Acidic Microenvironment Research Articles

A Novel CRISPR/Cas9‐Encapsulated Biomimetic Manganese Sulfide Nanourchins for Targeted Magnetic Resonance Contrast Enhancement and Self‐Enhanced Chemodynamics‐Gene‐Immune Synergistic Tumor Therapy

AbstractCRISPR/Cas9 gene editing systems are widely studied in cancer therapy, however, it is still challenging to improve the gene editing efficiency and enhance the anticancer efficacy. Herein, a novel self‐assembled manganese sulfide nanourchin with CRISPR/Cas9 and hybrid membrane encapsulation (MCRT) is developed to highlight the synergistic mechanism of hydrogen sulfide (H2S) and CRISPR/Cas9 in cancer therapy. The special structure and cationic surface of nanourchins facilitate the loading of CRISPR/Cas9 with the large size and the encapsulation of erythrocyte‐tumor cell hybrid membrane, endowing the nanoplatform with long circulation and precise tumor targeting. MCRT with pH‐response can effectively decompose into H2S and Mn2+, and release CRISPR/Cas9 in acidic tumor microenvironment. H2S can up‐regulate intracellular hydrogen peroxide level by inhibiting the activity of catalase, which enables self‐enhanced chemodynamic therapy (CDT) of Mn2+. In addition, H2S can synergize with the released CRISPR/Cas9, co‐downregulating the level of intracellular anti‐apoptotic protein survivin, achieving self‐enhanced gene therapy. Furthermore, systemic delivery of MCRT displays significantly tumor magnetic resonance imaging (MRI) contrast enhancement. Importantly, H2S‐enhanced CDT‐gene therapy simultaneously induces immunogenic cell death and triggered a robust anti‐tumor immune response. Thus, MCRT has a potential application for the targeted MRI and self‐enhanced CDT‐gene‐immune synergistic tumor therapy.

Folate-Mediated Targeting and Controlled Release: PLGA-Encapsulated Mesoporous Silica Nanoparticles Delivering Capecitabine to Pancreatic Tumor.

The discovery of specifically tailored therapeutic delivery systems has sparked the interest of pharmaceutical researchers considering improved therapeutic effectiveness and fewer adverse effects. The current study concentrates on the design and characterization of PLGA (polylactic-co-glycolic acid) capped mesoporous silica nanoparticles (MSN)-based systems for drug delivery for pH-sensitive controlled drug release in order to achieve a targeted drug release inside the acidic tumor microenvironment. The physicochemical properties of the nanoformulations were analyzed using TEM, zeta potential, AFM, TGA, FTIR, and BET analyses in addition to DLS size. The final formed PLGA-FoA-MSN-CAP and pure MSN had sizes within the therapeutic ranges of 164.5 ± 1.8 and 110.7 ± 2.2, respectively. Morphological characterization (TEM and AFM) and elemental analysis (FTIR and XPS) confirmed the proper capping and tagging of PLGA and folic acid (FoA). The PLGA-coated FoA-MSN exhibited a pH-dependent controlled release of the CAP (capecitabine) drug, showing efficient release at pH 6.8. Furthermore, the in vitro MTT test on PANC1 and MIAPaCa-2 resulted in an IC50 value of 146.37 μg/ml and 105.90 μg/ml, respectively. Mitochondrial-mediated apoptosis was confirmed from the caspase-3 and annexin V/PI flow cytometry assay, which displayed a cell cycle arrest at the G1 phase. Overall, the results predicted that the designed nanoformulation is a potential therapeutic agent in treating pancreatic cancer.

Unveiling the immunosuppressive landscape of pancreatic ductal adenocarcinoma: implications for innovative immunotherapy strategies.

Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), stands as the fourth leading cause of cancer-related deaths in the United States, marked by challenging treatment and dismal prognoses. As immunotherapy emerges asapromising avenue for mitigating PDAC's malignant progression, a comprehensiveunderstanding of the tumor's immunosuppressive characteristics becomesimperative. This paper systematically delves into the intricate immunosuppressive network within PDAC, spotlighting the significant crosstalk between immunosuppressive cells and factors in the hypoxic acidic pancreatic tumor microenvironment. By elucidating these mechanisms, we aim to provide insights into potential immunotherapy strategies and treatment targets, laying the groundwork for future studies on PDAC immunosuppression. Recognizing the profound impact of immunosuppression on PDAC invasion and metastasis, this discussion aims to catalyze the development of more effective and targeted immunotherapies for PDAC patients.

