Acute lymphoid leukemia cells with greater stem cell antigen-1 (Ly6a/Sca-1) expression exhibit higher levels of metalloproteinase activity and are more aggressive in vivo.

Stem cell antigen-1 (Ly6a/Sca-1) is a gene that is expressed in activated lymphocytes, hematopoietic stem cells and stem cells of a variety of tissues in mice. Despite decades of study its functions remain poorly defined. These studies explored the impact of expression of this stem cell associated gene in acute lymphoid leukemia. Higher levels of Ly6a/Sca-1 expression led to more aggressive leukemia growth in vivo and earlier death of hosts. Leukemias expressing higher levels of Ly6a/Sca-1 exhibited higher levels of matrix metalloproteinases. The results suggest the hypothesis that the more aggressive behavior of Ly6a/Sca-1 expressing leukemias is due at least in part to greater capacity to degrade microenvironmental stroma and invade tissues

Higher levels of Ly6a/Sca-1 expression can be induced by interferon-gamma in Ly6a/Sca-1-knockdown leukemias and in low Ly6a/Sca-1 clones.

<p>Cells were treated with interferon-gamma 10 ng/ml for 24 hours. Controls were cultured 24 hours without interferon-gamma. Ly6a/Sca-1 was measured by flow cytometry. Filled histograms are controls without interferon while the unshaded histograms are the leukemias treated with interferon-gamma. (A) ASLN-lentiviral-Ly6a/Sca-1 knockdown. (B) NSTY-lentiviral-Ly6a/Sca-1 knockdown. (C) Low Ly6a/Sca-1 NSTY clone 8.</p

Leukemia cells with higher levels of Ly6a/Sca-1 expression exhibit greater invasiveness in Matrigel assays.

<p>In each assay the invasion of leukemia cells in the experimental group with higher levels of Ly6a/Sca-1 is expressed as percent relative to number of invading cells in the control group with low levels of Ly6a/Sca-1 expression. The control is represented as 100%. Averages and sem are displayed. (A) “C1498-Ly6a/Sca+” is C1498-Ly6a/Sca-1 compared to C1498-vector only. P0.0151 by t-test. N3 per group. (B) “NSTY” is the NSTY leukemia carrying the lentivirus control vector. “NSTY-Ly6a/Sca KD” is NSTY transduced with the anti-Ly6a/Sca-1 shRNA vector. “NSTY clone 8 Low Ly6a/Sca” is the NSTY leukemia clone 8 isolated by limiting dilution that has low levels of Ly6a/Sca-1 while “NSTY clone 5 High Ly6a/Sca” “ is clone 5 which has high levels of Ly6a/Sca-1. One way ANOVA was performed and generated a p<0.0001 for comparison of all groups. Planned t-tests between clone 8 and clone 5 yielded p<0.001 and between NSTY and the knockdown yielded p<0.001. N = 3 per group. (C) “ASLN” is the ASLN line carrying the vector only, while “ASLN-Ly6a/Sca KD” is ASLN transduced with the anti-Ly6a/Sca-1 shRNA vector. P0.0179 by t-test. N = 3. (D) Effect of the metalloproteinase inhibitor, doxycycline, on invasiveness in vitro. Matrigel invasion assays using C1498-Ly6a/Sca+ and control C1498-vector only cells were performed in the presence or absence of doxycycline 10 µg/ml in the medium. N4 per group. Doxycycline significantly reduced invasiveness in the C1498-Ly6a/Sca leukemia (p0.0018 by t-test). Doxycycline did not significantly reduce invasiveness in the control C1498-vector only leukemia (p0.199 by t-test, n = 4).</p

Leukemia cells expressing higher levels of Ly6a/Sca-1 grow more aggressively in vivo and lead to earlier death.

<p>Panels A – D depict experiments with NSTY, while panels E – I represent C1498. <u>NSTY</u>. (A) Flow cytometry for Ly6a/Sca-1 expression in clones 5 and 8 at the time of challenge. (B) In vitro growth of NSTY leukemia clones measured in MTT assays. Wells were plated in triplicate. Error bars represent standard deviation. P0.72 by t test. (C) NSTY-clone 5 which expresses high levels of Ly6a/Sca-1 grows more aggressively in vivo in syngeneic mice. Survival curves are presented. N = 10 per group. Animals challenged with NSTY clone 5 with high levels of L6a/Sca-1 expression had significantly shorter survival (p<0.001 by logrank test). (D) Ly6a/Sca-1 expression on NSTY clonal leukemias reisolated from mice in vivo. At time of euthanasia marrow samples were collected from mice and analyzed by flow cytometry for Ly6a/Sca-1 expression. Cells were gated on the leukemia blast population as defined by forward scatter and GFP expression. The filled histogram is the NSTY clone 8 low Ly6a/Sca-1 leukemia while the unfilled histogram is the clone 5 high Ly6a/Sca-1 leukemia. Representative examples are presented. <u>C1498</u>. (E) Flow cytometry compares surface Ly6a/Sca-1 expression on C1498-Ly6a/Sca-1 to that on control C1498-vector only cells. (F) In vitro growth of C1498-Ly6a/Sca-1 and C1498-vector only control cells as assessed by MTT assays. Wells were plated in triplicate. Error bars represent standard deviation. P0.15 by t test. (G) C1498-Ly6a/Sca-1 grows more aggressively in syngeneic mice. Syngeneic C57BL/6 mice were intravenously injected with 1×10⁵ leukemia cells and survival monitored. N = 5 per group. Mice challenged with C1498-Ly6a/Sca-1 exhibited significantly shorter survival (p0.0026 by logrank test). (H) C1498-Ly6a/Sca-1 grows more aggressively in allogeneic bone marrow transplant recipients. C57BL/6 mice underwent allogeneic bone marrow transplant using C3.SW donors. 1×10⁶ C1498 leukemia cells were mixed with the allogeneic hematopoietic cells and both were infused iv together. N = 10 per group. Mice receiving C1498-Ly6a/Sca-1 leukemia exhibited significantly shorter survival (p<0.001 by logrank test). (I) Ly6a/Sca-1 expression on C1498 leukemias reisolated from mice in vivo. At time of euthanasia marrow samples were collected from mice and analyzed by flow cytometry for Ly6a/Sca-1 expression. Cells were gated on the leukemia blast population as defined by forward scatter and GFP expression. The filled histogram is the C1498-vector only leukemia while the unfilled histogram is the C1498-Ly6a/Sca-1 leukemia. Representative examples are presented.</p

