Glioblastoma chemoattract migratory interneuron precursors modified to deliver therapeutic proteins

Glioblastoma chemoattract migratory interneuron precursors modified to deliver therapeutic proteins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Glioblastoma chemoattract migratory interneuron precursors modified to deliver therapeutic proteins Thomas De Raedt, Stephanie Brosius, William Manley, Kyra Harvey, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5493594/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Glioblastoma has a poor prognosis with limited therapeutic options, in part due to the presence of the blood brain barrier, which often prevents the delivery of therapeutic agents, and the immune suppressive tumor environment (TME), which is a challenge for T-cell based immunotherapies. We developed a cellular delivery vector where implanted modified post-mitotic Migratory Inhibitory Interneuron Precursors (MIPs) are chemoattracted to high-grade glioma (HGG), independent of expressed antigens and unhindered by the TME, to deliver therapeutic proteins of choice. Biological sciences/Cancer/Cancer therapy Biological sciences/Biotechnology/Cell delivery Interneuron glioblastoma cellular therapy glioma Bispecific T-cell Engager Figures Figure 1 Figure 2 Introduction Glioblastoma (GBM) is a high-grade neoplasm of the brain with a 5% five-year overall survival 1 affecting both adult 2 and pediatric populations 3 . The delivery of therapeutic agents to HGG remains a major challenge 4 , in part due to the brain’s privileged space. To circumvent these obstacles, we established a cellular delivery system that harnesses the migratory capacity of MIPs to deliver therapeutic proteins to HGG. Many MIPs originate in the Medial-Ganglionic Eminence (MGE) of the embryonic brain as NKX2.1 positive cells and migrate to the cerebral cortex or striatum as post-mitotic LHX6 positive cells over 4–6 weeks in human (Fig. 1 a) 5 – 8 . This migration is guided by gradients of chemoattractants like CXCL12, NRG1, and HGF 6 . While these gradients are largely absent in the post-natal brain, these factors are often secreted by HGG. Remarkably, transplanted MIPs retain their migratory capacity in the adult brain 8 – 11 . The migratory capacity of MIP, together with their ability to integrate into host circuitry, has garnered further interest to use MIPs as a cellular therapy for epilepsy (clinical trial: NCT05135091). Here we demonstrate that MIPs migrate to HGG, independent of unique tumor antigens, both in vitro and in vivo . As proof of concept, we modified MIPs to deliver a Bispecific T-cell Engager (BiTE) targeting the tumor, prolonging survival in orthotopic xenograft models of HGG. Results We hypothesized that proteins secreted by HGGs would generate a gradient capable of attracting migratory cells native to the brain in an antigen independent manner, and that we could use these cells to deliver therapeutic proteins. Analyzing publicly available mRNA expression data of pediatric HGG and normal cortex revealed 35 proteins (Extended Data Table 1) involved in chemoattraction and migration that could establish this gradient. Intriguingly, several of these factors are ligands of CXCR4 12 (CXCL12, MIF and HMGB1) and chemoattract macrophages 13 , microglia, and MIPs 6 , 13 , 14 . Since macrophages/microglia contribute to HGG pathology we focused on MIPs. Other factors that chemoattract MIPs (HGF 15 and NRG1 6,16 ) were also identified in our analysis (Fig. 1 b and Extended Data Table 1). Given that many HGGs highly express multiple MIP chemoattractants, we postulated that transplanted MIPs would exhibit targeted migration to HGGs. In our assays we used mouse MGE-derived MIPs and two human ES-derived MIP lines (NKX2.1 cells derived from ES line HES3 expressing GFP under the NKX2.1 promotor and LHX6 cells derived from ES line H9 expressing Citrine under the LHX6 promotor). Cellular identity and appropriate progression through differentiation was verified on a weekly basis via immunocytochemistry for NKX2.1 and LHX6 (Fig. 1 c, Extended Data Fig. 1 a) and/or qPCR (Extended Data Fig. 1 b). Given the near uniform expression of NKX2.1 or LHX6 in our cultures (Fig. 1 c), we conclude that our cells are highly pure and committed to the MIP lineage. MIPs potently migrate to HGG in vitro and in vivo In an initial qualitative spheroid assay, we show that human MIPs migrate to and encase HGG spheres (Fig. 1 d and Extended Data Video 1). We subsequently showed that 8/13 pediatric and adult HGG lines chemoattract human MIPs using a transwell migration assay (Fig. 1 e, Extended Data Table 2, Extended Data Fig. 1 c). Next, we studied MIP-to-HGG migration in vivo (Fig. 2 a-e, Extended Data Fig. 2 a). Pediatric and adult HGG lines were