Borneol-Modified Chitosan Coating with Antibacterial Properties via Layer-by-Layer Strategy

Chitosan exhibits remarkable broad-spectrum antibacterial activity, especially in acidic environments. However, its poor solubility in water and significantly decreased antibacterial performance after membrane formation greatly limit its extensive application. To address this issue, glycol chitosan-4-pyridylcarboxaldehydeborneol (GCBP) was synthesized by conjugating glycol chitosan (GC) with 4-pyridylcarboxaldehydeborneol ester (BP) through dynamic Schiff base bonds utilizing a layer-by-layer self-assembly strategy. When bacteria come into contact with the surface, the local acidic microenvironment triggers the cleavage of the Schiff base, resulting in the release of bactericidal BP and GC for combined sterilization. In vitro results demonstrated that the antibacterial properties of GCBP were positively related to the modification layers. The excellent antibacterial performance of the GCBP modification demonstrates not only great potential for clinical urinary catheters but also for broad antibacterial applications in the medical field.

Abstract 4162: Peptide-targeted probe identifies pancreatic cancer using optoacoustic imaging

Abstract Introduction: Advanced pancreatic ductal adenocarcinoma (PDAC) suffers from inadequate early detection, inoperability, and poor outcomes. While tumor identication in an intraoperative setting largely utilize ultrasound, ultrasound suffers from insufficient resolution and lacks the ability to identify molecular features to differentiate tumor from non-tumor. To overcome these limitations, we utilized optoacoustic imaging combined with a pH-sensitive peptide-based probe, V3, to identify pancreatic cancer in orthotopic mouse models. Methods: The V3 peptide was synthesized using microwave chemistry which resulted in a peptide of >90% purity. The V3 peptide was conjugated to a fluorescent dye and dialyzed to make the V3-750 probe. S2VP10 cancer cell line was plated in a 6-well plate and acclimated to 7.4, 6.8, and 6.6 pH media to mimic a healthy microenvironment and the acidic tumor microenvironment found in pancreas cancer. Cells were treated with 1 micromolar of V3-750 probe for 1 hour, then imaged on the near-infrared fluorescence imager. Treated cells were loaded into tissue mimicking phantoms and imaged using multispectral optoacoustic tomography. Athymic mice were orthotopically implanted with S2VP10 cells and tumor grew to 3 mm. V3-750 was intraveneously injected and mice were imaged 3h after injection with MSOT. Results: Signal values for the samples displayed a trend that the V3-750 probe has higher signal values in the 6.8 and 6.6 pH as compared to the 7.4 pH. MSOT data supported this trend, with the signal values from the 6.8 and 6.6 pH being greater than the 7.4 pH by 10-fold (p<0.05). Biodistribution demonstrates peak tumor-specific uptake at 3h post injection (3.1 a.u., 0.003 a.u. liver, 0.002 a.u. kidney). Conclusion: The heightened signal values for the acidic pH’s from the fluorescence imaging and the MSOT suggest that the V3-750 probe can contrast acidic cancer cells from healthy cells because it targets the acidic cancer microenvironment. Citation Format: Emma Sanderson, Tereza Husarova, Lacey McNally. Peptide-targeted probe identifies pancreatic cancer using optoacoustic imaging [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 4162.

Abstract 462: Developing the miR-888 cluster as a therapeutic target for prostate cancer

Abstract Prostate Cancer (PCa) is a serious health crisis in the United States. 20% of men diagnosed with PCa will progress to fast-growing, advanced disease. There are no curative treatment options for PCa that has spread to distant sites and the 5-year survival rate for metastatic disease remains 30%. Recognizing the need for better PCa therapeutics, we are developing novel anti-microRNA delivery reagents targeted against the noncoding RNA miR-888 cluster to block prostate tumor progression. Our lab identified these oncomiRs in a biomarker discovery screen as elevated in human metastatic PCa cell lines and prostatic fluids from patients with high-grade PCa. We noted that over-expression of miR-888 cluster members (miR-888, miR-891a) promoted prostate cell growth and induced invasiveness in vitro. miR-888 and miR-891a also accelerated xenograft tumor growth in mice. Treatment of aggressive PCa cells in culture with synthetic antisense inhibitors against individual cluster members reversed these growth and invasion phenotypes, highlighting their clinical potential. We are testing pH Low Insertion Peptide (pHLIP)-Peptide Nucleic Acid (PNA)-based antimiR conjugates against miR-888 and miR-891a. The delivery vehicle pHLIP is a 36-amino acid peptide that at low pH (< pH 6.5) adopts an alpha-helical conformational change and facilitates the insertion of its C-terminus across lipid bilayers to transport conjugated anti-miRNA cargo into cells. When administered systemically, pHLIP effectively targets solid tumors with acidic microenvironments. We found that these reagents are readily internalized by human PCa cells cultured at pH 6.0 and in prostate xenograft tumors when delivered systemically in mice. Treatment significantly repressed miR-888 and miR-891a and acted to slow prostate cell growth in vitro. We are currently moving these studies into mouse prostate models. Furthermore, we are investigating the downstream signaling pathways for the miR-888 cluster. Our work indicates that the miR-888 cluster network coordinates the repression of the Tissue Inhibitors of Metalloproteinase (TIMP) and SMAD4 anti-metastatic pathways to drive extracellular matrix degradation and prostate cell invasion, and as a consequence, induces matrix metalloproteinase (MMP) activity. Expression profiling arrays and luciferase reporter assays indicated that multiple members of the miR-888 cluster co-suppress TIMPs in the prostate and similarly elevate multiple MMPs as well as cadherins, fibronectin and heparinase, and co-modulated several immune response genes. We are currently studying the consequence of various CRISPR-mediated miR-888 cluster deletions to understand how this non-coding RNA cluster functionally overlaps to promote aggressive PCa. This work will establish new clinical tools for aggressive PCa and optimize patient treatment management. Citation Format: Katherine Routon, Trevor Fachko, Vishal Kasina, Raman Bahal, Aurora Esquela Kerscher. Developing the miR-888 cluster as a therapeutic target for prostate cancer [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 462.

