Shift from stochastic to spatially-ordered expression of serine-glycine synthesis enzymes in 3D microtumors

Abstract

Cell-to-cell differences in protein expression in normal tissues and tumors are a common phenomenon, but the underlying principles that govern this heterogeneity are largely unknown. Here, we show that in monolayer cancer cell-line cultures, the expression of the five metabolic enzymes of serine-glycine synthesis (SGS), including its rate-limiting enzyme, phosphoglycerate dehydrogenase (PHGDH), displays stochastic cell-to-cell variation. By contrast, in cancer cell line-derived three-dimensional (3D) microtumors PHGDH expression is restricted to the outermost part of the microtumors’ outer proliferative cell layer, while the four other SGS enzymes display near uniform expression throughout the microtumor. A mathematical model suggests that metabolic stress in the microtumor core activates factors that restrict PHGDH expression. Thus, intracellular enzyme expression in growing cell ecosystems can shift to spatially ordered patterns in 3D structured environments due to emergent cell-cell communication, with potential implications for the design of effective anti-metabolic cancer therapies.

Modeling HIV-1 neuropathogenesis using three-dimensional human brain organoids (hBORGs) with HIV-1 infected microglia

“…Based on the results obtained with our mixed differentiation of brain cells from primary NPCs in 2D monolayers, we next developed a 3D culture platform to generate brain organoids. We adapted a hydrogel microwell platform previously described for generation of uniform size microtumors to generate uniform size brain organoids in a high throughput manner [41][42][43][44][45] . NPCs were seeded onto the hydrogel devices containing multiple 600 μm microwells that enabled formation of aggregates, referred henceforth as neurospheres (NS) within 24-48 h ( Fig.…”

“…Fabrication of hydrogel microwell arrays. Non-adhesive hydrogel microwell arrays containing 70-80 microwells per 1 × 1 cm 2 were microfabricated using polyethylene glycol dimethacrylate (PEGDMA, 1000 Da, Sigma) and polydimethyl siloxane (PDMS) molds as described previously [41][42][43][44][45] . Each microwell was 600 µm in diameter and 600 µm in depth.…”

Targeting the Temporal Dynamics of Hypoxia-Induced Tumor-Secreted Factors Halts Tumor Migration

“…Typical in vitro three-dimensional (3D) cultures such as spinner flasks (22-24) generate a broad size distribution of multicellular aggregates (microtumors), which present a technical difficulty for systematically studying the effect of tumor sizeinduced hypoxic microenvironment. To engineer controlled local microenvironment that is purely defined by microtumor size and tumor-secreted factors, we adopted microfabricated hydrogel microwells to generate hundreds of microtumors of uniform yet precisely controlled sizes from multiple cancer cell lines including breast, prostate and, head and neck cancer (24)(25)(26)(27)(28). Precise control over microtumor size translated in controlled hypoxic and metabolic microenvironments (25)(26)(27)(28).…”

“…To engineer controlled local microenvironment that is purely defined by microtumor size and tumor-secreted factors, we adopted microfabricated hydrogel microwells to generate hundreds of microtumors of uniform yet precisely controlled sizes from multiple cancer cell lines including breast, prostate and, head and neck cancer (24)(25)(26)(27)(28). Precise control over microtumor size translated in controlled hypoxic and metabolic microenvironments (25)(26)(27)(28). By manipulating microtumor size alone and without any genetic manipulations or exogenous stimuli, we reproducibly generated two distinct phenotypes from the same noninvasive parent breast cancer cells: small nonhypoxic microtumors represent nonmigratory phenotype whereas large hypoxic microtumors exhibit migratory phenotype (25,26,28).…”

PHGDH as a mechanism for resistance in metabolically-driven cancers

“…As previously described, serine supports a variety of cellular processes, including amino acid, nucleotide, and lipid synthesis, increased DNA methylation, and indirect αKG generation. De novo serine biosynthesis can in turn drive the synthesis of glycine, as well as the synthesis of cysteine from homocysteine within the methionine cycle, supporting protein synthesis [ 8 , 52 ] . Glycine is also directly incorporated into purine nucleotides.…”