partial response to therapy during their treatment course, whereas stable disease was identified in the remaining 2 patients (25%). One patient experienced mild disease progression after completing a course of trametinib monotherapy, but ultimately stabilized after a period of close observation. Another patient experienced tumor progression while on dabrafenib, but subsequently responded to dual therapy with dabrafenib and trametinib. The most common adverse reactions to targeted therapy were cutaneous toxicity (100%) and diarrhea (50%). CONCLUSIONS: Targeted therapies have the potential to become a standard treatment option for pLGG due to their favorable toxicity profile and oral route of administration. This case series provides preliminary evidence that targeted therapies can induce an early disease response as a first-line adjuvant treatment; however, large-scale studies are required to assess long-term durability and safety.

The human cathelicidin peptide LL-37 inhibits pancreatic cancer growth by suppressing autophagy and reprogramming of the tumor immune microenvironment.

Pancreatic cancer is amongst the most lethal malignancies, while its poor prognosis could be associated with promotion of autophagy and the tumor immune microenvironment. Studies have confirmed the pro-tumorigenic nature of the cathelicidin family of peptide LL-37 in several types of cancer. However, at higher doses, LL-37 exerts significant cytotoxicity against gastrointestinal cancer cells. In our study, we investigated the anti-tumorigenic potential of LL-37 in pancreatic cancer and the underlying mechanisms. Our results have shown that LL-37 inhibited the growth of pancreatic cancer both in vitro and in vivo. Mechanistic studies have demonstrated that LL-37 induced DNA damage and cell cycle arrest through induction of reactive oxygen species (ROS). Further study indicates that LL-37 suppressed autophagy in pancreatic cancer cells through activation of mTOR signaling, leading to more accumulation of ROS production and induction of mitochondrial dysfunctions. With combined treatment of LL-37 with the mTOR inhibitor rapamycin, LL-37-induced ROS production and cancer cell growth inhibition were attenuated. Subsequent in vivo study has shown that LL-37 downregulated the immunosuppressive myeloid-derived suppressor cells and M2 macrophages while upregulated the anti-cancer effectors CD8+ and CD4+ T cells in the tumor microenvironment. By using an in vitro co-culture system, it was shown that promotion of M2 macrophage polarization would be suppressed by LL-37 with inhibition of autophagy, which possessed significant negative impact on cancer growth. Taken together, our findings implicate that LL-37 could attenuate the development of pancreatic cancer by suppressing autophagy and reprogramming of the tumor immune microenvironment.

The Pioneer Transcription Factor Foxa2 Modulates T Helper Differentiation to Reduce Mouse Allergic Airway Disease.

Foxa2, a member of the Forkhead box (Fox) family of transcription factors, plays an important role in the regulation of lung function and lung tissue homeostasis. FOXA2 expression is reduced in the lung and airways epithelium of asthmatic patients and in mice absence of Foxa2 from the lung epithelium contributes to airway inflammation and goblet cell hyperplasia. Here we demonstrate a novel role for Foxa2 in the regulation of T helper differentiation and investigate its impact on lung inflammation. Conditional deletion of Foxa2 from T-cells led to increased Th2 cytokine secretion and differentiation, but decreased Th1 differentiation and IFN-γ expression in vitro. Induction of mouse allergic airway inflammation resulted in more severe disease in the conditional Foxa2 knockout than in control mice, with increased cellular infiltration to the lung, characterized by the recruitment of eosinophils and basophils, increased mucus production and increased production of Th2 cytokines and serum IgE. Thus, these experiments suggest that Foxa2 expression in T-cells is required to protect against the Th2 inflammatory response in allergic airway inflammation and that Foxa2 is important in T-cells to maintain the balance of effector cell differentiation and function in the lung.

Integrated DNA Copy Number and Expression Profiling Identifies IGF1R as a Prognostic Biomarker in Pediatric Osteosarcoma.

