Intercellular communication between T cells and cancer cells plays a pivotal role in determining cancer cell survival or death. Yet, our understanding of this interaction remains incomplete. Methods to study heterotypic cell interactions are either limited to targeted studies relying on predefined set of proteins, or require cell separation, thus disrupting the native environment. Stable isotope labeling by amino acids in cell culture (SILAC) enables proteome distinction in heterologous co-cultures without the need for physical separation. But dynamic studies remain constrained by the need for numerous mass spectrometry (MS) runs, the challenges in detecting low-abundant proteins, particularly in immune cells and the limited data completeness due to the use of data-dependent MS1-based precursor quantification. To overcome these limitations, we evaluate the integration of SILAC with tandem mass tag (TMT) multiplexing and SILAC-directed real-time search (RTS). TMT labeling enables simultaneous analysis of multiple samples, while RTS-MS3 acquisition using SILAC-induced mass shifts as fixed modifications triggers MS3 scans for specific proteome populations within a mixed cell system, improving quantitative accuracy and proteome coverage for target protein populations. We benchmarked our acquisition methods using SILAC-labeled samples mixed at defined ratios and validated the approach in biologically relevant co-culture experiments. Additionally, we introduced a carrier channel to enhance detection of lower-abundant T cell proteins, while maintaining acceptable quantitative precision. Our results demonstrate that the combined SILAC-TMT-RTS strategy dramatically improves proteome depth, temporal resolution, and cell-type specificity for short-term co-culture interaction proteomics studies. In co-culture samples of T cells with non-small cell lung cancer cell lines that were either sensitive or resistant to T cell killing, our method revealed candidate mechanisms underlying their differential sensitivity. Our integrated approach combining SILAC, TMT and RTS to resolve cell-specific proteome dynamics in co-culture represents a novel and powerful advance.

In a groundbreaking proteogenomic study, researchers have unveiled the profound impact of missense mutations on protein abundance within prostate cancer tissues, employing a sophisticated Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC)-based quantitative proteomics approach. This meticulous analysis juxtaposed malignant prostate samples against adjacent healthy tissues, revealing a complex landscape where genetic alterations directly reshape the tumor proteome, offering new vistas in understanding tumor biology and potential clinical applications.

Microphysiological systems, such as multicellular spheroids, hold great promise for drug screening experiments. Spheroids may be dosed statically, where the drug is introduced to the growing chamber at one time point, or dynamically, where the drug is introduced via a fluidic component. Dynamic dosing can generate pharmacokinetic curves that more closely represent those seen in vivo than static dosing. In this work, we demonstrate the dynamic dosing of colorectal cancer spheroids in a 3D printed fluidic device with liposomal doxorubicin. Spheroids are valuable models to evaluate dynamic dosing, as they recapitulate the nutrient, oxygen, and pH gradients of solid tumors. Spheroids feature distinct cellular populations with a necrotic core, quiescent middle layer, and proliferative outer layer. Drug and liposome penetration are tracked with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and fluorescence imaging, showing that liposomal doxorubicin is stable to fluidic dosing and penetrates spheroids after 48 h. To provide a comprehensive pharmacodynamic profile of the distinct cellular regions within spheroids, we employ spatially stable isotopic labeling by amino acids in cell culture (spatial SILAC) proteomics to isotopically label the core and outer layers. Proteomic analysis reveals 714 upregulated proteins in the core upon treatment and 30 in the outer layers, as well as 103 downregulated proteins in the core and 1276 in the outer layers. Spatial SILAC uncovers the differential regulation of proteins associated with glycolysis, the TCA cycle, and lipid synthesis upon drug treatment between the spheroid core and outer layers. Using MALDI MSI and spatial SILAC proteomics, we interrogate the effects of dynamic dosing with liposomal doxorubicin on spheroid regions that would be overlooked by bulk analysis.

Cell-cell interactions are critical for the growth of organisms and maintaining homeostasis. In the tumor microenvironment, these interactions promote cancer progression. Given their importance in hea...

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Relative quantitation, used by most MS-based proteomics laboratories to determine protein fold-changes, requires samples being processed and analyzed together for best comparability through minimizing...