Biomimetic Nanovesicles Target Senescent-Escape Cancer Stem Cells in Breast Cancer

Abstract

Although such progress has been achieved in breast cancer research, BC remains one of the most common causes of cancer-related deaths worldwide and most of these remain due to persisting CSCs that cause recurrence and metastasis. Recently, cellular senescence has emerged as a contributing factor to chemotherapy-induced increases in the population size of senescent-escape CSCs that exhibit immune evasion with enhanced chemoresistance. This paper reports a novel therapeutic approach using pH-responsive biomimetic nanovesicles, covered with single-chain variable fragment anti-CD276, for selective targeting and elimination of senescent-escape CSCs. The nanovesicles are designed to transport siRNA targeting caldesmon (siCALD1) and Adriamycin (ADR), inducing apoptosis within CSCs and suppressive immune checkpoint molecules CD276 and PD-L1. In addition, Mn2+ addition contributes to raising the level of immune activation, hence favoring CD8+ T cell infiltration and lowering immune evasion. Assaying the compound in humanized mice models for breast cancer confirmed highly significant inhibition of tumor growth in the context of primary, recurrent, metastatic, and abscopal conditions. This study opens the potential of the dual-targeting approach for overcoming chemoresistance and improving the efficacy of immunotherapy, hence offering a promising novel avenue toward improved outcomes in breast cancer treatment.

Introduction

One of the most common cancers affecting millions of women every year, breast cancer is a major threat worldwide. Recurrence and metastasis, despite advances in therapy comprising chemotherapy, targeted therapies, and immunotherapy, still pose considerable challenges in advanced breast cancer. The existence of cancer stem cells (CSCs) -- a small subpopulation within the tumors that exhibits stem-like properties, including self-renewal, differentiation, and chemoresistance a critical factor behind these problems. This latter capacity for treatment resistance and dormancy usually causes the disease to recur eventually.

Increasingly documented as a double-edged sword in the concept of breast cancer, chemotherapy-induced cellular senescence is a phenomenon that transiently suppresses tumor progression but at the same time enhances the proportion of CSCs, which have a high survival and evasion capacity from immune escape even with aggressive treatment strategies. Thus, being one of the main causes of recurrence and metastasis, selectively killing these ESCs highlights one of the major goals for changing clinical outcomes.

Recent findings have shown that multicellular drug-resistant breast cancer cells showed overexpression of CD276, more commonly known as B7-H3, a molecule that promotes the immunoevasion process mediated by senescent-escape CSCs. The overexpression of the caldesmon (CALD1) gene in Adriamycin (ADR)-resistant breast cancer cells promotes the survival and chemoresistance of these CSCs, due to its antiapoptotic effect and promotion of CD276 and PD-L1 expression levels that decrease the activity of the immune system.

In this article, we describe a promising therapeutic approach targeting the selective killing of senescent-escape CSCs. The strategy comprises a thermosensitive, controlled-release hydrogel filled with pH-sensitive biomimetic nanovesicles coated with scFV against CD276. The former facilitated the concurrent delivery of CALD1 siRNA (siCALD1) and ADR, which led to the induction of apoptosis in the CSCs and reduced their immune evasion capability, thereby enhancing immune surveillance. This might bring a new light to the treatment of primary, recurrent metastatic, and abscopal breast cancers and maybe improve the effectiveness of immunotherapy.

The Challenge of Senescent-Escape Cancer Stem Cells (CSCs) in Breast Cancer

Probably the most significant challenge in the management of breast cancer is the potential of CSCs to avoid standard therapies, including chemotherapy and targeted therapies. Self-renewal and the ability to differentiate into diverse cell types within a tumor are the hallmarks of CSCs. Such an ability propels heterogeneity within the tumor, recurrence, and metastasis. Furthermore, CSCs exhibit profound resistance to chemotherapy as well as radiation when compared to their non-CSC counterparts, which explains why they remain an ongoing threat to long-term remission.

The latest experiments revealed that chemotherapy-induced senescence state in which cancer cells stop proliferating but remain metabolically active paradoxically increases the proportion of CSCs within a tumor. Although it appears to temporarily halt a tumor's growth, senescence eventually facilitates CSCs to exist in a dormant state that allows them to survive and evade immune surveillance. In the end, such senescent-escape CSCs re-enter the cell cycle, culminating in recurrence and metastasis of the tumor.

This process of how senescent cells escape and become reprogrammed to become more aggressive CSCs is not well understood, but alterations in the tumor microenvironment and immune surveillance machinery could be involved. Senescent-escape CSCs are better adapted to an immunologically ignorant environment. Important candidates for this are the overexpression of immune checkpoint molecules, including CD276 and PD-L1, which inhibit the ability of the immune system to detect and remove such cells.

CD276 and CALD1: Drivers of Immune Evasion and Chemoresistance

CD276, also known as B7-H3, is an immune checkpoint protein that is overexpressed in many solid tumors, including breast cancer. High levels of CD276 expression are associated with poor prognosis, increased tumor aggressiveness, and resistance to therapy. In multidrug-resistant breast cancer cells, CD276 expression is particularly high (95.60%), allowing senescent-escape CSCs to evade immune detection and continue proliferating.

Another critical player in the survival of senescent-escape CSCs is caldesmon (CALD1), a gene involved in cytoskeletal regulation. CALD1 is upregulated in ADR-resistant breast cancer cells, where it promotes the survival of CSCs in an anti-apoptotic state. This gene not only enhances chemoresistance but also upregulates CD276 and PD-L1, further enabling immune evasion.

