Orthotopic Cancer Model Research Articles

Enzymatically catalyzed molecular aggregation.

The dynamic modulation of the aggregation process of small molecules represents an important research objective for scientists. However, the complex and dynamic nature of internal environments in vivo impedes controllable aggregation processes of single molecules. In this study, we successfully achieve tumor-targeted aggregation of an aggregation-induced emission photosensitizer (AIE-PS), TBmA, with the catalysis of a tumor-overexpressed enzyme, γ-Glutamyl Transferase (GGT). Mechanistic investigations reveal that TBmA-Glu can be activated by GGT through cleavage of the γ-glutamyl bond and releasing TBmA. The poor water solubility of TBmA induces its aggregation, leading to aggregation-enhanced emission and photodynamic activities. The TBmA-Glu not only induces glutathione (GSH) depletion through GGT photo-degradation but also triggers lipid peroxidation accumulation and ferroptosis in cancer cells through photodynamic therapy. Finally, the in vivo studies conducted on female mice using both tumor xenograft and orthotopic liver cancer models have also demonstrated the significant anti-cancer effects of TBmA-Glu. The exceptional cancer-targeting ability and therapeutic efficiency demonstrated by this GGT activatable AIE-PS highlights enzymatic-mediated modulation as an effective approach for regulating small molecule aggregation intracellularly, thereby advancing innovative therapeutic strategies for various diseases.

Salmonella Biomimetic Nanoparticles for Photothermal-Chemotherapy of Colorectal Cancer.

Nanomedicines have been widely used in colorectal cancer treatment, but their suboptimal targeting and deficient penetration capabilities remain obstacles in the delivery of therapeutics. In this study, inspired by the natural tumor tropism and intestinal invasion of Salmonella, we engineered highly biomimetic nanoparticles (SM-AuNRs) utilizing a Salmonella membrane to coat bacilliform Au nanorods. The engineered SM-AuNRs were able to mimic the germ's morphology and biological surface. SM-AuNRs containing the specific proteins inherited from the Salmonella membrane facilitated specific targeting and internalization into tumor cells. Meanwhile, SM-AuNRs with the rod-shaped morphology effectively traversed mucus barriers and tumor stroma. Due to the superior biological barrier penetrating and tumor targeting capabilities, doxorubicin-loaded SM-AuNRs with near-infrared laser irradiation displayed remarkable photothermal-chemotherapeutic antitumor effects in mouse orthotopic colorectal cancer models. Our findings pave the way for the design of bacteria-mimicking nanoparticles, presenting a promising avenue for the targeting and efficient treatment of colorectal cancer.

Galectin-3 induces pathogenic immunosuppressive macrophages through interaction with TREM2 in lung cancer

BackgroundHigh infiltration of tumor-associated macrophages (TAMs) is associated with tumor promotion and immunosuppression. The triggering receptor expressed on myeloid cells 2 (TREM2) is emerged as a key immunosuppressive regulator for TAMs, however, how TREM2-expressing TAMs are recruited and what ligands TREM2 interacts with to mediate immunosuppression is unknown.MethodsFlow cytometry and single-cell RNA sequencing were used to analyze TREM2 expression. Mechanistically, mass spectrometry and immunoprecipitation were employed to identify proteins binding to TREM2. Phagocytosis and co-culture experiments were used to explore the in vitro functions of galectin3-TREM2 pair. Establishment of TREM2f/f-Lyz2-cre mice to validate the role of TREM2 signaling pathway in lung carcinogenesis. GB1107 were further supplemented to validate the therapeutic effect of Galectin3 based on TREM2 signaling regulation.ResultsThis study identified that abundant TREM2+ macrophages were recruited at the intra-tumor site through the CCL2-CCR2 chemotactic axis. Galectin-3 impaired TREM2-mediated phagocytosis and promoted the conversion of TREM2+ macrophages to immunosuppressive TAMs with attenuated antigen presentation and co-stimulatory functions both in vitro both in vivo, and galectin-3 is a potential ligand for TREM2. Genetic and pharmacological blockade of TREM2 and galectin-3 significantly inhibited lung cancer progression in subcutaneous and orthotopic cancer models by remodeling the tumor immune microenvironment.ConclusionOur findings revealed a previously unknown association between galectin-3 and TREM2 in TAMs of lung cancer, and suggested simultaneous inhibition of galectin3 and TREM2 as potent therapeutic approach for lung cancer therapy.Graphical

Sono-blasting Triggered Cascading-Amplification of Oxidative Stress for Enhanced Interventional Therapy of Hepatocellular Carcinoma.

