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Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand–receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo.Implications:Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Glypican-1 and Syndecan-1 are identified as heparan sulfate proteoglycans expressed on GBM43.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand–receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. Implications: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Surgical Hip Dislocation (SHD) is a powerful tool in the armamentarium of any surgeon treating conditions affecting the hips of children presenting with sequelae of a number of common conditions including Legg-CalvéPerthes disease (LCPD) and slipped capital femoral epiphysis (SCFE). Risks associated with the procedure are well described. We investigated to assess if SHD is associated with significant surgical risk and if it improved clinical outcomes for patients.We conducted a prospective cohort study. We reviewed 18 (11 males and 7 females; mean age 13.7 years (6-17) with symptomatic hip pathology, secondary to femoroacetabular impingement (FAI) between 2017 and 2021. All patients underwent a surgical hip dislocation approach and femoral head-neck osteochondroplasty, Head Split osteotomy or both. Clinical improvement was assessed using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index. The minimum follow-up was 6 months (mean, 22 months; range, 6-42 months).WOMAC scores improved at final follow-up from 10 to 3 for pain, 33 to 10 for function, and 4 to 2 for the stiffness subscales. All radiographic measures improved significantly of the postoperative X-rays. No patients developed osteonecrosis, implant failure, deep infection, or nonunion.Surgical Hip Dislocation, in the short term, we found improvement in WOMAC scores and radiographic indices with a low complication rate.

Extracellular sulfatases (SULF1 and SULF2) selectively remove 6-O-sulfate groups (6OS) from heparan sulfate proteoglycans (HSPGs) and by this process control important interactions of HSPGs with extracellular factors including morphogens, growth factors, and extracellular matrix (ECM) components. The expression of SULF1 and SULF2 is dynamically regulated during development and is altered in pathological states such as glioblastoma (GBM), a highly malignant and highly invasive brain cancer. SULF2 protein is increased in an important subset of human GBM and it helps regulate receptor tyrosine kinase (RTK) signaling and tumor growth in a murine model of the disease. By altering ligand binding to HSPGs SULF2 has the potential to modify the extracellular availability of factors important in a number of cell processes including proliferation, chemotaxis, and migration. Diffuse invasion of malignant tumor cells into surrounding healthy brain is a characteristic feature of GBM that makes therapy challenging. Here, we describe methods to assess SULF2 expression in human tumor tissue and cell lines and how to relate this to tumor cell invasion.

