Tumor-associated macrophages (TAMs) play a role in both pro- and anti-tumor functions; and the targeted polarization from M2 to M1 TAMs has become an effective therapy option. Although detection of M1 TAMs is imperative to assess cancer immunotherapeutic efficacy, existing optical probes suffer from shallow tissue penetration depth and poor specificity toward M1 TAMs. Herein, we report a tandem-locked NIR chemiluminescent (CL) probe for specific detection of M1 TAMs. Through a combined molecular engineering approach via both atomic alternation and introduction of electron-withdrawing groups, near-infrared (NIR) chemiluminophores are screened out to possess record-long emission (over 800 nm), record-high CL quantum yield (2.7 % einstein/mol), and prolonged half-life (7.7 h). Based on an ideal chemiluminophore, the tandem-locked probe (DPDGN) is developed to only activate CL signal in the presence of both tumour (γ-glutamyl transpeptidase) and M1 macrophage biomarkers (nitric oxide). Such a tandem-lock design ensures its high specificity towards M1 macrophages in the tumor microenvironment over those in normal tissues or peripheral blood. Thus, DPDGN permits noninvasive imaging and tracking of M1 TAM in the tumor of living mice during R837 treatment, showing a good correlation with ex vivo methods. This study not only reports a new molecular approach towards highly efficient chemiluminophores but also reveals the first tandem-locked CL probes for enhanced imaging specificity.

Eosinophils are important immune effector cells that affect T cell-mediated antitumor immunity. However, the low frequency and restrained activity of eosinophils restricted the outcome of cancer immunotherapies. We herein report an eosinophil-activating semiconducting polymer nanoparticle (SPNe) to improve photodynamic tumor immunogenicity, modulate eosinophil chemotaxis, and reinvigorate T-cell immunity for activated cancer photo-immunotherapy. SPNe comprises an amphiphilic semiconducting polymer and a dipeptidyl peptidase 4 (DPP4) inhibitor sitagliptin via a 1O2-cleavable thioketal linker. Upon localized NIR photoirradiation, SPNe generates 1O2 to elicit immunogenic cell death of tumors and induce specific activation of sitagliptin. The subsequent inhibition of DPP4 increases intratumoral CCL11 levels to promote eosinophil chemotaxis and activation. SPNe-mediated photo-immunotherapy synergized with immune checkpoint blockade greatly promotes tumor infiltration and activation of both eosinophils and T cells, effectively inhibiting tumor growth and metastasis. Thus, this study presents a generic polymeric nanoplatform to modulate specific immune cells for precision cancer immunotherapy.

Tumor-associated mast cells (TAMCs) have been recently revealed to play a multifaceted role in the tumor microenvironment. Noninvasive optical imaging of TAMCs is thus highly desired to gain insights into their functions in cancer immunotherapy. However, due to the lack of a single enzyme that is specific to mast cells, a common probe design approach based on single-enzyme activation is not applicable. Herein, we reported a bienzyme-locked molecular probe (THCMC) based on a photoinduced electron transfer-intramolecular charge-transfer hybrid strategy for in vivo imaging of TAMCs. The bienzyme-locked activation mechanism ensures that THCMC exclusively turns on near-infrared (NIR) fluorescence only in the presence of both tryptase and chymase specifically coexpressed by mast cells. Thus, THCMC effectively distinguishes mast cells from other leukocytes, including T cells, neutrophils, and macrophages, a capability lacking in single-locked probes. Such a high specificity of THCMC allows noninvasive tracking of the fluctuation of TAMCs in the tumor of living mice during cancer immunotherapy. The results reveal that the decreased intratumoral signal of THCMC after combination immunotherapy correlates well with the reduced population of TAMCs, accurately predicting the inhibition of tumor growth. Thus, this study not only presents the first NIR fluorescent probe specific for TAMCs but also proposes a generic bienzyme-locked probe design approach for in vivo cell imaging.

Combination cancer immunotherapy based on electromagnetic energy and immunotherapy shows potent anti-cancer efficacy. However, as a factor that mediates tumor metastasis and immune suppression, the impact of tumor exosomes on therapy under electromagnetic energy stimulation remains unclear. Herein, findings indicate that sonodynamic therapy (SDT) increases serum exosome levels by inducing apoptotic exosomes and loosening the tumor extracellular matrix, promoting lung metastasis. To address this problem, an exosome-inhibiting polymeric sonosensitizer (EIPS) selectively inhibiting tumor exosome generation in response to the tumor biomarker is synthesized. EIPS consists of a semiconducting polymer backbone capable of inducing SDT and a poly(ethylene glycol) layer conjugated with a tumor-specific enzyme-responsive exosome inhibitor prodrug. After being cleaved by tumor Cathepsin B, EIPS releases active exosome inhibitors, preventing tumor exosome-mediated immune suppression and lung metastasis. As a result, EIPS elicits robust antitumor effects through the synergistic effect of SDT and tumor exosome inhibition, completely preventing lung metastasis and establishing a long-term immune memory effect. This is the first example showing that combining SDT with tumor-specific exosome inhibition can elicit a potent immune response without the help of typical immune agonists.

