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Procell Inc aml cell lines
Aml Cell Lines, supplied by Procell Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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aml cell lines - by Bioz Stars, 2026-05

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FLT3-ITD AML cells are resistant to FLT3 inhibitors in hypoxia, associated with post-translational FLT3-ITD downregulation. A. Primary FLT3-ITD AML blasts from two patients were cultured for 48 hours under normoxia (21%O 2 ) or hypoxia (<1% O 2 ) with the FLT3 inhibitors gilteritinib or quizartinib at increasing concentrations and cytotoxicity was measured using the WST-1 assay. Cytotoxicity of both gilteritinib and quizartinib was reduced in hypoxia (red lines) compared with normoxia (black lines). Data represent means of three replicate wells. B. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of FLT3, p-STAT5, STAT5 and vinculin loading control. Bands were quantified by densitometry and FLT3, p-STAT5 and STAT5 were normalized to vinculin and to Time 0. Immunoblots and graphs are shown. FLT3-ITD and p-STAT5 were downregulated in hypoxia, but not in normoxia, while total STAT5 remained stable in both. C. FLT3 and GAPDH control mRNA was measured by RT-qPCR in blasts from three FLT3-ITD AML patients cultured for 0, 48 and 96 hours in hypoxia and normoxia. Graphs of FLT3 mRNA, normalized to GAPDH mRNA, show no decrease in FLT3 mRNA. D. FLT3-ITD AML patient blasts were treated with cycloheximide (CHX, 100 µg/mL) to block new protein translation, with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes to block proteasomal degradation, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was accelerated in hypoxia (1.0 vs. 2.5 hours), in the absence, but not presence, of MG-132, consistent with increased proteasomal degradation in hypoxia.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: FLT3-ITD AML cells are resistant to FLT3 inhibitors in hypoxia, associated with post-translational FLT3-ITD downregulation. A. Primary FLT3-ITD AML blasts from two patients were cultured for 48 hours under normoxia (21%O 2 ) or hypoxia (<1% O 2 ) with the FLT3 inhibitors gilteritinib or quizartinib at increasing concentrations and cytotoxicity was measured using the WST-1 assay. Cytotoxicity of both gilteritinib and quizartinib was reduced in hypoxia (red lines) compared with normoxia (black lines). Data represent means of three replicate wells. B. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of FLT3, p-STAT5, STAT5 and vinculin loading control. Bands were quantified by densitometry and FLT3, p-STAT5 and STAT5 were normalized to vinculin and to Time 0. Immunoblots and graphs are shown. FLT3-ITD and p-STAT5 were downregulated in hypoxia, but not in normoxia, while total STAT5 remained stable in both. C. FLT3 and GAPDH control mRNA was measured by RT-qPCR in blasts from three FLT3-ITD AML patients cultured for 0, 48 and 96 hours in hypoxia and normoxia. Graphs of FLT3 mRNA, normalized to GAPDH mRNA, show no decrease in FLT3 mRNA. D. FLT3-ITD AML patient blasts were treated with cycloheximide (CHX, 100 µg/mL) to block new protein translation, with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes to block proteasomal degradation, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was accelerated in hypoxia (1.0 vs. 2.5 hours), in the absence, but not presence, of MG-132, consistent with increased proteasomal degradation in hypoxia.

Article Snippet: The human FLT3-ITD AML cell lines MV4-11 and MOLM-14, with homozygous and heterozygous FLT3-ITD, respectively (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum, 1% penicillin-streptomycin and 2 mM L-glutamine, unless otherwise indicated, and were tested for Mycoplasma every six months ( ).

