Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept

Abstract

Hypoxia is a common characteristic of many solid tumors that has been associated with tumor aggressiveness. Limited diffusion of oxygen generates a gradient of oxygen availability from the blood vessel to the interstitial space and may underlie the recruitment of macrophages fostering cancer progression. However, the available data based on the recruitment of circulating cells to the tumor microenvironment has been so far carried out by conventional co-culture systems which ignore the hypoxic gradient between the vessel to the tumor interstitium. Here, we have designed a novel easy-to-build cell culture device that enables evaluation of cellular cross-talk and cell migration while they are being simultaneously exposed to different oxygenation environments. As a proof-of-concept of the potential role of differential oxygenation among interacting cells we have evaluated the activation and recruitment of macrophages in response to hypoxic melanoma, breast, and kidney cancer cells. We found that hypoxic melanoma and breast cancer cells co-cultured with normoxic macrophages enhanced their directional migration. By contrast, hypoxic kidney cells were not able to increase their recruitment. We also identified well-described hypoxia-induced pathways which could contribute in the immune cell recruitment (VEGFA and PTGS2 genes). Moreover, melanoma and breast cancer increased their proliferation. However, oxygenation levels affected neither kidney cancer cell proliferation nor gene expression, which in turn resulted in no significant changes in macrophage migration and polarization. Therefore, the cell culture device presented here provides an excellent opportunity for researchers to reproduce the in vivo hypoxic gradients in solid tumors and to study their role in recruiting circulating cells to the tumor in specific types of cancer.

Bioengineering the Oxygen-Deprived Tumor Microenvironment Within a Three-Dimensional Platform for Studying Tumor-Immune Interactions

“…Oxygen diffusibility and consumption within a 3D ECM niche was previously explored by Colom et al using a mathematical model (Colom et al, 2014). Crosstalk of macrophages and cancer cells was shown to be influenced by oxygen availability (Campillo et al, 2019). In another study, immune cell infiltration into 3D matrices was shown to vary significantly based on the oxygen content, when cancer cells and highly efficient chimeric antigen receptor T (CAR-T) cells were incorporated in vitro in a 3D micropattern within a photo-crosslinked hydrogel and coupled to a microfluidic hypoxia device (Ando et al, 2019).…”

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model

“…These results suggest that PnV induced an increase in blood monocytes, and these cells were directed preferentially to the tumor. Hypoxia is a common characteristic of many solid tumors and limited oxygen diffusion generates a gradient of oxygen availability from the blood vessel to the interstitial space and may underlie macrophages recruitment, promoting cancer progression 33 . The macrophages can be recruited to the tumor in PnV-treated animals because the tumor is highly necrotic and the environment is probably very poor of oxygen.…”

Bioengineering a novel 3D in-vitro model to recreate physiological oxygen levels and tumor-immune interactions

“…An adaptive, key hallmark of cancer is the tumor's capacity to accommodate to changeable states of oxygen deprivation. As oxygen content regulation is essential to maintaining cell homeostasis, small deviations in oxygen level in the TME can result in major changes in tumor cell functionality (34,(58)(59)(60). Physiological oxygen levels in solid tumors are very heterogeneous ranging from 0.5 -4% oxygen saturation compared to 4 -14% in healthy tissues (59,(61)(62)(63).…”

“…In the efforts to recreate oxygen deprivation-driven tumor immune evasion mechanisms in the laboratory, very few cancer in-vitro models adequately mimic physiological oxygen levels relevant to breast tissue and its tumor-immune interactions (32)(33)(34). Traditional two-dimensional (2D) culture models fail to generate oxygen gradients as such, and hence experiments using these models expose the cells to higher than physiological oxygen levels (35).…”