Systemic Delivery of a STING Agonist-Loaded Positively Charged Liposome Selectively Targets Tumor Immune Microenvironment and Suppresses Tumor Angiogenesis.

Although stimulator of interferon genes (STING) agonists has shown great promise in preclinical studies, the clinical development of STING agonist therapy is challenged by its limited systemic delivery. Here, positively charged fusogenic liposomes loaded with a STING agonist (PoSTING) are designed for systemic delivery and to preferentially target the tumor microenvironment. When PoSTING is administered intravenously, it selectively targets not only tumor cells but also immune and tumor endothelial cells (ECs). In particular, delivery of STING agonists to tumor ECs normalizes abnormal tumor vasculatures, induces intratumoral STING activation, and elicits robust anti-tumor T cell immunity within the tumor microenvironment. Therefore, PoSTING can be used as a systemic delivery platform to overcome the limitations of using STING agonists in clinical trials.

Deep learning model enables the discovery of a novel immunotherapeutic agent regulating the kynurenine pathway.

Kynurenine (Kyn) is a key inducer of an immunosuppressive tumor microenvironment (TME). Although indoleamine 2,3-dioxygenase (IDO)-selective inhibitors have been developed to suppress the Kyn pathway, the results were not satisfactory due to the presence of various opposing mechanisms. Here, we employed an orally administered novel Kyn pathway regulator to overcome the limitation of anti-tumor immune response. We identified a novel core structure that inhibited both IDO and TDO. An orally available lead compound, STB-C017 (designated hereafter as STB), effectively inhibited the enzymatic and cellular activity of IDO and TDO in vitro. Moreover, it potently suppressed Kyn levels in both the plasma and tumor in vivo. STB monotherapy increased the infiltration of CD8+ T cells into TME. In addition, STB reprogrammed the TME with widespread changes in immune-mediated gene signatures. Notably, STB-based combination immunotherapy elicited the most potent anti-tumor efficacy through concurrent treatment with immune checkpoint inhibitors, leading to complete tumor regression and long-term overall survival. Our study demonstrated that a novel Kyn pathway regulator derived using deep learning technology can activate T cell immunity and potentiate immune checkpoint blockade by overcoming an immunosuppressive TME.

High endothelial venule is a surrogate biomarker for T-cell inflamed tumor microenvironment and prognosis in gastric cancer.

BACKGROUND: High endothelial venule (HEV) is a specialized vasculature for lymphocyte trafficking. While HEVs are frequently observed within gastric cancer (GC), the vascular-immune interaction between HEV and tumor-infiltrating lymphocytes (TILs) has not been well elucidated. In this study, we aimed to unveil the potential value of HEVs as a surrogate marker for T-cell inflamed immune microenvironment in GC using a large number of prospectively collected surgical specimens of GC. METHODS: We included 460 patients with GC who underwent surgical resection. Nanostring PanCancer immune profiling was performed to evaluate the immunological phenotype of GCs. HEV density and three distinct patterns of TILs (Crohn-like lymphoid reaction, peritumoral lymphoid reaction, and intratumoral lymphoid reaction) were analyzed for their relationship and evaluated as prognostic factors for relapse-free survival (RFS) and overall survival (OS). RESULTS: HEV-high GC revealed increased infiltration by immune cell subsets, including dendritic cells, CD8+ cytotoxic T cells, and CD4+ helper T cells. In addition, HEV-high GC demonstrated increased immune-modulating chemokines, type I or II interferon pathway, and immune checkpoints, all of which indicate the inflamed tumor microenvironment (TME). All three distinct patterns of TILs were associated with HEV density. In survival analysis, patients with HEV-high GC displayed significantly longer RFS and OS than those with HEV-low GC (p<0.001 for RFS, p<0.001 for OS). Multivariate analysis demonstrated that HEV was the most significant immunological prognostic factor for RFS (patients with high HEV compared with those with low HEV; HR 0.412, 95% CI 0.241 to 0.705, p=0.001) and OS (HR 0.547, 95% CI 0.329 to 0.909, p=0.02) after adjustment for age, stage, and TIL. CONCLUSION: HEV is the most significant immunological prognosticator for RFS and OS in resected GC, indicating inflamed TME.

STING activation normalizes the intraperitoneal vascular-immune microenvironment and suppresses peritoneal carcinomatosis of colon cancer.