Src homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed non-receptor tyrosine phosphatase, plays a critical role in the regulation of the MAPK signaling pathway and cellular proliferation. Activating mutations in SHP2 are associated with the development of multiple malignancies including leukemia, lung cancer and neuroblastoma. In addition, SHP2 promotes the conversion of oncogenic KRAS to its active GTP-bound state and it’s inhibition can enhance efficacy of GDP-KRASG12C inhibitors as well as other MAPK pathway inhibitors (RAF, MEK and ERK) which have suboptimal clinical efficacy as single agents. As a result, inhibition of SHP2 through genetic manipulation or pharmacological means has been shown to suppress tumor growth and presents an attractive potential avenue for the treatment of malignancies as monotherapy or in combination with other MAPK/PI3K inhibitors. Here we describe BBP-398, a potent, orally bioavailable allosteric small molecule inhibitor of SHP2. BBP-398 displays high selectivity against other phosphatases, kinases, GPCRs, transporters and hERG. Predicted human PK properties show good oral bioavailability with half-life of ~12-16 hours enabling continuous daily dosing and optimal therapeutic index in combination with other targeted therapeutics. In cellular assays, BBP-398 demonstrates potent pERK/DUSP6 inhibition and loss of viability across a panel of cell lines with active MAPK signaling, such as mutant EGFR and KRASG12C. In vivo, BBP-398 strongly suppresses RAS-ERK signaling in RTK- or RAS-driven xenografts. In the EGFR-dependent non-small cell lung cancer (NSCLC) HCC827 and esophageal squamous cell carcinoma KYSE-520 xenograft models, BBP-398 drives dose dependent efficacy consistent with the level of target inhibition. Detailed analysis of tumor response shows that efficacy is driven by maintaining better than 50% inhibition of pERK for most of the dosing interval. In addition to its strong single agent activity, BBP-398 also leads to enhanced efficacy in vitro and in vivo when used in combination with targeted therapeutics against driver MAPK genetic alterations, such as KRAS, EGFR or MET. Combination targeting, such as with the GDP-KRASG12C inhibitor sotorasib in the NSCLC NCI-H358 xenograft model, or with the mutant EGFR inhibitor osimertinib in the HCC827 erlotinib resistant (ER) xenograft model, drives strong suppression of MAPK activity and results in tumor regressions. Collectively, these findings highlight that SHP2 inhibition is a promising molecular therapeutic strategy in cancer which can potentially strongly suppress tumor growth as a single agent or in combination with other MAPK pathway inhibitors. Given its preclinical properties and projected favorable clinical pharmacokinetic profile, BBP-398 is currently being evaluated in a Phase 1/1b trial in patients with advanced solid tumors (NCT04528836). Citation Format: James P. Stice, Sofia Donovan, Yuting Sun, Nancy Kohl, Barbara Czako, Faika Mseeh, Paul Leonard, Anna Wade, Justin Lim, Phil Jones, Eli Wallace, Kerstin Sinkevicius, Pedro Beltran. BBP-398, a potent, small molecule inhibitor of SHP2, enhances the response of established NSCLC xenografts to KRASG12C and mutEGFR inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P207.

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults and confers a poor prognosis due, in part, to diffuse invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycans, present on the cell surface and in the extracellular matrix, regulate cell signaling pathways and cell–microenvironment interactions. In GBM, the expression of HS glycosaminoglycans and the enzymes that regulate their function are altered, but the actual HS content and structure are unknown. However, inhibition of HS glycosaminoglycan function is emerging as a promising therapeutic strategy for some cancers. In this study, we use liquid chromatography–mass spectrometry analysis to demonstrate differences in HS disaccharide content and structure across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine GBM with enrichment of mesenchymal and proneural gene expression (mMES and mPN, respectively) markers. In GBM, the heterogeneous HS content and structure across patient-derived tumorsphere lines suggested diverse functions in the GBM tumor microenvironment. In GBM5 and mPN, elevated expression of sulfatase 2 (SULF2), an extracellular enzyme that alters ligand binding to HS, was associated with low trisulfated HS disaccharides, a substrate of SULF2. In contrast, other primary tumorsphere lines had elevated expression of the HS-modifying enzyme heparanase (HPSE). Using gene editing strategies to inhibit HPSE, a role for HPSE in promoting tumor cell adhesion and invasion was identified. These studies characterize the heterogeneity in HS glycosaminoglycan content and structure across GBM and reveal their role in tumor cell invasion. Implications: HS-interacting factors promote GBM invasion and are potential therapeutic targets. Mol Cancer Res; 15(11); 1623–33. ©2017 AACR.