The combined action of CD276 and CALD1 creates a powerful shield that allows CSCs to survive chemotherapy, escape immune surveillance, and contribute to tumor recurrence. These molecules represent promising therapeutic targets for selectively eradicating CSCs and overcoming chemoresistance in breast cancer.

Biomimetic Nanovesicles: A Precision Delivery System for Targeting CSCs

To effectively target senescent-escape CSCs and overcome the immune evasion driven by CD276 and CALD1, a novel delivery system is required. Researchers have developed a controlled-release, thermosensitive hydrogel containing pH-responsive biomimetic nanovesicles designed to selectively deliver therapeutic agents to CSCs. These nanovesicles are engineered to fuse with the cell membranes of senescent-escape CSCs, delivering a combination of siCALD1 and ADR.

The nanovesicles are coated with an anti-CD276 scFV, a small antibody fragment that selectively binds to CD276-expressing cells. This coating allows the nanovesicles to target senescent-escape CSCs with high specificity, sparing healthy cells and minimizing off-target effects. Once the nanovesicles bind to the CSCs, they fuse with the cell membrane, releasing their therapeutic payload in a controlled manner.

The pH-responsive nature of the nanovesicles ensures that the release of siCALD1 and ADR is triggered by the acidic microenvironment of the tumor, further enhancing the precision of this delivery system. This targeted approach maximizes the therapeutic impact on CSCs while minimizing systemic toxicity.

Dual Attack: siCALD1 and ADR Induce Apoptosis and Reverse Immune Evasion

The combination of siCALD1 with ADR delivered by biomimetic nanovesicles is so designed that it elicits apoptosis in CSCs while being integrated with the reversal of immune evasion. Targeting the CALD1 gene for a small interfering RNA in CSCs, siCALD1 disturbs the anti-apoptotic state, making them more susceptible to chemotherapy. Silencing CALD1 reduces the expression of CD276 and PD-L1, the two principal molecules used by CSCs for immune evasion.

Adriamycin (ADR) is one of the most commonly used chemotherapeutic agents, which synergizes with siCALD1 to induce apoptosis in CSCs, as ADR alone may not overcome resistance in CSCs, but siCALD1, along with ADR, enhances the cytotoxic effects of ADR into the selective killing of senescent-escape CSCs.

This therapy also upregulates major histocompatibility complex class I molecules on the surface of CSCs, thereby enhancing apoptosis. MHC I molecules provide key target sites for the recognition of carcinoma cells by cytotoxic CD8+ T cells. Thus, the treatment enhances the immune system's detection and elimination abilities in CSCs.

Mn2+ Enhancement: Boosting Immune Surveillance and T-Cell Infiltration

One of the key innovations in this approach is the incorporation of manganese (Mn2+) into the nanovesicle shell. Mn2+ plays a vital role in enhancing immune surveillance by activating the STING (stimulator of interferon genes) pathway, a critical component of the innate immune response. Activation of the STING pathway leads to the production of type I interferons, which stimulate the recruitment and activation of immune cells, including CD8+ T cells.

In the context of this therapeutic strategy, Mn2+ works synergistically with siCALD1 and ADR to promote CD8+ T cell infiltration into the tumor microenvironment. This enhanced immune response not only helps to eliminate CSCs but also creates a more hostile environment for the survival of any remaining tumor cells. By combining chemotherapy with immune activation, this approach offers a powerful one-two punch against breast cancer recurrence and metastasis.

Preclinical Evaluation: Humanized Mice Models for Primary, Recurrent, Metastatic, and Abscopal Breast Cancer

To assess the efficacy of this novel therapeutic approach, preclinical studies were conducted in humanized mice models representing various stages of breast cancer, including primary, recurrent, metastatic, and abscopal disease. Humanized mice are engineered to possess a functional human immune system, allowing for the evaluation of immune responses in a more clinically relevant setting.

In these models, the controlled-release hydrogel containing anti-CD276 scFV-coated nanovesicles was administered to mice bearing tumors derived from ADR-resistant breast cancer cells. The results demonstrated a significant reduction in tumor growth across all models, with notable improvements in survival and a decrease in metastatic spread.

In particular, the abscopal effect, where treatment of a primary tumor leads to the regression of distant, untreated tumors, was observed. This phenomenon is likely due to the enhanced immune activation achieved by the combination of siCALD1, ADR, and Mn2+, which primes the immune system to attack tumor cells throughout the body.

Clinical Implications and Future Directions

The development of this controlled-release, biomimetic nanovesicle strategy represents a breakthrough in the treatment of breast cancer. By selectively targeting senescent-escape CSCs and reversing immune evasion mechanisms, this approach offers a promising solution to one of the most difficult challenges in cancer therapy: the elimination of chemotherapy-resistant, immune-evasive cancer stem cells.

As this approach moves toward clinical trials, there are several key questions that will need to be addressed. These include optimizing the dosing and delivery of the nanovesicles, evaluating potential toxicities, and determining the long-term durability of the immune response. Additionally, it will be important to explore the potential of this strategy in combination with other immunotherapies, such as checkpoint inhibitors, to further enhance its efficacy.

Conclusion

In summary, the anti-CD276 scFV-coated biomimetic nanovesicles provide a novel route to overcome chemoresistance and immune evasion by breast carcinoma by effectively delivering siCALD1 and ADR. The described mechanism selectively targets senescent-escape CSCs that provide enhanced immunity for the treatment of primary, recurrent, metastatic, and abscopal breast carcinoma. It could completely change the approach to cancer treatment by integrating such a strategy with other immunotherapies by providing a longer survival period and lesser recurrence. This innovative therapy could be the cornerstone of the near-future personalized breast cancer treatment as research grows.

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