Quantitative Visualization of Hypoxia and Proliferation Gradients Within Histological Tissue Sections

Abstract

The formation of hypoxic microenvironments within solid tumors is known to contribute to radiation resistance, chemotherapy resistance, immune suppression, increased metastasis, and an overall poor prognosis. It is therefore crucial to understand the spatial and molecular mechanisms that contribute to tumor hypoxia formation to improve the efficacy of radiation treatment, develop hypoxia-directed therapies, and increase patient survival. The objective of this study is to present a number of complementary novel methods for quantifying tumor hypoxia and proliferation in multiplexed immunofluorescence images, especially in relation to the location of perfused blood vessels. A standard marker analysis strategy is to take a positive pixel count approach, in which a threshold for positive stain is used to compute a positive area fraction for hypoxia. This work is a reassessment of that approach, utilizing not only cell segmentation but also distance to nearest blood vessel in order to incorporate spatial information into the analysis. We describe a reproducible pipeline for the visualization and quantitative analysis of hypoxia using a vessel distance analysis approach. This methodological pipeline can serve to further elucidate the relationship between vessel distance and microenvironment-linked markers such as hypoxia and proliferation, can help to quantify parameters relating to oxygen consumption and hypoxic tolerance in tissues, as well as potentially serve as a hypothesis generating tool for future studies testing hypoxia-linked markers.

Hypoxia and the phenomenon of immune exclusion

“…Immunohistochemistry (IHC) can also be used to evaluate the concomitant expression of HIF-induced proteins and the infiltration of immune cells, in tumors [ 29 , 321 ]. Exogenously administered nitroimidazole-based components accumulate in hypoxic cells and they can be detected by IHC, PET or SPECT imaging.…”

“…Endogenous proteins such as HIF-1α can be tested by IHC, together with HIF-induced proteins. Such commonly used protein markers for hypoxia include CAIX, VEGFA, osteopontin, glucose transporter GLUT-1 and GLUT-3, BNIP3, PDK1, LDHA, LOX and EPO [ 321 , 333 , 337 – 339 ].…”

In Vitro Innovation of Tendon Tissue Engineering Strategies

“…For this reason, controlling oxygen tension from atmospheric (20%) to a more physiological level could be a good strategy to reach this goal. It is essential to note that tissue normoxia varies substantially in vivo, existing as gradients within tissue [258], whereby cell cultures could be influenced by oxygen concentrations [243] or time of exposure [259], and this means that the choice of adequate cell culture conditions is critical in order to develop effective cellular therapies.…”

Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter

“…The binding increases exponentially with decreasing oxygen concentration [ 29 ] below about 1.3% O 2 (10 mmHg) [ 30 , 31 ], although staining has been detected at higher concentrations up to about 9.2% O 2 (70 mmHg) [ 32 ]. A wide range of hypoxia levels can therefore be assessed, and a steady decrease in staining intensity away from necrosis in histological tumor sections has been shown ( Figure 3 B) [ 27 , 33 ]. By comparing this staining pattern with expression data of proteins like epidermal growth factor receptor (EGFR) and HIFA, molecular responses at different hypoxia levels have been assessed [ 34 , 35 ].…”