Background: The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) of preclinical models including patient-derived xenografts (PDX), organoids (PDOrg) and patient-derived cell cultures (PDC). Extensive clinical annotation and genomic datasets are available for these preclinical models. However, it is unclear if the molecular profiles of the corresponding patient tumors are stably propagated in these models. We have previously demonstrated that PDX models from the NCI PDMR faithfully represent the patient tumors both in terms of genomic stability and tumor heterogeneity. Here, we conduct an in-depth investigation of genomic representation of patient tumors in the PDOrgs and PDCs. Methods: PDOrgs (n=64) and PDCs (n=94) were established from tumor fragments (i.e., initiator specimens) obtained either from patient specimens or from PDX specimens of early passage. For some models (n=19), both PDOrgs and PDCs were generated from the same tumor tissue; in fewer cases (n=4), PDCs were established from organoids derived from patient specimens. Whole Exome Sequencing and RNA-Seq were performed on all PDCs and PDOrgs, and data were compared with patient specimens or early passage PDXs. Results: A majority of the PDOrgs and PDCs have stably inherited the genome of the corresponding patient specimens based on the following observations: (1) >87% of PDOrgs and PDCs maintained similar copy number alteration profiles compared with the initiator specimens of the preclinical model; (2) the variant allele frequency (VAF) of clinically relevant mutations remained consistent between the PDOrgs, PDCs, and the initiator specimens, with none of the PDCs or PDOrgs deviating by >15% VAF; and (3) clinically relevant biomarkers (e.g., MSI, LOH, mutational signatures etc.) are concordant amongst the PDOrgs, PDCs, and the initiator specimens. We observed that the majority of SNVs and indels present in the initiator specimens were also found in the PDOrgs and PDCs, suggesting almost all the tumor heterogeneity was preserved in these preclinical models. Conclusions: This large and histologically diverse set of PDOrgs and PDCs from the NCI PDMR exhibited genomic stability and faithfully represented the tumor heterogeneity observed in corresponding patient specimens. These preclinical models thus represent a valuable resource for researchers interested in pre-clinical drug or other studies. Citation Format: Biswajit Das, Yvonne A. Evrard, Li Chen, Rajesh Patidar, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm-Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Carrie A. Bonomi, Kelly M. Dougherty, Joseph P. Geraghty, Marion V. Gibson, Savanna S. Styers, Abigail J. Walke, Jenna E. Moyer, Anna Wade, Mariah L. Baldwin, Kaitlyn A. Arthur, Kevin J. Plater, Luke Stockwin, Matthew R. Murphy, Michael E. Mullendore, Dianne L. Newton, Melinda G. Hollingshead, Chris A. Karlovich, Paul M. Williams, James H. Doroshow. Patient-derived organoid and cell culture models from the NCI Patient-Derived Models Repository (NCI PDMR) preserve genomic stability and heterogeneity of patient tumor specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3916.

Cancer organoids are heterogeneous 3D cellular clusters with complexities that mimic some characteristics of tumors in situ. Thus, assays performed with cancer organoids might enable better predictions of in vivo drug responses than those performed with cell monolayers. The National Cancer Institute (NCI) is developing a national repository of Patient-Derived (PD) models comprised of clinically annotated and molecularly characterized PD xenografts (PDXs), PD tumor cell lines (PDCs), and PD cancer organoids (PDOrgs) (https://pdmr.cancer.gov/). We evaluated the therapeutic activity of a panel of FDA-approved and investigational anticancer agents, including carboplatin, gemcitabine, paclitaxel, SN38, 5-FU, adavosertib, erlotinib, trametinib, and vemurafenib, against a cohort of PDCs, PDOrgs, and PDXs from solid tumors including colon, gastroesophageal, head and neck, NSCLC, pancreatic, bladder, and uterine cancers. Our goal was to investigate whether drug sensitivities determined using PDCs and PDOrgs correlate with responses observed in the matching PDXs. Cultures were exposed to anticancer agents at concentrations ranging from 1 pM to 100 µM for periods of 4 or 6 days. The data indicated that the GI50 values for PDOrgs were in overall agreement with in vivo PDX drug responses measured as relative median to event free survival (RMEFS), where an event is the median time (days) from treatment initiation to tumor volume quadrupling, calculated as median time to tumor volume quadrupling for treated animals/median time to tumor volume quadrupling for control animals. For both paclitaxel and trametinib, responses in PDOrgs, from most sensitive to most resistant, were similar to the corresponding PDXs. Drug sensitivities determined in PDC monolayers were less clearly related to in vivo PDX responses; particularly for PDCs treated with carboplatin, gemcitabine, and SN-38. This work is part of a larger effort to provide a rigorous comparison between fully characterized and annotated PDCs-PDOrgs-PDXs to assess the value of different in vitro model systems for the prediction of PDX drug responses. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Petreena Campbell, Curtis Hose, Lara El Touny, Erik Harris, John Connelly, Carrie Bonomi, Kelly Dougherty, Savanna Styers, Abigail Walke, Jenna Moyer, Mariah Baldwin, Anna Wade, Michael Mullendore, Kaitlyn Arthur, Matthew Murphy, Kevin Plater, Marion Gibson, Joseph Geraghty, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Tiffanie Chase, John Carter, Howard Stotler, Debbie Trail, Luke Stockwin, Dianne Newton, Yvonne Evrard, Melinda Hollingshead, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, James H. Doroshow, Annamaria Rapisarda. Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3913.