Abstract 4158: Non-invasive mapping of acidosis and extracellular lactosis for assessing tumor invasion in vivo

Abstract Tumor cells typically display a hyperglycolytic metabolic phenotype characterized by increased lactate production, known as the Warburg effect. This drift in oxidative metabolism often results in a more acidic microenvironment. This adverse microenvironment represents a unique metabolic signature in cancer cells with more invasive phenotypes. However, to date, there are no effective methods available to evaluate cancer progression and metastatic potential in vivo. To bridge this gap, we have developed a molecular imaging technique (CEST-MRI) that can track extracellular pH (pHe; acidoCEST) and extracellular lactate levels (shiftCEST) simultaneously to discern different cancer phenotypes. In this study we have generated and selected in vivo two PyMT derived murine breast cancer cell lines, a parental and a G6 metastatic. We first characterized the cell’s metabolism by analyzing key biomarkers with qPCR, Western Blot and microscopy, and confirmed their distinct metabolic profiles with Seahorse and RNA Sequencing. Notably, the metastatic PyMT exhibited a more active metabolism, aligning with the invasive phenotype observed by spheroid invasion assay. Herein, we combined the injection of iopamidol and shift reagent (SR) to perform quasi-simultaneous acidoCEST and shiftCEST. First, we assessed the approach in vitro by introducing iopamidol and SR into phantoms containing the individual contrast agents, and the combination in pure water and cells supernatants at different pHe and lactate levels. We performed LDH assays to cross-validate the extracellular concentrations and monitored the pH with a micro pH meter (Mettler-Toledo). In vivo, the cells were injected orthotopically in the 4th mammary fat pad of C57BL/6N mice. Hybrid shift/acidoCEST sequences were optimized and acquired following the injection of the iopamidol+SR solution. Preclinical shift/acidoCEST enabled the simultaneous and accurate determination of extracellular acidosis and lactosis, revealing increased acidification in the metastatic models despite lower lactate secretion. Metastatic tumors exhibited a negative correlation between tumor volume and pHe, indicating the significance of acidity in mutagenesis and invasion. In contrast, no correlation was observed for the parental non-metastatic tumor. Additionally, the metastatic tumors had lower levels of lactate than the parental tumors, which could be attributed to their higher mitochondrial activity, reflecting a diverted metabolic fitness. In summary, our study highlights the efficacy of shift/acidoCEST as a reliable method for non-invasive metabolic imaging, enabling the simultaneous detection of extracellular acidity and lactate production by MRI. This research introduces a unique platform for assessing the metabolic aggressiveness of cancer, emphasizing the significance of acidity and lactate production as crucial biomarkers in cancer invasion. Citation Format: Anais Choffart, Remy Chiaffarelli, Max Zimmermann, Laura Kuebler, André Martins. Non-invasive mapping of acidosis and extracellular lactosis for assessing tumor invasion in vivo [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 4158.

Abstract 6340: Novel conditionally active tetravalent B7-H3 x CD3 T-cell engagers targeting solid tumors

Abstract B7-H3, also known as CD276, is an immune-checkpoint molecule and a type 1 transmembrane protein that exerts a variety of biological functions. In cancer biology, the overexpression of B7-H3 is correlated with tumor cell invasion and metastasis, decreased T-cell infiltration and function, poor prognosis, and resistance to therapy. As B7-H3 is expressed in multiple solid tumors and plays an important role in modulating anti-tumor immune response as well as promoting cancer cell formation, it became an important target for the development of antibody-based immunotherapy. Many forms of bispecific antibodies targeting B7-H3 have shown antitumor response in preclinical studies. However, bispecific T-cell engagers (TCE) targeting B7H3 and CD3 have been correlated with cytokine release syndrome and hepatic adverse events, a common toxicity observed using TCEs which has limited the development of this immunotherapeutic in clinic. We leveraged BioAtla’s Conditionally Active Biologic (CAB) platform (1) to develop a DualCAB T-cell engager targeting B7-H3 and CD3. This DualCAB-TCE molecule, BA3142, was engineered to bind with high affinity to both recombinant B7-H3 and CD3 receptor/antigens under conditions that mimic the acidic tumor microenvironment, but with low affinity in alkaline physiological conditions. In vitro and in vivo efficacy and toxicity data for the clone BA3142 will be presented. Our data demonstrated that BA3142 promotes cytotoxicity of B7-H3-expressing cancer cells in vitro and induces significant tumor regression in vivo. In Non-Human Primates BA3142 bispecific antibody was tolerated up to 25 mg/kg with minimal cytokine release and no clinical and histopathological signs of toxicities. The CAB technology offers the opportunity to expand the number of targets suitable for drug development in a new class of DualCAB TCEs with superior safety margin and potency in the clinic. (1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, and Short, J.M. Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118. Citation Format: Ana Paula Cugnetti, PhD, Haizhen Liu, Patricia McNeeley, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active tetravalent B7-H3 x CD3 T-cell engagers targeting solid tumors [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 6340.