Techniques: Cell Culture, WST-1 Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Blocking Assay

A. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of c-CBL, p-c-CBL (Y371) and vinculin loading control, and bands were quantified by densitometry. Expression of c-CBL and p-c-CBL was upregulated in hypoxia, but not in normoxia. B. c-CBL and GAPDH control mRNA was measured at 0, 48 and 96 hours by RT-qPCR, and graphs of c-CBL mRNA normalized to GAPDH control mRNA are shown. c-CBL mRNA expression did not decrease in cells cultured in hypoxia. C . MOLM-14 cells were treated with cycloheximide (CHX, 100 µg/mL), with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was similar in hypoxia and normoxia and the effect of MG-132 was also similar.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Blasts from three FLT3-ITD AML patients cultured in normoxia and hypoxia were harvested at 0, 48 and 96 hours and immunoblotted for expression of c-CBL, p-c-CBL (Y371) and vinculin loading control, and bands were quantified by densitometry. Expression of c-CBL and p-c-CBL was upregulated in hypoxia, but not in normoxia. B. c-CBL and GAPDH control mRNA was measured at 0, 48 and 96 hours by RT-qPCR, and graphs of c-CBL mRNA normalized to GAPDH control mRNA are shown. c-CBL mRNA expression did not decrease in cells cultured in hypoxia. C . MOLM-14 cells were treated with cycloheximide (CHX, 100 µg/mL), with or without addition of the proteasome inhibitor MG-132 (20 µmol/L) after 30 minutes, then harvested at 0, 1, 2, and 4 hours, starting 1 hour after addition CHX, and immunoblotted for FLT3 and vinculin control. Bands were quantified by densitometry and FLT3 protein half-lives were calculated by linear regression analysis. FLT3-ITD protein turnover was similar in hypoxia and normoxia and the effect of MG-132 was also similar.

Techniques: Cell Culture, Expressing, Control, Quantitative RT-PCR

A. Whole-cell lysates of MOLM-14 and MV4-11 FLT3-ITD AML cells and primary FLT3-ITD AML blasts (Patient 1) transfected with c-CBL siRNA or control (CTR) siRNA were immunoblotted and probed for c-CBL and vinculin loading control, and bands were quantified by densitometry. Immunoblots demonstrate c-CBL knockdown in cells transfected with c-CBL siRNA. B. MOLM-14 and MV4-11 FLT3-ITD AML cell lines and primary FLT3-ITD AML patient blasts transfected with c-CBL siRNA or CTR siRNA cultured in hypoxia were harvested at 0, 48, or 96 hours, and whole-cell lysates were probed for FLT3, c-CBL and vinculin loading control. Immunoblots and densitometric analysis are shown, demonstrating that c-CBL knockdown abrogates FLT3-ITD downregulation in hypoxia. C. c-CBL, FLT3 and GAPDH control mRNA expression was measured by RT-qPCR in MOLM-14, MV4-11 and primary FLT3-ITD AML patient blasts treated with c-CBL siRNA or CTR siRNA for 96 hours. Data are shown graphically, with bars showing mean fold expression (2^-ΔCt) ± SD in cells transfected with c-CBL siRNA, relative to CTR siRNA. c-CBL silencing decreases c-CBL, but not FLT3, mRNA, showing that, as expected, FLT3 downregulation in cells transfected with CTR siRNA is not transcriptional . D. MOLM-14 cells transfected with c-CBL siRNA or CTR siRNA were treated with CHX to inhibit new protein translation, with or without MG-132 to inhibit proteasomal degradation. FLT3 protein turnover was accelerated in cells transfected with control, but not c-CBL, siRNA, in the absence, but not presence, of MG-132, consistent with c-CBL silencing rescuing FLT3-ITD AML cells from increased FLT3-ITD proteasomal degradation in hypoxia.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Whole-cell lysates of MOLM-14 and MV4-11 FLT3-ITD AML cells and primary FLT3-ITD AML blasts (Patient 1) transfected with c-CBL siRNA or control (CTR) siRNA were immunoblotted and probed for c-CBL and vinculin loading control, and bands were quantified by densitometry. Immunoblots demonstrate c-CBL knockdown in cells transfected with c-CBL siRNA. B. MOLM-14 and MV4-11 FLT3-ITD AML cell lines and primary FLT3-ITD AML patient blasts transfected with c-CBL siRNA or CTR siRNA cultured in hypoxia were harvested at 0, 48, or 96 hours, and whole-cell lysates were probed for FLT3, c-CBL and vinculin loading control. Immunoblots and densitometric analysis are shown, demonstrating that c-CBL knockdown abrogates FLT3-ITD downregulation in hypoxia. C. c-CBL, FLT3 and GAPDH control mRNA expression was measured by RT-qPCR in MOLM-14, MV4-11 and primary FLT3-ITD AML patient blasts treated with c-CBL siRNA or CTR siRNA for 96 hours. Data are shown graphically, with bars showing mean fold expression (2^-ΔCt) ± SD in cells transfected with c-CBL siRNA, relative to CTR siRNA. c-CBL silencing decreases c-CBL, but not FLT3, mRNA, showing that, as expected, FLT3 downregulation in cells transfected with CTR siRNA is not transcriptional . D. MOLM-14 cells transfected with c-CBL siRNA or CTR siRNA were treated with CHX to inhibit new protein translation, with or without MG-132 to inhibit proteasomal degradation. FLT3 protein turnover was accelerated in cells transfected with control, but not c-CBL, siRNA, in the absence, but not presence, of MG-132, consistent with c-CBL silencing rescuing FLT3-ITD AML cells from increased FLT3-ITD proteasomal degradation in hypoxia.