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg). NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. These models are offered to the extramural community for research use (https://pdmr.cancer.gov), along with clinical annotation and molecular information (whole exome sequence, gene expression using RNASeq), via a publicly accessible database. Currently, over 200 PDX models, 50 PDC models, and 100 CAF models are available for distribution to the US research community. Approximately 50 PDOrg models will be released in early 2019. As part of its rare cancer initiative, the NCI is also targeting the collection of infrequently-observed tumor histologies to advance both biological investigations and drug development efforts for under-studied malignancies. Comparison of matched models, models where more than one model type are available (e.g., PDX and PDC), demonstrate a high degree of concordance across the model types. Genetic stability across the models is assessed using multiple criteria including genetic assessment of CNVs and presence of driver mutations. Optimal CNV assessment uses whole exome sequence data corrected for cellularity in the patient specimen using germline reads and corrected for cellularity in the PDX specimens by subtraction of the mouse reads. Histomorphologic comparison of PDXs and cell line xenografts (CLX) generated from in vitro PDCs and PDOrgs also overall show a high degree of concordance, though loss of features and dedifferentiation can be observed in some models. Overall these models demonstrate a high degree of conservation at the genetic and pathologic level when compared to the patient tumor. These models can provide researchers the ability to perform high- or mid-throughput screening in 2D or 3D culture followed by targeted selection of PDX models for in vivo studies. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Dianne Newton, Biswajit Das, Sergio Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Carrie Bonomi, Suzanne Borgel, John Carter, Tiffany Chase, Alice Chen, Lily Chen, Nikki E. Craig, Vivekananda Datta, Emily Delaney, Raymond Divelbiss, Kelly Dougherty, Thomas Forbes, Kyle Georgius, Joe Geraghty, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Kelly Hedger, Sierra Hoffman, Chris Karlovich, Wiem Lassoued, Shahanawaz Jiwani, Candace Mallow, Chelsea McGlynn, Mallorie Morris, Jenna Moyer, Mike Mullendore, Matt Murphy, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Nicki Scott, Luke H. Stockwin, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Tomas Vilimas, Anna Wade, Abigail Walke, Thomas Walsh, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Comparison of PDX, PDC, and PDOrg models from the National Cancer Institute’s Patient-Derived Models Repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4524.

Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour-microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets.

Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavan's disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca(2+)](i) in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies.

Platelet-derived growth factor alpha receptor (PDGFRA)/NG2-expressing glia are distributed throughout the adult CNS. They are descended from oligodendrocyte precursors (OLPs) in the perinatal CNS, but it is not clear whether they continue to generate myelinating oligodendrocytes or other differentiated cells during normal adult life. We followed the fates of adult OLPs in Pdgfra-creER(T2)/Rosa26-YFP double-transgenic mice and found that they generated many myelinating oligodendrocytes during adulthood;20% of all oligodendrocytes in the adult mouse corpus callosum were generated after 7 weeks of age, raising questions about the function of the late-myelinating axons. OLPs also produced some myelinating cells in the cortex, but the majority of adult-born cortical cells did not appear to myelinate. We found no evidence for astrocyte production in gray or white matter. However, small numbers of projection neurons were generated in the forebrain, especially in the piriform cortex, which is the main target of the olfactory bulb.