Abstract 3069: Targeting lactic acid sensing GPR132 in ccRCC

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, accounting for 70-80% of the cases. Despite the improvements in tumor therapeutics, patients with metastatic clear cell tumors still have a five-year survival rate lower than 10%. Most of the ccRCC tumors harbor loss of VHL tumor suppressor gene on chromosome 3, leading to constitutively stabilized HIF, activation of downstream pathways involved in glycolysis, angiogenesis and metastasis and a highly acidic, vascularized, and immunosuppressive tumor microenvironment (TME). Such molecular alterations lead to the buildup of lactic acid within the tumor microenvironment. Long has been considered as a byproduct of hypoxic tumor metabolism, but recent studies have demonstrated a pro-tumorigenic role of lactic acid. Therefore, elucidating the molecular mechanisms on how tumor cells take advantage of lactic acid can identify potential novel therapeutic options. To understand how ccRCC cells sense extracellular lactic acid, I explored the concept of a lactate sensor. The currently known lactate sensors are GPR81 and GPR132. By querying TCGA data, GPR81 is reduced in ccRCC tumors and does not predict patient outcome while GPR132 is elevated in ccRCC tumors compared to normal kidney and demonstrates a strong negative effect on survival. Therefore, GPR132 stands out as a potential lactate sensor in ccRCC with meaningful impact on patient survival. Previously, GPR132 has been shown to be involved in tumor associated macrophage M1 to M2 transition upon lactic acid exposure. However, GPR132 exerts a striking impact on ccRCC tumors. Depleting GPR132 significantly decreased the HIF2α level in multiple ccRCC cell lines, establishing a molecular connection between lactic acid, GPR132 and HIF2α. Furthermore, due to suppressed HIF2α signaling, GPR132 knockout led to decreased lactic acid uptake through MCT1. Such decreased lactic transporter MCT1 abrogated the elevated aggressive phenotype in ccRCC cells when exposed to lactic acid. Metabolically, GPR132 knockout led to decreased lipid deposition. In addition, such deprivation of lipid droplet is not replenished when cells are exposed to lactic acid. Furthermore, by conducting an isotype-tracing experiment, GPR132 knockout actively decreased lactic acid uptake by the tumors and limit their resources to regenerate TCA cycle intermediates and build up lipid droplets. More importantly, GPR132 knockout led to defective mitochondria activities and activation of apoptosis, both of which led to significantly suppressed ccRCC growth in vitro and in vivo. This suggested that GPR132 likely affects ccRCC fitness through multiple layers of signaling mechanisms. Overall, our study has revealed that lactate sensing GPR132 is a critical signaling pathway for ccRCC tumor development in a glycolytic feed-forward context. As a membrane bound receptor, GPR132 holds immense potential as a novel therapeutic target in ccRCC. Citation Format: Dazhi Wang. Targeting lactic acid sensing GPR132 in ccRCC [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 3069.

Targeting Vascular Destruction by Sonosensitizer‐Free Sonocatalytic Nanomissiles Instigates Thrombus Aggregation and Nutrition Deprivation to Starve Pancreatic Cancer

AbstractSonosensitizers in current sonodynamic therapy (SDT) often suffer from poor delivery efficiency, phototoxicity, and disputed safety. To overcome these issues, a sonosensitizers‐free sonocatalytic nanomissile is constructed, wherein PLGA nanoparticles as vehicles conjugate with L‐arginine (LA) and aptamer XQ2d capable of adsorbing CO2 and targeting CD71‐overexpressed pancreatic cancer, respectively. The adsorbed CO2 can respond to acidic tumor microenvironment and local ultrasound to release CO2 bubbles and enhance ultrasound‐triggered inertial cavitation, which can further split H2O and activate dissolved O2 to produce ·OH and 1O2, respectively, unlocking the sonosensitizers‐free sonocatalytic ROS birth. Moreover, such CO2 bubbles‐enhanced inertial cavitation also can target intratumoral vascular destruction, instigate thrombus aggregation, induce nutrition and oxygen deprivation, pose hypoxia and alter tumor metabolism, thus establishing an intratumoral vascular destruction‐targeted starvation therapy. The strategy is different from previous starvation therapy in which the presence of undamaged intratumoral blood vessels compromises their outcomes. Especially, the active targeting aptamer XQ2d allows more sonosensitizer‐free sonocatalytic nanomissiles to retain in pancreatic cancer, significantly magnifying the sonosensitizers‐free sonocatalytic therapy and starvation therapy against subcutaneous and orthotopic pancreatic cancers. Therefore, the research provides a promising route and therapeutic platform for clinical pancreatic cancer therapy.

