Biology subjects

Shpall, E. J.

Publications and source records attributed to Shpall, E. J..

7 recordsLinked to original sources

bioRxiv2026

MRI-Trackable Mesenchymal Stem Cells for Oncolytic Therapy: A Clinically Feasible Dual-Labeling Protocol

Non-invasive imaging of oncolytic virus-loaded mesenchymal stem cells (MSCs) could help evaluate cell trafficking and delivery in cancer therapy. Labeling MSCs with iron oxide particles (IOPs) enables in vivo tracking using magnetic resonance imaging (MRI). This study evaluated a dual-labeling strategy that loads MSCs with MegaPro IOPs and the oncolytic virus Delta-24-RGD (DNX-2401; tasadenoturev) and assessed cellular function and MRI detectability. MSCs were labeled with IOPs alone or in combination with the oncolytic virus. We compared cell viability and intracellular iron content among IOP-labeled, dual-labeled, and naive MSCs, and evaluated differentiation, invasion, and immunophenotype in naive and IOP-labeled MSCs. We evaluated phantoms containing varying numbers of IOP-labeled MSCs (9 x 103-1.5 x 105 cells) using R2-, R2*-, and R1-weighted MRI, and generated corresponding relaxation rate maps to assess concentration dependent contrast and detection sensitivity. Iron content was comparable between IOP-labeled and dual-labeled MSCs (9.2 {+/-} 2.6 and 11.6 {+/-} 1.4 pg/cell, respectively), with >91% viability across all groups. IOP-labeled MSCs retained invasion and differentiation potential. At the highest labeled-cell concentration, R2*, R2, and R1 increased approximately 4, 2.5, and 1.5 fold, respectively, relative to the agarose control. The estimated detection limits were approximately 2 x 103 cells for R2* and 6 x 103 cells for R2 and R1. These findings demonstrate that dual labeling of MSCs with IOPs and oncolytic virus preserves key cellular characteristics while providing MRI-detectable contrast, supporting further investigation of this approach for image-guided tracking of cell-based cancer therapies.

bioengineering↗

bioRxiv2026

Microbial valerate is associated with CAR T dysbiosis and its supplementation enhances CAR T function in B-cell lymphoma

Anaerobe-depleting antibiotic exposure is associated with inferior progression-free survival after CD19 CAR T-cell therapy in large B-cell lymphoma, yet the cellular mechanisms linking gut dysbiosis to the CAR T-cell product and whether this imprint is reversible have remained undefined. In two independent CAR-T candidate cohorts, low stool valerate at the time of CAR-T eligibility identified a multi-metabolite-deficient dysbiotic gut microbiome state marked by depletion of fiber-fermenting commensals and loss of carbohydrate-fermentation, SCFA-biosynthesis, and amino-acid metabolism pathways. Reanalysis of single-cell RNA sequencing from 42 lymphoma patients stratified by piperacillin-tazobactam/imipenem/meropenem (PIM) exposure revealed that PIM-exposed CAR T-cell products were CD4-skewed, with significantly elevated AP-1/immediate-early gene (IEG) and cellular activation signatures that together predicted inferior progression-free survival. Ex vivo conditioning of CAR T-cells with valerate produced a chromatin and transcription factor program distinct from butyrate or propionate, characterized by KLF/SP/EGR family engagement, KLF4 promoter opening, and broad induction of AP-1/IEG and MHC class II transcripts, whereas butyrate drove broader chromatin remodeling with TBX21/EOMES/NF-{kappa}B gains and KLF2 promoter closure, and propionate induced an NFY-centered program with preferential commitment to low-mitochondrial-content states. Untargeted metabolomics confirmed valerate uptake and mitochondrial {beta}-oxidation in CAR T-cells, while dietary sodium valerate supplementation in meropenem-treated mice bearing A20 lymphoma significantly reduced tumor burden and extended survival compared with CAR T-cells alone. These findings identify stool valerate as a bedside-deployable biomarker of dysbiosis-imprinted CAR T-cell dysfunction and support ex vivo or dietary valerate supplementation as a clinically tractable strategy to improve CAR-T anti-tumor function in patients with disrupted gut microbiomes.

