Target receptor identification and subsequent treatment of resected brain tumors with encapsulated and engineered allogeneic stem cells

Abstract

Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic "off-the-shelf" clinical-grade bifunctional mesenchymal stem cells (MSC^Bif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSC^Bif (EnMSC^Bif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSC^Bif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSC^Bif in primary and recurrent GBM patients.

Fig. 1. GBM patient roadmap and survival.

a Therapeutic roadmap representing the clinical standard of care for primary and recurrent GBM patients. b Kaplan–Meier survival plot showing 10-year survival data of 587 GBM patients from the TCGA portal. c Genomic mutation analysis of GBM patient datasets revealing the potential for the development of receptor-targeted therapies. d Illustration showing the diagnostic application of circulating tumor cells (CTC) based stratification for patients and the potential for integration into mainline treatment. e Photomicrographs of target receptors’ staining by immunocytochemistry of patient GBM tumor blocks. f Photomicrographs of target receptors’ staining by immunocytochemistry of mice bearing GBM xenografts corresponding to tumor blocks in e. g Plot showing a comparison of target receptor expression between human tissue blocks and GBM xenografts (MX) implanted in mice (n = 3 randomly chosen microscopic fields/ sample/ marker). Image analysis was done with ImageJ. Scale bars 100 μM (e, f); Fluorescence: mCherry expression of Xenografts; NB: Normal Brain. Data are shown as mean ± S.D.

Fig. 2. Detection of target receptor expression in circulating tumor cells.

a Illustration showing the experimental model for CTC analysis from mice bearing GBM xenografts. b Illustration showing the principle underlying CTC isolation from blood samples using CELLTRACKS system and subsequent analysis. c Plot showing CD105⁺/CD45⁻ GBM CTC isolated and enumerated from mice bearing GBM tumor xenografts harvested and analyzed at day 21 (n = 3 mice/group). d Photomicrographs showing CD105 and CD45 staining on single cells as obtained from the analyzer. e Photomicrographs revealing mCherry Fluorescence and DR5 staining on GBM-FmC-CTC isolated from mice. f Plot showing surface expression of DR5 on GBM cells and corresponding GBM-CTC by flow cytometry. g Plot showing surface expression of DR4 on GBM cells and their corresponding GBM-CTC. h Schematic depicting the workflow for CTC isolation and analysis for clinical blood samples. i Plot showing the number of CD146⁺/CD105⁺/CD45⁻ CTC isolated from various deidentified GBM patient samples prior to surgical debulking of tumor. j Plot showing cell surface DR5 receptor expression on clinical CTC samples by flow cytometry. Scale bars 50 μM (d) and 100 μM (e). Data are shown as mean ± S.E.M.

Fig. 3. Allogeneic MSC can be banked for “off-the-shelf” therapeutic use.

a Illustration depicting the MSC isolation and banking process. b Conventional karyotype analysis of MSC^Bif as compared with naive MSC showing no changes in the chromosomes post MSC transduction. c Plot showing surface expression analysis of naïve MSC and MSC^Bif using expression markers CD146, CD90, CD105, and CD45 revealing distinct MSC identity of MSC^Bif by flow cytometry. d Plot showing the quantification of various markers analyzed in c. e Immunogenicity profiling plots for MHC Class I and II antigen markers of MSC and MSC^Bif by flow cytometry. f Plot quantifying the immunogenicity profiling represented in e. Data shown are an aggregate of three biologically independent experiments. Data are shown as mean ± S.E.M.

Fig. 4. sECM kinetics is essential to MSC migration in vitro and facilitates EnMSC^Bif efficacy.