Techniques: Transfection, Control, Western Blot, Knockdown, Cell Culture, Expressing, Quantitative RT-PCR

Primary blasts from three patients with AML with WT FLT3 (Patients 7–9) cultured in hypoxia for 96 hours were immnunoblotted for FLT3, c-CBL and vinculin loading control. The findings suggest that, in contrast to FLT3-ITD AML, hypoxia does not uniformly induce FLT3 downregulation in FLT3-WT primary blasts, and that additional patient-specific factors may influence FLT3 stability under low-oxygen conditions.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: Primary blasts from three patients with AML with WT FLT3 (Patients 7–9) cultured in hypoxia for 96 hours were immnunoblotted for FLT3, c-CBL and vinculin loading control. The findings suggest that, in contrast to FLT3-ITD AML, hypoxia does not uniformly induce FLT3 downregulation in FLT3-WT primary blasts, and that additional patient-specific factors may influence FLT3 stability under low-oxygen conditions.

Techniques: Cell Culture, Control

A. Primary FLT3-ITD AML cells from four patients were cultured for 48 hours with the FLT3 inhibitors gilteritinib or quizartinib in increasing concentrations in normoxia or hypoxia in the presence or absence of 2 mM glutamine, and cytotoxicity was measured using the WST-1 assay. FLT3-ITD AML cells remained sensitive to FLT3 inhibitors in hypoxia in the absence of glutamine. B. Primary FLT3-ITD AML cells cultured in hypoxia in the presence or absence of 2 mM glutamine were harvested for immunoblotting at 0, 48 and 96 hours. FLT3-ITD and p-STAT5 expression decreased progressively in the presence, but not absence, of 2 mM glutamine, while total STAT5 expression did not change, demonstrating that FLT3-ITD and p-STAT5 downregulation in hypoxia is abrogated in the absence of glutamine. C. FLT3-ITD AML cells were treated with CHX to block new protein synthesis, with or without addition of the proteasome inhibitor MG-132, and cultured in hypoxia in the presence or absence of 2 mM glutamine. Increased FLT3-ITD proteasomal degradation of occurred in hypoxia in the presence, but not absence, of glutamine.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Primary FLT3-ITD AML cells from four patients were cultured for 48 hours with the FLT3 inhibitors gilteritinib or quizartinib in increasing concentrations in normoxia or hypoxia in the presence or absence of 2 mM glutamine, and cytotoxicity was measured using the WST-1 assay. FLT3-ITD AML cells remained sensitive to FLT3 inhibitors in hypoxia in the absence of glutamine. B. Primary FLT3-ITD AML cells cultured in hypoxia in the presence or absence of 2 mM glutamine were harvested for immunoblotting at 0, 48 and 96 hours. FLT3-ITD and p-STAT5 expression decreased progressively in the presence, but not absence, of 2 mM glutamine, while total STAT5 expression did not change, demonstrating that FLT3-ITD and p-STAT5 downregulation in hypoxia is abrogated in the absence of glutamine. C. FLT3-ITD AML cells were treated with CHX to block new protein synthesis, with or without addition of the proteasome inhibitor MG-132, and cultured in hypoxia in the presence or absence of 2 mM glutamine. Increased FLT3-ITD proteasomal degradation of occurred in hypoxia in the presence, but not absence, of glutamine.

