Integration of AIEgens into covalent organic frameworks for pyroptosis and ferroptosis primed cancer immunotherapy.

Immunogenic programmed cell death, such as pyroptosis and ferroptosis, efficiently induces an acute inflammatory response and boosts antitumor immunity. However, the exploration of dual-inducers, particularly nonmetallic inducers, capable of triggering both pyroptosis and ferroptosis remains limited. Here we show the construction of a covalent organic framework (COF-919) from planar and twisted AIEgen-based motifs as a dual-inducer of pyroptosis and ferroptosis for efficient antitumor immunity. Mechanistic studies reveal that COF-919 displays stronger near-infrared light absorption, lower band energy, and longer lifetime to favor the generation of reactive oxygen species (ROS) and photothermal conversion, triggering pyroptosis. Because of its good ROS production capability, it upregulates intracellular lipid peroxidation, leading to glutathione depletion, low expression of glutathione peroxidase 4, and induction of ferroptosis. Additionally, the induction of pyroptosis and ferroptosis by COF-919 effectively inhibits tumor metastasis and recurrence, resulting in over 90% tumor growth inhibition and cure rates exceeding 80%.

Activation of Pyroptosis Using AIEgen-Based sp2 Carbon-Linked Covalent Organic Frameworks.

Covalent organic frameworks (COFs) have emerged as a promising class of crystalline porous materials for cancer phototherapy, due to their exceptional characteristics, including light absorption, biocompatibility, and photostability. However, the aggregation-caused quenching effect and apoptosis resistance often limit their therapeutic efficacy. Herein, we demonstrated for the first time that linking luminogens with aggregation-induced emission effect (AIEgens) into COF networks via vinyl linkages was an effective strategy to construct nonmetallic pyroptosis inducers for boosting antitumor immunity. Mechanistic investigations revealed that the formation of the vinyl linkage in the AIE COF endowed it with not only high brightness but also strong light absorption ability, long lifetime, and high quantum yield to favor the generation of reactive oxygen species for eliciting pyroptosis. In addition, the synergized system of the AIE COF and αPD-1 not only effectively eradicated primary and distant tumors but also inhibited tumor recurrence and metastasis in a bilateral 4T1 tumor model.

CSRP2 promotes cell stemness in head and neck squamous cell carcinoma.

BACKGROUND: Cysteine-rich protein 2 (CSRP2) is discovered as oncogene. The study aims to investigate the clinical significance and potential mechanism of CSRP2 in head and neck squamous cell carcinoma (HNSCC). METHODS: CSRP2 expression was explored by immunohistochemistry tissue microarrays and Western blotting in HNSCC. The effect of CSRP2 on the cancer stemness and epithelial-to-mesenchymal transition (EMT) of HNSCC cells was investigated by sphere formation, wound healing, and transwell assays. The vitro and vivo experiments revealed that CSRP2 modulated cancer stemness and EMT phenotypes in HNSCC. RESULTS: CSRP2 was overexpressed in HNSCC patients and presented poor prognosis. CSRP2 knockdown inhibited the migration and invasion ability of the HNSCC cells. And CSRP2 expression was closely associated with CSCs markers, EMT-transcription factor, new oncoprotein, and immune checkpoint. CONCLUSION: The overexpression of CSRP2 indicates poor prognosis and plays a key role in maintaining the cancer cell stemness and EMT.

Targeting PCSK9 reduces cancer cell stemness and enhances antitumor immunity in head and neck cancer.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been demonstrated to play a critical role in regulating cholesterol homeostasis and T cell antitumor immunity. However, the expression, function, and therapeutic value of PCSK9 in head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. Here, we found that the expression of PCSK9 was upregulated in HNSCC tissues, and higher PCSK9 expression indicated poorer prognosis in HNSCC patients. We further found that pharmacological inhibition or siRNA downregulating PCSK9 expression suppressed the stemness-like phenotype of cancer cells in an LDLR-dependent manner. Moreover, PCSK9 inhibition enhanced the infiltration of CD8+ T cells and reduced the myeloid-derived suppressor cells (MDSCs) in a 4MOSC1 syngeneic tumor-bearing mouse model, and it also enhanced the antitumor effect of anti-PD-1 immune checkpoint blockade (ICB) therapy. Together, these results indicated that PCSK9, a traditional hypercholesterolemia target, may be a novel biomarker and therapeutic target to enhance ICB therapy in HNSCC.

CTLA-4 blockade induces tumor pyroptosis via CD8+ T cells in head and neck squamous cell carcinoma.