Interventional therapy is widely regarded as a highly promising treatment approach for nonsurgical liver cancer. However, the development of drug resistance and tolerance to hypoxic environments after embolization can lead to increased angiogenesis, enhanced tumor cell stemness, and greater invasiveness, resulting in metastasis and recurrence. To address these challenges, a novel approach involving the use of lecithin and DSPE-PEG comodified Ca2+ loaded (NH4)2S2O8 (LDCNSO) drug in combination with transcatheter arterial embolization (TAE) has been proposed. The sono-blasting effect of LDCNSO under ultrasound triggers a cascading amplification of oxidative stress, by releasing sulfate radical (·SO4-), hydroxyl radical (·OH), and superoxide (·O2-), inducing Ca2+ overload, and reducing glutathione (GSH) levels, which eventually leads to apoptosis. LDCNSO alongside TAE has demonstrated remarkable therapeutic efficacy in the rabbit orthotopic cancer model, resulting in significant inhibition of tumor growth. This research provides valuable insights for the effective treatment of orthotopic tumors.

Host derived macrophage migration inhibitory factor expression attenuates anti-tumoral immune cell accumulation and promotes immunosuppression in the tumor microenvironment of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif+/+ or Mif−/− mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif−/− mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif−/− mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8+ T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.

RhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors

BackgroundChimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy...

Diffuse reflectance spectroscopy for optical characterizations of orthotopic head and neck cancer models in vivo.

We demonstrated an easy-to-build, portable diffuse reflectance spectroscopy device along with a Monte Carlo inverse model to quantify tissue absorption and scattering-based parameters of orthotopic head and neck cancer models in vivo. Both tissue-mimicking phantom studies and animal studies were conducted to verify the optical spectroscopy system and Monte Carlo inverse model for the accurate extraction of tissue optical properties. For the first time, we reported the tissue absorption and scattering coefficients of mouse normal tongue tissues and tongue tumor tissues. Our in vivo animal studies showed reduced total hemoglobin concentration, lower tissue vascular oxygen saturation, and increased tissue scattering in the orthotopic tongue tumors compared to the normal tongue tissues. Our data also showed that mice tongue tumors with different sizes may have significantly different tissue absorption and scattering-based parameters. Small tongue tumors (volume was ∼60 mm3) had increased absorption coefficients, decreased reduced-scattering coefficients, and increased total hemoglobin concentrations compared to tiny tongue tumors (volume was ∼18 mm3). These results demonstrated the potential of diffuse reflectance spectroscopy to noninvasively evaluate tumor biology using orthotopic tongue cancer models for future head and neck cancer research.

TOP CAR with TMIGD2 as a safe and effective costimulatory domain in CAR cells treating human solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy shows impressive efficacy treating hematologic malignancies but requires further optimization in solid tumors. Here, we developed a TMIGD2 optimized potent/persistent (TOP) CAR that incorporated the costimulatory domain of TMIGD2, a T and NK cell costimulator, and monoclonal antibodies targeting the IgV domain of B7-H3, an immune checkpoint expressed on solid tumors and tumor vasculature. Comparing second- and third-generation B7-H3 CARs containing TMIGD2, CD28, and/or 4-1BB costimulatory domains revealed superior antitumor responses in B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells in vitro. Comparing these two constructs using in vivo orthotopic human cancer models demonstrated that B7-H3.TMIGD2 CAR-T cells had equivalent or superior antitumor activity, survival, expansion, and persistence. Mechanistically, B7-H3.TMIGD2 CAR-T cells maintained mitochondrial metabolism; produced less cytokines; and established fewer exhausted cells, more central memory cells, and a larger CD8/CD4 T cell ratio. These studies demonstrate that the TOP CAR with TMIGD2 costimulation offered distinct benefits from CD28.41BB costimulation and is effective against solid tumors.

