Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Mitochondrial ROS promote migration and dissemination of malignant lymphocytes (A) Schematic representation of workflow of mROS sorting and subsequent transwell migration and xenograft studies. (B) Transwell migration of Jurkat cells sorted for five different mROS levels ranging from low to high as indicated in different colors. (C) Representative images of 18 FDG-PET/CT studies and hematoxylin and eosin (H&E)-stained liver sections of hepatic dissemination of sorted mROS lo , mROS bulk , and mROS hi Hut78 cells. Liver outlined by dotted white lines in PET/CT images and infiltrating lymphocyte aggerates outlined in yellow in H&E images (n=4 mice per group in PET/CT studies). (D) Quantification of hepatic infiltration of sorted Hut78 cells in H&E sections shown as percent of liver section area (n=2 mice). (E) Quantification of organ infiltration after xenografting of HH cells. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=4 mice). (F) Hepatic infiltration after second-generation xenografting of isolated mROS lo and mROS hi xenograft tumor cells (n=3 mice/group). (G) Gene set enrichment analysis of FACS-sorted mROS lo and mROS hi Jurkat cells. (H) Effect of antioxidants, N-acetylcysteine (NAC) and mitoTEMPO (mitoT), on migration of Jurkat cells sorted for different mROS levels ranging from low to high. (I) Effect of N-acetylcysteine (NAC) and mitoTEMPO (mitoT) on transwell migration of Hut78 cells and freshly isolated leukemic cells from patients with CTCL (n=4), ALL (n=14), and CLL (n=11). (J) Hepatic dissemination of Hut78 cells pretreated with NAC and mitoT. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=3 mice/group). (K) Effect of mitoTEMPO on ex vivo migration of xenograft cells isolated from subcutaneous primary tumors (SQ CD45+) and liver infiltrates (Liver CD45+). (L) Effect of antioxidant mitoTEMPO on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=10). (M) Levels of mROS and transwell migration of Jurkat cells treated with increasing concentrations of phenformin (0, 1, 2, 4, 6, 10 uM), rotenone (0, 10, 50, 100, 200, 500 nM), and antimycin A (0, 25, 50, 100, 200, 400 nM) for 24 hours. (N) Effect of antioxidant mitoTEMPO on transwell migration of Jurkat cells treated with low (+) and high (++) concentrations of each phenformin (4 and 10 uM), rotenone (50 and 500 nM), and antimycin A (100 and 500 nM). (O) Hepatic dissemination of Hut78 cells pretreated with phenformin. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=4 mice/group). All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: HH, Jurkat, Myla, Daudi and Toledo were propagated in RPMI-1640 media (ATCC) and Hut78 was propagated in Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC).

Techniques: Migration, Positron Emission Tomography-Computed Tomography, Staining, Flow Cytometry, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Promotion of malignant lymphocyte spread by mROS is mediated through HIF-1a (A) Levels of HIF-1a in sorted mROS lo and mROS hi Hut78 cells detected by flow cytometry and immunoblotting. Immunoblots from nuclear extracts with antibody against H2A-Z used as loading control. (B) Flow-cytometric quantification of HIF-1a in mROS lo and mROS hi cells of indicated cell lines. HIF-1a levels shown in mean fluorescence intensity. (C) Increased HIF-1a levels in isolated leukemic cells from patients with CTCL (n=2), ALL (n=3), and CLL (n=3). (D and E) Effect of HIF-1a knockout in Hut78 cells on invasive potential in transwell assays (D) and hepatic infiltration in vivo (E; n=3 animals per group). (F) Effect of PX-478 on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=10). (G and H) Immunoblots (G) and corresponding transwell migration assay (H) of Jurkat cells transfected with either wild-type HA-tagged HIF-1a (HIF WT ) or mutant HA-tagged-HIF-1a P402A/P564A (HIF mut ) followed by treatment with antioxidants, NAC or mitoTEMPO. LE, low exposure, HE, high exposure. (I and J) Immunoblots (I) and corresponding transwell migration assays (J) of Jurkat cells treated with PHD inhibitor, IOX2, and either NAC or mitoTEMPO. LE, low exposure, HE, high exposure. (K and L) Immunoblots (K) and corresponding e x vivo transwell migration assays (L) of leukemic cells treated with IOX2 and either NAC or mitoTEMPO after isolation from patients with CTCL (n=3), ALL (n=8), CLL (n=8). LE, low exposure, HE, high exposure. (M) Gene set enrichment analysis of Jurkat cells treated with PX-478 and sorted into mROS lo and mROS hi subpopulations. (N) Effect of hypoxia (3% O 2 ) versus normoxia on transwell migration of Jurkat cells treated with mitoTEMPO or PX-478. All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: HH, Jurkat, Myla, Daudi and Toledo were propagated in RPMI-1640 media (ATCC) and Hut78 was propagated in Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC).

