Figure 1. Introduction of FLuc-transduced leukemia cell lines in vivo silences the FLuc transgene. (A) Pseudocolor representations of AML syngeneic cell lines transduced with lentivirus containing FLuc after addition of D-Luciferin (200 µg/mL) to cells in culture (Rad (radiance) = photons/s/cm2/sr). (B) Pseudocolor representations of intravital luminescence imaging of mice (n = 3) challenged intraperitoneally (i.p.) with C1498-FLuc cells after i.p. injection of D-Luciferin (150 mg/kg). Days 5 and 21 post-challenge are shown. (C) Kaplan–Meier survival curve of mice challenged intravenously (i.v.) via tail vein with parental C1498 or C1498-FLuc cells. Within each group mice were challenged with one of three different cell numbers: 5 × 104, 2.5 × 105, or 1 × 106 cells/mouse.

Figure 1. Introduction of FLuc-transduced leukemia cell lines in vivo silences the FLuc transgene. (A) Pseudocolor representations of AML syngeneic cell lines transduced with lentivirus containing FLuc after addition of D-Luciferin (200 µg/mL) to cells in culture (Rad (radiance) = photons/s/cm2/sr). (B) Pseudocolor representations of intravital luminescence imaging of mice (n = 3) challenged intraperitoneally (i.p.) with C1498-FLuc cells after i.p. injection of D-Luciferin (150 mg/kg). Days 5 and 21 post-challenge are shown. (C) Kaplan–Meier survival curve of mice challenged intravenously (i.v.) via tail vein with parental C1498 or C1498-FLuc cells. Within each group mice were challenged with one of three different cell numbers: 5 × 104, 2.5 × 105, or 1 × 106 cells/mouse.

Figure 2. 5-Aza treatment rescues bioluminescence ex vivo and in vivo in AML-FLuc cell lines. (A) C57Bl/6 mice were implanted i.v. with C1498-FLuc or FBL3-FLuc cell lines and AML was allowed to develop (C1498-FLuc: 8 × 104 cells for 18 days, FBL3-FLuc: 5 × 104 cells for 6 days). Blood was taken from challenged mice and grown in culture after RBC lysis until primary cells were eliminated. Cells were then incubated in increasing concentrations of 5-Aza for 24 and 48 h. D-luciferin was added at a concentration of 150µg/mL and luminescence was read. Bar graphs represent signal intensity as densitometry, n = 3 biological replicates/group. 2-way ANOVA followed by Tukey’s test for multiple comparisons, all statistics shown compared to group “0”, * p < 0.05, **** p < 0.0001. (B) Ex vivo cell viability was assayed by trypan-blue exclusion after incubation in increasing concentrations of 5-Aza for 24 and 48 h. 2-way ANOVA followed by Tukey’s test for multiple comparisons, all statistics shown compared to group “0”, **** p < 0.0001. (C) C57Bl/6 mice were challenged i.p. with 6 × 105 C1498-FLuc cells. After 12 days, three consecutive treatments of Aza (5 µg/kg) were given i.p. and intravital luminescence imaging was performed 24 h after the final treatment. Pseudocolor images are shown of representative mice, n = 4 mice per group. p = 0.0172, unpaired t-test with Welch’s correction.

Figure 2. 5-Aza treatment rescues bioluminescence ex vivo and in vivo in AML-FLuc cell lines. (A) C57Bl/6 mice were implanted i.v. with C1498-FLuc or FBL3-FLuc cell lines and AML was allowed to develop (C1498-FLuc: 8 × 104 cells for 18 days, FBL3-FLuc: 5 × 104 cells for 6 days). Blood was taken from challenged mice and grown in culture after RBC lysis until primary cells were eliminated. Cells were then incubated in increasing concentrations of 5-Aza for 24 and 48 h. D-luciferin was added at a concentration of 150µg/mL and luminescence was read. Bar graphs represent signal intensity as densitometry, n = 3 biological replicates/group. 2-way ANOVA followed by Tukey’s test for multiple comparisons, all statistics shown compared to group “0”, * p < 0.05, **** p < 0.0001. (B) Ex vivo cell viability was assayed by trypan-blue exclusion after incubation in increasing concentrations of 5-Aza for 24 and 48 h. 2-way ANOVA followed by Tukey’s test for multiple comparisons, all statistics shown compared to group “0”, **** p < 0.0001. (C) C57Bl/6 mice were challenged i.p. with 6 × 105 C1498-FLuc cells. After 12 days, three consecutive treatments of Aza (5 µg/kg) were given i.p. and intravital luminescence imaging was performed 24 h after the final treatment. Pseudocolor images are shown of representative mice, n = 4 mice per group. p = 0.0172, unpaired t-test with Welch’s correction.

