The quaternary state of polymerized human hemoglobin regulates oxygenation of breast cancer solid tumors: A theoretical and experimental study

Abstract

A major constraint in the treatment of cancer is inadequate oxygenation of the tumor mass, which can reduce chemotherapeutic efficacy. We hypothesize that polymerized human hemoglobin (PolyhHb) can be transfused into the systemic circulation to increase solid tumor oxygenation, and improve chemotherapeutic outcomes. By locking PolyhHb in the relaxed (R) quaternary state, oxygen (O2) offloading at low O2 tensions (<20 mm Hg) may be increased, while O2 offloading at high O2 tensions (>20 mm Hg) is facilitated with tense (T) state PolyhHb. Therefore, R-state PolyhHb may deliver significantly more O2 to hypoxic tissues. Biophysical parameters of T and R-state PolyhHb were used to populate a modified Krogh tissue cylinder model to assess O2 transport in a tumor. In general, we found that increasing the volume of transfused PolyhHb decreased the apparent viscosity of blood in the arteriole. In addition, we found that PolyhHb transfusion decreased the wall shear stress at large arteriole diameters (>20 μm), but increased wall shear stress for small arteriole diameters (<10 μm). Therefore, transfusion of PolyhHb may lead to elevated O2 delivery at low pO2. In addition, transfusion of R-state PolyhHb may be more effective than T-state PolyhHb for O2 delivery at similar transfusion volumes. Reduction in the apparent viscosity resulting from PolyhHb transfusion may result in significant changes in flow distributions throughout the tumor microcirculatory network. The difference in wall shear stress implies that PolyhHb may have a more significant effect in capillary beds through mechano-transduction. Periodic top-load transfusions of PolyhHb into mice bearing breast tumors confirmed the oxygenation potential of both PolyhHbs via reduced hypoxic volume, vascular density, tumor growth, and increased expression of hypoxia inducible genes. Tissue section analysis demonstrated primary PolyhHb clearance occurred in the liver and spleen indicating a minimal risk for renal damage.

Fig 1. Illustration of predicted performance of transfused high and low O₂ affinity HBOCs compared to a control of unsupplemented blood.

The magnitude of O₂ delivery to the tissue from each species is designated by the size of the arrow. Note that O₂ delivery should be elevated at all conditions with low O₂ affinity HBOCs. In contrast, O₂ delivery is elevated under hypoxic conditions for high O₂ affinity HBOCS.

Fig 2. Schematic of the tumor arteriole KTC model.

Fig 3. PolyhHb enhanced plasma viscosity and flow indices.

(A) The shear stress versus shear rate plot for human plasma, pure polyhHb, and 10–30% mixtures of PolyhHb in human plasma by volume at 37°C. (B) The flow consistency index as a function of the volume percent of PolyhHb in human plasma. The error bars here depict the standard deviation (n = 3).

Fig 4. Velocity profiles, apparent viscosity and wall shear stress.

(A) Velocity gradients represented by a color map through a 10 μm diameter arteriole and a 50 μm thick tissue space with an inlet average velocity of 0.3 mm/s. Note there are two color gradients for flow in the arteriole and flow in the tissue space. The white lines in the tissue space denote the flow paths for interstitial flow. (B) Apparent viscosities at varying diameters and flow rates with increasing top load adminstration of PolyhHb. (C) Radial velocity profile in the arteriole for a 10 μm diameter arteriole with v_avg = 0.1 cm/s. Wall shear stress calculated for varying top load transfusion percentages from 0 to 40% with PolyhHb at (D) 0.01 cm/s, (E) 0.1 cm/s and (F) 1 cm/s for arteriole diameters of 7, 10, 25, and 50 μm and a hematocrit of 40%.

Fig 5. Amount of each oxygenated species at the arteriole inlet as a function of inlet O₂ tension.

These figures show the amount of each species (dissolved O₂ in plasma, oxygenated Hb inside RBCs, oxygenated 35:1 T-/ 30:1 R-state PolyhHb) and total species as a function of the inlet O₂ tension in the arteriole. (A) shows the equilibrium profile for unsupplemented blood, (B) shows the equilibrium profile for a 20% 30:1 R-state PolyhHb top load transfusion, (C) shows the equilibrium profile for a 20% 35:1 T-state PolyhHb top load transfusion, and (D) shows the total amount of O₂ available from each O₂ containing species at the inlet of the arteriole for varying levels of top load transfusions.

Fig 6. Overall O₂ transfer rate as a function of inlet O₂ tension at varying volumetric flow rate.

This figure shows the overall O₂ transfer rate calculated for 30:1 R-state PolyhHb, 35:1 T-state PolyhHb, unsupplemted blood and Ringer’s lactate solution for (A) all inlet pO_2,in (1–90 mm Hg) and (B) for normoxic pO_2,in (40–90 mm Hg). For all simulations the percent top load was set to 20% and the maximum rate of O₂ consumption was set to 50 μM/s.

Fig 7. O₂ consumption rate (OCR) as a function of inlet O₂ tension.

The OCR and individual contributions resulting from the various O₂ carrying species in solution (i.e. dissolved O₂ in plasma, oxygenated Hb inside RBCs, and oxygenated 35:1 T-/ 30:1 R-state PolyhHb) as a function of inlet O₂ tension. OCR profiles are shown for (A) unsupplemented blood, (B) 20% blood volume top load of 30:1 R-state PolyhHb, (C) 20% blood volume top load of 35:1 T-state PolyhHb. For each of these simulations, v_avg = 0.03 cm/s, V_max = 40 μM/s, HCT = 40% and t_tissue = 50 μm.