Abstract 5294: Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy

Abstract Introduction: Tumor-specific recruitment of co-stimulatory bispecific antibodies (bsAbs) has emerged as a promising therapeutic strategy. Here, we investigated pH-selective CD28xVISTA bsAbs to act selectively within the acidic tumor microenvironment (TME). Our CD28xVISTA bsAbs are designed for tripartite “trans-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. Experimental Procedures: We evaluated various CD28xVISTA bsAbs, focusing on a prototype 1+2 format with monovalent CD28 binding, bivalent VISTA binding and Fc receptor interaction null mutations (BS2). BS2 was tested for T-cell trans-activation using Jurkat-IL-2-luciferase reporter cells in the presence of HEK293 cells expressing membrane bound OKT3-scFv and CHO cells expressing human VISTA. BS2’s potency was further evaluated in an xCelligence-based human T-cell killing assay that dynamically monitors growth of LNCaP prostate cancer cells co-cultured with VISTA+ Kasumi-3 cells, alone or in combination with a CD3xPSMA bispecific T-cell engager. T-cell activation and proliferation were also measured using flow cytometry with CD3, CD4, CD8 and CD25 markers. Additionally, we modified BS2 to introduce pH-selective VISTA binding, limiting its activity to the TME, and tested this version in a syngeneic mouse model in combination with anti-murine PD-1 (anti-mPD-1). Finally, cytokine release from human PBMCs, co-cultured with human umbilical vein endothelial cells (HUVECs) and treated with the pH-selective BS2 (or TGN1412 as a positive control), was measured using a bead-based multiplex immune assay. Results: Our data show that the combination of anti-mPD-1 and our prototype CD28xVISTA bsAb (BS2) with pH-selective VISTA binding significantly inhibited tumor growth in vivo. In vitro, CD28xVISTA co-stimulation in trans by BS2 potentiated the activity of a CD3xPSMA bispecific T-cell engager in our human T-cell killing assay. Cytokine release assessments demonstrated negligible induction of inflammatory cytokines, indicating a favorable safety profile for this antibody. Conclusion: Our study demonstrates the feasibility of cis and trans CD28 co-stimulation using a CD28xVISTA bsAb (BS2). This approach, which bypasses the need for tumor-associated antigens (TAAs) required by other CD28xTAA bispecifics in development, suggests a potentially safer alternative for T-cell engagement and stimulation. Moreover, our novel myeloid-directed TME-selective co-stimulation strategy with a CD28xVISTA bsAb may broaden the potential for T-cell engagement approaches in solid tumors by enabling rational combinations with existing CD3xTAA T-cell engagers without competing for the same target. Citation Format: Thomas Thisted, Zhi-Gang Jiang, Zuzana Biesova, Adejumoke Onumajuru, Yuliya Kleschenko, Kanam Malhotra, Vikas Saxena, Arnab Mukherjee, F. Donelson Smith, Edward H. van der Horst. Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy [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 5294.

Abstract 4163: Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors

Abstract Objective: Current intraoperative imaging modalities such as near-infrared fluorescent imaging (NIR) or ultrasound are limited due to operator experience and depth, and targeted imaging of pancreatic tumors would be beneficial. However, specific pancreatic tumor biomarkers available for targeting in a novel imaging protocol are lacking. As such, we focus on developing a novel targeted imaging paradigm utilizing multispectral optoacoustic imaging (MSOT) to target the acidic microenvironment of pancreatic tumors. Peptides know as pH low insertion peptides (pHLIPs) when bound to dye allow for contrasted imaging selectively based on pH using MSOT. We have identified the need for a pHLIP with improved dynamic range to improve imaging accuracy. Here, we synthesize the novel pHLIP V7FS for comparison to V3 with aims of improving the dynamic range of imaging with MSOT in pancreatic cancer. Methods: Using microwave chemistry, V7FS and V3 were synthesized with >90% purity. Peptides were conjugated to HiLyte 750 dye, and peptide-dye conjugation confirmed via spectroscopy. Intraoperative measurements of human pancreatic tumor and surrounding uninvolved tissue were obtained using a microsensor. Near-infrared fluorescent imaging was used to measure probe uptake in S2VP10 and S2013 pancreatic cancer cells lines grown in pH specific media (7.4, 6.8, 6.6) in replicates. MSOT was then used to image probes in phantoms mimicking tissue and then in mice with pancreatic tumors from orthotopically implanted S2VP10. Statistical analysis was performed using ANOVA. Results: Compared to surrounding uninvolved pancreatic tissue, human pancreatic tumor was more acidic with pH 6.2-6.7 vs 7.2-7.3 (p<0.01). Signal at pH 7.4, 6.8 and 6.6 was higher for V7FS-750 compared to V3-750 (p=0.0105). Signal for V7FS-750 was centered around pH 6.6 and signal for V3-750 centered around pH 6.8. Interaction between probe and pH did not reach significance (p=0.745). Peak uptake in orthotopic murine models was observed at 3 hours following intravenous injection. Conclusions: V7FS-750 was shown to have higher signal accumulation compared to V3-750 favoring a more acidic environment (pH 6.6 vs pH 6.8). This novel pHLIP was demonstrated to be viable for use in an imaging paradigm utilizing MSOT in an orthotopic murine model. Continued improvement in the dynamic range of these probes is promising for the use of pHLIPS in imaging of pancreatic cancer with MSOT. Citation Format: Ryan Bynum, Emma Sanderson, Barish H. Edil, Ajay Jain, Lacey R. McNally. Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors [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 4163.