cancer biology↗

bioRxiv2024

NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model

NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 expression in cancer cells induces DNA damage which leads to cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1R/ KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung metastases were developed from KRAS/STK11mt/aPD1R A549 cells in humanized mice and treated with NPRL2 gene-loaded cationic lipid nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting that an immune response contributed to antitumor activity. NPRL2 combined with pembrolizumab was not synergistic in the KRAS/STK11mt/aPD1R tumors but was synergistic in the KRASwt/aPD1S H1299 tumors. Consistent with the A549 humanized mouse model, NPRL2 showed a significantly strong antitumor effect on KRASmt/aPD1R LLC2 syngeneic tumors, whereas aPD1 was ineffective. The antitumor effect of NPRL2 was correlated with increased infiltration of human cytotoxic immune cells and Ag-presenting HLA-DR+ DC, CD11c DC, and downregulation of myeloid and regulatory T cells in the TME. The antitumor effect of NPRL2 was significantly abolished upon in-vivo depletion of CD8 T, macrophages, and CD4 T cells. However, the antitumor effect remained unaffected upon in-vivo depletion of NK cells. A distinct pattern of gene expression profile was found in lung met after NPRL2 treatment in humanized mice. The expression of genes associated with T cell functions, including IFN{gamma}, CD8b, CD7, TNFSF18, ITGA1, GATA3, and TBX21 was significantly increased, whereas the expression of genes associated with negative regulation of T cell functions, including FOXP3, TGFB1, TGFB2, and IL-10RA were strongly inhibited upon NPRL2 treatment. NPRL2 downregulated the expression of T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Tumors established from NPRL2 stably expressing cells in humanized mice exhibited significantly slower growth compared to controls. TME analysis showed an increased presence of human CD45+, CD3+ T, CD8+ T cells, and HLA-DR+ dendritic cells and a decreased percentage of Treg, CD3+PD1+T cells, MDSC, and CD163+ TAM in NPRL2-expressing tumors. In-vitro, NPRL2 stably expressing cells showed a substantial increase in colony formation inhibition and heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of MAPK and AKT-mTOR growth signaling through inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, and pS6 expression. Taken together, these data suggest that NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation.

cancer biology↗

bioRxiv2021

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones were used to model osimertinib acquired resistance in humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation were found in resistant clones. Resistant tumors in humanized mice were initially partially responsive to osimertinib, then aggressive tumor regrowth occurred accompanied by an immunosuppressive tumor microenvironment. 3-phosphoinositide-dependent kinase 1 (PDK1) was identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitized resistant clones and a patient derived xenograft (PDX) with acquired resistance to osimertinib. PDK1 knock-out dysregulated PI3K/Akt/mTOR signaling, promoted cell cycle arrest at the G1 phase, and inhibited nuclear translocation of yes-associated protein (YAP). Higher expression of PDK1 was found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

cancer biology↗

bioRxiv2021

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment

Not all cancer patients with severe neutropenia develop fever, and the fecal microbiome may play a role. In neutropenic hematopoietic cell transplant patients (n=119), 63 (53%) developed a subsequent fever and had increased fecal Akkermansia muciniphila, a mucus-degrading bacteria (p=0.006, corrected for multiple comparisons). In mouse models, two therapies, irradiation and melphalan, similarly expanded A. muciniphila. Dietary restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Azithromycin treatment depleted A. muciniphila and preserved colonic mucus. Dietary restriction raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila with lower pH and increased propionate prevented utilization of mucin. Treating irradiated mice with azithromycin or propionate preserved the mucus layer, lessened hypothermia, and reduced inflammatory cytokines in the colon. These results suggest that diet, metabolites and colonic mucus link the microbiome to neutropenic fever, and could guide future microbiome-based preventive strategies.

microbiology↗

bioRxiv2020

Generation of glucocorticoid resistant SARS-CoV-2 T-cells for adoptive cell therapy

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expanded SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observed that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions resulted in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs were directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells could not be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we developed an efficient strategy to inactivate the glucocorticoid receptor gene (NR3C1) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

immunology↗

bioRxiv2020

CIS checkpoint deletion enhances the fitness of cord blood derived natural killer cells transduced with a chimeric antigen receptor

Immune checkpoint therapy has produced remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor (CAR) engineering of natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that combining CIS checkpoint deletion with CAR engineering promotes the metabolic fitness of NK cells in an otherwise suppressive tumor microenvironment. This approach, together with the prolonged survival afforded by CAR modification, represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

immunology↗