Subcutaneous Tumor-bearing Mice Research Articles

Antitumor Cream: Transdermal Hydrogel Containing Liposome-Encapsulated Ruthenium Complex for Infrared-Controlled Multimodal Synergistic Therapy.

A transdermal drug delivery cream, which is non-invasive and painless, containing a liposome-encapsulated Ru(II) complex (LipoRu) is created for the treatment of skin cancer. This formulation capitalizes on the synergistic antitumor effects of two-photon excited photodynamic therapy (PDT), photothermal therapy (PTT), and chemotherapy. LipoRu exhibits effective tumor accumulation, efficient cellular uptake, pH-sensitive and infrared-accelerated release, and dual localization to the nucleus and mitochondria. The released Ru(II) complexes within cells exert multiple antitumor mechanisms, such as DNA topoisomerase and RNA polymerase inhibition, Type I and II PDT, PTT, DNA photodamage, and apoptosis and ferroptosis induction. The biodistribution and therapeutic efficacy of LipoRu in vivo are systematically compared via three distinct administration routes: intratumoral injection, intravenous injection, and transdermal delivery through topical cream application. The positive therapeutic effects of the LipoRu cream fabricated here in subcutaneous tumor-bearing mice offer optimistic potential for the painless and non-invasive treatment of both early-stage and advanced skin cancers, as well as superficially located solid tumors.

Nanozyme Decorated Metal-Organic Framework Nanosheet for Enhanced Photodynamic Therapy Against Hypoxic Tumor.

Photodynamic therapy (PDT) has attracted increasing attention in the clinical treatment of epidermal and luminal tumors. However, the PDT efficacy in practice is severely impeded by tumor hypoxia and the adverse factors associated with hydrophobic photosensitizers (PSs), including low delivery capacity, poor photoactivity and limited ROS diffusion. In this study, Pt nanozymes decorated two-dimensional (2D) porphyrin metal-organic framework (MOF) nanosheets (PMOF@HA) were fabricated and investigated to conquer the obstacles of PDT against hypoxic tumors. PMOF@HA was synthesized by the coordination of transition metal iron (Zr4+) and PS (TCPP), in situ generation of Pt nanozyme and surface modification with hyaluronic acid (HA). The abilities of hypoxic relief and ROS generation were evaluated by detecting the changes of O2 and 1O2 concentration. The cellular uptake was investigated using flow cytometry and confocal laser scanning microscopy. The SMMC-7721 cells and the subcutaneous tumor-bearing mice were used to demonstrate the PDT efficacy of PMOF@HA in vitro and in vivo, respectively. Benefiting from the 2D structure and inherent properties of MOF materials, the prepared PMOF@HA could not only serve as nano-PS with high PS loading but also ensure the rational distance between PS molecules to avoid aggregation-induced quenching, enhance the photosensitive activity and promote the rapid diffusion of generated radical oxide species (ROS). Meanwhile, Pt nanozymes with catalase-like activity effectively catalyzed intratumoral overproduced H2O2 into O2 to alleviate tumor hypoxia. Additionally, PMOF@HA, with the help of externally coated HA, significantly improved the stability and increased the cell uptake by CD44 overexpressed tumor cells to strengthen O2 self-supply and PDT efficacy. This study provided a new strategy of integrating 2D porphyrin MOF nanosheets with nanozymes to conquer the obstacles of PDT against hypoxic tumors.

