Mitoxantrone–Napabucasin Co-Nanoformulation Activates cGAS-STING in HCC Therapy

Introduction

Hepatocellular carcinoma (HCC) is one of the most common aggressive forms of liver cancer and is ranked as the sixth most prevalent cancer around the world. Although treatment has improved, HCC remains one of the leading causes of death in cancer patients mainly due to poor prognosis coupled with resistance towards traditional therapies. Through immune checkpoint inhibitors, specifically anti-PD-1 therapy, some cancers have been identified for potential treatment. However, these treatments have seen very minimal success in HCC because the TME is immunosuppressive.

TME in HCC forms a complex network that ultimately guards the cancer cells from being identified and targeted by the immune system. It has emerged as an important hindrance to the effectiveness of ICIs and there is an urgent need for novel strategies to reverse this immunosuppression. Chemotherapy, which was once thought of as direct cytotoxic activity against cancerous cells, has also proven to be an exciting way to remodel TME in an attempt to increase its immunogenicity.

Current research is directed towards activation of the cGAS-STING pathway, a key innate immunity mediator, for overcoming the defenses of TME. In this regard, the chemotherapeutic drug MIT activates the cGAS-STING pathway. This paper explains how a targeted co-delivery system using nanotechnology would make way in the immunosuppressive environment of HCC by combining MIT and a STAT3 inhibitor, napabucasin (NAP), to elevate the efficacy of immunotherapy.

Tumor Microenvironment and Immune Resistance

The tumor microenvironment (TME) in HCC is composed of tumor cells, immune cells, stromal cells, and the extracellular matrix. The TME is not a passive participant in the progression of cancer but is itself an active participant that causes factors conducive to tumor growth and invasion as well as immune evasion. Immune suppression is an intrinsic feature of the TME that allows tumor cells to evade detection by the immune response and proliferate consequently.

Normally, in a healthy immune response, the immunity cells of the body and more so the cytotoxic T cells should identify and kill the abnormal cells. However, in HCC, TME creates an immunosuppressive barrier, which has the features of such regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Such cells monitor the action of cytotoxic T cells, thereby becoming ineffective in the battle against the tumor.

There is also the immune checkpoint molecule PD-1 expressed on T cells and its ligand PD-L1 present on tumor cells, which further adds to suppression. The ICIs may block these interactions; yet, up to now, the successes with HCC have been poor because of the strongly immunosuppressive nature of TME. Breaking this resistance is crucial for ICI-based treatments to become more potent for HCC.

cGAS-STING Signaling Pathway

The cGAS-STING pathway is one of the key mechanisms designed by the innate immune system to detect foreign DNA within the cytoplasm of cells. With the detection of DNA from pathogens or damaged cells, the enzyme cGAS synthesizes a second messenger cyclic GMP-AMP (cGAMP). This molecule interacts with the STING receptor and initiates a signaling cascade that results in the production of type I interferons and pro-inflammatory cytokines.

These immune-stimulating molecules recruit and activate immune cells, such as dendritic cells and T cells, to spearhead an adaptive immunity response against the cancerous cells. The activation of the cGAS-STING pathway is hence crucial in initiating sufficient anti-tumor immune responses. However, many cancers downregulate this pathway as part of the means of escaping immune surveillance; HCC is not an exception.

More recent studies have also revealed some chemotherapeutic agents, such as mitoxantrone (MIT), which reactivates the cGAS-STING pathway to turn the suppressive TME that favors a tumor around. In doing so, it restores the immunity of the immune system in recognition and attacks against cancer cells, which indicates hopeful applications with immunotherapy.

The Role of Chemotherapy in Enhancing Immunotherapy

While chemotherapy has traditionally targeted rapidly proliferating cancer cells through its cytotoxic mechanism, this objective is changing with its role. In immunotherapy, chemotherapy may be used as an immunomodulatory agent to make the tumor more susceptible to immune system attacks.

The chemotherapeutic agent MIToxantrone (MIT) has been reported to trigger the cGAS-STING pathway in cancer cells. Activation leads to the expression of interferons and other immune-stimulatory molecules which can counteract such immunity-related evasion offered by HCC. In this regard, by induced expression of these molecules, MIT reorients the TME to be more permissive for infiltration and activation of immune cells in situ.

However, MIT may not be enough to fully counterbalance the immunosuppressive forces present within the TME. That is where NAP entry is a STAT3 inhibitor. A transcription factor that is overactivated within cancer cells, STAT3 promotes tumor growth, survival, and evasion of immunity. Inhibiting STAT3 further opens up the defenses of the TME for the immune response initiated by MIT.

