RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature

• Main Authors: Vinay P. Jani, Robert Asaro, Bryan Oronsky, Pedro Cabrales
• Format: Article
• Language: English
• Published: MDPI AG 2021-04-01
• Series: International Journal of Molecular Sciences
• Subjects: RRx-001; tumor microenvironment; RBC adhesion; shear stress
• Online Access: https://www.mdpi.com/1422-0067/22/9/4713
• ISSN: 1661-6596; 1422-0067

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (^99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNF<inline-formula>α<math display="inline"><semantics><mi mathvariant="sans-serif"></mi></semantics></math></inline-formula>) to mimic the known inflammation in the tumor microenvironment. One hour following administration of ^99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in ^99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined <inline-formula><math display="inline"><semantics><mrow><msub><mi mathvariant="sans-serif">τ</mi><mrow><mn>50</mn></mrow></msub></mrow></semantics></math></inline-formula>, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, <inline-formula><math display="inline"><semantics><mrow><msub><mi mathvariant="sans-serif">τ</mi><mrow><mn>50</mn></mrow></msub></mrow></semantics></math></inline-formula> was determined to be 4.5 dynes/cm² (95% CI: 4.3–4.7 dynes/cm²) for RBCs treated with 10 <inline-formula>μ<math display="inline"><semantics><mi mathvariant="sans-serif"></mi></semantics></math></inline-formula>M RRx-001, which was significantly different (<i>p</i> < 0.05) from <inline-formula><math display="inline"><semantics><mrow><msub><mi mathvariant="sans-serif">τ</mi><mrow><mn>50</mn></mrow></msub></mrow></semantics></math></inline-formula> in the absence of treatment. Under hypoxic conditions, the difference of <inline-formula><math display="inline"><semantics><mrow><msub><mi mathvariant="sans-serif">τ</mi><mrow><mn>50</mn></mrow></msub></mrow></semantics></math></inline-formula> with (4.8 dynes/cm²; 95% CI: 4.6–5.1 dynes/cm²) and without (2.6 dynes/cm²; 95% CI: 2.4–2.8 dynes/cm²) 10 <inline-formula>μ</inline-formula>M RRx-001 treatment was exacerbated (<i>p</i> = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.