Multimodal enhancement of ferroptosis for synergistic cascade colorectal cancer therapy

Emerging research positions ferroptosis as a formidable anti-tumor modality for colorectal cancer (CRC). However, the current pathways to potentiate ferroptosis through a singular or dual approach have fallen short in maximizing treatment efficacy. Herein, a multimodal enhancement strategy of ferroptosis is achieved for synergistic cascade colorectal cancer therapy, via clinical-related drug repurposing and prodrug self-assembly of SN38 and sulfasalazine (SAS). In this process, the SN38 or SAS prodrug is synthesized by conjugating ferrocene via disulfide bonds to SN38 or SAS, then the two prodrugs self-assemble into a hybrid nanomedicine (FSS). FSS exhibited pronounced stability under physiological conditions, while demonstrating rapid disassembly via glutathione-responsive break of disulfide bonds in tumor microenvironments. Then, a multimodal enhancement strategy of cancer ferroptosis is achieved via the SAS-induced regulation of amino acid metabolism, SN38-resulted regulation of glutathione synthesis, accumulation of ferroptosis-related lipid signaling, and ferrocene-directed iron overload in tumors. The potent ferroptosis-based CRC anticancer effects of FSS were systematically explored and confirmed by comprehensive in vitro and in vivo analyses, including cellular experiments, subcutaneous and orthotopic tumor-bearing mouse models, and RNA-sequencing transcriptomic studies. This study lays the groundwork for the clinical translation of multimodal ferroptosis inducers, offering an effective and safe approach to cancer treatment.

Tetradecanol-wrapped, CpG-loaded porous Prussian blue nanoimmunomodulator for photothermal-responsive in situ anti-tumor vaccine-like immunotherapy

Therapeutic vaccine becomes a promising strategy to fight cancer by enhancing and sustaining specific anti-tumor immune responses. However, its efficacy is often impeded by low immunogenicity, the immunosuppressive tumor microenvironment (TME), and immune-related adverse events. Herein, we introduce 1-tetradecanol (TD)-wrapped, CpG-loaded porous Prussian blue nanoparticles (pPBNPs-CpG@TD) as a nanoimmunomodulator to initiate photothermal-induced immunogenic cell death (ICD) and photothermal-responsive release of CpG for augmenting the ICD effect. It was revealed that the dual-photothermal action significantly potentiated the in situ anti-tumor vaccine-like immunotherapy in terms of enhanced immunogenicity, promoted dendritic cell maturation, and increased T lymphocyte infiltration, consequently eliciting a robust immune response for inhibiting both primary and rechallenge tumors on a subcutaneous 4T1 tumor-bearing mouse model. The development and use of photoactive nanoimmunomodulators represents a novel and effective strategy to boost immunogenicity and counteract immunosuppressive TME, marking a significant advancement in the realm of ICD-driven in situ anti-tumor vaccine-like immunotherapy.

Salphen-Based Fe-N2O2@C Nanomaterial Applied in Synergistic Sonodynamic and Chemodynamic Therapy of Tumors

To synthesize a Salphen-based Fe-N2O2@C material with high peroxidase (POD)-mimicking activity and sonosensitivity for the synergistic sonodynamic (SDT) and chemodynamic (CDT) therapy of tumors. Fe-N2O2 was synthesized via the hydrothermal method, and Fe-N2O2@C was prepared by incorporating a ketjen black substrate. The morphology, structure, composition, enzyme mimic activity for reactive oxygen species (ROS) production, and sonosensitivity of the material were characterized. The ability and mechanism of Fe-N2O2@C to perform synergistic SDT and CDT killing of 4T1 mouse breast cancer cells were explored through in vitro experiments. The in vivo tumor-killing ability of Fe-N2O2@C combined with ultrasound irradiation was investigated using a subcutaneous 4T1 tumor-bearing mouse model. FFe-N2O2 and Fe-N2O2@C were both irregularly shaped nanospheres with average particle sizes of 25.9 nm and 36.2 nm, respectively. XRD, FTIR, and XPS analyses confirmed that both Fe-N2O2 and Fe-N2O2@C possessed a Salphen covalent organic framework structure with M-N2O2 coordination, and the ketjen black loading had no significant impact on this structure. Compared to Fe-N2O2, Fe-N2O2@C exhibited high POD-mimicking activity (with K m reduced from 19.32 to 5.82 mmol/L and v max increased from 2.51×10-8 to 8.92×10-8 mol/[L·s]) and sonosensitivity. Fe-N2O2@C in combination with ultrasound irradiation could produce a large amount of ROS within cells and a subsequent significant decrease in mitochondrial membrane potential, thereby inducing TEM-observable mitochondrial damage and causing cell apoptosis and death. In addition, in vivo experiments showed that Fe-N2O2@C in combination with ultrasound irradiation could effectively inhibit tumor growth in a 4T1 subcutaneous tumor-bearing mouse model without significant in vivo toxicity. In this study, we prepared a Salphen-based Fe-N2O2@C material with good biocompatibility, which can be used in combination with ultrasound irradiation to achieve SDT and CDT synergistic killing of tumor cells and inhibit tumor growth. This Salphen-based Fe-N2O2@C nanomaterial shows promising potential for multimodal tumor therapy.

