Jung-Joon Min

Targeted therapeutics have transformed cancer treatment by selectively eliminating malignant cells while limiting systemic toxicity. L-asparaginase (L-ASNase), which induces metabolic stress by depleting asparagine (Asn), is clinically used for hematological malignancies but shows limited activity against solid tumors due to poor delivery and an immunosuppressive microenvironment. We previously developed CRT3LP, a calreticulin (CRT)-targeting monobody-L-ASNase fusion protein, designed to exploit immunogenic cell death (ICD); however, its therapeutic potential is constrained by insufficient immune activation. Here, we show that CRT3LFP, a multifunctional fusion protein incorporating the flagellin B subunit (FlaB) into the CRT3LP scaffold, effectively promotes M2-to-M1 macrophage polarization while maintaining tumor-selective metabolic disruption. In combination with the tumor-colonizing bacterial strain CNC018, which induces surface-exposed CRT, CRT3LFP achieves precise tumor localization. This synergistic approach significantly inhibits tumor growth and reshapes the tumor microenvironment, characterized by enhanced macrophage polarization, dendritic cell maturation, and CD8 ⁺ T-cell expansion. Additionally, CD47 blockade further potentiates this effect, fostering the formation of durable immune memory. Together, our findings establish CRT3LFP as a tumor-targeted immunometabolic platform that integrates metabolic deprivation with coordinated innate and adaptive immune activation to overcome resistance in solid tumors.

Photoacoustic microscopy (PAM) enables high‐resolution, label‐free imaging by detecting ultrasound generated from pulsed‐laser absorption. For high‐fidelity spatiospectral imaging with single‐raster scanning, two spectrally distinct pulses should arrive nearly simultaneously to preserve spatial–temporal correlation, and at least one wavelength should be tunable for exogenous agents. We present spatiospectral PAM (SS‐PAM) driven by a gain‐switched Ti:sapphire laser engineered for this purpose. The source provides 735–855 nm tunability spanning hemoglobin and NIR‐I agents (e.g., indocyanine green, ICG) and is intrinsically synchronized to a 532 nm pump with a ≈150 ns gain‐switch delay, producing dual pulses for spatiospectral excitation. Operating at 20–30 kHz with pulse energies up to 63 µJ, SS‐PAM yields temporally correlated, high‐contrast excitation beyond sequential or dual‐laser schemes. In vivo, we quantified hemoglobin oxygen saturation (sO ₂ ) under controlled hypoxia, tracked vascular transport after intravenous ICG, visualized peritumoral lymphatic drainage, and analyzed oxygen extraction in tumor‐associated microvasculature, establishing SS‐PAM as a robust platform for synchronized, vessel‐level functional and molecular imaging.

Rationale: The immunosuppressive tumor microenvironment (TME) remains a major barrier to the efficacy of immune checkpoint blockade (ICB) therapy, underscoring the need for strategies that can safely reprogram the TME to enhance cancer immunity.

Radiopharmaceutical therapy (RPT) represents a critical approach in oncology, nevertheless its efficacy may be limited by tumor resistance mechanisms associated with metabolic reprogramming. Enhancing tumor radiosensitivity remains a major challenge. Engineered bi-functional starvation probes (CRT3LP and CRT4LP) that simultaneously target ecto-CRT and exert L-ASNase activity are explored for disrupting tumor amino acid metabolism. Herein, we systematically evaluate the ability of targeted starvation probes to enhance antitumor efficacy in radioactive iodine (RAI) therapy. In vitro, the probes upregulated p53 expression while downregulating Rev1 and SOD₂, thereby impairing ROS scavenging and sensitizing tumor cells to RAI-induced oxidative stress. In vivo, the combination treatment elevated intratumoral ROS levels, increased CD4⁺ and CD8⁺ T cell infiltration, upregulated pro-inflammatory cytokines (IFN-γ and TNF-α), and reduced regulatory T cell populations. Additionally, markers of tumor proliferation (Ki67 and CD31) were suppressed, while apoptotic markers (TUNEL and p21) were increased. Co-administration of the immune checkpoint inhibitor αPD-L1 further improved therapeutic efficacy. These findings suggest that targeted tumor starvation probes boost radiosensitivity and anti-tumor immunity, and this strategy shows improved efficacy in combination with αPD-L1 therapy.

