Scaffold-mediated switching of lymphoma metabolism in culture

Abstract

Background: Diffuse large B cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and accounts for about a third of all NHL cases. A significant proportion (~40%) of treated DLBCL patients develop refractory or relapsed disease due to drug resistance which can be attributed to metabolomic and genetic variations amongst diverse DLBCL subtypes. An assay platform that reproduces metabolic patterns of DLBCL in vivo could serve as a useful model for DLBCL.

Methods: This report investigated metabolic functions in 2D and 3D cell cultures using parental and drug-resistant DLBCL cell lines as compared to patient biopsy tissue.

Results: A 3D culture model controlled the proliferation of parental and drug-resistant DLBCL cell lines, SUDHL-10, SUDHL-10 RR (rituximab resistant), and SUDHL-10 OR (obinutuzumab resistant), as well as retained differential sensitivity to CHOP. The results from metabolic profiling and isotope tracer studies with D-glucose-¹³C₆ indicated metabolic switching in 3D culture when compared with a 2D environment. Analysis of DLBCL patient tumor tissue revealed that the metabolic changes in 3D grown cells were shifted towards that of clinical specimens.

Conclusion: 3D culture restrained DLBCL cell line growth and modulated metabolic pathways that trend towards the biological characteristics of patient tumors. Counter-intuitively, this research thereby contends that 3D matrices can be a tool to control tumor function towards a slower growing and metabolically dormant state that better reflects in vivo tumor physiology.

Keywords: 3D model; DLBCL; Drug sensitivity; Lymphoma; Metabolic flux; Proliferation.

Fig. 1

Schematic representation of expansion and recovery of DLBCL cell lines in 3D hydrogel “VitroGel®”

Fig. 2

Cell proliferation profile of A SUDHL-10, B SUDHL-10 RR, and C SUDHL-10 OR and D doubling time variations of all three cell lines in 2D and 3D. Values indicate mean± SD. Experiments were done in triplicate. A 2-tailed t-test was used to determine significance between 2D and 3D denoted by *** (p<0.0001), ** (p<0.005), and * (p<0.05)

Fig. 3

Quantification of SUDHL-10 cell cycle analysis performed using flow cytometry from three independent experiments A overlay plot, day 1 (B) and day 5 (C). Values indicate mean ± SD. Experiments were done in triplicate. A 2-tailed t-test was used to determine significance between 2D and 3D denoted by *** (p<0.0001), ** (p<0.005) and * (p<0.05)

Fig. 4

Effect of CHOP on the viability of SUDHL-10 (A), SUDHL-10 RR (B), and SUDHL-10 OR (C) in 2D and 3D determined by CCK-8 assay. Cells were allowed to grow for 24 h and then treated with increasing concentrations of CHOP for 48 h. Error bars represent the standard deviation of means from replicates consisting of three independent experiments. A 2-tailed t-test was used to determine significance ** (p<0.005) and * (p<0.05) when compared to 2D

Fig. 5

Metabolic profiling of DLBCL cell lines in 2D vs 3D. Analysis of extracellular metabolites using Cedex Bioanalyzer: A glucose, B glutamine, and C lactate. Data is normalized to the ratio of starting to final cell densities. Data obtained from n=3 repeats, values indicate mean± SD. A 2-tailed t-test was used to determine significance ** (p<0.005) and * (p<0.05). D LC-MS analysis of intracellular metabolite exhibiting differential pattern between 2D vs 3D

Fig. 6

3D grown DLBCL cell lines show decreased nucleotide metabolism. A Total pool size of metabolites in SUDHL10, SUDHL10OR, and SUDHL10RR cells cultured in 2D vs 3D conditions for 48 h. B Summary of metabolic pathway changes represented by labels indicated in red (increased) or blue (decreased) in 3D cultured cells compared to 2D

Fig. 7

Comparison of DLBCL metabolite concentrations by quantitative mass spectrometry. Bar graphs represent the concentration of individual metabolites normalized per milligram of total protein (y-axis), in DLBCL tumors (n=10) and triplicates of SUDHL-10 cells cultured in 2D or 3D (x-axis). Error bars represent the standard deviation of means by replicates. Significant differences in metabolite concentration between the sample groups are denoted by *** (p<0.0001), ** (p<0.005), and * (p<0.05)