Abstract 3193: Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy

Abstract Many targeted therapies, such as antibody drug conjugates (ADCs) and chimeric antigen receptor (CAR) T cell therapies, rely on the identification of an optimal antigen as the cell marker for targeting cancer cells. However, there is a paucity of antigen candidates that are sufficiently specific to epithelial cancers. Therefore, technologies that can mark tumor cells for immune recognition could prove beneficial in the treatment of these solid cancers. pH low insertion peptides (pHLIPs) act as a unique delivery platform that can specifically target the acidic microenvironment of tumors, sparing healthy tissue in the process. We developed a pHLIP-peptide conjugate (pHLIP-SIINFEKL) to deliver the SIINFEKL peptide, an immunogenic fragment of ovalbumin, to investigate antigen delivery and recognition to tumors in vivo. When processed intracellularly, SIINFEKL is presented for immune recognition through the major histocompatibility complex (MHC) class I pathway. We observed selective delivery of pHLIP-SIINFEKL both in vitro and in vivo using fluorescently labeled constructs. In vitro, treatment of melanoma tumor cells with pHLIP-SIINFEKL resulted in recognition by SIINFEKL-specific T cells (OT1), leading to T cell activation and effector function. Mechanistically, we show that this recognition by OT1 cells was abrogated by siRNA knockdown of the β2 microglobulin (β2m) component of MHC class I in the target tumor cells, indicating that an intact antigen processing pathway in the cancer cells is necessary to mediate the effect of pHLIP-directed SIINFEKL delivery. In vivo, pHLIP-SIINFEKL treatment of tumor-bearing mice resulted in recruitment of OT1 T cells and suppression of tumor growth in two syngeneic tumor models in immunocompetent mice, with no effect when mutating either the pHLIP or SIINFEKL components of the conjugate. These results suggest that pHLIP-mediated peptide delivery can provide a novel approach to decorate solid tumors with engineered antigens for enhanced immune recognition and anti-tumor efficacy. Citation Format: Annali M. Yurkevicz, Yanfeng Liu, Peter M. Glazer. Utilizing a pH sensitive peptide (pHLIP) for antigen-directed T cell therapy [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 3193.

Acidity-Triggered "Sticky Spotlight": CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo.

Cancer which causes high mortality globally threatens public health seriously. There is an urgent need to develop tumor-specific near-infrared (NIR) imaging agents to achieve precise diagnosis and guide effective treatment. In recent years, imaging probes that respond to acidic environments such as endosomes, lysosomes, or acidic tumor microenvironments (TMEs) are being developed. However, because of their nonspecific internalization by both normal and tumor cells, resulting in a poor signal-to-noise ratio in diagnosis, these pH-sensitive probes fail to be applied to in vivo tumor imaging. To address this issue, a cholecystokinin-2 receptor (CCK2R)-targeted TME-sensitive NIR fluorescent probe R2SM was synthesized by coupling pH-sensitive heptamethine cyanine with a CCK2R ligand, minigastrin analogue 11 (MG11) for in vivo imaging, in which MG11 would target overexpressed CCK2Rs in gastrointestinal stromal tumors (GISTs). Cell uptake assay demonstrated that R2SM exhibited a high affinity for CCK2R, leading to receptor-mediated internalization and making probes finally accumulated in the lysosomes of tumor cells, which suggested in the tumor tissues, the probes were distributed in the extracellular acidic TME and intracellular lysosomes. With a pKa of 6.83, R2SM can be activated at the acidic TME (pH = 6.5-6.8) and lysosomes (pH = 4.5-5.0), exhibiting an apparent pH-dependent behavior and generating more intense fluorescence in these acidic environments. In vivo imaging showed that coupling of MG11 with a pH-sensitive NIR probe facilitated the accumulation of probe and enhanced the fluorescence in CCK2R-overexpressed HT-29 tumor cells. A high signal was observed in the tumor region within 0.5 h postinjection, indicating its potential application in intraoperative imaging. Fluorescence imaging of R2SM exhibited higher tumor-to-liver and tumor-to-kidney ratios (2.1:1 and 2.3:1, respectively), compared separately with the probes that are lipophilic, pH-insensitive, or MG11-free. In vitro and in vivo studies demonstrated that the synergistic effect of tumor targeting with pH sensitivity plays a vital role in the high signal-to-noise ratio of the NIR imaging probe. Moreover, different kinds of tumor-targeting vectors could be conjugated simultaneously with the NIR dye, which would further improve the receptor affinity and targeting efficiency.