Abstract 491: Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model

Abstract Purpose: Radiation is the most common modality for lung cancer treatment method. Radiationinduces apoptosis, causing cancer cells to be fragmented and expose the cancer-associatedantigens to the tumor microenvironment. These can be recognized by antigen-presenting cells(APC) to induce the antineoplastic effect by activating cytotoxic T cells. Our prior studies showadding immunoadjuvant like AntiCD40 with radiation can further activate the APCs in its anti-tumor (M1) format. Recent studies have shown that flavonoids like Hesperetin, an ACE2receptor agonist also can induce apoptosis in cancer cells whereas ACE2 receptors are abundant in lung cancer cells.This project aims to develop a novel treatment for non-small cell lung cancer (NSCLC) using nanotechnology for inhalation drug delivery Hesperetin, a flavonoid with antioxidant and pro-apoptotic activity; bound to PLGA-coated nanoparticles (Hesperetin Nanoparticles, HNPs) andAntiCD40 aiming for localized targeted dosing to reduce the treatment-related systemic toxicity.We plan to test the efficacy and safety of this aerosol spray. Methods: In-vitro studies were performed in human A549 (ATCC) and murine LLC1 (ATCC)lung cancer cell lines for growth (MTT) and clonogenic survival assays to demonstrate the anti-cancer cell activity of Hesperetin (Sigma). Next, HNP was prepared using NanoFabTx™ nano-formulation reagent kits (Millipore-Sigma). A syngeneic orthotopic murine model of Lungadenoma was generated in wild (+/+) C57/BL6 background mice with similar background LLC1cell lines. We developed syngeneic orthotopic murine lung tumors with luciferase genetransfected LL/2-Luc2 (ATCC) Lewis Lung Cancer cell line in wild-type C57BL/6 mice(Techonic). Lung tumor-bearing mice were treated with HNP, antiCD40, or both with aerosolspray with a control group treated with the solvent (dH2O) only. To assess the drug delivery withaerosol treatment ex-vivo lung tissue was analyzed for Fluorescence tagged antiCD40 andnanoparticle uptakes. A survival assay was performed to analyze the efficacy of aerosoltreatment of HNPs with or without AntiCD40. All cohorts were also analyzed for body score, body weight, and liver and kidney functions. Results: We were able to successfully develop an aerosol drug delivery model to administer bothAnti-CD40 and HNP in a murine lung cancer model. In our analysis of an orthotopic murinelung cancer model, we demonstrate a high intake of the HNP and AntiCD40 by the cancer cellssparing the normal lung tissue. Moreover, the highest percentage of survival rate was observed with the combination aerosol treatment with HNP+AntiCD40 (p<0.001), compared to CD40alone (p<0.01) or HNP (p<0.01) alone. Comments: This treatment model will allow us to make the lung cancer treatment method easilyavailable for the mass population without having hazardous radiation treatment in the lungcancer model. Citation Format: Sayeda Yasmin-Karim, Geraud Richard, Amanda Fam, Alina-Marissa Ogurek, G. Mike Makrigiorgos. Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 491.

Abstract 485: Cancer killing effect of ultrasound-responsive mechanophore nanoparticles

