(533f) Polymerized Hemoglobin Facilitated Modulation of Tumor Oxygenation Is Dependent on Tumor Oxygenation Status and Oxygen Affinity of the Hemoglobin-Based Oxygen Carrier

Background: Hemoglobin (Hb) based oxygen (O₂) carriers (HBOCs) are a promising class of O₂ therapeutic that can relieve chronic hypoxia in the tumor microenvironment, which may increase the effects of chemotherapy. Previous studies have demonstrated that HBOC O₂ affinity significantly affects how HBOC treated tumors respond to chemotherapy. However, there is no consensus concerning whether high or low O₂ affinity HBOCs results in greater chemosensitization. To address these concerns, we investigated how a low-O₂-affinity tense (T) quaternary state polymerized hemoglobin (PolyHb) and a high-O₂-affinity relaxed (R) quaternary state PolyHb impact tumor oxygenation and sensitivity to chemotherapeutics. These effects were evaluated with a combination of in silico and in vivo methods.

Methodology: For this study, we synthesized a library of low molecular weight (MW) PolyHb in the T- and R-state. The following biophysical properties of these materials were measured: O₂ affinity, gaseous ligand binding kinetics, MW distribution, and hydrodynamic diameter. With these properties, we simulated O₂ transport within a three dimensional vascularized tumor construct. After simulating the tumor, we used principal component analysis to examine which properties influence tumor oxygenation. We examined how the T-state and R-state PolyHb modulates O₂ delivery to hypoxic and normoxic human melanoma xenografts in a murine window chamber model. We examined microcirculatory fluid flow via video shearing optical microscopy, and oxygen distributions via phosphorescence quenching microscopy. Additionally, we examined how weekly infusion of a 20% top-load dose of PolyHb influences tumor growth rate, vascularization, and regional blood flow. To test the chemosensitizing potential, we administered an R-state PolyHb to three different non-small cell lung cancer cell lines cultured in vitro and implanted in vivo into healthy mice.

Results and Conclusions: In general, we found that infusion of T-state PolyhHb is more likely to decrease tissue hypoxia and modulate the metabolic rate of O₂ consumption. Infusion of T-state and R-state PolyHbs led to worse outcomes in normoxic tumors. Of particular concern was the high-affinity R-state PolyHb, which led to no improvement in hypoxic tumors and significantly worsened outcomes in normoxic tumors. Taken together, the results of this study indicate that the tumor oxygen status is a primary determinant of the potency and outcomes of infused PolyhHb. The R-state PolyHb administered in this study is unable to deliver O₂ unless under severe hypoxia, which significantly limits its oxygenation potential. In vitro sensitivity studies indicate that the administration of PolyHb increases the effectiveness of cisplatin under hypoxic conditions. Additional animal studies revealed that co-administration of R-state PolyHb with cisplatin attenuated tumor growth without alleviating hypoxia. Analysis of reactive O₂ species production in the presence of hypoxic culture indicates that exogenous ROS production by oxidized PolyHb may provide the mechanism of chemosensitization. This ROS mechanism, coupled with oxygenation, may be a potential chemosensitizing strategy for use in cancer treatment.