Immune checkpoint blockade (ICB) treatment has demonstrated excellent medical effects in oncology, and it is one of the most sought after immunotherapies for tumors. However, there are several issues with ICB therapy, including low response rates and a lack of effective efficacy predictors. Gasdermin-mediated pyroptosis is a typical inflammatory death mode. We discovered that increased expression of gasdermin protein was linked to a favorable tumor immune microenvironment and prognosis in head and neck squamous cell carcinoma (HNSCC). We used the mouse HNSCC cell lines 4MOSC1 (responsive to CTLA-4 blockade) and 4MOSC2 (resistant to CTLA-4 blockade) orthotopic models and demonstrated that CTLA-4 blockade treatment induced gasdermin-mediated pyroptosis of tumor cells, and gasdermin expression positively correlated to the effectiveness of CTLA-4 blockade treatment. We found that CTLA-4 blockade activated CD8+ T cells and increased the levels of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) cytokines in the tumor microenvironment. These cytokines synergistically activated the STAT1/IRF1 axis to trigger tumor cell pyroptosis and the release of large amounts of inflammatory substances and chemokines. Collectively, our findings revealed that CTLA-4 blockade triggered tumor cells pyroptosis via the release of IFN-γ and TNF-α from activated CD8+ T cells, providing a new perspective of ICB.

CD103 blockade impair anti-CTLA-4 immunotherapy in oral cancer.

OBJECTIVES: CD103+CD8+T cells is a subtype of T cells with excellent tumor killing ability and it could response to immune checkpoint blockade therapy in several types of cancer, but the phenotype, role and molecular mechanism CD103+CD8+T cells in the OSCC still unclear. MATERIALS AND METHODS: The distribution and phenotype of CD103+CD8+T cells were investigated by performing multiplexed immunohistochemistry on human OSCC tissue microarray and flow cytometric analysis of fresh OSCC tumor-infiltrating lymphocytes (TILs). By in vivo use of anti-CD103 monoclonal antibody (mAb) in the 4MOSC1 tumor-bearing mouse model, CD103+CD8+T cell infiltration and cytotoxicity was clarified. RESULTS: The majority of CD8+T cells in both human and animal OSCC intra-tumoral region were CD103+CD8+T cells with high expression levels of cytotoxic molecules, which can be impaired by CD103 blockade. In addition, combined use of anti-CD103 mAb with anti-CTLA-4 mAb displayed impaired immune checkpoint blockade therapy efficiency. CONCLUSION: CD103+CD8+T cells are the major intra-tumoral subset of CD8+T cells in both animal and human OSCC, and that CD103+CD8+T cells demonstrate remarkable tumor-infiltrating and tumor-killing properties, thereby CD103+CD8+T cells may critical for anti-CTLA-4 immunotherapy in OSCC.

Bioengineering of BRAF and COX2 inhibitor nanogels to boost the immunotherapy of melanoma via pyroptosis.

Glutathione-responsive nanogels (CDNPs) crosslinked via crosslinker DBHD with the BRAF inhibitor dabrafenib and the COX2 inhibitor celecoxib were fabricated. The CDNPs can effectively induce tumor cell pyroptosis to activate robust antitumor immunity. Additionally, CDNPs combined with αPD-1 antibody greatly inhibited tumor growth in a melanoma mouse model with a prolonged survival time.

NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays cell- and tissue-specific roles in cancer, meaning that its activation in different tumors or cells may play different roles in tumor progression. We have previously described the tumor-promoting function of tumor-intrinsic NLRP3/IL-1ß signaling in head and neck squamous cell carcinoma (HNSCC), but its role in immune cells remains unclear. In this study, we found that NLRP3 was highly expressed in tumor-associated macrophages (TAMs) in both mouse and human HNSCC, and the expression of NLRP3 was positively correlated with the density of TAMs according to immunohistochemistry, immunofluorescence, and flow cytometry analyses. Importantly, the number of NLRP3high TAMs was related to worse overall survival in HNSCC patients. Knocking out NLRP3 inhibited M2-like macrophage differentiation in vitro. Moreover, the carcinogenic effect induced by 4-nitroquinoline-1-oxide was decreased in Nlrp3-deficient mice, which had smaller tumor sizes. Genetic depletion of NLRP3 reduced the expression of protumoral cytokines, such as IL-1ß, IL-6, IL-10, and CCL2, and suppressed the accumulation of TAMs and myeloid-derived suppressor cells (MDSCs) in mouse HNSCC. Thus, activation of NLRP3 in TAMs may contribute to tumor progression and have prognostic significance in HNSCC.

