Engineering an Immunoactive Tumor Microenvironment Utilizing Cold-Responsive Nanoparticles for Cryoimmunotherapy of Breast Cancer

Introduction: Cancer immunotherapy represents a groundbreaking approach that leverages the body’s immune system for combating cancers1. However, its effectiveness is greatly compromised by the presence of an immunosuppressive tumor microenvironment (TME)2, impeding the immune cells’ tumor-eradicating function. Cryosurgery, a minimally invasive surgical technique employing “frostbite” temperature to induce tumor destruction and trigger immunogenic cell death-mediated immune response, shows promise in modulating the TME and generating cryoimmunotherapy to inhibit the growth of localized tumors. However, its potential in combating distant or metastatic tumors, either using cryosurgery alone or in combination with other therapies, is missing in literature. Furthermore, the effect of combining cryosurgery with cold-responsive nanoparticles, particularly those carrying immunotherapeutic agents, on distant or metastatic tumors has not been explored so far. In this study, we develop innovative cold-responsive nanoparticles (CRNPs) to transform the TME from immunosuppressive “cold” state to immunologically active “hot” state through co-delivery of chemo- and immunotherapeutic agents for combining with cryosurgery. This approach not only ensures potent cancer cell eradication throughout the frozen tumor iceball, including its periphery with a high subzero temperature ranging from ~ -4 to -20 °C where conventional cryosurgery alone may fail short, but also promotes the formation of an immune-active TME and cryo-immune response against both the primary tumor with the cryosurgery and distant tumors with no cryosurgery. Overall, this combinational strategy may be invaluable for converting the immunologically “cold” TME into immunologically “hot” one, eliciting potent tumor-specific immune responses in treating both primary and distant/metastatic tumors.

Materials and

Methods: Poly (N-isopropylacrylamide co-butyl acrylate) polymers with varying lower critical solution temperature (LCST) were synthesized by changing the molar ratios of N-isopropylacrylamide to butyl acrylate. The resulting polymers were utilized as the cold-responsive materials for the fabrication of CRNPs. These CRNPs (CPT&siR CRNPs) were loaded with irinotecan (CPT) and PD-L1 siRNA (siR) through a double-emulsion (water-in-oil-in-water) method (4,5). The cold-responsiveness of CRNPs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and a Linkam temperature-controlled microscope. The maturation of dendritic cells (DCs) after co-culture with the EO771 tumor cells treated with CRNPs (CPT: 10 µg/ml and siR: 50 nM) and subjected to treatment of cryo-temperature (-20°C, 10 min), the subsequent CD8+ cytotoxic T lymphocytes (CTLs) proliferation, activation, and the tumor attacking capability were assessed by flow cytometry and confocal microscopy. The in vivo therapeutic effect of CRNPs in combination with cryosurgery against primary and distant/metastatic tumors were evaluated using EO771 and 4T1 tumor models. The tumor size was measured every three days with caliper. The induction of antitumor cryo-immune response was investigated by analyzing the immune cell population (T cell, DC, macrophages, and monocytes) in the TME utilizing flow cytometry and confocal imaging.

Results, Conclusions, and Discussions: Results and

Discussion: The CPT&siR CRNPs with a diameter of 143.6 ± 6.6 nm, and encapsulation efficiency (EE) of 51.2 ± 8.2% for CPT and 73.4 ± 4.2% for siR, exhibit a core-shell structure as demonstrated by their TEM image (Fig. 1A, top). Upon incubation at -4 °C for 10 min followed by warming back to 22 °C, structural destruction of the CRNPs is observable (Fig. 1A, bottom), confirming the cold-responsiveness of the CRNPs. When incubated with EO771 tumor cells, CPT&siR CRNPs in combination with treatment at cryo-temperature (+C) enhance the release of damage-associated molecular patterns, fostering an immunologically active TME and facilitating DC maturation5. Subsequent co-culture with CD8+ T cells results in a remarkable increase in the percentage of T cell that proliferate, reaching ~79% (Fig. 1B). Notably, CD8+ T cells (indicated by arrows in Fig. 1C) activated from DCs co-cultured with tumor cells receiving the CPT&siR CRNPs+C treatment, exhibit high capability of recognizing and attacking the EO771 tumor cells (green) in vitro. In vivo therapeutic experiments reveal that mice treated with CPT&siR CRNPs combined with cryosurgery on primary tumors exhibit infiltration of cytotoxic CD8+ T cells expressing granzyme B (GZMB) not only in the tumor section of primary tumors with cryosurgery (Fig. 1D), but also distant tumors without cryosurgery (Fig. 1E). Additionally, the combinational treatment of cryosurgery with CPT&siR CRNPs significantly suppress the growth of both primary (Fig. F, top) and distant (Fig. F, bottom) tumors. These findings suggest that the potent antitumor immunity elicited by the combined action of cryosurgery and CPT&siR CRNPs has great potential to eliminate both primary and distant cancer cells through the CTLs.

Conclusions: We have demonstrated a cryo-immune engineering strategy that integrates cryosurgery and CPT&siR CRNPs to transform the TME into an immunologically “hot” state and trigger potent antitumor immune responses. Furthermore, this approach induces a strong antitumor response capable of destroying both primary and distant/metastatic tumors in vivo. This technology may be invaluable for addressing cancer and its metastasis, particularly in cases where conventional cancer immunotherapy approaches have been inadequate.

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