Biology subjects

Lum, J. J.

Publications and source records attributed to Lum, J. J..

7 recordsLinked to original sources

bioRxiv2025

Ambient mass spectrometry imaging enables spatial metabolomics of optimal cutting temperature compound (OCT)-embedded tumors

Mass spectrometry imaging (MSI) is emerging as a powerful tool for uncovering the distribution of metabolites in the tumor microenvironment and studying tumor metabolism in vivo. However, to date, MSI of primary patient biobanked tissues contextualized by patient data has been limited to peptides, proteins, and glycans - with few examples for metabolites. This is because most biobanked fresh-frozen tissue required for spatial metabolomics is embedded in optimal cutting temperature compound (OCT), which introduces high-abundance polymeric interferents. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to demonstrate the MSI of metabolites in OCT-embedded tissue. Metabolite coverage and sensitivity for prepared tissue mimetic homogenates embedded in OCT and an MSI-compatible material, carboxymethylcellulose (CMC), showed excellent agreement. We apply our ambient MSI workflow to detect changes in intratumoral methionine using a preclinical cancer mouse model undergoing adoptive T-cell therapy. Eight days after tumor incubation, lymphoma-bearing mice were maintained on a complete or methionine-restricted diet for 2 days. Nano-DESI MSI revealed a heterogeneous tumor microenvironment, with multiple methionine-cycle intermediates (S-adenosylmethionine, S-adenosylhomocysteine) and related metabolites, including known T-cell modulators (1-methylnicotinamide, polyamines) localizing to tumor subregions. Methionine-restricted tumors exhibited reduced methionine levels and elevated S-adenosylmethionine, relative to the control group. Overall, this work demonstrates spatial metabolomics on fresh-frozen OCT-embedded tissue, unlocking the wealth of information stored in primary tissue biobanks and consequently accelerating our understanding of cancer metabolism and treatment.

cancer biology↗

bioRxiv2025

Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (FR) CAR expression cassette into the locus of the essential autophagy gene ATG5, thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5-knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro, and exhibited superior and long-lasting tumor control against ovarian tumors in vivo. Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of FR CAR-T cells for ovarian cancer immunotherapy.

immunology↗

bioRxiv2025

Methionine regulates antitumor function of CD8⁺ T cells through polyamine synthesis

Methionine is an essential amino acid critical for T cell activation. While methionine restriction (MR) combined with immune checkpoint blockade has been shown to enhance T cell function, the impact of methionine on adoptive T cell therapies is largely unexplored. Here, we examined the functionality of T cells under MR and pharmaceutical inhibition of the methionine cycle (MAT2Ai), using primary T cells and a murine adoptive T cell therapy model. In vitro, transient MR or MAT2Ai treatment increased interferon gamma (IFN{gamma}) expression in CD8+ T cells, whereas sustained MR led to the upregulation of T cell exhaustion-associated markers. Mechanistically, transient MR suppressed the polyamine synthesis pathway, and supplementation with polyamines reversed MR-induced IFN{gamma} expression. Genetic ablation of s-adenosylmethionine decarboxylase, an enzyme in the polyamine synthesis pathway, recapitulated the effect of MR, indicating that transient MR enhances T cell function by inhibiting polyamine synthesis. Despite this, transient MR treatment of ovalbumin (OVA)-specific (OT-I) CD8+ T cells prior to adoptive transfer did not improve antitumor efficacy against EG7-OVA tumors in vivo. In contrast, sustained dietary MR accelerated EG7-OVA tumor growth in mice treated with OT-I T cells, demonstrating that methionine availability is essential for the activity of adoptively transferred T cells. These findings suggest that enhancing methionine availability in the tumor microenvironment may improve the efficacy of adoptive T cell therapies.

immunology↗

bioRxiv2023

MTOR as a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

Chimeric antigen receptors (CARs) reprogram T cells to recognize and target cancer cells. Despite remarkable responses observed with CAR-T cell therapy in patients with hematological malignancies, CAR-T cell engineering still relies mostly on randomly integrating vectors, limiting the possibilities of fine-tuning T cell function. Here, we designed a CRISPR-based marker-free selection strategy to simultaneously target a therapeutic transgene and a gain-of-function mutation to the MTOR locus to enrich cells resistant to rapamycin, a clinically used immunosuppressant. We readily engineered rapamycin-resistant (RapaR) CAR-T cells by targeting CAR expression cassettes to the MTOR locus. Using in vitro cytotoxicity assays, and a humanized mouse model of acute lymphoblastic leukemia, we show that RapaR-CAR-T cells can efficiently target CD19+ leukemia cells in presence of immunosuppressing doses of rapamycin. Furthermore, our strategy allows multiplexed targeting of rapamycin-regulated immunoreceptors complexes (DARICs) to the MTOR and TRAC loci to pharmacologically control CAR-T cells activity. We foresee that our approach could both facilitate the enrichment of CRISPR-engineered CAR-T cells ex vivo and in vivo while improving tumor eradication.

cancer biology↗

bioRxiv2021

Clinically-relevant T cell expansion protocols activate distinct cellular metabolic programs and phenotypes

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables including proliferation, differentiation, function, activation and mitochondrial phenotypes. T cells adapted their metabolism to match their media expansion condition as shown by glucose and glutamine uptake, and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Furthermore, T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. In one case, culturing in ascites resulted in increased glucose uptake which was insufficient to rescue T cell function. Thus, ex vivo T cell expansion conditions have profound impacts on metabolism and function.

immunology↗

bioRxiv2020

1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer

Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few notable exceptions, their identities remain largely unknown. We uncovered the immune regulatory metabolic states and metabolomes of sorted tumor and stromal, CD4+, and CD8+ cells from the tumor and ascites of patients with high-grade serous ovarian cancer (HGSC) using high-dimensional flow cytometry and metabolomics supplemented with single cell RNA sequencing. Flow cytometry revealed that tumor cells show a consistently greater uptake of glucose than T cells, but similar mitochondrial activity. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the gene encoding the enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Treatment of T cells with MNA resulted in an increase in T cell-mediated secretion of the tumor promoting cytokine tumor necrosis factor alpha. Thus, the TME-derived metabolite MNA contributes to an alternative and non-cell autonomous mechanism of immune modulation of T cells in HGSC. Collectively, uncovering the tumor-T cell metabolome may reveal metabolic vulnerabilities that can be exploited using T cell-based immunotherapies to treat human cancer.

immunology↗

bioRxiv2017

Accelerated human liver progenitor generation from pluripotent stem cells by inhibiting formation of unwanted lineages

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0{+/-}3.1% AFP+ human liver bud progenitors and 87.3{+/-}9.4% ALBUMIN+ hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5{+/-}3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.

cell biology↗