The effect of fetal hemoglobin on the survival characteristics of sickle cells

The determinants of sickle red blood cell (RBC) life span have not been well-defined but may include both intrinsic factors (eg, the tendency to sickle) and extrinsic factors (eg, the capacity of the reticuloendothelial system to remove defective RBCs). Fetal hemoglobin (HbF) is heterogeneously distributed among sickle RBCs; F cells contain 20% to 25% HbF, whereas the remainder have no detectable HbF (non-F cells). Autologous sickle RBCs were labeled with biotin and reinfused to determine overall survival, non–F- and F-cell survival, and time-dependent changes in HbF content (%HbF) for the surviving F cells. A total of 10 patients were enrolled, including 2 who were studied before and after the percentage of F cells was increased by treatment with hydroxyurea. As expected, F cells survived longer in all subjects. Non–F-cell survival correlated inversely with the percentage of F cells, with the time for 30% cell survival ranging from 6 days in patients with more than 88% F cells to 16 days in patients with less than 16% F cells. As the biotin-labeled RBCs aged in the circulation, the HbF content of the surviving F-cell population increased by 0.28%/d ± 0.21%/d, indicating that within the F-cell population those with higher HbF content survived longer

Targeting and Cytotoxicity of SapC-DOPS Nanovesicles in Pancreatic Cancer

<div><p>Only a small number of promising drugs target pancreatic cancer, which is the fourth leading cause of cancer deaths with a 5-year survival of less than 5%. Our goal is to develop a new biotherapeutic agent in which a lysosomal protein (saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS) are assembled into nanovesicles (SapC-DOPS) for treating pancreatic cancer. A distinguishing feature of SapC-DOPS nanovesicles is their high affinity for phosphatidylserine (PS) rich microdomains, which are abnormally exposed on the membrane surface of human pancreatic tumor cells. To evaluate the role of external cell PS, <i>in vitro</i> assays were used to correlate PS exposure and the cytotoxic effect of SapC-DOPS in human tumor and nontumorigenic pancreatic cells. Next, pancreatic tumor xenografts (orthotopic and subcutaneous models) were used for tumor targeting and therapeutic efficacy studies with systemic SapC-DOPS treatment. We observed that the nanovesicles selectively killed human pancreatic cancer cells <i>in vitro</i> by inducing apoptotic death, whereas untransformed cells remained unaffected. This <i>in vitro</i> cytotoxic effect correlated to the surface exposure level of PS on the tumor cells. Using xenografts, animals treated with SapC-DOPS showed clear survival benefits and their tumors shrank or disappeared. Furthermore, using a double-tracking method in live mice, we showed that the nanovesicles were specifically targeted to orthotopically-implanted, bioluminescent pancreatic tumors. These data suggest that the acidic phospholipid PS is a biomarker for pancreatic cancer that can be effectively targeted for therapy utilizing cancer-selective SapC-DOPS nanovesicles. This study provides convincing evidence in support of developing a new therapeutic approach to pancreatic cancer.</p></div

Effect of SapC-DOPS nanovesicles on pancreatic tumor growth.

<p>Subcutaneous tumors (MiaPaCa-2 cell line) treated with SapC-DOPS nanovesicles had significantly reduced tumor end-volume (A) and end-weight (B) compared to controls.</p

Localization of fluorescently labeled SapC-DOPS in normal mouse tissue.

<p>The organ distribution of fluorescently labeled SapC-DOPS 1, 12 and 24 hours after injection is shown in (A), (B), and (C) respectively. The liver and spleen were the only organs that had detectable fluorescent signal 1 and 12 hours post-injection for all animals. This signal was no longer present by 24 hours post-injection. The lung, heart, and kidney showed no detectable fluorescent signal. SapC = 3.2 mg/kg, DOPS = 1.8 mg/kg, CVM = 6 µM.</p

Survival of SapC-DOPS-treated mice with orthotopically implanted pancreatic tumors.

<p>(A) Survival of SapC-DOPS-treated mice was significantly greater than controls. Insert panel: bioluminescence confirms hidden implanted pancreatic tumor on live imaging (mouse on right). (B) Tumor weight at time of death is shown. No tumor was noted in the four surviving mice at post-treatment day 110, as documented by the absence of bioluminescence in those mice (insert panel).</p

Targeting of subcutaneous tumor xenografts by fluorescently labeled SapC-DOPS nanovesicles.

<p>Heterotopic MiaPaCa-2 tumors (circled) were generated by subcutaneous injection in the upper flank of nude mice. (A) Mice 1 & 2 were tumor-bearing mice, injected with fluorescently labeled SapC-DOPS nanovesicles; Mouse 3 was non-tumor-bearing, PBS injected. Mice 1 and 2 both demonstrate localization of the fluorescent label to the tumor site. Fluorescence also localizes to the liver by 2 hours, but is gone by 24 hours. (B) Tumor-bearing mice 4, 5, and 6 were injected with non-complexed SapC and fluorescently labeled DOPS, fluorescently labeled DOPS only, and PBS, respectively. There is no fluorescence localized to tumor in any of these mice. Like the SapC-DOPS (in mice 1 and 2), non-complexed SapC and fluorescently labeled DOPS and fluorescently labeled DOPS alone (in mice 4 and 5, respectively) did localize to the liver, but quickly dissipated (C) Subcutaneous tumors created using cfPac1-Luc3 pancreatic tumor cells that were and were not pretreated with PS-specific binding proteins (Lactadherin-C2 [left upper panel] and Beta-GP-1 [left lower panel]) display bioluminescence on live imaging. After administration of CVM fluorescently labeled SapC-DOPS nanovesicles, the tumors that were not pretreated demonstrated fluorescence, while the tumors that had been pretreated did not demonstrate any fluorescence (right upper and lower panels). (D) Presence of bioluminescence (upper panel) confirms presence of orthotopic pancreatic tumor, and co-localized fluorescence (bottom panel) confirms targeting by fluorescently labeled SapC-DOPS nanovesicles.</p

Microscopic inspection of cells with and without SapC-DOPS treatment.

<p>The tumor cells (PANC-1, Capan-1 and MiaPaCa-2 in (B), (C), and (D), respectively) had morphological features consistent with apoptotic death after treatment with SapC-DOPS, while the untransformed HPDE cells (A) appeared normal.</p

Dose-dependent inhibition of pancreatic tumor growth by SapC-DOPS in xenograft model.

<p>PANC-1 xenograft tumors in nude mice were treated every other day with 1, 4, or 8 mg/kg of SapC-DOPS, or with PBS control. Tumor sizes at high doses (4 mg/kg and 8 mg/kg) were significantly smaller than control and 1 mg/kg groups (p = 0.003* and 0.00015**, respectively).</p

Targeting of fluorescently-labeled SapC-DOPS nanovesicles to pancreatic tumor cells <i>in vivo</i>.

<p>The nanovesicles were rapidly targeted to the palpable tumor within 5 minutes (data not shown). The fluorescence persisted for over 1 (A) and 4 (B) days. SapC = 3.2 mg/kg, DOPS = 1.8 mg/kg, CVM = 6 µM.</p

Hypothetical diagram of cancer-selective targeting and apoptosis induction by SapC-DOPS nanovesicles.

<p>Hypothetical diagram of cancer-selective targeting and apoptosis induction by SapC-DOPS nanovesicles.</p