Heparan sulfate (HS) / Heparan sulfate proteoglycans (HSPGs) are implicated in tumor malignancy based on their ability to promote cell signaling and tumor invasion. The mechanisms by which they do this in an individual tumor type, however, are often poorly defined. Using both patient-derived glioblastoma (GBM) tumorsphere lines and a murine model for GBM, we investigate how cell surface HSPGs modulate cell signaling in infiltrating astrocytoma. In human tumorsphere lines, chemical ablation of cell surface HS reduced the phosphorylation of several receptor-tyrosine kinases (RTKs) including platelet-derived growth factor receptor α (PDGFRα). The reduction in PDGFRA activation was associated with decreased binding of tagged PDGF-BB ligand to the cell surface suggesting HS can promote ligand retention and activity at the cell surface. To ablate all HS production we established HS-deficient murine tumor-prone cells by knockout of Ext1, a glycosyltransferase required for HS chain elongation. HS-deficient cells had decreased growth in vitro, decreased invasion in a 3D spheroid invasion assays, and reduced activation of RTKs as detected by Western blotting compared to control cells. Preliminary in vivo experiments suggest HS-deficient cells also exhibit reduced growth in vivo and the structural role for HS in RTK signaling in glioma can now be addressed.

Cancer Organoids are discrete multicellular structures that recapitulate tumor microanatomy (1). These reagents can be generated by extended culture of partially or fully dissociated tumor samples in three-dimensional matrices. By maintaining tumor and accessory cells in an appropriate context, they provide a biosimilar platform for studying disease pathogenesis and cellular pharmacology (2). Similarly, cancer organoid culture is useful for propagating slow growing tumors or those requiring heterotypic cell-cell interactions. Here, preliminary data will be presented regarding generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiative. This initiative aims to develop a national repository of patient-derived cancer models (PDMs) consisting of clinically annotated patient-derived xenografts (PDXs) and patient-derived tumor cell cultures (PDCs) prepared from primary and metastatic tumors (3). A standardized panel of different organoid media formulations was constructed to optimize culture conditions for disease subsets. Using this approach, organoids were generated for colon, prostate, pancreatic, breast, melanoma, NSCLC, and bladder tumors. Although some samples were refractory to organoid generation, in several instances, samples that failed to generate 2D cultures thrived as organoids. A further finding was that direct implantation of organoid cultures was an efficient means of generating xenografts. Indeed, work will be presented detailing the exact number of organoids required to establish xenograft tumors. Protocols were developed for routine culture, passaging and long-term storage in liquid nitrogen. Similarly, organoids were amenable to characterization by FACS analysis, ICC/IHC and qRT-PCR to evaluate metrics such as tumor type, histological similarity with patient tumor, cell viability, percentage stroma and whether mouse cells persist in PDX-derived organoids. In summary, growth, expansion, analysis and storage of tumor organoids is feasible for a wide range of tumor types. Importantly, for certain samples, generation of cancer organoids appears to be a useful intermediary step for subsequent PDX model and 2D culture generation. Funded by NCI Contract No. HHSN261200800001E. References: 1. Baker LA, Tiriac H, Clevers H, Tuveson DA. Modeling pancreatic cancer with organoids. Trends Cancer. 2016;2:176-90. 2. Cantrell MA, Kuo CJ. Organoid modeling for cancer precision medicine. Genome Med. 2015;7. 3. Doroshow J, Hollingshead M, Evrard Y, Williams M, Datta V, Das B, et al. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA). Mol Cancer Ther. 2015;14(12 Suppl 2). Citation Format: Luke H. Stockwin, Jenna Moyer, Anna Wade, Carrie Bonomi, Kelly Dougherty, John Carter, Jesse Stottlemeyer, Kaitlyn Arthur, Vivekananda Datta, Lindsay Dutko, Michael Mullendore, James H. Doroshow, Melinda G. Hollingshead, Dianne L. Newton. Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4827. doi:10.1158/1538-7445.AM2017-4827

BackgroundNeural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism.Scope of reviewIn this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior.Major conclusionsThe NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors.General significanceThe factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.