Abstract Radiotherapy, a mainstay in cancer treatment, often inadvertently damages healthy tissues while targeting malignancies with free radicals. As an alternative, Photodynamic Therapy (PDT) employs non-ionizing photons to generate reactive oxygen species (ROS) in tumors, offering precise targeting with minimal long-term effects. However, its efficacy is constrained to superficial tumors due to the limited penetration of light. Sonodynamic Therapy (SDT), an evolution of PDT, uses ultrasound to enhance tissue penetration while maintaining its non-invasive nature. Initial SDT studies suggest that ultrasound can activate PDT's photosensitizers to produce ROS, offering a potential pathway for tumor eradication. Nevertheless, the principal mechanisms of ROS generation in SDT, namely pyrolysis and sonoluminescence, raise concerns about potential tissue injury. In our research, we introduce an innovative approach employing mechanophores into nanoparticles. Mechanophores, molecular units within polymers sensitive to mechanical stress, undergo transformations under such conditions, including the release of small molecules or free radicals. We have developed injectable nano-sized mechanophore nanoparticles, designed to respond to high-frequency ultrasound (>100 kHz) more efficiently and with reduced energy requirements. This study focuses on nano-engineering the echogenicity of these mechanophore nanoparticles to enhance ROS generation. When agitated with focused ultrasound (FUS), these nanoparticles successfully induced ROS, leading to significant cell death, a result not observed with FUS alone in the absence of nanoparticles. In vivo experiments using mouse orthotopic cancer models demonstrated these nanoparticles' ability to inhibit tumor growth effectively. Under biocompatible ultrasound conditions, the nanoparticles cause effective cancer cell eradication with minimal impact on surrounding healthy tissues. Our findings not only introduce a novel approach to cancer treatment but also pave the way for broader applications of mechanophore nanoparticles in biomedicine. This study underscores the potential of ultrasound-activatable mechanophore nanoparticles as a versatile, effective, and safe option for cancer therapy. Citation Format: Jian Wang, Shensheng Zhao, Junxi Yi, Yunyan Sun, Megha Agrawal, King Li, Jeffrey S. Moore, Michael Oelze, Yun-Sheng Chen. Cancer killing effect of ultrasound-responsive mechanophore nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 485.

Abstract 4184: Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice

Abstract (a) To investigate novel therapeutic agents in pancreatic cancer, animal models that recapitulate the features of the human immune system are necessary. We aim to analyze an orthotopic model in CD34+HSC-based humanized mice and to prove that is an indispensable model for immune-oncology therapy research. (b) We established an orthotopic tumor model using luciferase-expressing BxPC3 cells in humanized NSG mice. After inoculation of tumor cells, anti-PD-1 Ab was administered to mice 5 times a week by I.P. injection. Tumor formation and progression were monitored using IVIS. To compare the subsets and immune-check point receptors of human immune cells in blood and tumor tissue, Flow cytometry and immunohistopathology were used. Additionally, we compared the distribution of various cells included in the tumor and the mRNA expression level of each cell through a single cell RNA sequence. We also establish primary cell lines and organoids with the humanized mouse tumor for further evaluation. (c) After inoculation of BxPC3_Luc cell line into mice pancreas, we monitored tumor growth using a noninvasive imaging method of IVIS. When we compared the tumor growth between the anti-PD-1 treatment and control, there was no significant difference. Using FACS, we analyzed the various markers of human immune cells in mouse blood and tumor tissue. In blood, PD-1 expression of CD8+ T cells decreased, and Tim-3 expression increased in anti-PD-1 group. And, PD-1 expression of CD4+ T cells decreased in anti-PD-1 group. In tumor tissue, we also verified the decrease of PD-1 expression on tissue infiltrating CD8+ T cells and CD4+ T cells in the anti-PD-1 group. Especially in the more responsive mouse among anti-PD-1 group, activation markers in cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (CD4+FOXP3+CD25+) increased, and also, M1 macrophage (CD86+) expression was increased. Furthermore, we compared the mRNA expression levels of various cells in the tumor microenvironment according to anti-PD-1 Ab treatment through single cell RNA seq analysis. It was confirmed that the expression of genes related to proliferation and growing decreased, while the expression of genes related to apoptosis increased in cancer cells. Finally, we constructed primary cancer cells and organoids from tumor tissue and confirmed that the immune-oncological characters of these models are maintained after passaging. (d) We successfully established a pancreatic orthotopic model in a humanized mouse and analyzed the changes in immune cells and tumor cells after anti-PD-1 treatment. In addition, by obtaining tumor tissue-based preclinical models, we laid the foundation for future immune-oncologic experiments. We hope that these attempts and results will help in the development and drug verification of new immunotherapy agents for refractory solid tumors such as pancreatic cancer. Citation Format: Sujin Park, Eun-young Koh, Inha Yoo, Ji Hye Jeong, Eunsung Jun. Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4184.