Polyprodrug nanomedicine for chemiexcitation-triggered self-augmented cancer chemotherapy and gas therapy

Carbon monoxide (CO) has emerged as a potential antitumor agent by inducing the dysfunction of mitochondria and the apoptosis of cancer cells. However, it remains challenging to deliver appropriate amount of CO into tumor to ensure efficient tumor growth suppression with minimum side effects. Herein we developed a CO prodrug-loaded nanomedicine based on the self-assembly of camptothecin (CPT) polyprodrug amphiphiles. The polyprodrug nanoparticles readily dissociate upon exposure to endogenous H2O2 in the tumor, resulting in rapid release of CPT and generation of high-energy intermediate dioxetanedione. The latter can transfer the energy to neighboring CO prodrugs to activate CO production by chemiexcitation, while CPT promotes the generation of H2O2 in tumors, which in turn facilitates cascade CPT and CO release. As a result, the polyprodrug nanoparticles display remarkable tumor suppression in both subcutaneous and orthotopic breast tumor-bearing mice owing to the self-augmented CPT release and CO generation. In addition, no obvious systemic toxicity was observed in mice treated with the metal-free CO prodrug-loaded nanomedicine, suggesting the good biocompatibility of the polyprodrug nanoparticles. Our work provides new insights into the design and construction of polyprodrug nanomedicines for synergistic chemo/gas therapy.

Discovery of cinnamylaldehyde-derived mono-carbonyl curcumin analogs as anti-gastric cancer agents via suppression of STAT3 and AKT pathway

The structural modification of curcumin has always been a hotspot in drug development. In this paper, a class of cinnamylaldehyde-derived mono-carbonyl curcumin analogs (MCAs) with 7-carbon-links were designed and synthesized and their anticancer properties were evaluated. Through screening anti-gastric cancer activity of these compounds, H1 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest in vitro (SGC-7901 and AGS gastric cancer cells). Moreover, the SGC-7901 subcutaneous tumor-bearing mice studies revealed that H1 significantly inhibited the tumor growth of gastric cancer. We explored the possible potential targets of H1 through network pharmacology. Mechanistically, our results demonstrated that H1 showed potential anti-gastric cancer activity through suppression of the STAT3 and AKT signaling pathway in vitro and in vivo, which was validated by molecular docking. Overall, our results indicate the potential of H1 as a potent chemotherapeutic drug against gastric cancer.

Interferon Gamma Induces Higher Neutrophil Extracellular Traps Leading to Tumor-Killing Activity in Microsatellite Stable Colorectal Cancer.

