Need final proposal done for cancer biology

Use of hemoglobin-based oxygen carries derived from high-flying birds to target hypoxia

induced pathways of non-small cell lung cancer as a mechanism for treatment.

Department of Biology, Kennesaw State University

BIOL 4550: Cancer Biology

Dr. Selma Cuya-Smith

July 28, 2022

Description of Proposed Study

A. Scientific Basis. Hypoxia is a condition common to many progressed and solid malignant

tumors [1]. A hypoxic environment forms a complex but defective vascularization network

from the primary cancer tissue, resulting in decreased blood flow and oxygen circulation to

the affected tissue. It is caused by abnormal angiogenesis, poor blood flow, uncontrolled

proliferation of the tumor cells, and oxygen consumption surpassing the oxygen supply [2].

The hypoxic milieu significantly contributes to tumor resistance to targeted radio- and

chemotherapies, anti-tumor drugs, and metastasis progression. Non-small cell lung cancer

(NSCLC) is the most common cause of cancer-related deaths, with hypoxia being the most

common characteristic of the tumor microenvironment [3]. Targeted therapies for NSCLC

are available and have successfully retarded cancer progression. However, these anti-cancer

treatments “preferentially target [rapidly proliferating] tumor cells” and are unable to target

cells in highly hypoxic regions, which are “quiescent, low-proliferating, stem-cell-like cell

fractions” [1]. Therefore, these therapies can still only be considered adjuvant and

alleviating. For the NSCLC tumor cells, mitigation of the hypoxic condition offers a

promising target for anti-cancer treatments to increase the vulnerability of malignant tumor

cells to existing and developing therapies.

One of many methods for improving tumor hypoxia has focused on the transportation

of oxygen-blood circulation [2]. Hyperbaric oxygen therapy (HBO) has yielded mixed results

for in vivo and in vitro models, improving the amount of oxygen in blood plasma and tumor

tissue sensitivity to therapeutic effects of cancer-fighting treatments. In combination with this

treatment, hemoglobin-based oxygen carriers (HBOCs) are undergoing clinical trials to

accommodate for the oxygen affinity, longevity, and covalent bonding properties of

hemoglobulin through genetic engineering of the red blood cell carrying the hemoglobin

protein (Hb). So far, red blood cell substitutes are sourced from human red blood cells, E.

coli, and bovine red blood cells. However, little research has been conducted using high-

flying birds with Hb higher oxygen affinity than any mammalian species [4]. Additionally,

studies have supported that avian species have a very low tendency to develop malignant

cancers [5].

The development of hypoxia-inducible factor (HIF) inhibitors has been of particular

interest for further pharmacological development as a positive correlation between high

expression of the 1-alpha protein subunit of HIF (HIF-1a) and the aggressive, treatment-

resistant, stem-cell-like NSCLC cells [6]. HIF fosters an epithelial-to-mesenchymal transition

in cell phenotype, allowing tumorigenesis through increased cell motility, alteration of

morphology and cell adhesion, and promoting angiogenesis, migration, and invasion abilities.

“Patients with [solid tumor cancer] demonstrating high expression of HIF-1a had a lower

chance to achieve complete remission after irradiation” [1]. The HIF pathway responds as a

survival pathway and is not customary to normoxic tissues. Consequently, control of this low

oxygen-dependent factor through HIF inhibitor therapy poses an opportunity to control the

spread and advancement of malignant NSCLCs. HIF-1a can be used not only as a target for

future therapies but also as a predictive biological marker for tracking the progression of

NSCLC cells.

B. Significance. Hypothetically, HBO and using genetically engineered HBOCs from a species

with high-performance success in hypoxic conditions can inhibit HIF-1alpha expression and,

ultimately, progression of NSCLC cells. Additionally, we hypothesize that this treatment will

be as effective, if not more effective, than current HIF inhibitors in increasing the

vulnerability of these tumor cells to radiotherapy, chemotherapy, and anti-tumor drugs. This

approach could produce a less invasive and more targeted approach to cancer treatment.