Techniques: Cell Culture, WST-1 Assay, Western Blot, Expressing, Blocking Assay

FLT3-ITD AML blasts were cultured in hypoxia for 96 hours in medium with or without 2 mM L-glutamine and immunoblotted for expression of c-CBL and vinculin loading control. Immunoblot and graphic representation are shown. c-CBL protein expression was increased in cells cultured with, but not without, glutamine supplementation.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: FLT3-ITD AML blasts were cultured in hypoxia for 96 hours in medium with or without 2 mM L-glutamine and immunoblotted for expression of c-CBL and vinculin loading control. Immunoblot and graphic representation are shown. c-CBL protein expression was increased in cells cultured with, but not without, glutamine supplementation.

Techniques: Cell Culture, Expressing, Control, Western Blot

A. Primary FLT3-ITD AML cells cultured in hypoxia with and without glutamine (2 mM), with telaglenastat (100 nM) and with glutamine and telaglenastat and harvested at 0, 48 and 96 hours were immunoblotted for c-CBL, FLT3, p-STAT5, STAT5 and vinculin expression, and bands were quantified by densitometry. Immunoblots and densitometric analysis are shown. B. MOLM-14 cells and primary FLT3-ITD AML cells from two patients were cultured in hypoxia in the presence of glutamine with telaglenastat and the FLT3 inhibitors gilteritinib or quizartinib at diverse concentrations. Drug combination effects were analyzed using the Chou-Talalay method. Combination index <1, =1 or >1 indicated synergy, additivity and antagonism, respectively. Synergistic effects were seen.

Journal: bioRxiv

Article Title: Glutamine-Dependent Downregulation of FLT3-ITD is a Mechanism of FLT3 Inhibitor Resistance in FLT3-ITD AML in Hypoxia

doi: 10.64898/2026.05.02.722336

Figure Lengend Snippet: A. Primary FLT3-ITD AML cells cultured in hypoxia with and without glutamine (2 mM), with telaglenastat (100 nM) and with glutamine and telaglenastat and harvested at 0, 48 and 96 hours were immunoblotted for c-CBL, FLT3, p-STAT5, STAT5 and vinculin expression, and bands were quantified by densitometry. Immunoblots and densitometric analysis are shown. B. MOLM-14 cells and primary FLT3-ITD AML cells from two patients were cultured in hypoxia in the presence of glutamine with telaglenastat and the FLT3 inhibitors gilteritinib or quizartinib at diverse concentrations. Drug combination effects were analyzed using the Chou-Talalay method. Combination index <1, =1 or >1 indicated synergy, additivity and antagonism, respectively. Synergistic effects were seen.