Huntington's disease (HD) is a progressive neurological disorder characterised by motor impairments caused by degeneration in the striatum. The mechanism by which the HD mutation leads to the neurodegenerative pathology of HD is still unknown. Recently it was shown that, in HD patients, early pathological changes in white matter precede selective cell death in the striatum. We wondered whether axonal pathology is also an early pathological feature in a transgenic mouse model carrying the HD mutation (R/2 line). R6/2 mice show brain atrophy, a progressive neurological deterioration and skeletal muscle atrophy that resemble those seen in HD patients. However, there is very little neuronal cell loss seen in these animals, even when they show severe symptoms. Here we used sciatic nerve to look for evidence of early neurodegenerative changes in axons of the R6/2 mouse at an ultrastructural level. We observed ultrastructural changes that preferentially affected large myelinated fibres of the sciatic nerve in 10-week-old asymptomatic R6/2 mice. The changes included a significant decrease in the axoplasm diameter of myelinated neurons and an increase in the number of degenerating myelinated fibres compared to age-matched wild type littermates. Myelin thickness and unmyelinated fibre diameter were not affected. The abnormalities described here precede the appearance of overt motor symptoms in the R6/2 mouse and occur in parallel with pathophysiological changes at the neuromuscular junction. We suggest that degenerative changes in axons are likely to contribute to the early pathological phenotype in HD, even in the absence of frank neuronal cell loss.

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BACKGROUND: Extracellular proteoglycans, including heparan sulfate proteoglycan (HSPG), play critical roles in the regulation of neural stem cell signaling and progenitor cell migration via their interactions with extracellular ligands, matrix, and cell receptors and may serve similar purposes in oncogenic states. In glioblastoma (GBM), our studies suggest that proteoglycans regulate critical oncogenic signaling pathways. Here we assess the function of proteoglycans and their alterations that drive GBM, and test the hypothesis that these may represent novel therapeutic targets and biomarkers of disease. METHODS: To investigate the role for proteoglycans in tumorigenesis we used a combination of primary human GBM, primary human GBM cultures, and a murine model for malignant glioma based on the genetic manipulation of neural progenitor cells. GAG structure was elucidated and quantified using liquid chromatography-mass spectrometry (LCMS), an ultra-sensitive and high-resolution technique that can overcome problems associated with low availability of biological samples. RESULTS: In GBM we identified patterns of altered expression of HSPG protein cores and their modifying enzymes relative to non-tumor brain, including the extracellular enzyme SULF2. Indeed, SULF2 is overexpressed in many human GBM and ablation of SULF2 function results in decreased activation of PDGFRα signaling and in decreased tumor growth in vivo. Consistent with a role for SULF2 in PDGFRA signaling in human GBM, SULF2 is upregulated in PDGFRA-amplified compared to non-amplified tumors (p < 0.05, n = 40 amplified, 332 non-amplified). SULF2 reduced HS sulfation, specifically the HS6S component of heparin, as determined by liquid chromatography-mass spectrometry (LCMS) and immunohistochemistry. As HS6S sulfation is an important determinant of HS-ligand binding, we investigated whether SULF2 could alter PDGF binding to HS. While ligand binding was similar, SULF2 was associated with altered expression and cell surface localization of PDGFRA. Thus, SULF2, and possibly HS6S, may regulate PDGFRA signaling via two distinct mechanisms. As HS structure is critical for normal neural stem cell proliferation and differentiation, we analyzed HS structure over tumor progression using LCMS. We demonstrate that tumor progression is associated with changes in GAG structure, and we are now using HS mimetics to try to inhibit the oncogenic role for proteoglycans in GBM. CONCLUSIONS: Our data demonstrate the functional importance of proteoglycans in GBM and suggest GAG structure may be particularly important for oncogenic signaling. While therapeutic targeting of single RTKs has been difficult in GBM, a drug targeting HS has the potential to simultaneously inhibit multiple oncogenic pathways in tumor cells and disrupt critical tumor-microenvironment interactions. SECONDARY CATEGORY: n/a.