David Taylor, Ph.D., a scientist in Cayman's Assay Research & Development group, discussed how to modulate ENPP1 expression and STING activation in vitro with LipidLaunch™ loadable lipid nanoparticles (LNPs) in a recent webinar. He also explored the contribution of ovarian cancer cell-derived extracellular vesicles (EVs) in the tumor microenvironment. 

We have summarized four key takeaways from this webinar here.

Key Takeaways

• Loadable LNPs enabled effective siRNA-mediated ENPP1 knockdown in PA-1, SK-OV-3, and CaoV-3 cell lines. 

• ENPP1 associated with EVs may exert paracrine immunosuppressive functions in the tumor microenvironment.

• Loadable LNPs enhanced 2'3'-cGAMP delivery to and STING activation in THP-1 macrophages.
  

• PA-1-derived EVs containing active ENPP1 inhibited 2'3'-cGAMP-induced cytokine release in THP-1 macrophages.
  
  

Background

LipidLaunch™ Loadable LNPs

LipidLaunch™ Research-Ready LNPs & Reagent Kits

Loadable LNPs are one of four available options from Cayman's line of LipidLaunch™ research-ready LNPs and reagent kits. Available in various formats, they offer researchers flexibility in cargo selection, LNP formulation, and application.

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The cGAS-STING Pathway in Cancer

Cyclic GMP-AMP synthase (cGAS) produces the cyclic dinucleotide (CDN) second messenger 2’3’-cGMP in response to cytosolic DNA. 2’3’-cGMP can be exported out of cells, where it is taken up by neighboring cells to activate stimulator of interferon genes (STING), leading to initiation of the type 1 interferon (IFN) response and antitumor immunity. 

Figure 1. 2'3'-cGAMP is produced by cGAS in response to cytosolic DNA. 2'3'-cGAMP can be taken up by healthy host cells in the tumor microenvironment, leading to STING activation and antitumor immunity. 

ENPP1 in Cancer

Cancerous cells can also express ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a membrane-bound 2'3'-cGAMP hydrolase, which leads to immune suppression by suppressing STING activation. ENPP1 is upregulated in several cancers, including ovarian, liver, breast, thyroid, and pancreatic cancers, and is associated with poor prognosis.

Figure 2. ENPP1 degrades extracellular 2'3'-cGAMP to inhibit paracrine STING signaling and antitumor immunity.

Our Findings

1.  Loadable LNPs enabled effective siRNA-mediated ENPP1 knockdown in PA-1, SK-OV-3, and CaoV-3 cell lines.

ENPP1 knockdown using siRNA delivered via loadable LNPs was achieved in PA-1, SK-OV-3, and CaoV-3 ovarian cancer cell lines. In PA-1 cells, ENPP1 silencing enhanced 2'3'-cGAMP levels in culture media, indicating the ENPP1 silencing enhanced 2'3'-cGAMP export from PA-1 cells.  

2.  ENPP1 associated with extracellular vesicles (EVs) may exert paracrine immunosuppressive functions in the tumor microenvironment.

PA-1 cells were found to secrete ENPP1-containing extracellular vesicles (EVs), which were able to degrade extracellular 2'3'-cGAMP. siRNA-mediated silencing of ENPP1 via loadable LNPs also reduced ENPP1 expression and activity in PA-1 cell-derived EVs.

3. Loadable LNPs enhanced 2'3'-cGAMP delivery and STING activation in THP-1 macrophages.

Loadable LNPs containing 2'3'-cGAMP were found to enhance 2'3'-cGAMP uptake in THP-1 macrophages, resulting in STING activation and production of IL-6 and IL-1β. 

4. PA-1-derived EVs containing active ENPP1 inhibited 2'3'-cGAMP-induced cytokine release in THP-1 macrophages.

When EVs containing ENPP1 were administered with 2'3'-cGAMP directly in cell media, there was no change in IL-6 or IL-1β production. However, EVs containing ENPP1 reduced the production of IL-6 and IL-1β induced by 2'3'-cGAMP delivered via LNPs. This finding suggests that there is the possibility of an interaction occurring between the EVs and LNPs, reducing the level of 2'3'-cGAMP being delivered to THP-1 macrophages. 

Conclusions

PA-1 cancer cells express high levels of ENPP1 and export ENPP1 in EVs, resulting in lower levels of 2'3'-cGAMP. When ENPP1 is silenced in PA-1 cancer cells with LNP-mediated siRNA knockdown, there is reduced expression of ENPP1, resulting in higher concentrations of 2'3'-cGAMP in the media, which is immunostimulatory. 

Figure 3. siRNA-mediated ENPP1 knockdown with LipidLaunch™ loadable LNPs increases 2'3'-cGAMP levels in cancer cells. 

LNPs can be used to deliver 2'3'-cGAMP to THP-1 macrophages, promoting STING activation. However, this response could be attenuated by PA-1-derived EVs, which is likely attributable to ENPP1 activity in these EVs. siRNA-mediated ENPP1 knockdown with LipidLaunch™ loadable LNPs is a potential approach to restoring antitumor immunity in the tumor microenvironment.
 

Figure 4. siRNA-mediated ENPP1 knockdown with LipidLaunch™ loadable LNPs increases 2'3'-cGAMP levels, restoring antitumor immunity in the tumor microenvironment. 

Cayman Products Used in this Study