Diselenide-Based Dual-Responsive Prodrug as Pyroptosis Inducer Potentiates Cancer Immunotherapy.

Pyroptosis is demonstrated to trigger antitumor immunity and represents a promising new strategy to potentiate cancer immunotherapy. The number of potent pyroptosis inducers, however, is limited and without tumor-targeting capability, which inevitably causes damage in normal tissues. Herein, a small molecular prodrug of paclitaxel-oxaliplatin is rationally synthesized, which can be covalently self-assembled with diselenide-containing cross-linking (Dse11), producing a diselenide nanoprodrug (DSe@POC) to induce pyroptosis for the first time. The diselenide bonds within DSe@POC can be split by high glutathione in the tumor microenvironment (TME) and reactive oxygen species induced by photodynamic therapy, thus possessing excellent TME on-target effects. Additionally, DSe@POC is able to elicit intense pyroptosis to remodel the immunostimulated TME and trigger a robust immune response. Furthermore, combined αPD-1 therapy effectively inhibits the growth of remote tumors through the abscopal effect, amplifies a long-term immune memory response to reject rechallenged tumors, and prolongs survival. Collectively, DSe@POC, as the first TME dual-responsive diselenide-based pyroptosis inducer, will open up an attractive approach for cancer immunotherapy.

pH-responsive nanoprodrugs combining a Src inhibitor and chemotherapy to potentiate antitumor immunity via pyroptosis in head and neck cancer.

As the prominent feature of the development and progression of head and neck squamous cell carcinoma (HNSCC) is immunosuppression, therapeutic strategies to restore antitumor immunity have shown promising prospects. The efficacy of chemotherapy, a mainstay in HNSCC treatment, is exemplified by cytotoxic effects as well as immunostimulation, whereas compensatory activation of prosurvival signals in tumor tissues may compromise its efficacy. Aberrant activation of Src is present in many human malignancies including HNSCC, and is implicated in chemotherapy resistance. In this regard, tumor-microenvironment-responsive prodrug nanomicelles (PDO NPs) are rationally designed to combine chemotherapy (oxaliplatin, OXA) and Src inhibitors (dasatinib, DAS) for HNSCC therapy. PDO NPs are constructed by chemically modifying small-molecule prodrugs (DAS-OXA) loaded in block copolymer iPDPA with pH-triggered transforming capability. PDO NPs can controllably release drugs in response to tumor acidity, thus increasing tumor accumulation and therapeutic efficacy. Moreover, PDO NPs can elicit pyroptosis of tumor cells and induce T-cell-mediated antitumor immunity in murine HNSCC models. In summary, nanoprodrugs integrating Src inhibitors enhance the immunological effects of chemotherapy and provide insight into promising approaches for augmenting immunochemotherapy for HNSCC. STATEMENT OF SIGNIFICANCE: In this study, pH-responsive nanomicelles (PDO NPs) were constructed by loading a small molecular prodrug synthesized by the Src inhibitor dasatinib and the chemotherapy drug oxaliplatin into the amphiphilic block copolymer iPDPA to improve the immunological effects of chemotherapy for HNSCC. These nanomicelles can efficiently accumulate in tumor cells and achieve pH-responsive drug release. The PDO NPs can induce pyroptosis of tumor cells and potentiate antitumor immunity in subcutaneous and syngenetic orthotopic HNSCC mouse models, which may present a promising strategy to enhance immunochemotherapy for HNSCC.

Engineering Multienzyme-Mimicking Covalent Organic Frameworks as Pyroptosis Inducers for Boosting Antitumor Immunity.

The engineering of a series of multienzyme-mimicking covalent organic frameworks (COFs), COF-909-Cu, COF-909-Fe, and COF-909-Ni, as pyroptosis inducers, remodeling the tumor microenvironment to boost cancer immunotherapy, is reported. Mechanistic studies reveal that these COFs can serve as hydrogen peroxide (H2 O2 ) homeostasis disruptors to elevate intracellular H2 O2 levels, and they not only exhibit excellent superoxide dismutase (SOD)-mimicking activity and convert superoxide radicals (O2 •- ) to H2 O2 to facilitate H2 O2 generation, but also possess outstanding glutathione peroxidase (GPx)-mimicking activity and deplete glutathione (GSH) to alleviate the scavenging of H2 O2 . Meanwhile, the outstanding photothermal therapy properties of these COFs can accelerate the Fenton-like ionization process to facilitate their chemodynamic therapy efficiency. One member, COF-909-Cu, can robustly induce gasdermin E (GSDME)-dependent pyroptosis and remodel the tumor microenvironment to trigger durable antitumor immunity, thus promoting the response rate of αPD-1 checkpoint blockade and successfully restraining tumor metastasis and recurrence.