orthotopically xenografted in nude mice and allowed to establish tumors over the course of a week. MIPs were grafted approximately 2 mm apart from HGG cells and allowed to migrate for 7 days for mouse MIPs and 14 days for human MIPs. RFP-positive murine MIPs potently migrated to HGG U87 (Fig. 2 a), not only reaching the edge of the main neoplasm (Fig. 2 b) but also encapsulating satellite lesions (Fig. 2 c). Implanted human stem cell-derived MIPs effectively targeted 7316 − 1746 cells as well as U87s in vivo (Fig. 2 d-e, Extended Data Fig. 2 a). MIPs deliver a therapeutic protein to HGG MIPs can be equipped with a range of therapeutic proteins. As proof of principle, we stably transduced LHX6-reporter human stem cell line derived MIPs to express a Bispecific T-cell Engager (BiTE) targeting wild-type EGFR or a non-targeting CD19 control (Extended Data Fig. 2 b). BiTEs are an adaptor protein made of two single-chain variable antibody fragments recognizing a tumor antigen and CD3 (T-cell receptor) and allowing bystander T-cells to target tumor cells that had previously evaded the immune system. EGFR BiTE-expressing MIPs kill 4/8 HGG cell lines in vitro (Extended Data Fig. 2 c-f). To evaluate the efficacy of BiTE-expressing MIPs to treat pediatric HGG orthotopic xenograft models, we co-injected the 3 different cell types in a single location since (1) sequential transplantation of HGG cells, MIPs, and intraventricular cannulation to deliver exogenous T-cells to immunocompromised mice would require three surgeries within a three-week window, and (2) a tumor would inevitably grow into the MIP injection site in a treatment time period (defeating the purpose of injecting MIPs at a distance). Mice that received EGFR BiTE-expressing MIPs but not the CD19 BiTE control, maintained a subthreshold luciferase signal at 8 weeks post-transplant of 7316 − 3058 HGG cells (Fig. 2 f, Extended Data Fig. 2 g) and had greatly improved median overall survival (p = 0.015). These results were confirmed with HGG line 7316 − 6439 (p = 0.025, Extended Data Fig. 2 g). We also evaluated if recurrent T-cell dosing might amplify the anti-tumor response in established tumors by orthotopically allografting 7316 − 3058 HGG cells with EGFR or CD19 BiTE-expressing MIPs and implanting an intraventricular cannula three weeks later for weekly T-cell administration. Mice that received EGFR BiTE-expressing MIPs had markedly extended survival (p = 0.02) versus CD19 BiTE controls (Fig. 2 g). Furthermore, one EGFR-BiTE mouse had no evidence of residual tumor on necropsy five months after initial xenografting. Collectively, these proof of principle experiments suggest we can modify MIPs to secrete therapeutic proteins at levels sufficient to induce HGG regression and significantly prolong survival. Discussion For glioblastoma, the standard of care consists of maximal safe surgical resection followed by radiation and adjuvant chemotherapy 17 , 18 . Although some targeted, cellular, and immunotherapies have shown promise in early trials, most fail to make a substantial impact on patient survival. The inability of many agents to reach the glioma in adequate concentrations remains a bottleneck. 4 Here we show that three independent MIP lines have tropism to a large percentage of HGG, and that these MIPs are capable of delivering/secreting a therapeutic cargo. Unlike CAR-T this migration is independent of specific antigens and seemingly not affected by the immune suppressive tumor microenvironment. As a therapy, we envision that MIPs could be injected into the wall of the resection cavity either at time of diagnosis or at recurrence. There MIPs can track and clear residual, highly infiltrative HGG cells. MIPs could also be directly injected into or near residual tumor for patients in which gross-total resection is not possible. We used BiTEs as one example of a therapeutic protein that can be delivered by MIPs. Other therapeutic agents (anti-tumor antibodies, peptides modulating the tumor microenvironment, or oncolytic viruses) might eventually prove more appropriate, especially given the immune suppressive microenvironment of HGG and the reliance on T-cells for any BiTE strategy. A clinical trial using injected inhibitory MIPs for refractory epilepsy is ongoing (NCT05135091). This trial uses transplants of inhibitory MIPs into the epileptic focus to reduce seizures and provides proof of concept that sufficient human stem cell-derived MIPs can be generated under GMP conditions to treat patients. To avoid the need to give patients immunosuppressants, MIPs will likely require HLA matching in a trial to treat HGG. Not every injected MIP will reach the HGG, and these MIPs can eventually integrate in the normal circuitry of the brain. In animals, negative