Many patients with colorectal cancer do not respond to immune checkpoint blockade (ICB) therapy, highlighting the urgent need to understand tumor resistance mechanisms. Recently, the link between the IFNγ signaling pathway integrity and ICB resistance in the colorectal cancer tumor microenvironment has been revealed. The immunosuppressive microenvironment poses a significant challenge to antitumor immunity in colorectal cancer development. Tumor-associated neutrophils found in tumor tissues exhibit an immunosuppressive phenotype and are associated with colorectal cancer patient prognosis. Neutrophil extracellular traps (NET), DNA meshes containing cytotoxic enzymes released into the extracellular space, may be promising therapeutic targets in cancer. This study showed increased NETs in tumor tissues and peripheral neutrophils of high levels of microsatellite instability (MSI-H) patients with colorectal cancer compared with microsatellite stable (MSS) patients with colorectal cancer. IFNγ response genes were enriched in MSI-H patients with colorectal cancer compared with patients with MSS colorectal cancer. Co-culturing neutrophils with MSI-H colorectal cancer cell lines induced more NET formation and higher cellular apoptosis than MSS colorectal cancer cell lines. IFNγ treatment induced more NET formation and apoptosis in MSS colorectal cancer cell lines. Using subcutaneous or orthotopic CT-26 (MSS) tumor-bearing mice models, IFNγ reduced tumor size and enhanced PD-1 antibody-induced tumor-killing activity, accompanied by upregulated NETs and cellular apoptosis. These findings suggest that IFNγ could be a therapeutic strategy for MSS colorectal cancer.

Camouflaging attenuated Salmonella by cryo-shocked macrophages for tumor-targeted therapy

Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed “dead” yet “functional” Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained “dead” macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This “Trojan horse”-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.

High-speed adaptive photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) is capable of observing the distribution of optical absorbers inside bio-tissues with a high spatial resolution of micrometers. Unfortunately, due to the employment of a tight optical focus, it suffers from a limited depth of field (DOF), making it challenging to achieve high-resolution imaging of targets with arbitrary surfaces. Here, we propose a high spatiotemporal adaptive photoacoustic focusing mechanism through integrating a high-speed optical focuser, a time-of-flight contour deriving algorithm, and the rotary-scanning photoacoustic microscopy. The developed system, named high-speed adaptive photoacoustic microscopy (HA-PAM), features an ultrashort focus-shifting time of 5 ms and an enlarged DOF of up to 5 mm. With the assistance of the proposed mechanism, we can achieve a homogeneous lateral resolution of 6 μm over a 10 mm circular imaging domain within 5 s. We demonstrate the advantages of HA-PAM through imaging phantoms with curved surfaces, subcutaneous tumor-bearing mice, resected rabbit kidneys, and pulsating mouse brains. The imaging results suggest that this approach provides a high and consistent spatial resolution for imaging bio-tissues with arbitrary surfaces without sacrificing the imaging speed, and has the potential to extend the fundamental and clinical applications of OR-PAM.

Intratumoral Lactate Depletion Based on Injectable Nanoparticles-Hydrogel Composite System Synergizes with Immunotherapy against Postablative Hepatocellular Carcinoma Recurrence.

Thermal ablation is a crucial therapeutic modality for hepatocellular carcinoma (HCC), but its efficacy is often hindered by the high recurrence rate attributed to insufficient ablation. Furthermore, the residual tumors following insufficient ablation exhibit a more pronounced immunosuppressive state, which accelerates the disease progression and leads to immune checkpoint blockade (ICB) resistance. Herein, evidence is presented that heightened intratumoral lactate accumulation, stemming from the augmented glycolytic activity of postablative residual HCC cells, may serve as a crucial driving force in exacerbating the immunosuppressive state of the tumor microenvironment (TME). To address this, an injectable nanoparticles-hydrogel composite system (LOX-MnO2 @Gel) is designed that gradually releases lactate oxidase (LOX)-loaded hollow mesoporous MnO2 nanoparticles at the tumor site to continuously deplete intratumoral lactate via a cascade catalytic reaction. Using subcutaneous and orthotopic HCC tumor-bearing mouse models, it is confirmed that LOX-MnO2 @Gel-mediated local lactate depletion can transform the immunosuppressive postablative TME into an immunocompetent one and synergizes with ICB therapy to significantly inhibit residual HCC growth and lung metastasis, thereby prolonging the survival of mice postablation. The work proposes an appealing strategy for synergistically combining antitumor metabolic therapy with immunotherapy to combat postablative HCC recurrence.

