Role of Heme in Non-Small Cell Lung Cancer Growth and Tumor Vascular Oxygenation

Abstract

Despite a decline in deaths caused by lung cancer, it remains the leading cause of cancer-related death in the U.S. Of these, about 84% are cases of non-small cell lung cancer (NSCLC). Despite the advent of various targeted therapies and immunotherapies; the 5-year survival rate for lung cancer is very low. Therefore, alternative therapeutic strategies should be explored to improve therapeutic outcomes for patients of lung cancer. Numerous studies have demonstrated that elevated levels of mitochondrial respiration or oxidative phosphorylation (OXPHOS) are a key feature of NSCLC cells. Recent studies in our lab show that NSCLC cells demonstrate elevated levels of heme via increased heme uptake and synthesis, as well as elevated mitochondrial respiration/oxidative phosphorylation (OXPHOS) which fuels NSCLC growth and progression. Also, limiting heme via inhibition of heme synthesis with Cyclopamine tartrate (CycT) or inhibition of heme uptake with heme sequestering peptides (HSP2) delays the growth and progression of NSCLC tumors. The tumor microenvironment (TME) influences tumor heterogeneity, invasion, metastasis, and is a major contributor to cancer progression. Tumor vasculature and oxygenation are important aspects of TME; however, faulty tumor vascular oxygenation is a marker for poor prognosis in cancer. Given the central role of heme in oxygen transport, utilization, and storage, it is important to investigate the effect of limiting heme on tumor vascular oxygenation. This study aimed to gain insights into how limiting heme using heme targeting drugs (CycT or HSP2) inhibits tumor growth and progression as well as how this influences the vascularization status of NSCLC tumors. Here, we used subcutaneous/orthotopic lung tumor xenografts models of NSCLCs expressing luciferase in NOD-SCID mice to monitor tumor growth after treatment with CycT or HSP2 using bioluminescence imaging (BLI). Also, mice were subjected to multispectral optoacoustic tomography (MSOT) to visualize tumor vasculature, oxygenation, and hypoxia. MSOT data were acquired to calculate total hemoglobin concentrations (THb) and oxygenation (sO2). Tissue homogenates of resected tumors were used to determine oxygen consumption rates and ATP levels. Tumors/lung tissues were harvested, processed and paraffinembedded for immunohistochemistry (IHC) and histology experiments. BLI data, tumor volumes, masses, and hematoxylin and eosin (H&E) show that CycT or HSP2 treatment suppresses the growth of subcutaneous/lung orthotopic tumor xenografts. CycT or HSP2 treated tumors also show reduced oxygen consumption and ATP levels compared to control. MSOT data show that THb levels decrease when treated with CycT or HSP2. OE-MSOT (Oxygen enhanced MSOT) data show increased levels of sO2 MSOT in HSP2- or CycT-treated tumors compared to control tumors. IHC data for various hypoxia and angiogenesis markers confirm that limiting heme effectively normalizes tumor vascular oxygenation. This study demonstrates that targeting heme holds immense potential as a therapeutic strategy that can effectively inhibit angiogenesis, normalize tumor vasculature, and alleviate tumor hypoxia in NSCLC tumors thereby delaying lung tumor growth and progression.