Real‐time in vivo imaging of RNA can enhance the understanding of physio‐pathological processes. However, most nucleic acid‐based sensors have poor resistance to nucleases and limited photophysical properties, making them suboptimal for this purpose. To address this, a semiconducting polymer nanospherical nucleic acid probe (SENSE) for transcriptomic imaging of cancer immunity in living mice is developed. SENSE comprises a semiconducting polymer (SP) backbone covalently linked with recognition DNA strands, which are complemented by dye‐labeled signal DNA strands. Upon detection of targeted T lymphocyte transcript (Gzmb: granzyme B), the signal strands are released, leading to a fluorescence enhancement correlated to transcript levels with superb sensitivity. The always‐on fluorescence of the SP core also serves as an internal reference for tracking SENSE uptake in tumors. Thus, SENSE has the dual‐signal channel that enables ratiometric imaging of Gzmb transcripts in the tumor of living mice for evaluating chemo‐immunotherapy; moreover, it has demonstrated sensitivity and specificity comparable to flow cytometry and quantitative polymerase chain reaction, yet offering a faster and simpler means of T cell detection in resected tumors. Therefore, SENSE represents a promising tool for in vivo RNA imaging.

The efficacy of combination immunotherapy has been limited by tumor specificity and immune-related adverse events (irAEs). Herein, we report the development of polymeric STING pro-agonists (PSPA), whose sono-immunotherapeutic efficacy is activated by sono-irradiation and elevated glutathione (GSH) within the tumor microenvironment (TME). PSPA is composed of sonosensitizers (semiconducting polymer) and STING agonists (MSA-2) via the GSH-activatable linkers. Under sono-irradiation, PSPA serves as a sonosensitizer to generate 1 O2 and induce immunogenic cell death (ICD) of malignant tumor cells. Furthermore, MSA-2 is released specifically in tumor microenvironment with highly expressed GSH, minimizing off-target side effects. The activation of the STING pathway elevates the interferon-β level and synergizes with SDT to enhance the anti-tumor response. Therefore, this work proposes a universal approach for spatiotemporal regulation of cancer sono-immunotherapy.

Cytokine therapy mediates the interaction between immune cells and non-immune cells in the tumor microenvironment (TME), forming a promising approach in cancer therapy. However, the dose-dependent adverse effects and non-selective stimulation of cytokines limit their clinical use. We herein report a sonodynamic cytokine nano-immunocomplex (SPNAI ) that specifically activates effector T cells (Teffs) for antitumor immunotherapy. By conjugating anti-interleukin-2 (anti-IL-2) antibodies S4B6 on the semiconducting polymer nanoparticles to afford SPNA , this nanoantibody SPNA can bind with IL-2 to form SPNAI which can block the interaction between IL-2 and regulatory T cells (Tregs), selectively activating Teffs in TME. Moreover, SPNAI generates 1 O2 to trigger immunogenic cell death of cancer cells upon sono-irradiation, which promotes the maturation of dendritic cells and the proliferation of Teffs. This SPNAI -mediated combination sonodynamic immunotherapy thus elevates the ratio of Teffs/Tregs in TME, resulting in inhibition of tumor growth, suppression of lung metastasis and prevention of tumor relapse.

Sono-immunotherapy holds great potential for deep tumor inhibition; however, smart sono-therapeutic agents to simultaneously eliminate 'domestic' tumor cells and regulate the 'community' tumor immune microenvironment have rarely been developed. Herein, we report a spatiotemporally controllable semiconducting iron-chelated nano-metallomodulator (SINM) for hypersensitive sono-metallo-immunotherapy of cancer. SINM consists of a semiconducting polymer (SP) backbone chelating iron ions (Fe3+ ) with thiophene-based Schiff base structure, and a hydrophilic side chain. Upon accumulation in tumors after systemic administration, SINM specifically arouses ferroptosis and M1 macrophage polarization due to its response toward the tumor redox environment; meanwhile, the chelation of Fe3+ enhances the sono-sensitizing effect of SPs, leading to enhanced generation of reactive oxygen species for immunogenic cell death. Such combined sonodynamic metallo-immunotherapy of SINM efficiently ablates deep tumor and spatiotemporally regulates immunophenotypes.