consequences of MIP transplantation are not evident, and any adverse events observed with the MIP clinical trial for epilepsy will be informative. To improve safety and protect patients from adverse effects, a kill switch like inducible Caspase 9, can be engineered similar to CAR-T (clinical trial NCT04377932) 19 , 20 . We believe that, with the appropriate safeguards in place, MIP therapy can be safely administered to brain tumor patients. Notably, there is precedent for a parallel approach using fetal neural stem cells as a delivery vector in glioblastoma with several ongoing clinical trials (NCT01478852, NCT03072134, NCT05139056) 21 – 25 . Injected neural stem cells undergo a period of expansion (14 days post-injection); however, few transplanted cells persist beyond 30 days, with very few observed 79 days post-transplant in one post-mortem sample 25 . The need for frequent re-dosing has been a substantial limitation of neural stem cells in current trials as repeated intratumoral dosing via catheter has been limited by scarring at the catheter site 23 . MIP therapies offer several advantages to treat HGG: (1) Migration is antigen independent cicumventing potential issues with tumor heterogeneity and antigen escape seen in T-cell based therapies. (2) MIPs are capable of long-term survival following transplant; our and other investigators data show persistence for at least 9 months 7 , 26 . Therefore, MIPs provide a stable delivery system that may require less frequent dosing. (3) MIPs are post-mitotic and incapable of expansion post-transplant, mitigating concerns for aberrant proliferation and potential tumor formation. (4) MIPs can be derived from iPSCs 27 , circumventing both the ethical concerns regarding the use of fetal cells in therapies as well as the potential need for immunosuppression with transplant when using “off the shelf” MIPs or MIPs generated from patients. (5) Therapeutic proteins like bispecific engagers are notoriously unstable, requiring continuous infusion 28 which poses a high risk for infection, whereas MIPs will continuously secrete these proteins at the tumor. (6) Therapeutic proteins often have limited diffusion capabilities and may not reach the tumor through intraventricular delivery, thereby benefiting from local MIP delivery. (7) Local secretion can limit on-target, off-tumor side effects allowing potential targeting of antigens unique to brain but not to other tissues. (8) MIPs are inhibitory neurons, which integrate in the host circuitry. Glutamatergic signaling from excitatory neurons leads to a peritumoral hyperexcitability driving HGG proliferation 29 . This raises the possibility that MIP-based therapies may inhibit the aberrant neuronal-glial communication that stimulates HGG growth. Loss of peritumoral inhibitory neurons can also induce seizures 30 which could be significantly reduced with a MIP transplant. There are potential limitations to MIP-based therapies such as the presence of multifocal, bulky disease diluting the number of MIPs reaching each lesion. The minimum number of MIPs required to effectively clear a given tumor remains unknown. The optimal method of MIP delivery remains to be evaluated; intraventricular rather than intracerebral injections would greatly simplify transplant procedures 31 . Additional studies are also required to determine if MIPs retain their migratory capacity and survive with standard of care therapy, which will inform the timepoint MIPs can be administered within a treatment plan. Finally, we have shown that the majority of glioblastoma and HGG attract these MIPs in vitro ; however, the exact molecular mechanisms mediating MIP-to-HGG migration have yet to be elucidated. We expect that multiple chemokines play a role in the MIP chemoattraction, likely complicating these experiments. In conclusion, transplanted human stem cell derived MIPs are chemoattracted to HGGs both in vitro and in vivo and can be engineered to deliver anti-tumor agents. This honing is independent of antigens or TME. 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F. Implications of Extended Inhibitory Neuron Development. Int J Mol Sci 22, doi: 10.3390/ijms22105113 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files BrosiusInterneuronNatBio241120extendeddata.docx ExtendedDataVideo1.mp4 Human MIPs migrate to 7316-1746 RFP positive tumor spheres in a spheroid assay. ExtendedDataVideo2.mp4 Control CD19-BiTE expressing 293Ts co-cultured CD8 T-cells fail to eliminate glioblastoma 7316-1746 (red). ExtendedDataVideo3.mp4 Targeting EGFR-BiTE expressing 293Ts co-cultured CD8 T-cells potently eliminate glioblastoma 7316-1746 (red). Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5493594","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":385801215,"identity":"80983e70-454d-4c7d-9a06-40a8ab4883c7","order_by":0,"name":"Thomas De Raedt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDCCAyCCjYGBn4EHzGdsIFKLgYRkA8laDA4Qq4XveO/DDx/K/tQZHz978HEBg43shgMEtEieOW4sOeOcgYTZmbxk4xkMacYEtRjcSGOQ5m0DarnBYybNw3A4kbCW+8+Yf/8FajGeAdbynwgtN9jYpBmBWgwkwFoOENYieSaNzbLnHNA/Z3KMjWcYJBvPJKSF7/gx5hs/yuT4+dvPGD4uqLCT7SOkBQUwMxiQohyiZRSMglEwCkYBFgAAGPpAAhWP0X0AAAAASUVORK5CYII=","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"De Raedt","suffix":""},{"id":385801216,"identity":"348ce743-b170-46a8-9f3e-6bb79663e0d8","order_by":1,"name":"Stephanie Brosius","email":"","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Brosius","suffix":""},{"id":385801217,"identity":"639f4d18-f7bd-47eb-a5ed-cc1899b4be83","order_by":2,"name":"William Manley","email":"","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Manley","suffix":""},{"id":385801218,"identity":"29954b26-cc40-4571-ac52-bc6785b84489","order_by":3,"name":"Kyra Harvey","email":"","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":false,"prefix":"","firstName":"Kyra","middleName":"","lastName":"Harvey","suffix":""},{"id":385801219,"identity":"188c4a01-7b3a-47c5-a04a-0205769b8a3f","order_by":4,"name":"Jamie Galanaugh","email":"","orcid":"https://orcid.org/0000-0001-9358-7928","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Jamie","middleName":"","lastName":"Galanaugh","suffix":""},{"id":385801220,"identity":"dc15b959-9ef4-4056-b311-4739e57a778e","order_by":5,"name":"Deborah Rohacek","email":"","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":false,"prefix":"","firstName":"Deborah","middleName":"","lastName":"Rohacek","suffix":""},{"id":385801221,"identity":"f572827c-c1d9-4d8e-ab1d-1b2949d06259","order_by":6,"name":"Stewart Anderson","email":"","orcid":"","institution":"CHOP/UPenn","correspondingAuthor":false,"prefix":"","firstName":"Stewart","middleName":"","lastName":"Anderson","suffix":""}],"badges":[],"createdAt":"2024-11-20 23:10:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5493594/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5493594/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70634800,"identity":"48a207c1-e38c-45f3-9fce-0eece3ef6e4e","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":820866,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of MGE derived MIP migration (b) Violin plots comparing expression (transcripts per million, TMP) of known MIP chemoattractants and CXCR4 ligands in pediatric HGG (pHGG) versus normal cortex. (c) Human MIP immunofluorescence staining with LHX6 and NKX2-1 of LHX6 reporter MIPs. As expected at the 7-week time point shown, nearly all cells are either NKX2-1 (red), or LHX6 (green) positive prior to exposure to mitotic inhibitors, suggesting all cells are committed to the MIP lineage. (d) Human MGE-derived interneurons (green) are attracted to a 7316-1746 HGG sphere (red) when the glioma spheres are encased in a Matrigel matrix containing MIPs (green). Images at day 0 and 5 post plating. (e) Transwell migration assay demonstrating chemoattraction to HGG-conditioned media. Data are plotted as fold change number of cells on the bottom of the membrane compared to unconditioned media control. *p\u0026lt;0.02, **p\u0026lt;0.005\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/d6cc56f5fe1b054ae0c41e22.png"},{"id":70634801,"identity":"5ec1fb6a-2df2-47e6-ad40-843010ed0dc8","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":938806,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Mouse MIPs migrate to U87 HGG cells with a stream of MIPs between tumor and injection site. (b) Mouse MIPs (RFP) arrive at the location of the tumor (DAPI, U87) in 7 days and wrap around the primary tumor (green=CXCL12). (c) Mouse MIPs also display significant honing capabilities and encapsulate satellite lesions. (d) NKX2.1 human MIPs (green) migrate to the site of a glioblastoma within 14 days when injected 2mm away. (e) Magnification of D showing MIPs (GFP) wrapping around the 7316-1746 tumor (RFP). (f) Mice were co-injected with 7316-3058 tumor cells, BiTE expressing MCIPs, and CD8 T-cells (500k, 100k, and 1million cells, respectively) and monitored for tumor establishment. Luminescence signal demonstrates tumor in CD19-BiTE MIP controls, while the signal is below detection threshold with EGFR-BiTE MIPs 8 weeks post-transplant. (g) Schematic of treatment where 6 mice per cohort were co-injected with 7316-3058 tumor cells and MIPs. A tumor was allowed to prior to placement of interventricular cannula for weekly CD8 T-cell delivery. Survival curve of the 7316-3058 xenografts showing extended survival in EGFR-BiTE treated (p=0.02, log-rank).