Abstract
The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors, and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate, simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems, but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments, with a focus on lung cancer cells, their interactions with macrophages and T cells, and their response to modulators in the immune microenvironment. We describe the experimental set up, illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages, and outline strategies to address the heterogeneous TME.
Highlights
Free versus bound concentrations of metabolites can be discriminated in cells by fluorescence lifetime imaging microscopy (FLIM) [158,194,195]
Microscopy-based metabolic imaging requires the use of fluorescent probes such as NBD-glucose for measuring glucose uptake [58,59,60], with the exception of a few metabolites that have intrinsic fluorescence (e.g., FADH2, NAD(P)H) [196]
Not directly useful for metabolic imaging, the emerging Digital Spatial Profiling (DSP) technique can be used to measure the expression of metabolic genes and proteins in situ at single cell type resolution
Summary
Most tissues and solid tumors are highly heterogeneous at the molecular, cellular, and regional levels, as well as across entire organ. With the recent development of Digital Spatial Profiling (DSP), multiplexing bar-coded antibodies or oligonucleotide probes each for a different protein or mRNA target is employed to obtain the spatial distribution of multiple targets in tissues at single cell type resolution [64,65,66] (e.g., Figure 1B). There are substantial advantages in developing multiplexed SIRM (mSIRM) approaches where multiple precursors containing different tracer atoms such as 13 C, 15 N, and D are used together These include large expansion of metabolic network coverage without interferences from sample batch variations while greatly reducing sample requirement; the latter is crucial to studies with very limited patient-derived (PD) materials such as PD organotypic tissue culture (OTC) or organoid (PDO) studies. We have begun such development, as described in Fan et al [107]
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