Reexamining the effects of drug loading on the in vivo performance of PEGylated liposomal doxorubicin.

Higher drug loading employed in nanoscale delivery platforms is a goal that researchers have long sought after. But such viewpoint remains controversial because the impacts that nanocarriers bring about on bodies have been seriously overlooked. In the present study we investigated the effects of drug loading on the in vivo performance of PEGylated liposomal doxorubicin (PLD). We prepared PLDs with two different drug loading rates: high drug loading rate, H-Dox, 12.9% w/w Dox/HSPC; low drug loading rate, L-Dox, 2.4% w/w Dox/HSPC (L-Dox had about 5 folds drug carriers of H-Dox at the same Dox dose). The pharmaceutical properties and biological effects of H-Dox and L-Dox were compared in mice, rats or 4T1 subcutaneous tumor-bearing mice. We showed that the lowering of doxorubicin loading did not cause substantial shifts to the pharmaceutical properties of PLDs such as in vitro and in vivo stability (stable), anti-tumor effect (equivalent effective), as well as tissue and cellular distribution. Moreover, it was even more beneficial for mitigating the undesired biological effects caused by PLDs, through prolonging blood circulation and alleviating cutaneous accumulation in the presence of pre-existing anti-PEG Abs due to less opsonins (e.g. IgM and C3) deposition on per particle. Our results warn that the effects of drug loading would be much more convoluted than expected due to the complex intermediation between nanocarriers and bodies, urging independent investigation for each individual delivery platform to facilitate clinical translation and application.

Automated Synthesis and Preclinical Evaluation of Optimized Integrin α6-Targeted Positron Emission Tomography Imaging of Pancreatic Cancer.

Integrin α6 has been considered a promising biomarker, is overexpressed in many tumors, and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we identified a novel high-affinity integrin α6-targeted peptide named RD2 (Arg-Trp-Tyr-Asp-PEG4)2-Lys-Lys and developed a 18F-radiolabeled peptide tracer ([18F]-AlF-NOTA-RD2) and evaluated its potential application in positron emission tomography (PET) imaging of pancreatic cancer. [18F]-AlF-NOTA-RD2 was produced using GMP (Good Manufacturing Practice of Medical Products)-compliant automatic radiosynthesis on a single GE FASTLab2 cassette-type synthesis module. The stability of [18F]-AlF-NOTA-RD2 was analyzed in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). The cell uptake assay of the tracer was assessed using PANC-1 cells. In addition, small-animal PET imaging and biodistribution studies of [18F]-AlF-NOTA-RD2 were performed in pancreatic cancer subcutaneous tumor-bearing mice. The PET tracer [18F]-AlF-NOTA-RD2 was obtained with a radiochemical yield of 23.7 ± 4.7%, radiochemical purity of >99%, and molar activity of 165.7 ± 59.1 GBq/μmol. [18F]-AlF-NOTA-RD2 exhibited good in vitro stability in PBS and FBS. LogP octanol water value for the tracer was -2.28 ± 0.05 (n = 3). The binding affinity of RD2 to the integrin α6 protein (Kd = 0.13 ± 3.65 μM, n = 3) was significantly higher than that of the RWY (CRWYDENAC) (Kd = 6.97 ± 1.44 μM, n = 3). Small-animal PET imaging and biodistribution also revealed that [18F]-AlF-NOTA-RD2 displayed rapid and good tumor uptake and lower liver background uptake in PANC-1 tumor-bearing mice. [18F]-AlF-NOTA-RD2 showed significant radioactivity accumulation in tumors and was successfully blocked by NOTA-RD2. Compared with [18F]-FDG, [18F]-AlF-NOTA-RD2 PET imaging and biodistribution studies in PANC-1 xenograft tumor-bearing mice confirmed a good tumor-to-muscle ratio (8.69 ± 2.03 vs 1.41 ± 0.23, respectively) at 0.5 h and (2.99 ± 3.02 vs 1.43 ± 0.17, respectively) at 1 h post injection. Autoradiography of human pancreatic cancer tumor tissues further confirmed high accumulation of [18F]-AlF-NOTA-RD2. In summary, we developed an optimized integrin α6-targeted imaging tracer and obtained high radioactivity products with a cassette-type synthesis module; moreover, the tracer exhibited good binding affinity with integrin α6 and good target specificity for PANC-1 cells in xenograft pancreatic tumor-bearing mice, demonstrating its promising application as a noninvasive PET radiotracer of integrin α6 expression in pancreatic cancer.