Fig 8. Unsupplemented blood normalized O₂ consumption rate.

This figure shows the O₂ consumption rate normalized against unsupplemented blood (horizontal dashed line) for 30:1 R-state PolyhHb (top) and 35:1 T-state PolyhHb (bottom). For each species, the dose (left column), average blood velocity through the arteriole (middle collumn) and the arteriole diameter (right column) were varied. Other parameters were maintained as follows: (V_max = 40 μM/s, v_avg = 0.03 cm/s, D = 10 μm, and t_tissue = 50 μm).

Fig 9. OCR as a function of inlet O₂ tension, average inlet blood velocity, and maximum rate of O₂ consumption.

These figures show the OCR for 30:1 R-state PolyhHb, 35:1 T-state PolyhHb, and unsupplemented blood. The average inlet blood velocity was varied from 0.01 to 1 cm/s (rows). The maximum rate of O₂ consumption was varied from 20 to 80 μM/s (columns). For all PolyhHb simulations, the top-load was set to 20% of the blood volume. (t_tissue = 50 μm.).

Fig 10. False detection rate (FDR) sensitivity to variable model parameters for k₀ and OCR.

These figures depict the FDR for (A) k₀ and (B) OCR for 35:1 T-state PolyhHb, 30:1 R-state PolyhHb, and unsupplemented blood at varying pO_2,in values. Below each column depicts if the effect of increasing the parameter is positive (+), negative (-), or mixed (m) for the T-state PolyhHb, R-state PolyhHb, and non-supplemented blood.

Fig 11. Minimum inlet O₂ tension as a function of the amount of PolyhHb remaining in the plasma at varying tissue layer thicknesses.

The figures show the minimum inlet O₂ tension that is observed before a hypoxic (< 5 mm Hg) region is observed. The thickness of the tissue region was varied from (A) 50 μm, (B) 65 μm, and (C) 80 μm. The amount of PolyhHb transfused was varied from 10 to 30% top load. The percentage of initially administered PolyhHb remaining in the circulation was decreased. Unsupplemented blood was used as a control. For this simulation, the hematocrit was set to 0.45, and the flow rate was set to 0.03 cm/s.

Fig 12. Biomarkers for tumor growth and oxygenation.

(A) Immunohistochemical staining using antibodies specific for the detection of hypoxic adducts (hypoxyprobe) (top panel) (scale bar = 200 μm) and CD31 (bottom panel) (scale bar = 50 μm) on tissue sections of MDA-MB-231 orthotopic breast tumors. Arrows indicate vessels in the CD31 stained images. (B-D) Images were deconvoluted and a threshold was created for each stain to determine percent hypoxic area (B) (mean ± SEM, n = 3–4 mice × 4 images), percent area of CD31 stain (C) (mean ± SEM, n = 3–4 mice × 4 images) or number of blood vessels (D) (mean ± SEM, n = 3–4 mice × 4 images). (E) Tumor weights at end of trial (mean ± SEM, n = 3–4). (F) Hypoxia-inducible gene expression in tumors treated with either the control (saline), T-state PolyhHb or R-state PolyhHb. mRNA levels of P4HA1, PGK1, LDHA and BNIP3 were analyzed in tumors established with MDA-MB-231 cells normalized to the control (mean ± SEM, n = 3–4 mice × 3 replicates). (* p < 0.05, ANOVA with post-hoc correction).

Fig 13. Tissue markers of iron accumulation following PolyhHb treatment.

(A) Light microscopy images of representative Perls DAB stained tissue sections from control mice and mice after treatment with PolyhHb in the R- and T-states. Control, spleen shows the characteristic brown staining pattern of red pulp (red arrow) and white pulp (blue arrow). Tumor and kidney tissue are absent of iron accumulation with or without PolyhHb treatment, while the liver shows iron accumulation in macrophage and monocyte cells (black arrows). Expansion of red pulp regions in the spleen (red arrows) is observed following both R- and T-state PolyhHb treatment. Images were obtained at (100 and 200× magnification (objective 10×, 20×) and scale bars represent 100 μm for the tumor, liver and spleen and 50 μm for the kidney. (B) Western Blot analysis is shown for tumor, kidney, liver and spleen HO-1 and ferritin H protein expression. (C) Densitometry analysis of (B) showing significantly increased splenic HO-1 (P = 0.0329 *, T-state PolyhHb vs. control, n = 3–4) and (P = 0.0013*, R-state PolyhHb vs. control, (n = 3–4). An increase in ferritin H was also observed in the spleen following PolyhHb treatment versus the control (P = 0.0471*, n = 3–4), by ANOVA with a Multiple Comparisons Test.

Fig 14. Retrospective analysis of expected increase in OCR from previous experimental models.

This figure shows the OCR normalized against unsuplemented blood (horizontal solid line) compared to computational estimates of HBOC-201, carbogen gas, or HBOC-201/carbogen gas co-therapeutic as described in a 1993 study by Teicher et al. (9L Gliosarcoma) and a 2005 study by Gottschalk et al. (R1H Rhabdomyosarcoma). (V_max = 40 μM/s, v_avg = 0.03 cm/s, D = 10 μm, and t_tissue = 50 μm).