Overexpression of CD168 is related to poor prognosis in oral squamous cell carcinoma.

OBJECTIVES: Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) is also known as CD168. This study proposed to elucidate the prognostic and clinicopathological significance of CD168 expression in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Immune staining of a human tissue microarray and Western blot were used to reveal the expression level of CD168 in OSCC. Correlations between clinicopathological indexes and CD168 expression in OSCC patients were assessed. RESULTS: Increased expression of CD168 was detected in OSCC tissues. High expression of CD168 indicated worse survival of patients (p < .05). Furthermore, high expression of CD168 was related to pathological grade in OSCC (p < .05). CD168 expression was positively related to programmed death ligand 1 (PD-L1), CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6), B7 homology 4 protein (B7-H4), CD44, CD133, and Slug expression in OSCC. CONCLUSION: This study revealed the overexpression of CD168 in OSCC and shed light on the prognostic significance of CD168 expression in OSCC patients.

Three-Dimensional Covalent Organic Frameworks with Cross-Linked Pores for Efficient Cancer Immunotherapy.

We report the design and synthesis of a series of three-dimensional (3D) covalent organic frameworks (COFs) as immunogenic cell death (ICD) inducers for cancer immunotherapy. Three triple-topic amine building blocks, inactive to inducing ICD, were used to construct three COFs, COF-607, COF-608, and COF-609, with outstanding ICD eliciting efficiency. Mechanism studies revealed that after linking these ICD inert monomers into the COF backbone, the optical properties of these COFs could be systematically tuned to achieve excellent reactive oxygen species (ROS) production performance. This combined with 3D cross-linked pores, mimicking lung structure, favor the exchange and diffusion of oxygen and ROS, leading to excellent inducing ICD efficacy. One member, COF-609, is capable of triggering abscopal and long-lasting immune memory effects in a mouse model of breast cancer with >95% mice survival after being treated with COF-609+αCD47 for 110 days.

Overexpression of PREX1 in oral squamous cell carcinoma indicates poor prognosis.

Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins control many fundamental cellular functions including cell migration, actin cytoskeletal rearrangement and adhesion in many cancers. However, P-Rex1 expression and its prognostic effect and possible clinical value are not clearly elucidated in human oral squamous cell carcinoma (OSCC). Here, OSCC tissue microarrays were used to verify the expression levels of P-Rex1, coinhibitory immune checkpoints and tumor associated macrophage (TAM) markers, and to analyze the relationship between PREX1 expression levels and clinicopathological characteristics in OSCC. The study found that P-Rex1 expression was elevated in OSCC compared to dysplasia and normal mucosa (P < 0.0001). In addition, patients who expressed high PREX1 had a poorer prognosis than those who expressed low PREX1 (P = 0.0070). Furthermore, positive correlations were found between P-Rex1 expression and the immune checkpoints PD-L1, Galectin-9 and B7-H4, and the TAM markers CD68, CD206 and CD163. In short, these findings implicated that overexpression of P-Rex1 may predict a poor prognosis in human OSCC.

Overexpression of ATAD2 indicates Poor Prognosis in Oral Squamous Cell Carcinoma.

ATPase family AAA domain-containing protein 2 (ATAD2) is highly expressed in a variety of malignancies and can promote the proliferation of tumor cells and inhibit their differentiation. However, the expression of ATAD2 and its related mechanism in oral squamous cell carcinoma (OSCC) are still unknown. Immunohistochemical staining of ATAD2, cancer stem cells (CSCs) markers and immune checkpoint molecules was conducted on human OSCC specimens to determine the expression levels of these proteins and their correlations with the clinicopathological characteristics of ATAD2 in OSCC. Moreover, the role of ATAD2 in cell proliferation, apoptosis, migration and epithelial-mesenchymal transition (EMT) were assessed by silencing ATAD2 in vitro. Immunohistochemical analysis revealed that ATAD2 expression in OSCC tissues was markedly higher than that in adjacent dysplastic tissues and normal mucosal tissues. Overexpression of ATAD2 was related to poor overall survival in OSCC patients. In addition, the protein expression of ATAD2 was notably correlated with the expression of B7-H4, PD-L1, CMTM6, Slug and ALDH1 in human OSCC. ATAD2 knockdown arrested the cell cycle, promoted the apoptosis, and inhibited the proliferation, migration, and EMT of OSCC cells. In conclusion, these findings revealed that ATAD2 is highly expressed in OSCC and can act as a poor prognostic indicator.