Bioorthogonal Glycoengineering-Mediated Multifunctional Liquid Metal Nanoprobes for Highly Efficient Photoacoustic Imaging-Guided Photothermal/Chemotherapy of Tumor.

The development of a multifunctional cancer diagnosis and treatment platform offers excellent prospects for the effective eradication of malignant solid tumors. Herein, a doxorubicin hydrochloride (DOX)-loaded tannic acid (TA)-coated liquid metal (LM) multifunctional nanoprobe was synthesized and applied as a highly efficient platform for the photoacoustic (PA) imaging-guided photothermal/chemotherapy of tumor. The multifunctional nanoprobes exhibited strong near-infrared absorption, a remarkable photothermal conversion efficiency (PCE) of 55%, and high DOX loading capacity. Combined with the large intrinsic thermal expansion coefficient of LM, highly efficient PA imaging and effective drug release were realized. The LM-based multifunctional nanoprobes were specifically adsorbed into the cancer cells and tumor tissues via glycoengineering biorthogonal chemistry. The in vitro and in vivo photothermal/chemo-anticancer activity confirmed their promising potential in cancer treatment. The subcutaneous breast tumor-bearing mice completely recovered in 5 days under light illumination with clear PA imaging presentation, which showed better antitumor outcomes than single-mode chemotherapy or photothermal therapy (PTT), while keeping side effects at a minimum. Such an LM-based PA imaging-guided photothermal/chemotherapy strategy provided a valuable platform for resistant cancer precise treatment and intelligent biomedicine.

A programmable oral bacterial hydrogel for controllable production and release of nanovaccine for tumor immunotherapy

Oral protein vaccines are mainly used to prevent the infection of intestinal pathogens in clinic due to their high safety and strong compliance. However, it is necessary to design the efficient delivery systems to overcome the harsh gastrointestinal environment in the application process. Here we established a programmable oral bacterial hydrogel system for spatiotemporally controllable production and release of nanovaccines. The system was divided into three parts: (1) Engineered bacteria were encapsulated in chitosan-sodium alginate microcapsules, which offered protection against the extreme acid conditions in the stomach. (2) Microcapsules were dissolved, and then engineered bacteria were released and colonized in the intestine. (3) The release of nanovaccines was controlled periodically by a synchronous lysis genetic circuit for tumor immunotherapy. Compared to control groups, tumor volume of subcutaneous tumor-bearing mice treated with bacterial microgels releasing optimized nanovaccine was almost inhibited by 75% and T cell response was activated at least 2-fold. We believe that this programmable bacterial hydrogel will offer a promising way for the application of oral nanovaccines.