Techniques: Cell Culture, Expressing, Western Blot

(A) Long-term killing assay of TIM-3.CAR-CIK cells against four primary AML samples compared with NT cells. Blasts survival was assessed by flow cytometry (E:T 1:10 and 1:50, n = 8 donors). See also Figure S2D . (B) Survival of TIM-3 + primary AML blasts (n=3) after 7-day co-culture with TIM-3.CAR-CIK or NT cells (E:T 1:10 and 1:50, n = 4 donors). See also Figure S2E . (C) Recovery of the LSC-enriched CD34 + CD38 - population (n = 9) and ( D ) of GPR56 + blasts (n = 6) after long-term co-culture. (E) Proliferation of TIM-3.CAR-CIK cells assessed by Ki67 staining after 72 hours co-culture with AML blasts (E:T 1:1, n = 7). See also Figure S2G . (F) Cytokine production (IFN-γ, IL-2) after 5 hours co-culture of TIM-3.CAR-CIK or NT cells with primary AML blasts (E:T 1:3, n = 9). See also Figure S2H . Data are presented as individual values and mean ± SD. Statistics were calculated with repeated-measures two-way ANOVA with Bonferroni’s post hoc test. ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. See also Figure S2 for TIM-3.CAR validation in KASUMI-3, AML cell line.

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Long-term killing assay of TIM-3.CAR-CIK cells against four primary AML samples compared with NT cells. Blasts survival was assessed by flow cytometry (E:T 1:10 and 1:50, n = 8 donors). See also Figure S2D . (B) Survival of TIM-3 + primary AML blasts (n=3) after 7-day co-culture with TIM-3.CAR-CIK or NT cells (E:T 1:10 and 1:50, n = 4 donors). See also Figure S2E . (C) Recovery of the LSC-enriched CD34 + CD38 - population (n = 9) and ( D ) of GPR56 + blasts (n = 6) after long-term co-culture. (E) Proliferation of TIM-3.CAR-CIK cells assessed by Ki67 staining after 72 hours co-culture with AML blasts (E:T 1:1, n = 7). See also Figure S2G . (F) Cytokine production (IFN-γ, IL-2) after 5 hours co-culture of TIM-3.CAR-CIK or NT cells with primary AML blasts (E:T 1:3, n = 9). See also Figure S2H . Data are presented as individual values and mean ± SD. Statistics were calculated with repeated-measures two-way ANOVA with Bonferroni’s post hoc test. ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. See also Figure S2 for TIM-3.CAR validation in KASUMI-3, AML cell line.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Co-Culture Assay, Staining, Biomarker Discovery

(A) TIM-3 expression on KASUMI-3 cells, primary AML blasts, and healthy immune subsets (CIK cells, monocytes, NK cells) assessed by flow cytometry using QuantiBRITE beads. REH (ALL cell line) served as negative control. (B) Short-term killing assay of TIM-3.CAR-CIK cells against CIK (n = 11) or KASUMI-3 (n = 8) cells compared with NT cells. Target cell lysis was evaluated by flow cytometry (E:T 5:1). (C) Short-term killing assay of TIM-3.CAR-CIK cells against monocytes (n = 8) or NK cells (n = 8) compared with NT (E:T 5:1). KASUMI-3 (n = 4) were included as positive control. (D) Immunoblot analysis of TIM-3 in lysates from monocytes, CIK cells, and KASUMI-3 cells following enzymatic treatment with PNGase F or broad neuraminidase, probed with a commercial anti–TIM-3 antibody (TIM-3-cmAb). GAPDH, loading control. Glycan symbols follow SNFG. (E) TIM-3 immunoprecipitates from monocytes, CIK cells, and KASUMI-3 cells treated with PNGase F or O- glycosidase and analyzed by immunoblot with TIM-3-cmAb and lectin far-western with Aleuria aurantia lectin (AAL; fucosylated epitopes). TGX stain-free total protein signal is shown as a loading/normalization control. (F) KASUMI-3 cells treated with vehicle (mock) or the fucosylation inhibitor 2F-peracetyl-fucose (SGN-2FF), followed by PNGase F or neuraminidase treatment and immunoblot/lectin probing with TIM-3-cmAb and AAL. See also Figure S3A . (G) Short-term killing assay of TIM-3.CAR-CIK cells against untreated or SGN-2FF-treated KASUMI-3 cells at various E:T ratios (5:1, 1:1, 0.5:1, 0.25:1 and 0.125:1, n = 8). (H) Affinity kinetics (left) and binding avidity at 1000 pN force (right) of TIM-3.CAR-CIK cells to untreated or defucosylated KASUMI-3 by LUMICKS analysis (n = 6). Immunoblot experiments (D-F) were repeated in three independent biological replicates with similar results. Data are presented as individual values and mean ± SD. Statistical significance was determined with repeated-measures two-way ANOVA with Bonferroni’s post hoc test (B, C) or using paired t test (G, H). ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. Illustrations were created with Biorender.com. See also Figure S3 for loading-matched TIM-3 immunoprecipitation controls.