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/de295728b8117546d78582bc.png"},{"id":103503775,"identity":"86b4be65-0dbf-4373-9079-4586108f33ca","added_by":"auto","created_at":"2026-02-26 12:57:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2482347,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/a1fb7c37-8c94-4b68-9059-25c6ee69a142.pdf"},{"id":70634804,"identity":"901528ba-ca24-4373-838d-54a77c02c363","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1080740,"visible":true,"origin":"","legend":"","description":"","filename":"BrosiusInterneuronNatBio241120extendeddata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/03a97202a0f17cd73b6a0370.docx"},{"id":70634803,"identity":"dc686f8f-49a6-4722-bb3f-4ea21ddb23c9","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3295423,"visible":true,"origin":"","legend":"\u003cp\u003eHuman MIPs migrate to 7316-1746 RFP positive tumor spheres in a spheroid assay.\u003c/p\u003e","description":"","filename":"ExtendedDataVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/f08d5d60fc905c7b5595234c.mp4"},{"id":70634805,"identity":"dd2969a6-e90f-4a6f-b45b-9ba26c5547cc","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3623574,"visible":true,"origin":"","legend":"\u003cp\u003eControl CD19-BiTE expressing 293Ts co-cultured CD8 T-cells fail to eliminate glioblastoma 7316-1746 (red).\u003c/p\u003e","description":"","filename":"ExtendedDataVideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/79c776187a99d2341600986f.mp4"},{"id":70634802,"identity":"0f3b0508-d409-453f-a49a-445d9d19c831","added_by":"auto","created_at":"2024-12-05 06:19:40","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2044277,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting EGFR-BiTE expressing 293Ts co-cultured CD8 T-cells potently eliminate glioblastoma 7316-1746 (red).\u003c/p\u003e","description":"","filename":"ExtendedDataVideo3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5493594/v1/3eb6c7cac1357f399416fb92.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Glioblastoma chemoattract migratory interneuron precursors modified to deliver therapeutic proteins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma (GBM) is a high-grade neoplasm of the brain with a 5% five-year overall survival\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e affecting both adult\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and pediatric populations\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The delivery of therapeutic agents to HGG remains a major challenge\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, in part due to the brain\u0026rsquo;s privileged space. To circumvent these obstacles, we established a cellular delivery system that harnesses the migratory capacity of MIPs to deliver therapeutic proteins to HGG. Many MIPs originate in the Medial-Ganglionic Eminence (MGE) of the embryonic brain as NKX2.1 positive cells and migrate to the cerebral cortex or striatum as post-mitotic LHX6 positive cells over 4\u0026ndash;6 weeks in human (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This migration is guided by gradients of chemoattractants like CXCL12, NRG1, and HGF\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While these gradients are largely absent in the post-natal brain, these factors are often secreted by HGG. Remarkably, transplanted MIPs retain their migratory capacity in the adult brain\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The migratory capacity of MIP, together with their ability to integrate into host circuitry, has garnered further interest to use MIPs as a cellular therapy for epilepsy (clinical trial: NCT05135091).\u003c/p\u003e \u003cp\u003eHere we demonstrate that MIPs migrate to HGG, independent of unique tumor antigens, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. As proof of concept, we modified MIPs to deliver a Bispecific T-cell Engager (BiTE) targeting the tumor, prolonging survival in orthotopic xenograft models of HGG.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe hypothesized that proteins secreted by HGGs would generate a gradient capable of attracting migratory cells native to the brain in an antigen independent manner, and that we could use these cells to deliver therapeutic proteins. Analyzing publicly available mRNA expression data of pediatric HGG and normal cortex revealed 35 proteins (Extended Data Table\u0026nbsp;1) involved in chemoattraction and migration that could establish this gradient. Intriguingly, several of these factors are ligands of CXCR4\u003csup\u003e12\u003c/sup\u003e (CXCL12, MIF and HMGB1) and chemoattract macrophages\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, microglia, and MIPs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Since macrophages/microglia contribute to HGG pathology we focused on MIPs. Other factors that chemoattract