Abstract 1275: In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis

Abstract Metastasis is responsible for about 90% of cancer-related mortality. Throughout the metastatic cascade, cancer cells requires substantial metabolic rewiring to adapt the nutrient micro-environmental changes enabling dissemination and seeding in distant organs. Recent insights into importance of the tumor-intrinsic metabolic reprograming has been implicated in immune escape, yet altered immune responses imposed by tumor metabolism during metastasis are poorly understood. Here, we generated genetic perturbations in triple-negative breast cancer cells using metabolism-focused CRISPR knockout library and performed in vivo screens in both immunocompetent and immunodeficient mice. By comparing enriched sgRNAs in lung metastases or primary tumors, we identify loss of one gene related to long chain fatty acid (LCFA) β-oxidation (FAO) potentiates metastatic dissemination in immunocompetent mice but no in immunodeficient mice, suggesting that impaired FAO in tumor cells promotes breast cancer lung metastasis through an lymphocytes-dependent manner. We confirmed individual sgRNAs targeting this FAO gene from the primary screen dramatically accelerate metastasis in different breast cancer orthotopic models. Specifically, we find that low expression of this gene in primary tumors of advanced breast cancer patients with high grade tumors and positive lymph nodes (LN+) is associated with reduced overall survival. In addition, deletion of this gene generates an immunosuppressive microenvironment in the lung metastatic tumors with increased neutrophils, monocytes and decreased TNFα+-IFNγ+ CD8+ T cells. Mechanistically, impaired FAO in tumor cells causes mitochondria imbalance under lipid metabolism stress, consequently triggers inflammatory response on establishing a neutrophil-rich metastatic niche via mitochondrial nucleic acid-driven innate immunity. Altogether, our data provides a new insight on how tumor cell lipid metabolism impacting immune interplay during metastatic progression, nominating potential anti-tumor immunotherapies. Citation Format: Xiaoyong Wang, Shu-ting Chou, Verra Ngwa, Yoonha Hwang, Breelyn Karno, Deanna Edwards, Jin Chen. In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1275.

The EIF3H-HAX1 axis increases RAF-MEK-ERK signaling activity to promote colorectal cancer progression

Eukaryotic initiation translation factor 3 subunit h (EIF3H) plays critical roles in regulating translational initiation and predicts poor cancer prognosis, but the mechanism underlying EIF3H tumorigenesis remains to be further elucidated. Here, we report that EIF3H is overexpressed in colorectal cancer (CRC) and correlates with poor prognosis. Conditional Eif3h deletion suppresses colorectal tumorigenesis in AOM/DSS model. Mechanistically, EIF3H functions as a deubiquitinase for HAX1 and stabilizes HAX1 via antagonizing βTrCP-mediated ubiquitination, which enhances the interaction between RAF1, MEK1 and ERK1, thereby potentiating phosphorylation of ERK1/2. In addition, activation of Wnt/β-catenin signaling induces EIF3H expression. EIF3H/HAX1 axis promotes CRC tumorigenesis and metastasis in mouse orthotopic cancer model. Significantly, combined targeting Wnt and RAF1-ERK1/2 signaling synergistically inhibits tumor growth in EIF3H-high patient-derived xenografts. These results uncover the important roles of EIF3H in mediating CRC progression through regulating HAX1 and RAF1-ERK1/2 signaling. EIF3H represents a promising therapeutic target and prognostic marker in CRC.

ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

There are significant commonalities among several pathologies involving fibroblasts, ranging from auto-immune diseases to fibrosis and cancer. Early steps in cancer development and progression are closely linked to fibroblast senescence and transformation into tumor-promoting cancer-associated fibroblasts (CAFs), suppressed by the androgen receptor (AR). Here, we identify ANKRD1 as a mesenchymal-specific transcriptional coregulator under direct AR negative control in human dermal fibroblasts (HDFs) and a key driver of CAF conversion, independent of cellular senescence. ANKRD1 expression in CAFs is associated with poor survival in HNSCC, lung, and cervical SCC patients, and controls a specific gene expression program of myofibroblast CAFs (my-CAFs). ANKRD1 binds to the regulatory region of my-CAF effector genes in concert with AP-1 transcription factors, and promotes c-JUN and FOS association. Targeting ANKRD1 disrupts AP-1 complex formation, reverses CAF activation, and blocks the pro-tumorigenic properties of CAFs in an orthotopic skin cancer model. ANKRD1 thus represents a target for fibroblast-directed therapy in cancer and potentially beyond.

CSN6-SPOP-HMGCS1 Axis Promotes Hepatocellular Carcinoma Progression via YAP1 Activation.

Cholesterol metabolism has important roles in maintaining membrane integrity and countering the development of diseases such as obesity and cancers. Cancer cells sustain cholesterol biogenesis for their proliferation and microenvironment reprograming even when sterols are abundant. However, efficacy of targeting cholesterol metabolism for cancer treatment is always compromised. Here it is shown that CSN6 is elevated in HCC and is a positive regulator of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1) of mevalonate (MVA) pathway to promote tumorigenesis. Mechanistically, CSN6 antagonizes speckle-type POZ protein (SPOP) ubiquitin ligase to stabilize HMGCS1, which in turn activates YAP1 to promote tumor growth. In orthotopic liver cancer models, targeting CSN6 and HMGCS1 hinders tumor growth in both normal and high fat diet. Significantly, HMGCS1 depletion improves YAP inhibitor efficacy in patient derived xenograft models. The results identify a CSN6-HMGCS1-YAP1 axis mediating tumor outgrowth in HCC and propose a therapeutic strategy of targeting non-alcoholic fatty liver diseases- associated HCC.

Significance of Second Near-Infrared Region (NIR-II) guided tumour detection and surgical treatment using Carcinoembryonic Antigen-Related Cell Adhesion Molecule (CEACAM) targetted probe in colorectal surgery.

Respected Ma’am, According to WHO, cancer alone killed 10 million people in 2018., making it a leading cause of mortality worldwide (1). Near-infrared fluorescence-guided surgery, a rapidly evolving technique, enables surgeons to assess tumour borders of various cancer types and lesions of other prevalent illnesses with great precision. Near-Infrared Region (NIR) radiation's ability to absorb energy at one wavelength and remit it at a different, longer wavelength stimulates substances to fluorescence, aiding the complete surgical removal of tumour tissues with negative margins, thus, improving the disease’s prognosis and decreasing chances of reoccurrence (2). Conventional fluorescence-guided surgery focuses on the first near-infrared window (NIR-I, 700–900 nm), whose tissue penetration is limited to a depth of only 1-6 mm. In contrast, the second near-infrared window (NIR-II, 1000-1700 nm) significantly reduces tissue absorption, autofluorescence, and photon scattering, enabling a deep penetration (up to 20 mm), micron-scale resolution, and an increased tumor-to-normal tissue ratio (T/NT) leading to more precise tumour removal (1–3). A recent study in Beijing, China (3), has explored the value of the second near-infrared window (NIR-II, 1000-1700 nm) in image guided surgeries that uses 2D5-IRDye800CW, as a targeted NIR-II probe formed by the conjugation of 2D5-CEACAM5 nanobody (2D5)34 with IRDye800CW dye for intraoperative navigation in colorectal cancer which is the type of cancer known to have one of the highest mortality rates. To compare the efficacy of NIR-11 with that of NIR-1comparative studies were also conducted on mouse CA colon, orthotopic cancer and metastasis models. Compared to NIR-I, NIR-2 fluorescence had a much greater tumor to background ratio, the difference was statistically significant (p<0.0001) proving that the imaging probe was more sensitive in detecting tumours. This technique detected and resected tumours smaller than 2 mm, making it the most suitable option for cancer diagnosis and treatment (3). Henceforth, it can be deduced that 2D5-IRDye800CW and NIR-II fluorescence collectively have the potential to act as a way of improvement in surgery of colorectal cancer. Studies have also demonstrated the use of NIR-II systems for imaging the vasculature, detecting sentinel lymph nodes, image-guided cancer surgery, and fluorescence endoscopy (4). Its other good attributes include high precision during image-guided procedures, over currently used NIR-I radiations (3). Finally, NIR-II fluorescence technique's crucial role in detecting and treating tumours, highlights its importance and warrants further advancement.