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) TIM-3 expression on KASUMI-3 cells, primary AML blasts, and healthy immune subsets (CIK cells, monocytes, NK cells) assessed by flow cytometry using QuantiBRITE beads. REH (ALL cell line) served as negative control. (B) Short-term killing assay of TIM-3.CAR-CIK cells against CIK (n = 11) or KASUMI-3 (n = 8) cells compared with NT cells. Target cell lysis was evaluated by flow cytometry (E:T 5:1). (C) Short-term killing assay of TIM-3.CAR-CIK cells against monocytes (n = 8) or NK cells (n = 8) compared with NT (E:T 5:1). KASUMI-3 (n = 4) were included as positive control. (D) Immunoblot analysis of TIM-3 in lysates from monocytes, CIK cells, and KASUMI-3 cells following enzymatic treatment with PNGase F or broad neuraminidase, probed with a commercial anti–TIM-3 antibody (TIM-3-cmAb). GAPDH, loading control. Glycan symbols follow SNFG. (E) TIM-3 immunoprecipitates from monocytes, CIK cells, and KASUMI-3 cells treated with PNGase F or O- glycosidase and analyzed by immunoblot with TIM-3-cmAb and lectin far-western with Aleuria aurantia lectin (AAL; fucosylated epitopes). TGX stain-free total protein signal is shown as a loading/normalization control. (F) KASUMI-3 cells treated with vehicle (mock) or the fucosylation inhibitor 2F-peracetyl-fucose (SGN-2FF), followed by PNGase F or neuraminidase treatment and immunoblot/lectin probing with TIM-3-cmAb and AAL. See also Figure S3A . (G) Short-term killing assay of TIM-3.CAR-CIK cells against untreated or SGN-2FF-treated KASUMI-3 cells at various E:T ratios (5:1, 1:1, 0.5:1, 0.25:1 and 0.125:1, n = 8). (H) Affinity kinetics (left) and binding avidity at 1000 pN force (right) of TIM-3.CAR-CIK cells to untreated or defucosylated KASUMI-3 by LUMICKS analysis (n = 6). Immunoblot experiments (D-F) were repeated in three independent biological replicates with similar results. Data are presented as individual values and mean ± SD. Statistical significance was determined with repeated-measures two-way ANOVA with Bonferroni’s post hoc test (B, C) or using paired t test (G, H). ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. Illustrations were created with Biorender.com. See also Figure S3 for loading-matched TIM-3 immunoprecipitation controls.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Expressing, Flow Cytometry, Negative Control, Lysis, Positive Control, Western Blot, Control, Glycoproteomics, Staining, Binding Assay, Immunoprecipitation

(A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Western Blot, Recombinant, Derivative Assay, Control, Quantitative RT-PCR, Expressing, Glycoproteomics

(A) Schematic of the xenograft KASUMI-3 AML model. (B) Representative flow cytometry plots of hCD45 + CD33 + (up) and of hCD45 + TIM-3 + cells (down) in the BM of CTR or TIM-3.CAR treated mice at sacrifice. ( C-E ) Frequencies of hCD33 + and hTIM-3 + cells in the (C) BM, (D), spleen and (E) peripheral blood (PB) at sacrifice. Illustrations were created with Biorender.com. Results represent three independent experiments using TIM-3.CAR-CIK cells generated from 3 different donors. Data are presented as individual values and mean ± SD. Statistical significance was determined by unpaired t test. *p = 0.01, **p < 0.001 and ****p < 0.0001.