The H2O2 Self-Sufficient 3D Printed β-TCP Scaffolds with Synergistic Anti-Tumor Effect and Reinforced Osseointegration.

Tumor recurrence and massive bone defects are two critical challenges for postoperative treatment of oral and maxillofacial tumor, posing serious threats to the health of patients. Herein, in order to eliminate residual tumor cells and promote osteogenesis simultaneously, the hydrogen peroxide (H2O2) self-sufficient TCP-PDA-CaO2-CeO2 (TPCC) scaffolds are designed by preparing CaO2 or/and CeO2 nanoparticles (NPs)/chitosan solution and modifying the NPs into polydopamine (PDA)-modified 3D printed TCP scaffolds by rotary coating method. CaO2 NPs loaded on the scaffolds can release Ca2+ and sufficient H2O2 in the acidic tumor microenvironment (TME). The generated H2O2 can further produce hydroxyl radicals (·OH) under catalysis effect by peroxidase (POD) activity of CeO2 NPs, in which the photothermal effect of the PDA coating enhances its POD catalytic effect. Overall, NPs loaded on the scaffold chemically achieve a cascade reaction of H2O2 self-sufficiency and ·OH production, while functionally achieving synergistic effects on anti-tumor and bone promotion. In vitro and in vivo studies show that the scaffolds exhibit effective osteo-inductivity, induced osteoblast differentiation and promote osseointegration. Therefore, the multifunctional composite scaffolds not only validate the concept of chemo-dynamic therapy (CDT) cascade therapy, but also provide a promising clinical strategy for postoperative treatment of oral and maxillofacial tumor.

Advances in tumor vascular growth inhibition.

Tumor growth and metastasis require neovascularization, which is dependent on a complex array of factors, such as the production of various pro-angiogenic factors by tumor cells, intercellular signaling, and stromal remodeling. The hypoxic, acidic tumor microenvironment is not only conducive to tumor cell proliferation, but also disrupts the equilibrium of angiogenic factors, leading to vascular heterogeneity, which further promotes tumor development and metastasis. Anti-angiogenic strategies to inhibit tumor angiogenesis has, therefore, become an important focus for anti-tumor therapy. The traditional approach involves the use of anti-angiogenic drugs to inhibit tumor neovascularization by targeting upstream and downstream angiogenesis-related pathways or pro-angiogenic factors, thereby inhibiting tumor growth and metastasis. This review explores the mechanisms involved in tumor angiogenesis and summarizes currently used anti-angiogenic drugs, including monoclonal antibody, and small-molecule inhibitors, as well as the progress and challenges associated with their use in anti-tumor therapy. It also outlines the opportunities and challenges of treating tumors using more advanced anti-angiogenic strategies, such as immunotherapy and nanomaterials.

Breaking Physical Barrier of Fibrotic Breast Cancer for Photodynamic Immunotherapy by Remodeling Tumor Extracellular Matrix and Reprogramming Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) assist in breast cancer (BRCA) invasion and immune resistance by overproduction of extracellular matrix (ECM). Herein, we develop FPC@S, a photodynamic immunomodulator that targets the ECM, to improve the photodynamic immunotherapy for fibrotic BRCA. FPC@S combines a tumor ECM-targeting peptide, a photosensitizer (protoporphyrin IX) and an antifibrotic drug (SIS3). After anchoring to the ECM, FPC@S causes ECM remodeling and BRCA cell death by generating reactive oxygen species (ROS) in situ. Interestingly, the ROS-mediated ECM remodeling can normalize the tumor blood vessel to improve hypoxia and in turn facilitate more ROS production. Besides, upon the acidic tumor microenvironment, FPC@S will release SIS3 for reprograming CAFs to reduce their activity but not kill them, thus inhibiting fibrosis while preventing BRCA metastasis. The natural physical barrier formed by the dense ECM is consequently eliminated in fibrotic BRCA, allowing the drugs and immune cells to penetrate deep into tumors and have better efficacy. Furthermore, FPC@S can stimulate the immune system and effectively suppress primary, distant and metastatic tumors by combining with immune checkpoint blockade therapy. This study provides different insights for the development of fibrotic tumor targeted delivery systems and exploration of synergistic immunotherapeutic mechanisms against aggressive BRCA.