Abstract B149: Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity

Abstract Beta-catenin represents a significant challenge as a therapeutic target due to its multifaceted role in tumor development as well as normal cell homeostasis. Dysregulation of the Wnt/beta-catenin signaling pathway is implicated in various cancers, including colorectal, hepatocellular, breast, ovarian, and pancreatic cancers. We developed ST316 as a peptide antagonist of the interaction of beta-catenin with BCL9, a co-activator implicated in oncogenic beta-catenin signaling. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) that is currently enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/beta-catenin signaling pathway. In vitro studies indicate that ST316 selectively attenuates Wnt transcriptional activity in beta-catenin-dependent but not -independent cell lines. Gene expression profiling using RNA sequencing in beta-catenin mutant HCT116 cells and APC mutant Colo320DM cells demonstrates a significant downregulation of Wnt pathway target genes, oncogenic signature genes and pro-tumor immunologic gene sets within 24 hours of ST316 treatment. Consequently, ST316 induced a dose-dependent decrease in viability of beta-catenin-dependent cells while beta-catenin-independent cells were resistant, indicating that the anticancer effects of ST316 are mediated by inhibition of the Wnt/beta-catenin pathway. To evaluate the impact of ST316 in vivo, a 4T1 triple negative breast cancer orthotopic tumor model was employed. Once weekly injection of ST316 (5mg/kg SC) over eight weeks suppressed expression of Wnt target genes CDK4 and cMyc and resulted in a significant 84.3% inhibition of tumor growth compared to the control group. In addition to direct anti-tumor activity, ST316 triggers a pro-inflammatory immune microenvironment. ST316 induces polarization of human macrophages derived from peripheral blood mononuclear cells, resulting in >100-fold shift from immunosuppressive M2 macrophages towards the anti-tumor M1 phenotype. Co-cultures of M2 macrophages with T cells treated with ST316 demonstrate a significant three-fold increase in T-cell activation, as indicated by intracellular IFN-γ staining. Finally, ST316 enhances the anti-tumor activity of anti-PD-1 therapy in a syngeneic orthotopic 4T1 mouse model, where combination ST316 and anti-PD-1 antibody results in enhanced tumor growth inhibition compared to either single agent alone (p<0.01). These findings demonstrate the antitumor and immunostimulatory effects of ST316 and highlight its potential as a therapeutic agent for targeting cancers with aberrantly activated Wnt signaling pathways. Citation Format: Lila Ghamsari, Claudio Scuoppo, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Zachary Mattes, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein, Jim Rotolo. Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B149.

Targeted delivery of genistein for pancreatic cancer treatment using hyaluronic-coated cubosomes bioactivated with frankincense oil