BACKGROUND: Peritoneal carcinomatosis is a fatal clinical presentation of colon cancer, characterized by unresponsiveness to conventional anticancer therapies, including immune checkpoint inhibitors. Here, we elucidated the immune-evasion mechanisms during the peritoneal carcinomatosis of colon cancer and developed a novel immunotherapy by activating the stimulator of interferon genes (STING) pathway. METHODS: We generated a syngeneic peritoneal carcinomatosis model of colon cancer. Mice were intraperitoneally treated with either STING agonist (MIW815, also known as ADU-S100) or PD-1 blockade or both. The tumor microenvironment was comprehensively analyzed using multiplexed immunofluorescence imaging, flow cytometry, and NanoString immune profiling. RESULTS: Intraperitoneal colon cancer cells induce a massive influx of immunosuppressive M2-like macrophages, upregulate immune checkpoints, and impair effector T cell functions during peritoneal dissemination; these collectively create a highly angiogenic and immunosuppressive milieu that is resistant to anti-PD-1 monotherapy. Intraperitoneal administration of a STING agonist suppressed aberrant angiogenesis, increased pericyte coverage, and normalized tumor vessels, thereby facilitating the infiltration of activated CD8+ T cells into peritoneal tumor nodules. Moreover, STING activation reprogramed tumor-associated macrophages toward the M1 phenotype. STING activation converted immunologically cold peritoneal tumors into T-cell-inflamed tumors in a type-I interferon-dependent manner. Lastly, the STING agonist synergistically cooperated with PD-1 and/or COX2 blockade to further suppress the peritoneal dissemination of colon cancer, resulting in complete eradication of tumor and ascites, and inducing durable antitumor immunity. CONCLUSIONS: STING activation can normalize the peritoneal vascular and immune microenvironment, providing a rationale for a novel combination therapeutic strategy for peritoneal carcinomatosis in colon cancer.

Antitumor effects of IL-12 and GM-CSF co-expressed in an engineered oncolytic HSV-1.

Oncolytic viruses selectively replicate and destroy cancer cells while sparing normal cells, prompting their recognition as promising antitumor agents. Herpes simplex virus (HSV) is suitable as an anticancer agent, given its considerable therapeutic gene capacity and excellent safety profile in clinical trials. Interleukin (IL)-12 induces a Th1-type immune response that mediates interferon (IFN)-γ release from natural killer (NK), CD4+ and CD8+ T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the generation of antigen-presenting cells and promotes dendritic cell differentiation. We established a novel oncolytic HSV-1 (∆6/GM/IL12) co-expressing IL-12 and GM-CSF and tested its effects against a B16-F10 murine melanoma model. ∆6/GM/IL12 administration diminished tumor growth and prolonged survival compared to treatment with ∆6/GM or ∆6/IL12 expressing each individual cytokine. Flow cytometry and histological analysis showed increased activation of CD4+ and CD8+ T cells in ∆6/GM/IL12-treated mice. Enzyme-linked immunosorbent spot assay showed an increase in the phenotypically characterized IFN-γ-producing cell population in ∆6/GM/IL12-treated mice. Moreover, ∆6/GM/IL12 induced a B16-F10-specific cytotoxic immune response that enhanced IFN-γ production by CD3+CD8+ T cells. Therefore, IL-12 and GM-CSF from an engineered oncolytic HSV have a synergistic effect, boosting the immune response to increase their antitumor effects.

Oncolytic vaccinia virus reinvigorates peritoneal immunity and cooperates with immune checkpoint inhibitor to suppress peritoneal carcinomatosis in colon cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) is a common and devastating manifestation of colon cancer and refractory to conventional anticancer therapeutics. During the peritoneal dissemination of colon cancer, peritoneal immunity is nullified by various mechanisms of immune evasion. Here, we employed the armed oncolytic vaccinia virus mJX-594 (JX) to rejuvenate the peritoneal antitumor immune responses in the treatment of PC. METHODS: PC model of MC38 colon cancer was generated and intraperitoneally treated with JX and/or anti-programmed cell death protein 1 (PD-1) antibody. The peritoneal tumor burden, vascular leakage, and malignant ascites formation were then assessed. Tumors and peritoneal lavage cells were analyzed by flow cytometry, multiplex tissue imaging, and a NanoString assay. RESULTS: JX treatment effectively suppressed peritoneal cancer progression and malignant ascites formation. It also restored the peritoneal anticancer immunity by activating peritoneal dendritic cells (DCs) and CD8+ T cells. Moreover, JX selectively infected and killed peritoneal colon cancer cells and promoted the intratumoral infiltration of DCs and CD8+ T cells into peritoneal tumor nodules. JX reinvigorates anticancer immunity by reprogramming immune-related transcriptional signatures within the tumor microenvironment. Notably, JX cooperates with immune checkpoint inhibitors (ICIs), anti-programmed death-1, anti-programmed death-ligand 1, and anti-lymphocyte-activation gene-3 to elicit a stronger anticancer immunity that eliminates peritoneal metastases and malignant ascites of colon cancer compared with JX or ICI alone. CONCLUSIONS: Intraperitoneal immunotherapy with JX restores peritoneal anticancer immunity and potentiates immune checkpoint blockade to suppress PC and malignant ascites in colon cancer.

Targeting Actomyosin Contractility Suppresses Malignant Phenotypes of Acute Myeloid Leukemia Cells.