Figure 3. 5-Aza treatment rescues expression of genes related to antigen presentation. (A) C1498-Fluc, (B) FBL3-FLuc, (C) HL-60, and (D) Jurkat cells were treated for 48 h in culture with vehicle (DMSO) and 1 μM 5-Aza (A,B) or 5 μM 5-Aza (C,D). RNA was isolated and amplified by qPCR with primers for H2d, β2m, Tap1, and Lmp2 (A,B) or HLA-A, HLA-C, huβ2m, huTap1, huLmp2 (C,D). n = 3 technical replicates per group. ** p < 0.01, *** p < 0.001, **** p < 0.0001, 2-way ANOVA followed by Sidak’s test for multiple comparisons. (E) C1498-Fluc and (F) FBL3-Fluc cells were treated in culture with vehicle (DMSO) or 5 μM 5-Aza for 24 h before washing and co-culture without splenocytes (-sc) or with splenocytes harvested from mice treated with a single dose of vehicle (+ sc DMSO) or 5mg/kg Aza (+sc 5-Aza) for 24 h. Viability of the culture was measured using MTT and absorbance was read at 590 nm. Viability relative to culture without splenocytes is shown (with viability from splenocytes cultured alone subtracted). * p < 0.05, ** p < 0.01, unpaired t-test.

Figure 3. 5-Aza treatment rescues expression of genes related to antigen presentation. (A) C1498-Fluc, (B) FBL3-FLuc, (C) HL-60, and (D) Jurkat cells were treated for 48 h in culture with vehicle (DMSO) and 1 μM 5-Aza (A,B) or 5 μM 5-Aza (C,D). RNA was isolated and amplified by qPCR with primers for H2d, β2m, Tap1, and Lmp2 (A,B) or HLA-A, HLA-C, huβ2m, huTap1, huLmp2 (C,D). n = 3 technical replicates per group. ** p < 0.01, *** p < 0.001, **** p < 0.0001, 2-way ANOVA followed by Sidak’s test for multiple comparisons. (E) C1498-Fluc and (F) FBL3-Fluc cells were treated in culture with vehicle (DMSO) or 5 μM 5-Aza for 24 h before washing and co-culture without splenocytes (-sc) or with splenocytes harvested from mice treated with a single dose of vehicle (+ sc DMSO) or 5mg/kg Aza (+sc 5-Aza) for 24 h. Viability of the culture was measured using MTT and absorbance was read at 590 nm. Viability relative to culture without splenocytes is shown (with viability from splenocytes cultured alone subtracted). * p < 0.05, ** p < 0.01, unpaired t-test.