Pancreatic cancer is an aggressive malignancy that remains a major cause of cancer-related deaths. Research for innovative anticancer therapeutic options is thus imperative. In this regard, phytotherapeutics offer great promise as efficient treatment modalities, especially leveraging nanodrug delivery. Herein, we innovatively coloaded the flavonoid genistein (Gen) and frankincense essential oil (FO) within cubosomes, which were then coated with the bioactive ligand hyaluronic acid (HA/Gen-FO-Cub) for active-targeting of pancreatic cancer. The novel HA/Gen-FO-Cub displayed optimum nanosize (198.2 ± 4.5 nm), PDI (0.27 ± 0.01), zeta-potential (−34.7 ± 1.2 mV), Gen entrapment (99.3 ± 0.01 %), and controlled Gen release (43.7 ± 1.2 % after 120 h). HA/Gen-FO-Cub exerted selective anticancer activity on pancreatic cancer cells (PANC-1; 8-fold drop in IC50), cellular uptake and anti-migratory effect compared to Gen solution. HA/Gen-FO-Cub revealed prominent cytocompatibility (100 ± 5.9 % viability of human dermal fibroblast). Moreover, HA/Gen-FO-Cub boosted the in vivo anticancer activity of Gen in an orthotopic cancer model, affording tumor growth suppression (2.5-fold drop) and downregulation of NFκB and VEGF (2.9- and 1.8-fold decrease, respectively), compared to Gen suspension. Antimetastatic efficacy and Bcl-2-downexpression was histologically confirmed. Our findings demonstrate the promising anticancer aptitude of HA/Gen-FO-Cub as an effective phytotherapeutic nanodelivery system for pancreatic cancer therapy.

Abstract PO-084: Deletion of macrophage migration inhibitory factor promotes antitumoral T cell infiltration and inhibits MDSC recruitment to the head and neck cancer microenvironment

Abstract Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to worse prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif+/+ or Mif− / − mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We found that Mif− / − mice have decreased tumor growth and burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased T cell recruitment to the HNSCC tumor microenvironment, within the tumors of Mif− / − mice. MIF deletion also enhanced the effector function of anti-tumoral Th1 cells and decreased the accumulation of granulocytic MDSC in the tumor microenvironment. Furthermore, we show that isolated MDSC chemotactically respond to MIF in a dose dependent manner which is inhibited when MIF is depleted. Interestingly, MDSCs of tumor bearing Mif− / − mice expressed increased levels of PDL1 compared to Mif+/+ mice. However, Mif− / − and Mif+/+ MDSCs have comparable abilities to suppress T cell proliferation. Herein we describe a chemotactic and immunomodulatory role for MIF in the context of HNSCC. MIF targeted immunomodulation remains a potentially viable cancer therapeutic, however more remains to be understood about the cellular interaction of MIF within the tumor microenvironment before these targeted therapies can be effectively employed Citation Format: Steve Oghumu. Deletion of macrophage migration inhibitory factor promotes antitumoral T cell infiltration and inhibits MDSC recruitment to the head and neck cancer microenvironment [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-084.

Saikosaponin d modulates the polarization of tumor-associated macrophages by deactivating the PI3K/AKT/mTOR pathway in murine models of pancreatic cancer.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) poses a major obstacle to traditional and immunomodulatory cancer therapies and is closely associated with macrophage polarization. Saikosaponin d (SSd), a major active component of triterpene saponins derived from Bupleurum falcatum, has anti-inflammatory and antitumor activities. However, whether SSd can regulate immune cells during the development of the TME in PDAC remains unknown. In the present study, we aimed to analyze the role of SSd in regulating immune cells in the PDAC TME, especially the polarization of macrophages, and examine the related mechanisms. An orthotopic PDAC cancer model was used to investigate the antitumor activities and the regulation of immune cells in vivo. In vitro, bone marrow mononuclear (BM-MNC) cells and RAW 264.7 cells were used to induce the M2 macrophage phenotype and examine the effects and molecular mechanism of SSd on M2 macrophage polarization. The results revealed that SSd could directly inhibit the apoptosis and invasion of pancreatic cancer cells, modulate the immunosuppressive microenvironment and reactivate the local immune response, especially by decreasing the shift toward M2 macrophage polarization by downregulating phosphorylated STAT6 levels and the PI3K/AKT/mTOR signaling pathway. Furthermore, 740-Y-P (PI3K activator) was used to verify that SSd inhibited M2 polarization in RAW264.7 cells via the PI3K/AKT/mTOR signaling pathway. In conclusion, this study provided experimental evidence of the antitumor effect of SSd, especially in the regulation of M2 macrophage polarization, and demonstrated that SSd may be a promising therapeutic agent in PDAC.