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Schematic of the xenograft KASUMI-3 AML model. (B) Representative flow cytometry plots of hCD45 + CD33 + (up) and of hCD45 + TIM-3 + cells (down) in the BM of CTR or TIM-3.CAR treated mice at sacrifice. ( C-E ) Frequencies of hCD33 + and hTIM-3 + cells in the (C) BM, (D), spleen and (E) peripheral blood (PB) at sacrifice. Illustrations were created with Biorender.com. Results represent three independent experiments using TIM-3.CAR-CIK cells generated from 3 different donors. Data are presented as individual values and mean ± SD. Statistical significance was determined by unpaired t test. *p = 0.01, **p < 0.001 and ****p < 0.0001.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Generated

(A) Schematic of IF-BETTER gate strategy showing dual antigen recognition of CD33 + /TIM-3 + target cell by CD33.CAR/TIM-3.CCR and TIM3.CAR/CD33.CCR-CIK cells. (B) Co-distribution of CD33 and TIM-3 expression (MFI) on bulk AML (top) and LSC-enriched CD34 + CD38 - population (bottom). Each dot represents a distinct patient (n=44 patients). (C) Schematics of next-generation Dual CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR constructs. CAR molecules are second-generation, carrying CD28 co-stimulatory domain, while CCR molecules present 4-1BB as co-stimulus. Both constructs were cloned into a pT4-transposon vector. See also Figure S4A, B . (D) Long-term killing assay (E:T 1:10) of all CAR-CIK cells against primary AML blasts (n=8 blasts) compared to NT cells. Blasts survival was determined by flow cytometry (n=13 donors). (E) Recovery of LSC-enriched CD34 + CD38 - population (n=8 patient samples) after 7 days co-culture with all CAR-CIK cells (E:T 1:10), compared to NT cells (n=13). Data are presented as individual values and the mean ± SD. Statistical significance was determined by one-way ANOVA test. ** p<0.01, **** p<0.0001. Illustrations were created with Biorender.com. See also Figure S4 for expression and phenotypic characterization of Dual CAR constructs, Figure S5 for Dual CAR-CIK cell activity against AML cell lines and Figure S6 for Dual CAR-CIK cell off-tumor toxicity against healthy immune and hematopoietic cells.

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Schematic of IF-BETTER gate strategy showing dual antigen recognition of CD33 + /TIM-3 + target cell by CD33.CAR/TIM-3.CCR and TIM3.CAR/CD33.CCR-CIK cells. (B) Co-distribution of CD33 and TIM-3 expression (MFI) on bulk AML (top) and LSC-enriched CD34 + CD38 - population (bottom). Each dot represents a distinct patient (n=44 patients). (C) Schematics of next-generation Dual CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR constructs. CAR molecules are second-generation, carrying CD28 co-stimulatory domain, while CCR molecules present 4-1BB as co-stimulus. Both constructs were cloned into a pT4-transposon vector. See also Figure S4A, B . (D) Long-term killing assay (E:T 1:10) of all CAR-CIK cells against primary AML blasts (n=8 blasts) compared to NT cells. Blasts survival was determined by flow cytometry (n=13 donors). (E) Recovery of LSC-enriched CD34 + CD38 - population (n=8 patient samples) after 7 days co-culture with all CAR-CIK cells (E:T 1:10), compared to NT cells (n=13). Data are presented as individual values and the mean ± SD. Statistical significance was determined by one-way ANOVA test. ** p<0.01, **** p<0.0001. Illustrations were created with Biorender.com. See also Figure S4 for expression and phenotypic characterization of Dual CAR constructs, Figure S5 for Dual CAR-CIK cell activity against AML cell lines and Figure S6 for Dual CAR-CIK cell off-tumor toxicity against healthy immune and hematopoietic cells.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Expressing, Construct, Clone Assay, Plasmid Preparation, Flow Cytometry, Co-Culture Assay, Activity Assay

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