Osteosarcoma is a primary malignant bone tumor arising from bone-forming mesenchymal cells in children and adolescents. Despite efforts to understand the biology of the disease and identify novel therapeutics, the survival of osteosarcoma patients remains dismal. We have concurrently profiled the copy number and gene expression of 226 osteosarcoma samples as part of the Strategic Partnering to Evaluate Cancer Signatures (SPECS) initiative. Our results demonstrate the heterogeneous landscape of osteosarcoma in younger populations by showing the presence of genome-wide copy number abnormalities occurring both recurrently among samples and in a high frequency. Insulin growth factor receptor 1 (IGF1R) is a receptor tyrosine kinase which binds IGF1 and IGF2 to activate downstream pathways involved in cell apoptosis and proliferation. We identify prevalent amplification of IGF1R corresponding with increased gene expression in patients with poor survival outcomes. Our results substantiate previously tenuously associated copy number abnormalities identified in smaller datasets (13q34+, 20p13+, 4q35-, 20q13.33-), and indicate the significance of high fibroblast growth factor receptor 2 (FGFR2) expression in distinguishing patients with poor prognosis. FGFR2 is involved in cellular proliferation processes such as division, growth and angiogenesis. In summary, our findings demonstrate the prognostic significance of several genes associated with osteosarcoma pathogenesis.

3D patient-derived tumor models to recapitulate pediatric brain tumors In Vitro.

Brain tumors are the leading cause of cancer-related deaths in children. Tailored therapies need preclinical brain tumor models representing a wide range of molecular subtypes. Here, we adapted a previously established brain tissue-model to fresh patient tumor cells with the goal of establishing3D in vitro culture conditions for each tumor type.Wereported our findings from 11 pediatric tumor cases, consisting of three medulloblastoma (MB) patients, three ependymoma (EPN) patients, one glioblastoma (GBM) patient, and four juvenile pilocytic astrocytoma (Ast) patients. Chemically defined media consisting of a mixture of pro-neural and pro-endothelial cell culture medium was found to support better growth than serum-containing medium for all the tumor cases we tested. 3D scaffold alone was found to support cell heterogeneity and tumor type-dependent spheroid-forming ability; both properties were lost in 2D or gel-only control cultures. Limited in vitro models showed that the number of differentially expressed genes between in vitro vs. primary tissues, are 104 (0.6%) of medulloblastoma, 3,392 (20.2%) of ependymoma, and 576 (3.4%) of astrocytoma, out of total 16,795 protein-coding genes and lincRNAs. Two models derived from a same medulloblastoma patient clustered together with the patient-matched primary tumor tissue; both models were 3D scaffold-only in Neurobasal and EGM 1:1 (v/v) mixture and differed by a 1-mo gap in culture (i.e., 6wk versus 10wk). The genes underlying the in vitrovs. in vivo tissue differences may provide mechanistic insights into the tumor microenvironment. This study is the first step towards establishing a pipeline from patient cells to models to personalized drug testing for brain cancer.

Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers.

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.

Sonic Hedgehog signalling in the regulation of barrier tissue homeostasis and inflammation.

Epithelial barrier tissues such as the skin and airway form an essential interface between the mammalian host and its external environment. These physical barriers are crucial to prevent damage and disease from environmental insults and allergens. Failure to maintain barrier function against such risks can lead to severe inflammatory disorders, including atopic dermatitis and asthma. Here, we discuss the role of the morphogen Sonic Hedgehog in postnatal skin and lung and the impact of Shh signalling on repair, inflammation, and atopic disease in these tissues.

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.

Systemic Pharmacological Smoothened Inhibition Reduces Lung T-Cell Infiltration and Ameliorates Th2 Inflammation in a Mouse Model of Allergic Airway Disease.

Allergic asthma is a common inflammatory airway disease in which Th2 immune response and inflammation are thought to be triggered by inhalation of environmental allergens. Many studies using mouse models and human tissues and genome-wide association have indicated that Sonic Hedgehog (Shh) and the Hedgehog (Hh) signaling pathway are involved in allergic asthma and that Shh is upregulated in the lung on disease induction. We used a papain-induced mouse model of allergic airway inflammation to investigate the impact of systemic pharmacological inhibition of the Hh signal transduction molecule smoothened on allergic airway disease induction and severity. Smoothened-inhibitor treatment reduced the induction of Shh, IL-4, and IL-13 in the lung and decreased serum IgE, as well as the expression of Smo, Il4, Il13, and the mucin gene Muc5ac in lung tissue. Smoothened inhibitor treatment reduced cellular infiltration of eosinophils, mast cells, basophils, and CD4+ T-cells to the lung, and eosinophils and CD4+ T-cells in the bronchoalveolar lavage. In the mediastinal lymph nodes, smoothened inhibitor treatment reduced the number of CD4+ T-cells, and the cell surface expression of Th2 markers ST2 and IL-4rα and expression of Th2 cytokines. Thus, overall pharmacological smoothened inhibition attenuated T-cell infiltration to the lung and Th2 function and reduced disease severity and inflammation in the airway.