Abstract 581: Peptide-based theranostics targeting ANTXR1 in cancer stroma

Abstract Background: ANTXR1 is one of the pancreatic ductal adenocarcinoma (PDAC) stroma-specific cell surface markers (Ref 1), which is also overexpressed in the stroma of various cancer types. Radiotheranostics refers to the combination of molecular imaging with targeted radionuclide therapy. Recent success of radiotheranostic agents such as 177Lu-DOTATATE and 177Lu-PSMA617 heralds the vibrant expansion of radiotheranostics. Herein, we sought to develop ANTXR1 targeting peptide as a cancer stroma-targeting radiotheranostic agent. Methods: We selected A549 and NCI-H460 as ANTXR1 positive cell lines and NCI-H1437 and NCI-H358 as negative cell lines based on a proteomics database for cancer cell lines. ANTXR1-targeting peptide (PC01) was discovered using Chemistry-based Ultra-high-throughput Screening (CUSTM) platform. PC01 was conjugated with either fluorescence dye (Cy5-PC01) or chelator (DOTA-PC01). DOTA-PC01 was labeled with 68Ga. Flow cytometry and confocal microscopy were utilized to assess specific binding of the peptide. Subcutaneous tumor-bearing mice were intravenously injected with Cy5-PC01 or 68Ga-DOTA-PC01. In vivo/ex vivo fluorescence imaging (IVIS) and positron emission tomography (PET) imaging were performed. Results: In western blot analysis, H460 and A549 demonstrated high levels of expression of ANTXR1, while H1437 and H358 showed negligible levels of expression, consistent with the proteomics database. The mean fluorescence intensity of H460 was significantly higher than that of H1437 after incubation with Cy5-PC01 in flow cytometry. In confocal microscopy, the two ANTXR1 positive cell lines demonstrated higher fluorescence signals compared to the two ANTXR1 negative cell lines, which indicates the specific binding of PC01 to ANTXR1. As for in vivo experiments, timepoints of image acquisition for both IVIS and PET were 0h, 1h, 2h, and 4h after injection of the peptide. In in vivo IVIS images, tumor uptake was more prominent in H460 tumors than in H1437 tumors. Furthermore, tumor/kidney uptake ratio and tumor/liver uptake ratio of Cy5-PC01 were significantly higher in H460 tumors compared to those of H1437 tumors (P < 0.05, P < 0.05, respectively). Radiolabeling efficiency of 68Ga-DOTA-PC01 was measured to be over 95% by thin-layer chromatography. Serial PET images of 68Ga-DOTA-PC01 demonstrated an initial intense renal uptake and subsequent fast renal clearance, which is a typical biodistribution of peptide-based radiotheranostic agents. Also, the tumor uptake was more pronounced in the H460 tumors than in the H1437 tumors. Conclusion: We developed an ANTXR1 targeting peptide conjugated with DOTA for radiotheranostics. We found that PC01 demonstrated favorable biodistribution and specific in vitro/in vivo binding to ANTXR1, which prompts us to further optimize and develop a peptide-based ANTXR1 targeting radiotheranostic agent. Ref 1. Cancer Res (2022) 82 (12_Supplement): 5007 Citation Format: Shengjun Li, Jin Yeong Choi, Hye Sook Ha, Yoo Joung Oh, Song-Gil Lee, Hongyoon Choi, Hyung-Jun Im. Peptide-based theranostics targeting ANTXR1 in cancer stroma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 581.

Preclinical Dosimetry for Small Animal Radiation Research in Proton Therapy: A Feasibility Study.

To evaluate the feasibility of the three-dimensional (3D) printed small animal phantoms in dosimetric verification of proton therapy for small animal radiation research. Two different phantoms were modeled using the computed-tomography dataset of real rat and tumor-bearing mouse, retrospectively. Rat phantoms were designed to accommodate both EBT3 film and ionization chamber. A subcutaneous tumor-bearing mouse phantom was only modified to accommodate film dosimetry. All phantoms were printed using polylactic-acid (PLA) filament. Optimal printing parameters were set to create tissue-equivalent material. Then, proton therapy plans for different anatomical targets, including whole brain and total lung irradiation in the rat phantom and the subcutaneous tumor model in the mouse phantom, were created using the pencil-beam scanning technique. Point dose and film dosimetry measurements were performed using 3D-printed phantoms. In addition, all phantoms were analyzed in terms of printing accuracy and uniformity. Three-dimensionally printed phantoms had excellent uniformity over the external body, and printing accuracy was within 0.5 mm. According to our findings, two-dimensional dosimetry with EBT3 showed acceptable levels of γ passing rate for all measurements except for whole brain irradiation (γ passing rate, 89.8%). In terms of point dose analysis, a good agreement (<0.1%) was found between the measured and calculated point doses for all anatomical targets. Three-dimensionally printed small animal phantoms show great potential for dosimetric verifications of clinical proton therapy for small animal radiation research.