C. Project Design and Methods

Aim 1: Identify and isolate the genetic sites influencing the allosteric regulation of Hb to

oxygen affinity in high-flying birds through bottom-up mass spectroscopy in various

conditions. Bar-headed geese are notorious for their reduced metabolism and ability to support

hypoxic flight at extreme altitudes [4]. The migratory flight of these birds has been obtainable

due to their unique physiology and various adaptations. Primarily, the hemoglobulin of high-

flying avian species has a much higher affinity for oxygen binding. Preliminary data demonstrate

the isolation and purification of the Hb through anion exchange chromatography, washing with

carbon monoxide, and centrifugation [7]. The Hb will then be analyzed through bottom-up mass

spectrometry, x-ray diffraction, and proteome profiling using the spectral library assembled by

Dr. Onder and his associates [8]. Samples will be taken from geese treated with physoxia and

intermittent hypoxia environments induced by laboratory wind tunnels that simulate high and

low-altitude conditions [4]. The hypoxic sample group will further be divided into acute and

chronic hypoxic exposure conditions [1]. Intermittent acute hypoxia will be sampled after 15 to

30 minutes of hypoxic exposure, a recovery period of 1 hour, and re-exposure for 30 to 40

minutes. Chronic hypoxia will be sampled after two weeks of exposure, 2 hours of recovery, and

re-exposure for 2 to 5 weeks. Once the Hb is isolated and analyzed, the development of red

blood cell conjugate for the Hb can begin.

a. Anion exchange chromatography in a flow-through model uses a negatively charged

resin at a higher pH to bind the unneeded contents of the blood sample and elute the Hb

from the chromatography column [7]. This process is used to isolate and optimize the

purification of Hb from the blood of the bar-headed geese. The geese will not be

sacrificed for collection. Blood will be drawn from the brachial vein using a sterile

syringe [4].

b. Washing with carbon monoxide binds to Hb and removes plasma proteins that have

been absorbed into the protein [7].

c. Centrifugation of the sample to further separate the Hb from any remaining residues,

including the carbon monoxide using centrifugal force and separating particles with

different densities.

d. Bottom-up mass spectrometry is the large-scale analysis of purified proteins cleaved

using enzymes and then further analyzed by their molecular weight and charge. This

process is what allows for sequence identification.

e. X-ray diffraction is the irradiation of Hb material or the blood sample with x-rays at

varying angles, then measuring the intensities of the rays to determine the structure of a

material [9].

f. Proteome profiling uses a specific information system for the comparative functional

characterization of proteins. It can further identify or diagnose a disease or medical

condition [8].

Aim 2: Analyzing live NSCLC cell lines cultured from human subjects with varying stages

of progressed non-small cell lung cancer and using Seahorse to detect the degree of hypoxia

in cells. When the tumor microenvironment of a cell becomes exposed to hypoxic conditions, the

cell will undergo intracellular changes on both the proteomic and genomic levels to survive [3].

These changes often include stimulation of tumor growth, tissue invasion, metastasis,

angiogenesis, and anaerobic metabolism, which allow the cells to escape and survive their

hypoxic environment. When HIF-1 is upregulated in the cell by low oxygen conditions, the

genes involved in the anaerobic metabolism of glucose are transcriptionally upregulated for ATP

production. Using the Agilent Seahorse XF Extracellular Flux Analyzer and assay to obtain

metabolic measurements of human cancer cells in stage 0 – IV NSCLC cell lines, a baseline for

mitochondrial cellular respiration can be established. The live cells will be cultured into

microplates at a density of 10,000 to 20,000 cells per well (XF Hypoxia assays may require

fewer cells per well to prevent anoxia during the measurement period [10]).

Each cancer cell group will first be divided by stage of progression. The sample groups

will then be separated into a control group, an acute hypoxia exposure group, a chronic exposure

hypoxia exposure group, and an intermittent hypoxia exposure group to test mitochondrial

response. Finally, a hypoxic chamber on these cell lines will be used to provide the cultures with

appropriate oxygen exposure. The combined use of the Agilent Seahorse XF Analyzer Assay and

the hypoxic chambers will establish a foundation to find which cell lines are most responsive to

hypoxia and which will be most responsive to the application of the developed HBOCs.

Aim 3: Obtain in vivo models of NSCLC, asthmatic lungs (control), and normoxic lungs for

isolation and analysis of the Warburg effect in associated tissues of mice. Approximately 500

genetically engineered heterozygous HIF-1α+/− mice, specially adapted to hypoxic conditions,

will be purchased and directly injected with NSCLC into the lung parenchyma [3]. The NSCLC

cells will be of varying hypoxic exposures and stages of cancer. A specialized group consisting

of asthma-induced mice will act as the control. The use of anaerobic glycolysis over the usual

oxidative phosphorylation by mitochondria of cancer cells is referred to as the Warburg Effect.