Green Nanopesticide: pH Response and Molybdenum Selenide Carrier with Photothermal Effect to Transport Prochloraz to Inhibit Sclerotinia Disease.

Accurate pesticide delivery is a key factor in improving pesticide utilization, which can effectively reduce the use of pesticides and environmental risks. In this study, we developed a nanocarrier preparation method which can be controlled by pH/near-infrared response. Mesoporous molybdenum selenide (MoSe2) with a high loading rate was used as the core, poly(acrylic acid) (PAA) with acid response was used as the shell, and prochloraz (Pro) was loaded to form a pH-/near-infrared-responsive core-shell nanosystem (Pro@MoSe2@PAA NPs, abbreviated as PMP). Sclerotinia sclerotiorum infection secretes oxalic acid, forming an acidic microenvironment. In an acidic environment, PMP could quickly release Pro, and the cumulative release amount of Pro at pH = 5.0 was 3.1 times higher than that at pH = 7.4, and the efficiency of releasing Pro in the acidic environment was significantly enhanced. In addition, the release rate of PMP under near-infrared light irradiation was also significantly improved, and the cumulative release of Pro under simulated sunlight was 2.35 times higher than that under no light. The contact angles of PMP droplets on rapeseeds were reduced by 31.2 and 13.9% compared to Pro and MoSe2, respectively, which proved that the nanosystems had good wettability. In addition, PMP shows excellent adhesion and resistance to simulated rain washout. In the plate antibacterial experiment, the inhibitory effect of 0.5 μg/mL PMP on S. sclerotiorum was as high as 75.2% after 6 days, which showed a higher bactericidal activity than Pro. More importantly, PMP shows excellent biocompatibility and safety to plants, microorganisms, and cells. In a word, PMP is a green nanopesticide with a dual response of pH/near-infrared light, which provides a new strategy for the sustainable development of agriculture.

Ion Pairing Enables Targeted Prodrug Activation via Red Light Photocatalysis: A Proof-of-Concept Study with Anticancer Gold Complexes.

Photocatalysis has found increasing applications in biological systems, for example, in localized prodrug activation; however, high-energy light is usually required without giving sufficient efficiency and target selectivity. In this work, we report that ion pairing between photocatalysts and prodrugs can significantly improve the photoactivation efficiency and enable tumor-targeted activation by red light. This is exemplified by a gold-based prodrug (1d) functionalized with a morpholine moiety. Such a modification causes 1d to hydrolyze in aqueous solution, forming a cationic species that tightly interacts with anionic photosensitizers including Eosin Y (EY) and Rose Bengal (RB), along with a significant bathochromic shift of absorption tailing to the far-red region. As a result, a high photoactivation efficiency of 1d by EY or RB under low-energy light was found, leading to an effective release of active gold species in living cells, as monitored by a gold-specific biosensor (GolS-mCherry). Importantly, the morpholine moiety, with pKa ∼6.9, in 1d brings in a highly pH-sensitive and preferential ionic interaction under a slightly acidic condition over the normal physiological pH, enabling tumor-targeted prodrug activation by red light irradiation in vitro and in vivo. Since a similar absorption change was found in other morpholine/amine-containing clinic drugs, photocages, and precursors of reactive labeling intermediates, it is believed that the ion-pairing strategy could be extended for targeted activation of different prodrugs and for mapping of an acidic microenvironment by low-energy light.

Acidic/hypoxia dual-alleviated nanoregulators for enhanced treatment of tumor chemo-immunotherapy

Chemotherapy plays a crucial role in triple-negative breast cancer (TNBC) treatment as it not only directly kills cancer cells but also induces immunogenic cell death. However, the chemotherapeutic efficacy was strongly restricted by the acidic and hypoxic tumor environment. Herein, we have successfully formulated PLGA-based nanoparticles concurrently loaded with doxorubicin (DOX), hemoglobin (Hb) and CaCO3 by a CaCO3-assisted emulsion method, aiming at the effective treatment of TNBC. We found that the obtained nanomedicine (DHCaNPs) exhibited effective drug encapsulation and pH-responsive drug release behavior. Moreover, DHCaNPs demonstrated robust capabilities in neutralizing protons and oxygen transport. Consequently, DHCaNPs could not only serve as oxygen nanoshuttles to attenuate tumor hypoxia but also neutralize the acidic tumor microenvironment (TME) by depleting lactic acid, thereby effectively overcoming the resistance to chemotherapy. Furthermore, DHCaNPs demonstrated a notable ability to enhance antitumor immune responses by increasing the frequency of tumor-infiltrating effector lymphocytes and reducing the frequency of various immune-suppressive cells, therefore exhibiting a superior efficacy in suppressing tumor growth and metastasis when combined with anti-PD-L1 (αPD-L1) immunotherapy. In summary, this study highlights that DHCaNPs could effectively attenuate the acidic and hypoxic TME, offering a promising strategy to figure out an enhanced chemo-immunotherapy to benefit TNBC patients.