Synergistic Effects of Mitoxantrone and Napabucasin (STAT3 Inhibitor)

The combination of MIT and NAP represents a potent strategy for combating the immune resistance of HCC. While MIT activates the cGAS-STING pathway and induces an immune response, NAP amplifies this effect by blocking STAT3, a key regulator of immune suppression in the TME.

STAT3 plays a critical role in the recruitment and activation of immunosuppressive cells within the TME, including Tregs, MDSCs, and TAMs. By inhibiting STAT3, NAP reduces the influence of these cells, allowing for a more robust anti-tumor immune response. The combination of MIT’s immune activation and NAP’s immune suppression reversal creates a synergistic effect that can significantly enhance the efficacy of immunotherapy.

However, delivering these two drugs effectively to the tumor site is a challenge. Traditional methods of drug delivery often result in poor bioavailability, off-target effects, and toxicities. This has led researchers to explore the use of nanotechnology to improve drug delivery and maximize therapeutic outcomes.

Nanotechnology for Drug Delivery: AEAA-PEG-PLGA Nanocarrier

Nanotechnology offers a revolutionary approach to drug delivery in cancer therapy. Nanocarriers can encapsulate therapeutic agents, protecting them from degradation in the bloodstream and delivering them directly to the tumor site. This targeted approach reduces systemic side effects and ensures that higher concentrations of the drugs reach the tumor, enhancing their efficacy.

In this study, researchers developed an aminoethyl anisamide (AEAA)-targeted polyethylene glycol (PEG)-modified poly(lactic-co-glycolic acid) (PLGA)-based nanocarrier for the co-delivery of MIT and NAP. The nanocarrier is designed to target cancer cells more effectively, taking advantage of overexpressed receptors on the surface of HCC cells.

The PEG-modified PLGA ensures that the nanocarrier remains stable in the bloodstream, while the AEAA targeting moiety enhances its ability to bind to cancer cells. This precision targeting allows for the simultaneous delivery of MIT and NAP to the tumor site, maximizing their therapeutic effects.

Preclinical Evaluation: In Vivo Studies on Mice

In vivo studies were conducted using mice orthotopically grafted with HCC to evaluate the efficacy of the co-nanoformulation. The results were promising, demonstrating that the co-delivery of MIT and NAP via the nanocarrier significantly improved outcomes compared to either drug alone or traditional delivery methods.

The co-nanoformulation activated the cGAS-STING signaling pathway, leading to the production of immune-stimulating molecules that reshaped the immunosuppressive TME. This remodeling of the TME allowed for increased infiltration of immune cells into the tumor, enhancing the efficacy of anti-PD-1 therapy.

Mice treated with the co-nanoformulation showed a significant reduction in tumor growth, extended survival, and reduced tumor recurrence compared to control groups. These findings suggest that the combination of MIT and NAP delivered through a targeted nanocarrier, has the potential to transform the treatment of HCC by overcoming immune resistance and enhancing the effects of immunotherapy.

Clinical Implications and Future Directions

The findings from this study have significant implications for the future of HCC treatment. By combining chemotherapy with immunotherapy and leveraging nanotechnology for targeted drug delivery, researchers have developed a novel approach that addresses the immune resistance inherent in the TME of HCC.

While these results are promising, further research is needed to translate these findings into clinical practice. Human clinical trials will be essential to confirm the safety and efficacy of the MIT and NAP co-nanoformulation. Additionally, researchers may explore the potential for combining this approach with other immunotherapies or expanding its use to other cancers with similar immunosuppressive TMEs.

Conclusion

This study revealed the possibility that mitoxantrone (MIT) might unleash the cGAS-STING signaling pathway, thus reprogramming the suppressive hepatocellular carcinoma tumor microenvironment. A potential combination of MIT with a small-molecule STAT3 inhibitor napabucasin (NAP) might enhance the immune responses and improve the efficiency of therapies targeting immune checkpoints. MIT and NAP co-delivered using a targeted nanocarrier to ensure the drugs reach the tumor location most effectively and result in therapeutic benefits.

This is an important step in overcoming the problem of immune resistance in cancer therapy and improving the prospect of long-term survival in patients with HCC. Whether this new promising approach becomes a new standard of care for treating liver cancer remains to be seen by continuing research and clinical trials.

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