Mitochondria, which rely on oxygen to produce ATP, experience decreased activity when their

cell is deprived of oxygen. As performed in an experiment focused on intermittent hypoxia by

the Pritzker School of Medicine at the University of Chicago, seven-week-old adult (of both

female and male populations) mice will be acclimated to the laboratory setting and a 12-hour

light-dark cycle. After one week, the mice will be injected with the NSCLC cells. After the cells

are allowed to incubate in vivo for another week, we will isolate blood from the test subjects,

stain and isolate the mitochondria from the cancer cells, and use specific antibodies to spot high

oxidation levels. These mice will then be separated into their respective chambers, establishing

an asthmatic control group, normoxic exposure group, and three hypoxic exposure groups: acute,

chronic, and intermittent hypoxic exposure. Mitochondria autophagy from the live mice will be

analyzed using the Agilent Seahorse XF Analyzer and the XF Hypoxia Rate Calculator every

week for 5 to 8 weeks [10]. Tumor cell cultures will also be extracted from the live mice, and a

western blot assay will determine tumor progression using HIF-1α concentrations as a

biomarker. At the end of the eight weeks, the mice will be sacrificed, and the cancerous or non-

cancerous lung tissue cells cultured to compare mitochondria autophagy levels throughout the

groups and to compare the progression of tumor size. Further aims would be using HBO in this

NSCLC-injected mice species to evaluate the suppression of the hypoxia-induced Warburg

Effect through HIF-1α.

References.

[1] Muz, B., de la Puente, P., Azab, F., & Azab, A. K. (2015). The role of hypoxia in cancer

progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia (Auckland, N.Z.), 3,

83–92. https://doi.org/10.2147/HP.S93413

[2] Zhu, X. H., Du, J. X., Zhu, D., Ren, S. Z., Chen, K., & Zhu, H. L. (2020). Recent Research

on Methods to Improve Tumor Hypoxia Environment. Oxidative medicine and cellular

longevity, 2020, 5721258. https://doi.org/10.1155/2020/5721258

[3] Ziółkowska-Suchanek I. (2021). Mimicking Tumor Hypoxia in Non-Small Cell Lung Cancer

Employing Three-Dimensional In Vitro Models. Cells, 10(1), 141.

https://doi.org/10.3390/cells10010141

[4] Storz J. F. (2016). Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence

for an adaptive trend?. The Journal of experimental biology, 219(Pt 20), 3190–3203.

https://doi.org/10.1242/jeb.127134

[5] Erten, E. Y., Tollis, M., & Kokko, H. (2020). Bird size with dinosaur-level cancer defences:

can evolutionary lags during miniaturisation explain cancer robustness in birds? bioRxiv.

https://doi.org/10.1101/2020.10.22.345439

[6] Onnis, B., Rapisarda, A., & Melillo, G. (2009). Development of HIF-1 inhibitors for cancer

therapy. Journal of cellular and molecular medicine, 13(9A), 2780–2786.

https://doi.org/10.1111/j.1582-4934.2009.00876.x

[7] Andrade, C. T., Barros, L. A., Lima, M. C., & Azero, E. G. (2004). Purification and

characterization of human hemoglobin: effect of the hemolysis conditions. International journal

of biological macromolecules, 34(4), 233–240. https://doi.org/10.1016/j.ijbiomac.2004.05.003

[8] Song, Y., Laskay, Ü.A., Vilcins, IM.E. et al. Top-Down-Assisted Bottom-Up Method for

Homologous Protein Sequencing: Hemoglobin from 33 Bird Species. J. Am. Soc. Mass

Spectrom. 26, 1875–1884 (2015). https://doi.org/10.1007/s13361-015-1185-z

[9] Chandramouli, K., & Qian, P. Y. (2009). Proteomics: challenges, techniques and possibilities

to overcome biological sample complexity. Human genomics and proteomics : HGP, 2009,

239204. https://doi.org/10.4061/2009/239204

[10] Team, D. (2020, October 2). Targeting Energy Metabolism for Cancer Therapeutic

Discovery using Agilent Seahorse XF Technology. Drug Discovery World (DDW).

https://www.ddw-online.com/targeting-energy-metabolism-for-cancer-therapeutic-discovery-

using-agilent-seahorse-xf-technology-727-202008/