Sickle Cells Abolish Melanoma Tumorigenesis in Hemoglobin SS Knockin Mice and Augment the Tumoricidal Effect of Oncolytic Virus In Vivo

Abstract

Insights from the study of cancer resistance in animals have led to the discovery of novel anticancer pathways and opened new venues for cancer prevention and treatment. Sickle cells (SSRBCs) from subjects with homozygous sickle cell anemia (SCA) have been shown to target hypoxic tumor niches, induce diffuse vaso-occlusion, and potentiate a tumoricidal response in a heme- and oxidant-dependent manner. These findings spawned the hypothesis that SSRBCs and the vasculopathic microenvironment of subjects with SCA might be inimical to tumor outgrowth and thereby constitute a natural antitumor defense. We therefore implanted the B16F10 melanoma into humanized hemoglobin SS knockin mice which exhibit the hematologic and vasculopathic sequelae of human SCA. Over the 31-day observation period, hemoglobin SS mice showed no significant melanoma outgrowth. By contrast, 68–100% of melanomas implanted in background and hemoglobin AA knockin control mice reached the tumor growth end point (p < 0.0001). SS knockin mice also exhibited established markers of underlying vasculopathy, e.g., chronic hemolysis (anemia, reticulocytosis) and vascular inflammation (leukocytosis) that differed significantly from all control groups. Genetic differences or normal AA gene knockin do not explain the impaired tumor outgrowth in SS knockin mice. These data point instead to the chronic pro-oxidative vasculopathic network in these mice as the predominant cause. In related studies, we demonstrate the ability of the sickle cell component of this system to function as a therapeutic vehicle in potentiating the oncolytic/vasculopathic effect of RNA reovirus. Sickle cells were shown to efficiently adsorb and transfer the virus to melanoma cells where it induced apoptosis even in the presence of anti-reovirus neutralizing antibodies. In vivo, SSRBCs along with their viral cargo rapidly targeted the tumor and initiated a tumoricidal response exceeding that of free virus and similarly loaded normal RBCs without toxicity. Collectively, these data unveil two hitherto unrecognized findings: hemoglobin SS knockin mice appear to present a natural barrier to melanoma tumorigenesis while SSRBCs demonstrate therapeutic function as a vehicle for enhancing the oncolytic effect of free reovirus against established melanoma.

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

“…The SSRBC-mediated vessel occlusions were often clustered and tightly packed with erythrocytes of which nearly 100% exhibited a sickle cell morphology ( Figure 7C). Because only 5% of circulating nondeformable sickle cells in SSKI mice display the sickle morphology (15), it is likely that SSRBCs trapped in the injured tumor microvessels transitioned to the sickle shape as their HbS deoxygenated and polymerized. That therapy with SR and CA-4 in SSKI but not AAKI mice can induce tumor regression in vivo confirms the key role of SSRBCs in the vaso-oc- Figure 2.…”

“…Intravital microscopy observations show that infused SSRBCs home to established tumor, form aggregates in tumor vessels, and induce focal vessel closure. Together with pro-oxidants or oncogenic virus, SSRBCs also induce a therapeutic tumor growth delay but fail to produce complete tumor regressions (15,16).…”

Sickle cells produce functional immune modulators and cytotoxics

“…To this end, we showed that cytotoxic drugs could be physically encapsulated in SSRBCs and programed ex vivo to discharge 4 times more drug cargo into hypoxic tumors relative to normal RBCs and free drug . Likewise, SSRBCs loaded with oncolytic reovirus directed the virus to melanoma in vivo and exhibited increased tumoricidal effectiveness relative to similarly treated normal RBCs and free virus . While osmosis‐based encapsulation allows the entrapment of large quantities of biologics it also injures cell membranes and alters the circulatory kinetics of the modified RBCs.…”

“…9 Likewise, SSRBCs loaded with oncolytic reovirus directed the virus to melanoma in vivo and exhibited increased tumoricidal effectiveness relative to similarly treated normal RBCs and free virus. 10 While osmosisbased encapsulation allows the entrapment of large quantities of biologics it also injures cell membranes and alters the circulatory kinetics of the modified RBCs. Physical entrapment is confined to a limited group of agents and release at the targeted site can be unpredictable.…”

“…11,12 Likewise, virus adsorbed to SSRBCs runs the risk of exchange with higher affinity receptors and neutralization by seroreactive neutralizing antibodies. 10 Am J Hematol. 2017;92:981-988.…”