Ginsenoside Rk3 is a novel PI3K/AKT-targeting therapeutics agent that regulates autophagy and apoptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Ginsenoside Rk3, an important and rare saponin in heat-treated ginseng, is generated from Rg1 and has a smaller molecular weight. However, the anti-HCC efficacy and mechanisms of ginsenoside Rk3 have not yet been characterized. Here, we investigated the mechanism by which ginsenoside Rk3, a tetracyclic triterpenoid rare ginsenoside, inhibits the growth of HCC. We first explored the possible potential targets of Rk3 through network pharmacology. Both in vitro (HepG2 and HCC-LM3 cells) and in vivo (primary liver cancer mice and HCC-LM3 subcutaneous tumor-bearing mice) studies revealed that Rk3 significantly inhibits the proliferation of HCC. Meanwhile, Rk3 blocked the cell cycle in HCC at the G1 phase and induced autophagy and apoptosis in HCC. Further proteomics and siRNA experiments showed that Rk3 regulates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway to inhibit HCC growth, which was validated by molecular docking and surface plasmon resonance. In conclusion, we report the discovery that ginsenoside Rk3 binds to PI3K/AKT and promotes autophagy and apoptosis in HCC. Our data strongly support the translation of ginsenoside Rk3 into novel PI3K/AKT-targeting therapeutics for HCC treatment with low toxic side effects.

Bioengineered bacterial outer membrane vesicles encapsulated Polybia-mastoparan I fusion peptide as a promising nanoplatform for bladder cancer immune-modulatory chemotherapy.

Nanosized bacterial outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have emerged as a novel antitumor nanomedicine reagent due to their immunostimulatory properties. The encapsulated bacterial composition in OMVs can be edited via manipulating bioengineering technology on paternal bacteria, allowing us to design an ingenious antitumor platform by loading the Polybia-mastoparan I (MPI) fusion peptide into OMVs. OMVs containing the MPI fusion peptide were obtained from bioengineered Escherichia coli transformed with recombinant plasmid. The antitumor efficacy of bioengineered OMVs in vitro was verified by performing cell viability and wound-healing and apoptosis assays using MB49 and UMUC3 cells, respectively. Subcutaneous MB49 tumor-bearing mice were involved to investigate the tumor inhibition ability of bioengineered OMVs. Moreover, the activated immune response in tumor and the biosafety were also evaluated in detail. The resulting OMVs had the successful encapsulation of MPI fusion peptides and were subjected to physical characterization for morphology, size, and zeta potential. Cell viabilities of bladder cancer cells including MB49 and UMUC3 rather than a non-carcinomatous cell line (bEnd.3) were decreased when incubated with bioengineered OMVs. In addition, bioengineered OMVs restrained migration and induced apoptosis of bladder cancer cells. With intratumor injection of bioengineered OMVs, growths of subcutaneous MB49 tumors were significantly restricted. The inherent immunostimulation of OMVs was demonstrated to trigger maturation of dendritic cells (DCs), recruitment of macrophages, and infiltration of cytotoxic T lymphocytes (CTLs), resulting in the increased secretion of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). Meanwhile, several lines of evidence also indicated that bioengineered OMVs had satisfactory biosafety. Bioengineered OMVs fabricated in the present study were characterized by strong bladder cancer suppression and great biocompatibility, providing a new avenue for clinical bladder cancer therapy.

Intracellular Delivery of Anti-Kirsten Rat Sarcoma Antibodies Mediated by Polymeric Micelles Exerts Strong In Vitro and In Vivo Anti-Tumorigenic Activity in Kirsten Rat Sarcoma-Mutated Cancers.

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.