Recently, various attenuated bacteria have been studied as cancer therapies due to their unique characteristics, which include tumor-targeting bioactivity and immunogenicity. Previously, we reported a Salmonella typhimurium strain, CNC018, which is attenuated by 10⁵–10⁶-fold compared with the wild-type strain but retains tumor-targeting specificity. However, although these bacteria suppress tumors at the early stage in mice, the tumors often regrow at later stages. Therefore, to increase antitumor efficacy, we used a doxycycline-inducible system to engineer this strain (CNC018pCH) to secrete both cytolysin A (ClyA) and hyaluronidase (HysA), a pore-forming toxin that kills tumor cells and an enzyme that disrupts the tumor microenvironment, respectively. Local secretion of ClyA from CNC018pCH triggered tumor cell death through pyroptosis, apoptosis, and necrosis (PANoptosis) in a cholesterol-dependent manner, thereby releasing cellular contents and danger signals to activate the immune system. In addition, localized secretion of HysA degraded hyaluronic acid secreted by cancer cells, facilitated bacterial penetration into tumors, and inhibited metastasis by down-regulating the ribosomal S6 kinase (RSK)-related signaling pathway. These therapeutic payloads enhanced the ability of S. typhimurium to control tumor growth and metastasis in various murine tumor models. Notably, CNC018pCH also generated memory responses by protecting cured mice from tumor rechallenge. Taken together, these findings demonstrate that this engineered bacterium is a promising candidate for cancer treatment by reshaping the tumor microenvironment through the induction of tumor cell death and degradation of hyaluronic acid.

Recently, various attenuated bacteria have been studied as cancer therapies due to their unique characteristics, which include tumor-targeting bioactivity and immunogenicity. Previously, we reported a Salmonella typhimurium strain, CNC018, which is attenuated by 10⁵–10⁶-fold compared with the wild-type strain but retains tumor-targeting specificity. However, although these bacteria suppress tumors at the early stage in mice, the tumors often regrow at later stages. Therefore, to increase antitumor efficacy, we used a doxycycline-inducible system to engineer this strain (CNC018pCH) to secrete both cytolysin A (ClyA) and hyaluronidase (HysA), a pore-forming toxin that kills tumor cells and an enzyme that disrupts the tumor microenvironment, respectively. Local secretion of ClyA from CNC018pCH triggered tumor cell death through pyroptosis, apoptosis, and necrosis (PANoptosis) in a cholesterol-dependent manner, thereby releasing cellular contents and danger signals to activate the immune system. In addition, localized secretion of HysA degraded hyaluronic acid secreted by cancer cells, facilitated bacterial penetration into tumors, and inhibited metastasis by down-regulating the RSK-related signaling pathway. These therapeutic payloads enhanced the ability of S. typhimurium to control tumor growth and metastasis in various murine tumor models. Notably, CNC018pCH also generated memory responses by protecting cured mice from tumor rechallenge. Taken together, these findings demonstrate that this engineered bacterium is a promising candidate for cancer treatment by reshaping tumor microenvironment through induction of tumor cell death and degradation of hyaluronic acid.

Infections caused by nontuberculous mycobacteria, such as Mycobacterium avium and Mycobacteroides abscessus, are becoming increasingly prevalent, and rising antibiotic resistance poses a significant clinical challenge. However, the mechanisms by which the host defense system controls these infections remain poorly understood. Here we show that the autophagy-related protein ATG7 in innate immune cells plays an essential role in controlling nontuberculous mycobacterial infection and protecting lung tissue from pathological inflammation. Patients with nontuberculous mycobacterial pulmonary disease exhibit reduced ATG7 expression in blood mononuclear cells and decreased ATG7 levels in necrotic lesions at disease sites. Mice lacking Atg7 in innate immune cells display elevated bacterial loads, excessive inflammation, mitochondrial damage, and multiple forms of cell death in the lungs, including pyroptosis, necrosis, and apoptosis. Notably, neutrophil infiltration in the lungs of these mice plays a key role in driving exacerbated inflammation and gasdermin E-associated cell death, which precede bacterial overgrowth. In vitro, Atg7-deficient macrophages exhibit impaired antimicrobial responses and reduced phagolysosomal fusion, but only modest increases in inflammation and cell death. These findings underscore the critical role of ATG7 in innate immune cells in orchestrating an effective host defense against nontuberculous mycobacterial lung infection by mitigating neutrophil-driven pathological inflammation and associated cell death.

Synergistic combinations of immunotherapeutic agents can improve the performance of anti-cancer therapies but may lead to immune-mediated adverse effects. These side-effects can be overcome by using a tumor-specific delivery system. Here, we report a method of targeted immunotherapy using an attenuated Salmonella typhimurium (SAM-FC) engineered to release dual payloads: cytolysin A (ClyA), a cytolytic anti-cancer agent, and Vibrio vulnificus flagellin B (FlaB), a potent inducer of anti-tumor innate immunity. Localized secretion of ClyA from SAM-FC induces immunogenic cancer cell death and promotes release of tumor-specific antigens and damage-associated molecular patterns, which establish long-term antitumor memory. Localized secretion of FlaB promotes phenotypic and functional remodeling of intratumoral macrophages that markedly inhibits tumor metastasis in mice bearing tumors of mouse and human origin. Both primary and metastatic tumors from bacteria-treated female mice are characterized by massive infiltration of anti-tumorigenic innate immune cells and activated tumor-specific effector/memory T cells; however, the percentage of immunosuppressive cells is low. Here, we show that SAM-FC induces functional reprogramming of the tumor immune microenvironment by activating both the innate and adaptive arms of the immune system and can be used for targeted delivery of multiple immunotherapeutic payloads for the establishment of potent and long-lasting antitumor immunity.