Vascular Targeting Agent Research Articles

Combining Vascular Targeting Agents with Radiation: An Effective Anti-Tumor Treatment but Associated with Radiation-Induced Systemic Toxicity

This study investigated the effect of combining radiation with an angiogenesis inhibitor and vascular disrupting agent on tumor response and systemic toxicity. CDF1 mice with 200 mm3 foot implanted C3H mammary carcinomas were treated with TNP-470 (100 mg/kg every second day for 2 weeks; s.c.) and combretastatin A-4 phosphate (CA4P; 1 × 250 mg/kg, i.p.). Radiation (230-kV X-rays) was locally administered to tumors of restrained non-anesthetized mice. Response was tumor growth delay and change in mouse body weight. Radiation induced changes in serum levels of 10 cytokines up to 72-h after irradiation were measured using a Luminex assay. The results showed that TNP-470 (100 mg/kg × 7) or CA4P (250 mg/kg × 1) significantly (Student’s t-test; p < 0.05) inhibited tumor growth; the greatest effect when these two drugs were combined. TNP-470 and CA4P, alone or together, also significantly enhanced tumor response to radiation. No systemic toxicity occurred with drugs administered alone or in combination, but toxicity was observed when TNP-470 was combined with radiation. Serum cytokine levels only showed a significant transient increase in IL-6 1-h after irradiating. In conclusion, combining different acting vascular targeting agents with radiation increased anti-tumor activity. However, this benefit may sometimes be associated with a radiation-induced inflammatory response increasing systemic toxicity.

Targeting vascular disrupting agent-treated tumor microenvironment with tissue-penetrating nanotherapy

Cancer treatment with vascular disrupting agents (VDAs) causes rapid and extensive necrosis in solid tumors. However, these agents fall short in eliminating all malignant cells, ultimately leading to tumor regrowth. Here, we investigated whether the molecular changes in the tumor microenvironment induced by VDA treatment sensitize the tumors for secondary nanotherapy enhanced by clinical-stage tumor penetrating peptide iRGD. Treatment of peritoneal carcinomatosis (PC) and breast cancer mice with VDA combretastatin A-4 phosphate (CA4P) resulted in upregulation of the iRGD receptors αv-integrins and NRP-1, particularly in the peripheral tumor tissue. In PC mice treated with CA4P, coadministration of iRGD resulted in an approximately threefold increase in tumor accumulation and a more homogenous distribution of intraperitoneally administered nanoparticles. Notably, treatment with a combination of CA4P, iRGD, and polymersomes loaded with a novel anthracycline Utorubicin (UTO-PS) resulted in a significant decrease in the overall tumor burden in PC-bearing mice, while avoiding overt toxicities. Our results indicate that VDA-treated tumors can be targeted therapeutically using iRGD-potentiated nanotherapy and warrant further studies on the sequential targeting of VDA-induced molecular signatures.

Peptide-IR820 Conjugate: A Promising Strategy for Efficient Vascular Disruption and Hypoxia Induction in Melanoma.

Photothermal therapy (PTT) has emerged as a noninvasive and precise cancer treatment modality known for its high selectivity and lack of drug resistance. However, the clinical translation of many PTT agents is hindered by the limited biodegradability of inorganic nanoparticles and the instability of organic dyes. In this study, a peptide conjugate, IR820-Cys-Trp-Glu-Trp-Thr-Trp-Tyr (IR820-C), was designed to self-assemble into nanoparticles for both potent PTT and vascular disruption in melanoma treatment. When co-assembled with the poorly soluble vascular disrupting agent (VDA) combretastatin A4 (CA4), the resulting nanoparticles (IR820-C@CA4 NPs) accumulate efficiently in tumors, activate systemic antitumor immune responses, and effectively ablate melanoma with a single treatment and near-infrared irradiation, as confirmed by our in vivo experiments. Furthermore, by exploiting the resulting tumor hypoxia, we subsequently administered the hypoxia-activated prodrug tirapazamine (TPZ) to capitalize on the created microenvironment, thereby boosting therapeutic efficacy and antimetastatic potential. This study showcases the potential of short-peptide-based nanocarriers for the design and development of stable and efficient photothermal platforms. The multifaceted therapeutic strategy, which merges photothermal ablation with vascular disruption and hypoxia-activated chemotherapy, holds great promise for advancing the efficacy and scope of cancer treatment modalities.