MIPs (HGF\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and NRG1\u003csup\u003e6,16\u003c/sup\u003e) were also identified in our analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Extended Data Table\u0026nbsp;1). Given that many HGGs highly express multiple MIP chemoattractants, we postulated that transplanted MIPs would exhibit targeted migration to HGGs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn our assays we used mouse MGE-derived MIPs and two human ES-derived MIP lines (NKX2.1 cells derived from ES line HES3 expressing GFP under the NKX2.1 promotor and LHX6 cells derived from ES line H9 expressing Citrine under the LHX6 promotor). Cellular identity and appropriate progression through differentiation was verified on a weekly basis via immunocytochemistry for NKX2.1 and LHX6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and/or qPCR (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Given the near uniform expression of NKX2.1 or LHX6 in our cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), we conclude that our cells are highly pure and committed to the MIP lineage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMIPs potently migrate to HGG\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn an initial qualitative spheroid assay, we show that human MIPs migrate to and encase HGG spheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Extended Data Video 1). We subsequently showed that 8/13 pediatric and adult HGG lines chemoattract human MIPs using a transwell migration assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, Extended Data Table\u0026nbsp;2, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eNext, we studied MIP-to-HGG migration \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Pediatric and adult HGG lines were orthotopically xenografted in nude mice and allowed to establish tumors over the course of a week. MIPs were grafted approximately 2 mm apart from HGG cells and allowed to migrate for 7 days for mouse MIPs and 14 days for human MIPs. RFP-positive murine MIPs potently migrated to HGG U87 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), not only reaching the edge of the main neoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) but also encapsulating satellite lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Implanted human stem cell-derived MIPs effectively targeted 7316\u0026thinsp;\u0026minus;\u0026thinsp;1746 cells as well as U87s \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMIPs deliver a therapeutic protein to HGG\u003c/h2\u003e \u003cp\u003eMIPs can be equipped with a range of therapeutic proteins. As proof of principle, we stably transduced LHX6-reporter human stem cell line derived MIPs to express a Bispecific T-cell Engager (BiTE) targeting wild-type EGFR or a non-targeting CD19 control (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). BiTEs are an adaptor protein made of two single-chain variable antibody fragments recognizing a tumor antigen and CD3 (T-cell receptor) and allowing bystander T-cells to target tumor cells that had previously evaded the immune system. EGFR BiTE-expressing MIPs kill 4/8 HGG cell lines \u003cem\u003ein vitro\u003c/em\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f).\u003c/p\u003e \u003cp\u003eTo evaluate the efficacy of BiTE-expressing MIPs to treat pediatric HGG orthotopic xenograft models, we co-injected the 3 different cell types in a single location since (1) sequential transplantation of HGG cells, MIPs, and intraventricular cannulation to deliver exogenous T-cells to immunocompromised mice would require three surgeries within a three-week window, and (2) a tumor would inevitably grow into the MIP injection site in a treatment time period (defeating the purpose of injecting MIPs at a distance). Mice that received EGFR BiTE-expressing MIPs but not the CD19 BiTE control, maintained a subthreshold luciferase signal at 8 weeks post-transplant of 7316\u0026thinsp;\u0026minus;\u0026thinsp;3058 HGG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg) and had greatly improved median overall survival (p\u0026thinsp;=\u0026thinsp;0.015). These results were confirmed with HGG line 7316\u0026thinsp;\u0026minus;\u0026thinsp;6439 (p\u0026thinsp;=\u0026thinsp;0.025, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eWe also evaluated if recurrent T-cell dosing might amplify the anti-tumor response in established tumors by orthotopically allografting 7316\u0026thinsp;\u0026minus;\u0026thinsp;3058 HGG cells with EGFR or CD19 BiTE-expressing MIPs and implanting an intraventricular cannula three weeks later for weekly T-cell administration. Mice that received EGFR BiTE-expressing MIPs had markedly extended survival (p\u0026thinsp;=\u0026thinsp;0.02) versus CD19 BiTE controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Furthermore, one EGFR-BiTE mouse had no evidence of residual tumor on necropsy five months after initial xenografting. Collectively, these proof of principle experiments suggest we can modify MIPs to secrete therapeutic proteins at levels sufficient to induce HGG regression and significantly prolong survival.