Exogenous sickle erythrocytes combined with vascular disruption trigger disseminated tumor vaso-occlusion and lung tumor regression.

Hypoxic tumor niches are chief causes of treatment resistance and tumor recurrence. Sickle erythrocytes' (SSRBCs') intrinsic oxygen-sensing functionality empowers them to access such hypoxic niches wherein they form microaggregates that induce focal vessel closure. In search of measures to augment the scale of SSRBC-mediated tumor vaso-occlusion, we turned to the vascular disrupting agent, combretastatin A-4 (CA-4). CA-4 induces selective tumor endothelial injury, blood stasis, and hypoxia but fails to eliminate peripheral tumor foci. In this article, we show that introducing deoxygenated SSRBCs into tumor microvessels treated with CA-4 and sublethal radiation (SR) produces a massive surge of tumor vaso-occlusion and broadly propagated tumor infarctions that engulfs treatment-resistant hypoxic niches and eradicates established lung tumors. Tumor regression was histologically corroborated by significant treatment effect. Treated tumors displayed disseminated microvessels occluded by tightly packed SSRBCs along with widely distributed pimidazole-positive hypoxic tumor cells. Humanized HbS-knockin mice (SSKI) but not HbA-knockin mice (AAKI) showed a similar treatment response underscoring SSRBCs as the paramount tumoricidal effectors. Thus, CA-4-SR-remodeled tumor vessels license SSRBCs to produce an unprecedented surge of tumor vaso-occlusion and infarction that envelops treatment-resistant tumor niches resulting in complete tumor regression. Strategically deployed, these innovative tools constitute a major conceptual advance with compelling translational potential.

Sickle cells produce functional immune modulators and cytotoxics.

Sickle erythrocytes' (SSRBCs) unique physical adaptation to hypoxic conditions renders them able to home to hypoxic tumor niches in vivo, shut down tumor blood flow and induce tumoricidal responses. SSRBCs are also useful vehicles for transport of encapsulated drugs and oncolytic virus into hypoxic tumors with enhanced anti-tumor effects. In search of additional modes for arming sickle cells with cytotoxics, we turned to a lentiviral ß-globin vector with optimized Locus Control Region/ß-globin coding region/promoter/enhancers. We partially replaced the ß-globin coding region of this vector with genes encoding T cell cytolytics, perforin and granzyme or immune modulating superantigens SEG and SEI. These modified vectors efficiently transduced Sca+ ckit- Lin- hematopoietic stem cells (HSCs) from humanized sickle cell knockin mice. Irradiated mice reconstituted with these HSCs displayed robust expression of transgenic RNAs and proteins in host sickle cells that was sustained for more than 10 months. SSRBCs from reconstituted mice harboring SEG/SEI transgenes induced robust proliferation and a prototypical superantigen-induced cytokine reaction when exposed to human CD4+/CD8+ cells. The ß-globin lentiviral vector therefore produces a high level of functional, erythroid-specific immune modulators and cytotoxics that circulate without toxicity. Coupled with their unique ability to target and occlude hypoxic tumor vessels these armed SSRBCs constitute a potentially useful tool for treatment of solid tumors.

Red blood cell storage duration and trauma.

Numerous retrospective clinical studies suggest that transfusion of longer stored red blood cells (RBCs) is associated with an independent risk of poorer outcomes for certain groups of patients, including trauma, intensive care, and cardiac surgery patients. Large multicenter randomized controlled trials are currently underway to address the concern about RBC storage duration. However, none of these randomized controlled trials focus specifically on trauma patients with hemorrhage. Major trauma, particularly due to road accidents, is the leading cause of critical injury in the younger-than-40-year-old age group. Severe bleeding associated with major trauma induces hemodynamic dysregulation that increases the risk of hypoxia, coagulopathy, and potentially multiorgan failure, which can be fatal. In major trauma, a multitude of stress-associated changes occur to the patient's RBCs, including morphological changes that increase cell rigidity and thereby alter blood flow hemodynamics, particularly in the microvascular vessels, and reduce RBC survival. Initial inflammatory responses induce deleterious cellular interactions, including endothelial activation, RBC adhesion, and erythrophagocytosis that are quickly followed by profound immunosuppressive responses. Stored RBCs exhibit similar biophysical characteristics to those of trauma-stressed RBCs. Whether transfusion of RBCs that exhibit storage lesion changes exacerbates the hemodynamic perturbations already active in the trauma patient is not known. This article reviews findings from several recent nonrandomized studies examining RBC storage duration and clinical outcomes in trauma patients. The rationale for further research on RBC storage duration in the trauma setting is provided.

Sickle erythrocytes target cytotoxics to hypoxic tumor microvessels and potentiate a tumoricidal response.

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H(2)O(2) and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

Mechanisms of stroke in sickle cell disease: sickle erythrocytes decrease cerebral blood flow in rats after nitric oxide synthase inhibition.

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.

Anaplastic carcinomas in nude mice and in original donor strain rats inoculated with cultured oval cells.

Male Sprague-Dawley rats were fed for 4-5 weeks a choline-devoid diet containing 0.1% DL-ethionine. Preparations of nonparenchymal epithelial cells, enriched in oval cells, were isolated from the livers of these animals and were placed in culture. Six lines of hepatic epithelial cells were thus established. The lines underwent transformation after several passages, became tumorigenic in nude mice and 3 lines also in rats of the same strain of origin of the isolated cells. The tumors were uniformly highly anaplastic carcinomas. Preliminary morphologic, cytologic, and cytochemical results were consistent with the tumoral cells being hepatocytelike cells. These findings are viewed and discussed in terms of the cellular source, in vivo, of longterm cultures of rat liver epithelial cells, and in relation to a possible role of hepatic nonparenchymal epithelial cells in the process of hepatocellular tumor induction by chemical carcinogens.

Study of irreversibly sickled cells in an animal model.

Erythrocytes (RBCs) from six patients with sickle cell anemia were transfused to laboratory rats in order to study the intravascular survival of irreversibly sickled cells (ISCs). Fifteen minutes after transfusion, a mean of 48.8 percent (range 23-95 percent) of the ICSs injected were present in the rats' blood, a value that was significantly lower than that for the total population of sickle cell anemia erythrocytes transfused (mean 82.4 percent, range 36-114 percent). The intravascular half-life of ISCs was also lower (mean 0.83 hours ± 0.18 SD) than that observed for the total sickle cell anemia erythrocytes (mean 1.62 hours ± 0.19 SD) during the initial two hours of the transfusion experiments. The irreversibly sickled cells that remained in the rats' blood thereafter survived as well as those cells that were not irreversibly sickled. Severe hypoxia in the recipient animals did not appear to selectively remove ISCs from circulation. These data are consistent with heterogeneity of ISCs in terms of their intravascular viability. Some ISCs may have adapted to the stress of circulation despite their abnormal shape.