Targeting the Tumor Vascular Supply to Enhance Radiation Therapy Administered in Single or Clinically Relevant Fractionated Schedules.

This pre-clinical study was designed to demonstrate how vascular disrupting agents (VDAs) should be administered, either alone or when combined with radiation in clinically relevant fractionated radiation schedules, for the optimal anti-tumor effect. CDF1 mice, implanted in the right rear foot with a 200 mm3 murine C3H mammary carcinoma, were injected with various doses of the most potent VDA drug, combretastatin A-1 phosphate (CA1P), under different schedules. Tumors were also locally irradiated with single-dose, or stereotactic (3 × 5-20 Gy) or conventional (30 × 2 Gy) fractionation schedules. Tumor growth and control were the endpoints used. Untreated tumors had a tumor growth time (TGT5; time to grow to 5 times the original treatment volume) of around 6 days. This increased with increasing drug doses (5-100 mg/kg). However, with single-drug treatments, the maximum TGT5 was only 10 days, yet this increased to 19 days when injecting the drug on a weekly basis or as three treatments in one week. CA1P enhanced radiation response regardless of the schedule or interval between the VDA and radiation. There was a dose-dependent increase in radiation response when the combined with a single, stereotactic, or conventional fractionated irradiation, but these enhancements plateaued at around a drug dose of 25 mg/kg. This pre-clinical study demonstrated how VDAs should be combined with clinically applicable fractionated radiation schedules for the optimal anti-tumor effect, thus suggesting the necessary pre-clinical testing required to ultimately establish VDAs in clinical practice.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

Tumor vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Suppresses macrophage capping protein beyond STING activation

The vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) induces apoptosis in vascular endothelial cells and leads to tumor hemorrhagic necrosis. While DMXAA has been proven to be a potent agonist of murine stimulator of interferon genes (mSTING), it has little effect on human-STING (hSTING). This species selectivity of DMXAA may explain its effectiveness against solid tumors in mice and its failure in clinical trials. However, DMXAA did reduce tumor volume in some patients during clinical trials. These paradoxical results have prompted us to investigate the anti-tumor mechanism of DMXAA beyond STING in the destruction of tumor vasculature in humans. In this study, we demonstrated that DMXAA binds to both human and mouse macrophage capping protein (CapG), with a KD of 5.839 μM for hCapG and a KD of 2.867 μM for mCapG, as determined by surface plasmon resonance (SPR) analysis. Homology modeling and molecular docking analysis of hCapG indicated that the critical residues involved in the hydrogen bond interaction of DMXAA with hCapG were Arg153, Thr151, and GLN141, Asn234. In addition, electrostatic pi-cation interaction occurred between DMXAA and hCapG. Further functional studies revealed that CapG protein plays a crucial role in the effects of DMXAA on human umbilical endothelial vein cell (HUEVC) angiogenesis and migration, as well as the expression of cytoskeletal proteins actin and tubulin, and the invasion of A549 lung adenocarcinoma cells. Our study has originally uncovered a novel cross-species pathway underlying the antitumor vascular disruption of DMXAA extends beyond STING activation. This finding deepens our understanding of the multifaceted actions of flavonoid VDAs in animal models and in clinical settings, and may provide insights for the precise therapy of DMXAA based on the biomarker CapG protein.

A dual drug-loaded tumor vasculature-targeting liposome for tumor vasculature disruption and hypoxia-enhanced chemotherapy

Vascular disrupting agents (VDAs) can destroy tumor vasculature and lead to tumor ischemia and hypoxia, resulting in tumor necrosis. However, VDAs are easy to induce the upregulation of genes that are associated with cancer cell drug resistance and angiogenesis in tumor cells. Hypoxia-activated chemotherapy will be an ideal supplement to VDAs therapy since it can help to fully utilize the ischemia and hypoxia induced by VDAs to realize a synergistic antitumor therapeutic outcome. Here, we design a liposome whose surface is modified with a tumor-homing peptide Cys-Arg-Glu-Lys-Ala (CREKA, which can specifically target tumor vessels and stroma) and whose aqueous cavity and lipid bilayer are loaded by a hypoxia-activatable drug banoxantrone dihydrochloride (AQ4N) and a VDA combretastatin A4 (CA4), respectively. CA4 can selectively target vascular endothelial cells and destroy the tumor blood vessels, which will cause the rapid inhibition of blood flow in tumor and enhance the hypoxia in the tumor region. As a consequence, AQ4N can exert its boosted cytotoxicity under the enhanced hypoxic environment. The as-prepared liposome with a uniform particle size exhibits good stability and high cancer cell killing efficacy in vitro. In addition, in vivo experiments confirm the excellent tumor-targeting/accumulation, tumor vasculature-damaging, and tumor inhibition effects of the liposome. This work develops a liposomal which can achieve safe and effective tumor suppression without external stimulus excitation by only single injection, and is expected to benefit the future development of effective antitumor liposomal drugs.