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor glioblastoma, the standard of care consists of maximal safe surgical resection followed by radiation and adjuvant chemotherapy\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Although some targeted, cellular, and immunotherapies have shown promise in early trials, most fail to make a substantial impact on patient survival. The inability of many agents to reach the glioma in adequate concentrations remains a bottleneck.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Here we show that three independent MIP lines have tropism to a large percentage of HGG, and that these MIPs are capable of delivering/secreting a therapeutic cargo. Unlike CAR-T this migration is independent of specific antigens and seemingly not affected by the immune suppressive tumor microenvironment.\u003c/p\u003e \u003cp\u003eAs a therapy, we envision that MIPs could be injected into the wall of the resection cavity either at time of diagnosis or at recurrence. There MIPs can track and clear residual, highly infiltrative HGG cells. MIPs could also be directly injected into or near residual tumor for patients in which gross-total resection is not possible. We used BiTEs as one example of a therapeutic protein that can be delivered by MIPs. Other therapeutic agents (anti-tumor antibodies, peptides modulating the tumor microenvironment, or oncolytic viruses) might eventually prove more appropriate, especially given the immune suppressive microenvironment of HGG and the reliance on T-cells for any BiTE strategy.\u003c/p\u003e \u003cp\u003eA clinical trial using injected inhibitory MIPs for refractory epilepsy is ongoing (NCT05135091). This trial uses transplants of inhibitory MIPs into the epileptic focus to reduce seizures and provides proof of concept that sufficient human stem cell-derived MIPs can be generated under GMP conditions to treat patients. To avoid the need to give patients immunosuppressants, MIPs will likely require HLA matching in a trial to treat HGG. Not every injected MIP will reach the HGG, and these MIPs can eventually integrate in the normal circuitry of the brain. In animals, negative consequences of MIP transplantation are not evident, and any adverse events observed with the MIP clinical trial for epilepsy will be informative. To improve safety and protect patients from adverse effects, a kill switch like inducible Caspase 9, can be engineered similar to CAR-T (clinical trial NCT04377932)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We believe that, with the appropriate safeguards in place, MIP therapy can be safely administered to brain tumor patients.\u003c/p\u003e \u003cp\u003eNotably, there is precedent for a parallel approach using fetal neural stem cells as a delivery vector in glioblastoma with several ongoing clinical trials (NCT01478852, NCT03072134, NCT05139056)\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Injected neural stem cells undergo a period of expansion (14 days post-injection); however, few transplanted cells persist beyond 30 days, with very few observed 79 days post-transplant in one post-mortem sample\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The need for frequent re-dosing has been a substantial limitation of neural stem cells in current trials as repeated intratumoral dosing via catheter has been limited by scarring at the catheter site\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMIP therapies offer several advantages to treat HGG: (1) Migration is antigen independent cicumventing potential issues with tumor heterogeneity and antigen escape seen in T-cell based therapies. (2) MIPs are capable of long-term survival following transplant; our and other investigators data show persistence for at least 9 months\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Therefore, MIPs provide a stable delivery system that may require less frequent dosing. (3) MIPs are post-mitotic and incapable of expansion post-transplant, mitigating concerns for aberrant proliferation and potential tumor formation. (4) MIPs can be derived from iPSCs\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, circumventing both the ethical concerns regarding the use of fetal cells in therapies as well as the potential need for immunosuppression with transplant when using \u0026ldquo;off the shelf\u0026rdquo; MIPs or MIPs generated from patients. (5) Therapeutic