In vivo bioluminescent imaging demonstrates that C1498-Ly6a/Sca-1 disseminates more rapidly and creates greater total leukemia burden than the C1498-vector only control.

<p>Normal mice were intravenously injected with leukemia cells (5×10⁵) that had been transduced with a luciferase gene. Animals were imaged on days 10, 15 and 23 after injection. 5 animals in each group. Bioluminescent images at day 15 of (A) C1498-vector only, and (B) C1498-Ly6A/Sca challenged mice. (C) Number of discrete anatomic sites exhibiting luminescence. Animals were imaged and two independent investigators identified the number of discrete sites of luminescence in each animal. Symbols are the average number of sites and bars are standard errors of the mean. P values were calculated with a Wilcoxon test. (D) Total body leukemia burden measured by in vivo bioluminescence. Animals were imaged on days 10, 15 and 23 after intravenous injection with leukemia cells. Total luminescence for entire body was measured in photons/sec. Symbols represent the mean and bars the standard error of the mean. P values were calculated with a Wilcoxon test.</p

Comparison of in/Sca-1 knockdown leukemias.

<p>Mice were injected iv with 1×10⁶ leukemia cells and monitored daily for survival. “Control” were leukemia lines transduced with a control lentiviral vector containing only a puromycin resistance gene, while the “Sca-knockdown” had been transduced with a lentivirus containing an anti-Ly6a/Sca-1 sequence as well as the puromycin resistance gene. (A) ASLN leukemia. N5 per group. Control group had median survival of 13 days, while Sca-1 knockdown had median survival of 14 days. P0.14 by logrank test. (B) NSTY leukemia. N10 per group. Control group had median survival of 7 days while the Sca-knockdown had median survival of 9 days. P<0.0001 by logrank test.</p

Greater metalloproteinase activity is observed in leukemia cells with greater expression of Ly6a/Sca-1.

<p>In vitro proteinase assays were performed in triplicate on cell lysates and expressed as relative fluorescence units per minute per microgram of protein. Filled bars represent leukemia lines with greater Ly6a/Sca-1 expression while white bars are those with low Ly6a/Sca-1 expression. “C1498-VO” is the C1498-vector only control, while “C1498-Sca” is C1498-Ly6a/Sca-1. “NSTY#8” is the NSTY leukemia clone 8 isolated by limiting dilution that has low levels of Ly6a/Sca-1 while “NSTY#5” is clone 5 which has high levels of Ly6a/Sca-1. “NSTY” is the NSTY line transduced with the lentivirus control vector while “NSTY-KD” is NSTY transduced with the anti-Ly6a/Sca-1 shRNA vector. For the ASLN leukemia “ASLN” and “ASLN-KD” have similar meanings. Bars are average and the error bars are standard variation. N = 3 per sample. P values for each comparison were calculated with t-tests.</p

Higher levels of MMP-9 in Ly6a/Sca-1 expressing leukemias.

<p>Western blot for MMP-9. For the C1498 leukemia “VO Sca-” is the C1498-vector only control, while “Ly6a vector Sca+” is C1498-Ly6a/Sca-1. For the NSTY leukemia “cl#8 Sca Low” is clone 8 that has low levels of Ly6a/Sca-1, while “cl#5 Sca High” is clone 5 which has high levels of Ly6a/Sca-1. “Ctrl Sca High” is NSTY leukemia that was treated with a control shRNA vector, while “KD Sca Low” is NSTY after knockdown with an anti-Ly6A/Sca-1shRNA lentivirus vector. For ASLN “Ctrl Sca High” is ASLN leukemia that was treated with a control shRNA vector, while “KD Sca Low” is ASLN after knockdown with an anti-Ly6A/Sca-1shRNA lentivirus vector. ß-actin was used a control for assessing lane loading.</p