Abstract 5974: Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B

Abstract Anti-VEGF therapy is a key therapeutic in several cancer treatment regimens. However, none of the current therapeutics are directly cytotoxic against tumor cells. VEGF121 is the smallest isoform in the VEGF-A family of cytokines that binds to VEGF receptors R-1 and R-2. Both receptors are over-expressed on several tumor cells and the endothelium of tumor vasculature but not normal vasculature. We hypothesized that delivery of a cytotoxic payload through VEGFR-2 into tumor cells and tumor neovasculature would significantly enhance cancer therapy. Granzyme B is a key effector in immune-mediated cell killing through both caspase-dependent and -independent mechanisms. We previously reported the development of a fusion protein composed of granzyme B (GrB) and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein incorporating a human IgG heavy chain Fc fragment for dimerization to increase the molecular weight and improve in vivo circulation and targeting potential. We expressed and purified GrB-Fc-VEGF121 from HEK-293E cells under serum-free conditions with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. We characterized this construct against both human and mouse VEGFR+ cell lines as this ligand cross-reacts to both species. GrB-Fc-VEGF121 showed in vitro cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2, while receptor-negative cells demonstrated IC50 levels in the high micromolar range. GrB-Fc-VEGF121 internalized into VEGFR-2+ tumor and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR2+ cells at the respective IC50 dose resulted in &amp;gt;50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability studies indicated a gradual protein loss of GrB-Fc-VEGF121 with an overall stability of about 50% over 96 hours. No toxicity was observed in mice treated with a total dose of 475 mg/kg (IP, QOD x 5), indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model (20 mg/kg GrB-Fc-VEGF121 x 5) resulted in significant growth inhibition of established tumors compared to vehicle controls. The number of CD31+ blood vessels and Ki-67+ proliferating tumor cells decreased significantly in treated tumors compared to controls. Pharmacokinetic studies are underway and will be reported. Thus, GrB-Fc-VEGF121 significantly reduces tumor growth in vivo with no observed toxicity against normal tissues. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B [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 5974.

Multi-parametric investigations on the effects of vascular disrupting agents based on a platform of chorioallantoic membrane of chick embryos.

Vascular disrupting agents (VDAs) are known to specifically target preexisting tumoural vasculature. However, systemic side effects as safety or toxicity issues have been reported from clinical trials, which call for further preclinical investigations. The purpose is to gain insights into their non-specific off-targeting effects on normal vasculature and provide clues for exploring underlying molecular mechanisms. Based on a recently introduced platform consisting laser speckle contrast imaging (LSCI), chick embryo chorioallantoic membrane (CAM), and assisted deep learning techniques, for evaluation of vasoactive medicines, hemodynamics on embryonic day 12 under constant intravascular infusion of two VDAs were qualitatively observed and quantitatively measured in real time for 30 min. Blood perfusion, vessel diameter, vessel density, and vessel total length were further analyzed and compared between blank control and medicines dose groups by using multi-factor analysis of variance (ANOVA) analysis with factorial interactions. Conventional histopathology and fluorescent immunohistochemistry (FIHC) assays for endothelial cytoskeleton including ß-tubulin and F-actin were qualitatively demonstrated, quantitatively analyzed and further correlated with hemodynamic and vascular parameters. The normal vasculature was systemically negatively affected by VDAs with statistical significance (P<0.0001), as evidenced by four positively correlated parameters, which can explain the side-effects observed among clinical patients. Such effects appeared to be dose dependent (P<0.0001). FIHC assays qualitatively and quantitatively verified the results and exposed molecular mechanisms. LSCI-CAM platform combining with deep learning technique proves useful in preclinical evaluations of vasoactive medications. Such new evidences provide new reference to clinical practice.

Hindering the unlimited proliferation of tumor cells synergizes with destroying tumor blood vessels for effective cancer treatment.