Figure 4. 5-Aza decreases leukemic burden and extends survival in immune-competent mice. (A) C57Bl/6 mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and treated with vehicle (0mg/kg, n = 3), 1mg/kg 5-Aza (n = 5 mice), or 5 mg/kg 5-Aza (n = 5 mice) for three consecutive days beginning on day 4 post challenge. Percent survival by days post challenge is shown as a Kaplan–Meier curve. ** p < 0.01, Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. (B) Retro-orbital blood collection on mice from (A) was performed on Day 17 post-challenge. Blood was smeared on a microscope slide and Wright’s stain was used to differentiate red blood cells (R) from white blood cells including engrafted C1498-Fluc (C) and lymphocytes (L). Representative images of C1498-Fluc from in vitro culture and in the blood of engrafted mice are shown. All mice from 0 mg/kg and 5 mg/kg 5-Aza treatment groups were confirmed to have C1498 present in blood smears (table). (C) C1498-FLuc cells in vitro were treated with vehicle (DMSO) or 5-Aza (5 μM) for 48 h and stained with antibody recognizing Annexin V (AV) and propidium iodide (PI). Percentage out of total cells of viable (AV−/PI−), early apoptotic (AV+/PI−), and late apoptotic (AV+/PI+) cells are plotted as a bar graph. Necrotic cells (AV−/PI+) cells were negligible in all samples. n = 3 wells per group. **** p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test. (D) C1498-FLuc cells were implanted in C57Bl/6 mice as in (A) and treated with vehicle (DMSO) or 5-Aza (5 mg/kg). Forty-eight hours after the third treatment, blood, bone marrow, spleen, liver, and lung from mice were reduced to single cell suspension and stained intracellularly with antibody recognizing luciferase followed by anti-rabbit IgG fluorescent secondary antibody. Percentages of luciferase positive cells out of total cells were analyzed by flow cytometry and are presented as a bar graph. * p < 0.05, ** p < 0.01 unpaired t-test with Welch’s correction.

Figure 4. 5-Aza decreases leukemic burden and extends survival in immune-competent mice. (A) C57Bl/6 mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and treated with vehicle (0mg/kg, n = 3), 1mg/kg 5-Aza (n = 5 mice), or 5 mg/kg 5-Aza (n = 5 mice) for three consecutive days beginning on day 4 post challenge. Percent survival by days post challenge is shown as a Kaplan–Meier curve. ** p < 0.01, Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. (B) Retro-orbital blood collection on mice from (A) was performed on Day 17 post-challenge. Blood was smeared on a microscope slide and Wright’s stain was used to differentiate red blood cells (R) from white blood cells including engrafted C1498-Fluc (C) and lymphocytes (L). Representative images of C1498-Fluc from in vitro culture and in the blood of engrafted mice are shown. All mice from 0 mg/kg and 5 mg/kg 5-Aza treatment groups were confirmed to have C1498 present in blood smears (table). (C) C1498-FLuc cells in vitro were treated with vehicle (DMSO) or 5-Aza (5 μM) for 48 h and stained with antibody recognizing Annexin V (AV) and propidium iodide (PI). Percentage out of total cells of viable (AV−/PI−), early apoptotic (AV+/PI−), and late apoptotic (AV+/PI+) cells are plotted as a bar graph. Necrotic cells (AV−/PI+) cells were negligible in all samples. n = 3 wells per group. **** p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test. (D) C1498-FLuc cells were implanted in C57Bl/6 mice as in (A) and treated with vehicle (DMSO) or 5-Aza (5 mg/kg). Forty-eight hours after the third treatment, blood, bone marrow, spleen, liver, and lung from mice were reduced to single cell suspension and stained intracellularly with antibody recognizing luciferase followed by anti-rabbit IgG fluorescent secondary antibody. Percentages of luciferase positive cells out of total cells were analyzed by flow cytometry and are presented as a bar graph. * p < 0.05, ** p < 0.01 unpaired t-test with Welch’s correction.

Figure 5. 5-Aza extends survival in half of immuno-deficient mice. (A) NSG mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and treated with vehicle (0 mg/kg) (n = 3 mice) or 5 mg/kg 5-Aza (n = 4) for three consecutive days beginning on day 4 post-challenge. Percent survival by days post-challenge is shown as a Kaplan–Meier curve. (B) Comparison of survival between vehicle (0mg/kg) treated C57Bl/6 and NSG mice challenged with C1498-Fluc. (C) Comparison of survival between 5-Aza-treated (5 mg/kg) C57Bl/6 and NSG mice challenged with C1498-Fluc. * p < 0.05, Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test.