a Schematic representing the formulation of MSC^Bif with the hydrogel to obtain EnMSC^Bif. b Illustration detailing the virtual zoning strategy used for quantification of the migration assay and corresponding zones to quantitate migration. EnMSC^Bif was placed at the center and the migration of MSC over time was quantitated. c Plot showing migration of MSC into various zones corresponding to the illustration in b at specified time points. d Plot showing changes in viability of various GBM-FmC following EnMSC^Bif treatment and GCV activation of HSV-TK as compared to controls. G, GCV. Data shown for n = 3 biologically independent samples/tumor type/treatment. e Trypan blue assay revealing percent live: dead GBM cells following EnMSC^Bif treatment as compared to controls. G, GCV. f Western blot showing the activation of the extrinsic apoptotic cascade following EnMSC^Bif treatment of GBM8-FmC, GBM18-FmC, and GBM31R-FmC. TK, HSV-TK. g, h Illustration depicting the evaluation of EnMSC^Bif kinetics in 3D bio-printed brains (g) and bio-printed with GBM8-FmC tumors (h). i Photomicrograph of the bio-printed brain. j Photomicrographs showing changes in tumor and stem cell fluorescence over time in 3D printed brains. Statistical analyses were performed using two-way ANOVA with multiple comparisons (d, k). Data shown for n = 3 biologically independent samples/tumor type/treatment (k). Plot showing changes in GBM8-FmC survival over time following GBM resection and EnMSC^Bif implantation in 3D printed brains. Scale bars 100 μM (i), 50 μM (i inset) and 100 μM (j). Data are shown as mean ± S.E.M. ***p < 0.001.

Fig. 5. Computer-assisted modeling aids with estimating resection cavity volume and the safety profile of EnMSC^Bif in vivo reveals no abnormalities in major organs.

a Representative MR scan and computer modeling of tumor volume (green) and resection cavity volume (blue) using 3D slicer. b Plot representing tumor and resection cavity volumes obtained from retrospective analysis of ten deidentified GBM patients pre- and post-surgical debulking. c Illustration of the real-time formulation of GMP-MSC^Bif obtained from master cell bank post shipment. d Photograph of representative bags used for GMP-MSC^Bif shipment. e Plot showing the viability of MSC^Bif over time in 10%HSA. Data analyzed by comparing two groups using t test. f Plot showing gelation time of GMP-EnMSC^Bif with varying cell numbers. g Plot showing activation of HSV-TK kill switch in GMP-EnMSC^Bif following GCV administration. h Plot showing changes in tumor volume in mice bearing GBM tumors treated with varying doses of GMP-EnMSC^Bif post resection in a dose-escalation study. (n = 5 mice/ group). i Plot showing changes in weights of mice bearing GBM tumors treated with varying doses of GMP-EnMSC^Bif post resection over time. j Plot showing median gelation time of GMP-Hystem Hydrogel from the two different lots in the absence and presence of MSC. k Plot showing the viability of GBM cells following co-culture with GMP-EnMSC^Bif at specified time points. l Plot showing weights of mice (n = 5 mice/group) following administration of either GMP-Hystem hydrogel alone or GMP-EnMSC^Bif in NOD.SCID mice over time. m Photomicrographs of H&E stains of the mouse brain and other major organs following GMP-EnMSC^Bif administration in non-tumor-bearing mice. n Photomicrographs of brain tissues by expansion microscopy (dEXM) using GFAP and Nestin staining. Data shown for n = 3 biologically independent samples/dose (e–g). Statistical analyses were done using two-way ANOVA with multiple comparisons (h, k). Data are shown as mean ± S.E.M. Scale bars 200 μM (m), and 100 μM (n); **p < 0.05, ***p < 0.01.

Fig. 6. EnMSC^Bif increases survival of mice in a clinically relevant mouse model of GBM resection.

a Schematic representing the murine model of GBM resection and treatment. b Plot showing changes in GBM8-FmC tumor volume over time following EnMSC^Bif administration post tumor resection as compared to controls. (n = 5 mice/group. Data representative of three independent experiments; Statistical analyses were done using two-way ANOVA with multiple comparisons). c Kaplan–Meier survival analysis of EnMSC^Bif treatment in GBM8-FmC mouse model of resection. d PET images showing brain uptake of [¹⁸F] FHBG tracer in mice (n = 3 mice/group) administered with EnMSC^Bif. e Plot representing uptake of the PET tracer, [¹⁸F] FHBG in mice administered with EnMSC^Bif. f Photomicrographs of immunofluorescence imaging of mouse brain sections stained with Cl. Caspase 3 as compared to control. g Plot showing the number of Cl. Caspase 3 cells as compared to control. h Plot showing changes in tumor volumes of GBM31R-FmC bearing mice (n = 5 mice/group) over time following EnMSC^Bif administration post tumor resection as compared to controls. i Kaplan–Meier survival analysis of EnMSC^Bif treatment in GBM31R-FmC mouse model of resection. Data are shown as mean ± S.E.M. Scale bars 100 μM. ***p < 0.01, ****p < 0.001.