Poster C9 - Investigating the impact of lymphoid tumor microenvironment cues (Ly-TME) on the spatial organization of B cell Receptor signaling in Activated B cell Diffuse Large B cell Lymphoma

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Pratul Amineni (he/him/his)

Student
Georgia Institute of Technology
Canton, Georgia, United States

Co-Author(s)

DB

Deepali Balasubramani, DB

Graduate Student
Coulter Department of Biomedical Engineering at Emory University and Georgia Tech, United States

Primary Investigator(s)

AS

Ankur Singh

Professor
Georgia Insitute of Technology, United States

Introduction: Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), one of the more aggressive forms of non-Hodgkin lymphoma, accounts for one-third of all DLBCL cases. There is an urgent need to develop new therapeutic interventions, as 40% of patients either relapse after an initial response or do not respond to current therapies. ABC DLBCL depends on constitutive activation of nuclear factor κB (NF-κB) driven by B cell receptor (BCR), phosphoinositide 3-kinase (PI3K) and toll like receptor 9 (TLR9) pathways. Previous work, using our bioengineered lymphoma organoids, demonstrates that the cellular components and biophysical factors of the lymphoid tumor microenvironment (Ly-TME) enhances BCR- signaling and attenuates response to BCR pathway inhibitors in ABC DLBCL. However, the impact of Ly-TME cues on the spatial expression and colocalization of BCR signaling components in DLBCL is not known. With our bioengineered B cell lymphoma organoids, we further aim to investigate the role of Ly-TME factors and BCR targeted therapies on the colocalization of BCR signaling components. The proximity ligation assay (PLA) will be used to detect and visualize protein interactions between BCR signaling components within tens of nanometers from each other, which include Bruton's tyrosine kinase (BTK), Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), mammalian target of rapamycin (mTOR) etc.

Materials and

Methods: Hydrogel-based organoids were composed of 4 arm Maleimide functionalized polyethylene glycol (PEG-4-MAL) functionalized with integrin specific peptides and crosslinked with matrix metalloproteinase (MMP) degradable, VPM (GCRDVPM↓SMRGGDRCG peptide), and nondegradable, Dithiothreitol (DTT), peptides. VPM and DTT were incorporated at 1:1 ratio. 5wt% and 10 wt% PEG-4MAL organoids were functionalized with REDV oligopeptide (GREDVGC), a vascular cell adhesion molecule (VCAM-1) mimetic that binds to α4β1 integrin. ABC DLBCL cells (40K cells/organoid) were encapsulated in the organoids and were cultured for 24 hours prior to fixation with 4% paraformaldehyde (PFA) for 20 minutes and permeabilization with methanol for 10 minutes. The organoids were blocked for 30 minutes with the duolink blocking buffer and were incubated overnight with rabbit anti-human MALT1 and mouse anti human mTOR primary antibodies, diluted in Duolink Antibody Diluent. The next day, organoids were incubated with the secondary PLUS and MINUS probes for 1 hour. Ligation and amplification steps were completed using the Duolink in situ Detection Reagents Orange kit according to the manufacturer’s instructions. DAPI was added for 10 minutes and the organoids were washed and mounted with 10% glycerol. Z stacks images capturing DLBCLs were acquired using the Zeiss LSM 900 Confocal microscope and the number of punctas were quantified. PLA scores were determined by normalizing the number of PLA spots counted in each sample to the average number of PLA spots counted in the control sample, set to 1. Box and whisker plots demonstrate the median PLA score; unpaired t tests were used to compare groups.

Results, Conclusions, and Discussions: In our study, we focused on elucidating the role of stiffness of the Ly-TME on the spatial localization and interactions of two BCR components -MALT1 and mTOR. Organoid stiffness could be varied by increasing the PEGwt% of the PEG-4MAL organoids. From our preliminary results, we observed a significant increase in the number of MTOR-MALT1 punctas in the cytoplasm of ABC DLBCL cells encapsulated in 5wt% organoids compared to 10 wt%. This suggests that as the stiffness of the microenvironment increases, the colocalization of MALT1-mTOR decreases. Cells sense variations in Ly-TME stiffness through integrin associated signaling followed by conformational changes in focal adhesion kinase complexes, downstream F-actin polymerization and activation of transcription factors which can affect long term cell processes such as differentiation, proliferation etc. Changes in the cytoskeleton due to increased stiffness and increased cell-ECM interactions could attenuate the mobility of BCR signaling components and could result in decreased colocalization. Future studies involve exploring the role of stiffness on the colocalization of MALT1 with mTOR as well as BTK, to validate current findings. We also aim to explore the impact of other Ly-TME factors such as CD40L, survival signals from T follicular helper cells in the microenvironment, that have been shown to attenuate the effect of BCR targeted therapies such as MALT1i. We further aim to investigate the impact of single and combinatorial BCR targeted therapies on protein interactions.

Acknowledgements (Optional):