Figure 5. 5-Aza extends survival in half of immuno-deficient mice. (A) NSG mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and treated with vehicle (0 mg/kg) (n = 3 mice) or 5 mg/kg 5-Aza (n = 4) for three consecutive days beginning on day 4 post-challenge. Percent survival by days post-challenge is shown as a Kaplan–Meier curve. (B) Comparison of survival between vehicle (0mg/kg) treated C57Bl/6 and NSG mice challenged with C1498-Fluc. (C) Comparison of survival between 5-Aza-treated (5 mg/kg) C57Bl/6 and NSG mice challenged with C1498-Fluc. * p < 0.05, Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test.

Figure 6. 5-Aza treatment alters the immune microenvironment. (A) Two million C1498-Fluc cells were treated with 10 µM 5-Aza for 48 h in culture and stained with antibodies recognizing PD-1, PD-L1, and CTLA-4. Percentages of positive cells analyzed by flow cytometry are shown for vehicle (DMSO) and 5-Aza treated cells. (B) C57Bl/6 mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and AML was allowed to develop. On day 14 post-challenge, mice were given three consecutive, daily treatments of vehicle (DMSO) (n = 4 mice) or 5 mg/kg 5-Aza (5-Aza) (n = 5 mice). Forty-eight hours after the third treatment, blood, spleen, lung, and bone marrow was processed for flow cytometry. Cells were surface stained with antibodies recognizing PD-1 and PD-L1 as well as stained intracellularly for CTLA-4 and Luciferase. Percentages of PD-1, PD-L1 or CTAl-4 positive cells gated out of Luciferase positive cells analyzed by flow cytometry are shown. (C–F) Mice were treated as in (B) and processed blood samples were stained with antibodies recognizing CD4, CD8, PD-1, PD-L1, and CTLA-4 (C,D). One hundred thousand cells (±1%) were analyzed by flow cytometry and percentages of positive cells are shown out of total WBC or gated CD8+ or CD4+ positive cells. (E,F) Processed blood cells were stained with antibodies recognizing Ly6C, Ly6G, CD11c, MHCII, Mac3 (intracellular), PD-1, PD-L1, and CTLA-4 (intracellular). Percentages of positive cells analyzed by flow cytometry are shown out of total WBC or gated myeloid populations. For all panels (A–F) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired t-test with Welch’s correction.

Figure 6. 5-Aza treatment alters the immune microenvironment. (A) Two million C1498-Fluc cells were treated with 10 µM 5-Aza for 48 h in culture and stained with antibodies recognizing PD-1, PD-L1, and CTLA-4. Percentages of positive cells analyzed by flow cytometry are shown for vehicle (DMSO) and 5-Aza treated cells. (B) C57Bl/6 mice were challenged with 8 × 105 C1498-FLuc cells via tail vein and AML was allowed to develop. On day 14 post-challenge, mice were given three consecutive, daily treatments of vehicle (DMSO) (n = 4 mice) or 5 mg/kg 5-Aza (5-Aza) (n = 5 mice). Forty-eight hours after the third treatment, blood, spleen, lung, and bone marrow was processed for flow cytometry. Cells were surface stained with antibodies recognizing PD-1 and PD-L1 as well as stained intracellularly for CTLA-4 and Luciferase. Percentages of PD-1, PD-L1 or CTAl-4 positive cells gated out of Luciferase positive cells analyzed by flow cytometry are shown. (C–F) Mice were treated as in (B) and processed blood samples were stained with antibodies recognizing CD4, CD8, PD-1, PD-L1, and CTLA-4 (C,D). One hundred thousand cells (±1%) were analyzed by flow cytometry and percentages of positive cells are shown out of total WBC or gated CD8+ or CD4+ positive cells. (E,F) Processed blood cells were stained with antibodies recognizing Ly6C, Ly6G, CD11c, MHCII, Mac3 (intracellular), PD-1, PD-L1, and CTLA-4 (intracellular). Percentages of positive cells analyzed by flow cytometry are shown out of total WBC or gated myeloid populations. For all panels (A–F) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired t-test with Welch’s correction.