Techniques: Flow Cytometry, Western Blot, Control, Fluorescence, Isolation, Knock-Out, In Vivo, Ex Vivo, Migration, Transwell Migration Assay, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Glucose is an essential fuel for migration through activation of mROS/HIF-1a (A) Clustering heatmap of metabolomic profiles of sorted mROS lo and mROS hi Jurkat cells (n=3 independent samples analyzed in triplicates). (B) Ratio of mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in isolated mROS lo and mROS hi cells. (C and D) Glucose uptake observed in mROS lo and mROS hi cells isolated from mouse xenograft tumors (C; n=3 tumors of each cell line) and isolated leukemic cells (D; ALL, n=3 patients; CLL, n=3 patients). (E) Effect of glucose-limiting conditions or inhibition of glycolysis with 2-deoxyglucose (2-DG) on transwell migration. Cells were incubated with different concentrations of glucose or 2-DG (0, 2.5, 5, 10, 20, 25 mM for both) during migration in transwell chambers. (F) Effect of glucose-limiting conditions on migration of isolated mROS lo and mROS hi cells. (G and H) Effect of long-term culture in glucose-limiting media on transwell migration (G). Transwell assays were performed after 30 days in glucose-limiting conditions with unchanged glucose concentration (-Glc) or freshly added glucose during the 24 h migration studies (+Glc; H) (I) Gene set enrichment analysis of Jurkat cells in glucose-free conditions. (J and K) Representative immunoblots (J) and corresponding transwell migration assay (K) of Jurkat cells transfected with either wild-type HA-tagged HIF-1a (HIF WT ) or mutant HA-tagged-HIF-1a P402A/P564A (HIF mut ) in media containing 3 mM of glucose or 25 mM of 2-DG. (L and M) Representative immunoblots (L) and corresponding ex vivo transwell migration assays (M) of isolated leukemic cells treated with PHD inhibitor IOX2 and NAC or mitoTEMPO (ALL, n=4 patients; CLL, n=4 patients). LE, low exposure, HE, high exposure. (N) Glucose uptake observed in control and HIF1a-KO Hut78 cells. mROS lo in light blue bars; mROS hi in orange bars in (B-D, F, N). All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: HH, Jurkat, Myla, Daudi and Toledo were propagated in RPMI-1640 media (ATCC) and Hut78 was propagated in Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC).

Techniques: Migration, Activation Assay, Metabolomic, Isolation, Inhibition, Incubation, Concentration Assay, Western Blot, Transwell Migration Assay, Transfection, Mutagenesis, Ex Vivo, Control

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Metabolic control of migratory potential at the pyruvate branch point (A) Transwell migration of cells supplemented with 2.5, 5, 10 mM pyruvate in glucose-free media for 24 hours. (B) Levels of mROS in response to pyruvate supplementation (10mM) in glucose-free media. (C) Immunoblots of HIF-1a in Jurkat cells after pyruvate supplementation in glucose-free media. (D and E) Effect of mitoTEMPO (D) and PX-478 (E) on transwell migration in glucose-free media with and without supplemented pyruvate. (F) Effect of pyruvate supplementation on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PMBCs from healthy subjects (n=10) in glucose-free media. (G) Transwell migration assay of cells transfected with mitochondrial pyruvate carrier 1 siRNA (siMPC1), monocarboxylate transporter 1 siRNA (siMCT1) or scrambled siRNA (siSCR). (H) Effect of mitoTEMPO (mitoT) and PX-478 on migration of cells transfected with mitochondrial pyruvate carrier 1 siRNA (siMPC1) or scrambled siRNA (siSCR). (I) Gene set enrichment analysis of Jurkat cells treated with AZD3965 compared to untreated control. (J and K) Immunoblots (J) and corresponding transwell migration assays (K) of Jurkat cells treated with PHD inhibitor IOX2 and Oxamate, DCA, or AZD3965. (L) Representative images of livers of mice treated with AZD3965 versus control and corresponding quantitation of hepatic disease infiltration in xenograft studies of Hut78 cells (n=4 animals per group). (M) Effect of AZD3965 on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=13). (N) Schematic summary of oxidative versus glycolytic metabolic programming controlling dissemination potential of lymphoid cancer cells through redox signaling. All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: HH, Jurkat, Myla, Daudi and Toledo were propagated in RPMI-1640 media (ATCC) and Hut78 was propagated in Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC).

Techniques: Control, Migration, Western Blot, Ex Vivo, Isolation, Transwell Migration Assay, Transfection, Quantitation Assay