Nancy Andon, MSc
Scientists Develop 3D Platform using PBMCs from HemaCare
How a 3D model is helping establish a library of compounds that enhance cytotoxic T cell activity in vivo
An independent publication cites the use of HemaCare-sourced PBMC as starting material for an assay designed to screen for factors that enhance anti-tumor cytotoxic T cell activity.
The findings appeared in the journal Nature and reflected research by Novartis scientists in San Diego to develop a discovery platform that more closely mimics the in-vivo tumor microenvironment.
Anti-tumor T cell activity is a primary focus of cancer immunotherapy treatments. Unfortunately, some cancer cells can create a microenvironment that limits the effectiveness of T cell mediated immunotherapy.
The Novartis research group reasoned that if they were better able to mimic the tumor microenvironment, they would increase the chances of discovering clinically relevant factors capable of enhancing cytotoxic T cell function . 2D cell culture systems, while useful, fail to replicate some key features of tumors in vivo. The fact that tumors are three dimensional creates gradients of oxygen and nutrient penetration. Cells located further inside the center of the tumor display characteristic pathologies such as sections of hypoxia, and acidic conditions. These conditions make it more difficult for cytotoxic T cells to infiltrate the tumor and function efficiently.
The researchers made the decision to use a 3D cell culture platform as part of their strategy to model clinically relevant tumor physiology.  The scientists began their experiments by using cryopreserved HemaCare-sourced PBMCs collected from healthy donors (Fig. 1). Cellular starting material viability and purity are directly related to downstream efficacy, so raw material sourcing is an important consideration factor for this type of study.
PBMCs were thawed and cultured in the presence of specific peptides and cytokines known to favor the growth, activation, and expansion of CD8+/CD127+ cytotoxic memory T cells.
Once the PBMC culture consisted mainly of CD8+/CD127+ T cells, human colorectal tumor cells were plated into spheroid plates, allowing them to form 3D structures as they would in vivo. Examination confirmed that the tumor spheroids displayed substantial areas of hypoxia, necrotic cores, and acidic pH. CD8+/CD127+ T cells were added to the tumor spheroid cultures at a 3:1 ratio, along with the experimental test compounds. Results were collected over a period of 3 days.
The research group tested a total 1770 small molecule compounds which were known or suspected to enhance cytotoxic T cell activity. While many of the compounds demonstrated cytotoxicity, not all of this activity was T cell dependent. The study therefore focused solely on those compounds that displayed anti-tumor activity only in the presence of T cells, and only in an antigen-dependent manner. Compounds displaying strong anti-tumor activity meeting these criteria fell into a few distinct categories:
- Compounds that negatively affected NF-κB or IL-4 signaling
- Cyclin-dependent kinase inhibitors, specifically CDK4 and/or CDK6
- Bromodomain inhibitors
- Compounds targeting other proteins with previously unreported T cell modifying activities
Further studies on the leading compounds showed that CDK4/6 inhibitors may increase antigen presentation by target cells and antigen recognition by cytolytic T cells. Bromodomain inhibitors, on the other hand, functioned by enhancing expansion of cytotoxic CD8+ T cell populations, and increasing CD8+ T cell tumor infiltration, IFN gamma secretion, and degranulation. One compound with particularly strong anti-tumor cell activity was shown to become activated in hypoxic environments and exerted its effects by enhancing T cell proliferation and by upregulating the well-known checkpoint inhibitor PD-1. Checkpoint inhibitors are widely used in cancer immunotherapy to help prevent immune suppression by cancer cells.
Interestingly, by profiling PBMCs collected from 9 different HemaCare donors, the scientists were able to identify antigen-dependent responders, antigen-independent responders, and non-responders to various target compounds. T cell characterization of individual donors showed a tight correlation between activation of specific T cell cytotoxic markers and anti-tumor response. This finding indicates that it may be possible to maximize therapeutic responses by sorting patients into treatment groups according to their likelihood of responding to a particular therapy.
The Novartis scientists plan to continue using their new 3D tumor model discovery platform to establish a library of compounds that enhance cytotoxic T cell activity in vivo. They will also continue with more detailed studies on how their lead targets exert their effects.
To J., et al. A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function. Nature Communications. 1-4. Dec 2020.
Lagies S., et al. Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells. Nature Communications. 3(246). May 2020
This article was written by free-lance science writer Nancy Andon, MSc. It originally appeared on a blog managed by HemaCare, which Charles River acquired in 2020. HemaCare sells biological products such as T cells, stem cells, bone marrow, plasma, and other medical products.