Actomyosin-mediated contractility is required for the majority of force-driven cellular events such as cell division, adhesion, and migration. Under pathological conditions, the role of actomyosin contractility in malignant phenotypes of various solid tumors has been extensively discussed, but the pathophysiological relevance in hematopoietic malignancies has yet to be elucidated. In this study, we found enhanced actomyosin contractility in diverse acute myeloid leukemia (AML) cell lines represented by highly expressed non-muscle myosin heavy chain A (NMIIA) and increased phosphorylation of the myosin regulatory light chain. Genetic and pharmacological inhibition of actomyosin contractility induced multivalent malignancy- suppressive effects in AML cells. In this context, perturbed actomyosin contractility enhances AML cell apoptosis through cytokinesis failure and aryl hydrocarbon receptor activation. Moreover, leukemic oncogenes were downregulated by the YAP/TAZ-mediated mechanotransduction pathway. Our results provide a theoretical background for targeting actomyosin contractility to suppress the malignancy of AML cells.

STING signaling is a potential immunotherapeutic target in colorectal cancer.

Background: Stimulator of Interferon Genes (STING) is an innate immune sensor for cytosolic DNA. STING signaling activation is indispensable for type I interferon response and the anti-cancer immune response by CD8+ T cells. The aim of this study was to characterize intratumoral STING expression pattern and its clinical implication in colorectal cancer (CRC). Methods: We analyzed STING and CD8 expression in 225 CRC patients who underwent surgical resection. Clinicopathological variables and survival outcomes were analyzed according to STING expression levels. Mice with syngeneic MC38 tumors were also treated with a STING agonist, and tumor microenvironments were analyzed using immunofluorescent staining and flow cytometry. Results: Distinct STING expression was observed in the CRC tumor specimens. Patients with higher STING expression had early stage cancer with increased intratumoral CD8+ T cell infiltration and less frequent lymphovascular invasion. Compared to CRC patients with lower STING expression, those with higher STING expression had longer overall and recurrence-free survival. Multivariate Cox regression model also revealed higher STING expression to be an independent prognostic factor for better overall survival. When MC38 colon tumors were treated with intratumoral injection of STING agonist, tumor growth was remarkably suppressed with increased intratumoral CD8+ T cell infiltration. Moreover, T-cell activation markers, ICOS and IFN-γ, were also upregulated in CD8+ T cells, indicating enhanced effector T cell function after STING treatment. Conclusion: We confirmed the distinct STING expression in CRC and demonstrated its independent prognostic value in survival outcomes. STING could be a potential therapeutic target that enhances anti-cancer immune response in CRC.

STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade.

The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy. Endothelial STING expression was correlated with enhanced T-cell infiltration and prolonged survival in human colon and breast cancer. Intratumoral STING activation with STING agonists (cGAMP or RR-CDA) normalized tumor vasculatures in implanted and spontaneous cancers, but not in STING-deficient mice. These were mediated by upregulation of type I/II interferon genes and vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a). STING in non-hematopoietic cells is as important as STING in hematopoietic cells to induce a maximal therapeutic efficacy of exogenous STING agonist. Vascular normalizing effects of STING agonists were dependent on type I interferon signaling and CD8+ T cells. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors. Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy.

Tumor Microenvironment Remodeling by Intratumoral Oncolytic Vaccinia Virus Enhances the Efficacy of Immune-Checkpoint Blockade.

PURPOSE: Cancer immunotherapy is a potent treatment modality, but its clinical benefit depends on the tumor's immune profile. Here, we used mJX-594 (JX), a targeted and GM-CSF-armed oncolytic vaccinia virus, as a strategy to remodel the tumor microenvironment (TME) and subsequently increase sensitivity to αPD-1 and/or αCTLA-4 immunotherapy. EXPERIMENTAL DESIGN: The remodeling of the TME was determined using histologic, flow-cytometric, and NanoString immune profiling analyses. JX was intratumorally injected into implanted Renca kidney tumors or MMTV-PyMT transgenic mouse breast cancers with or without αPD-1 and/or αCTLA-4. Various combination regimens were used to evaluate immunotherapeutic anticancer responses. RESULTS: Intratumoral injection of JX remodeled the TME through dynamic changes in the immune system, as shown by increased tumor-infiltrating T cells and upregulation of immune-related gene signatures. This remodeling induced conversion of a noninflamed tumor into an inflamed tumor. JX virotherapy led to enhanced abscopal effects in distant tumors, with increased intratumoral infiltration of CD8+ T cells. A depletion study revealed that GM-CSF is an indispensable regulator of anticancer efficacy of JX. Dual-combination therapy with intratumoral JX and systemic αPD-1 or αCTLA-4 further enhanced the anticancer immune response, regardless of various treatment schedules. Of note, triple combination immunotherapy with JX, αPD-1, and αCTLA-4 elicited the most potent anticancer immunity and induced complete tumor regression and long-term overall survival. CONCLUSIONS: Our results show that intratumoral JX treatment induces dramatic remodeling of the TME and more potently suppresses cancer progression with immune-checkpoint blockades by overcoming resistance to immunotherapy.