proteins like bispecific engagers are notoriously unstable, requiring continuous infusion\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e which poses a high risk for infection, whereas MIPs will continuously secrete these proteins at the tumor. (6) Therapeutic proteins often have limited diffusion capabilities and may not reach the tumor through intraventricular delivery, thereby benefiting from local MIP delivery. (7) Local secretion can limit on-target, off-tumor side effects allowing potential targeting of antigens unique to brain but not to other tissues. (8) MIPs are inhibitory neurons, which integrate in the host circuitry. Glutamatergic signaling from excitatory neurons leads to a peritumoral hyperexcitability driving HGG proliferation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This raises the possibility that MIP-based therapies may inhibit the aberrant neuronal-glial communication that stimulates HGG growth. Loss of peritumoral inhibitory neurons can also induce seizures\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e which could be significantly reduced with a MIP transplant.\u003c/p\u003e \u003cp\u003eThere are potential limitations to MIP-based therapies such as the presence of multifocal, bulky disease diluting the number of MIPs reaching each lesion. The minimum number of MIPs required to effectively clear a given tumor remains unknown. The optimal method of MIP delivery remains to be evaluated; intraventricular rather than intracerebral injections would greatly simplify transplant procedures\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Additional studies are also required to determine if MIPs retain their migratory capacity and survive with standard of care therapy, which will inform the timepoint MIPs can be administered within a treatment plan. Finally, we have shown that the majority of glioblastoma and HGG attract these MIPs \u003cem\u003ein vitro\u003c/em\u003e; however, the exact molecular mechanisms mediating MIP-to-HGG migration have yet to be elucidated. We expect that multiple chemokines play a role in the MIP chemoattraction, likely complicating these experiments.\u003c/p\u003e \u003cp\u003eIn conclusion, transplanted human stem cell derived MIPs are chemoattracted to HGGs both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and can be engineered to deliver anti-tumor agents. This honing is independent of antigens or TME. As injected MIPs will not leave the brain and will locally deliver a therapy, proteins that would induce systemic toxicity can be a viable cargo for MIPs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOstrom, Q. 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Int J Mol Sci 22, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms22105113\u003c/span\u003e\u003cspan address=\"10.3390/ijms22105113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Interneuron, glioblastoma, cellular therapy, glioma, Bispecific T-cell Engager","lastPublishedDoi":"10.21203/rs.3.rs-5493594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5493594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma has a poor prognosis with limited therapeutic options, in part due to the presence of the blood brain barrier, which often prevents the delivery of therapeutic agents, and the immune suppressive tumor environment (TME), which is a challenge for T-cell based immunotherapies. We developed a cellular delivery vector where implanted modified post-mitotic Migratory Inhibitory Interneuron Precursors (MIPs) are chemoattracted to high-grade glioma (HGG), independent of expressed antigens and unhindered by the TME, to deliver therapeutic proteins of choice.\u003c/p\u003e","manuscriptTitle":"Glioblastoma chemoattract migratory interneuron precursors modified to deliver therapeutic proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-05 06:19:35","doi":"10.21203/rs.3.rs-5493594/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-neuroscience","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"neuro","sideBox":"Learn more about [Nature Neuroscience](http://www.nature.com/neuro/)","snPcode":"","submissionUrl":"","title":"Nature Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a68c4989-cf97-4c42-90bb-ab2eca3e53d6","owner":[],"postedDate":"December 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":41103526,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":41103527,"name":"Biological sciences/Biotechnology/Cell delivery"}],"tags":[],"updatedAt":"2026-03-08T14:50:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-05 06:19:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5493594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5493594","identity":"rs-5493594","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}