The rational combination of chemotherapy drugs can improve the curative effect of cancer treatment. As two early recognized tumor hallmarks, the limitless replicative potential of tumor cells is essential for the development of their malignant growth state, and sustained angiogenesis is a prerequisite to the rapid growth of tumors. Based on this, we propose a combination therapy that hinders the unlimited proliferation of tumor cells and destroys tumor blood vessels. Herein, 7-ethyl-10-hydroxycamptothecin (SN38), a typical topoisomerase I inhibitor, was bonded to poly(L-glutamic acid) (PLG) to prepare the nanodrug SN38-NPs, which hinders the unlimited proliferation of tumor cells. A poly(L-glutamic acid)-combretastatin A4 conjugate (CA4-NPs), a representative vascular disrupting agent (VDA), was used to selectively disrupt the tumor blood vessels, cutting off the necessary nutrients and oxygen for the proliferation of tumor cells. In the 4T1 tumor model with an initial volume of about 400 mm3, the combined treatment of SN38-NPs and CA4-NPs showed an excellent cancer treatment effect with a tumor suppression rate of 94.3% and a synergistic interaction (Q = 1.25). Our study provides a new combination therapy approach for chemotherapy, with the hope of further improving the curative effect of anti-cancer therapy.

Molecular Dynamics and Machine Learning reveal distinguishing mechanisms of Competitive Ligands to perturb α,β-Tubulin

The mechanisms of action of ligands competing for the Colchicine Binding Site (CBS) of the α,β-Tubulin are non-standard compared to the commonly witnessed ligand-induced inhibition of proteins. This is because their potencies are not solely judged by the binding affinity itself, but also by their capacity to bias the conformational states of the dimer. Regarding the latter requirement, it is observed that ligands competing for the same pocket that binds colchicine exhibit divergence in potential clinical outcomes. Molecular dynamics-based ∼5.2 µs sampling of α,β-Tubulin complexed with four different ligands has revealed that each ligand has its customized way of influencing the complex. Primarily, it is the proportion of twisting and/or bending characteristic of modes of the intrinsic dynamics which is revealed to be ‘fundamental’ to tune this variation in the mechanism. The milder influence of ‘bending’ makes a ligand (TUB092), better classifiable under the group of vascular disrupting agents (VDAs), which are phenotypically effective on cytoskeletons; whereas a stronger impact of ‘bending’ makes the classical ligand Colchicine (COL) a better Anti-Mitotic agent (AMA). Two other ligands BAL27862 (2RR) and Nocodazole (NZO) fall in the intermediate zone as they fail to explicitly induce bending modes. Random Forest Classification method and K-means Clustering is applied to reveal the efficiency of Machine Learning methods in classifying the Tubulin conformations according to their ligand-specific perturbations and to highlight the significance of specific amino acid residues, mostly positioned in the α-β and β-β interfaces involved in the mechanism. These key residues responsible to yield discriminative actions of the ligands are likely to be highly useful in future endeavours to design more precise inhibitors.

Preparation and Characterization Evaluation of Poly(L-Glutamic Acid)-g-Methoxy Poly(Ethylene Glycol)/Combretastatin A4/BLZ945 Nanoparticles for Cervical Cancer Therapy.

Cervical cancer (CC) is a highly vascularized tumor with abundant abnormal blood vessel, which could be targeted by therapeutic strategies. Poly(L-glutamic acid)-g-methoxy poly(ethylene glycol)/combretastatin A4 (CA4)/BLZ945 nanoparticles (CB-NPs) have shown great potential as nano vascular disrupting agents (VDAs) in the realm of synergistic cancer therapy. In this study, we investigated the nanocharacteristics of CB-NPs, focusing on active pharmaceutical ingredients (API), as well as lyophilized samples combining API with protective agents (PAs). The in vivo efficacy of final sample (API + PAs) was evaluated. The assembled sphere of API with complex core and thin-shell structure was confirmed. PAs were found to significantly influence in vivo efficacy. Collaborative efforts between API and PAs, namely mannitol and lactose, resulted in the most promising lyophilized sample, ie, the final sample (FS2) for CC therapy. Impressively, FS2 demonstrated an exceptional 100% cure rate on the CC U14-bearing mice model. FS2 has provided significant insights for cervical cancer therapy. It is also crucial to develop a comprehensive evaluation strategy for the formulation of nanomedicine, which has the potential to serve as a guideline for future clinical trials.

Synthesis and biological evaluation of structurally diverse 6-aryl-3-aroyl-indole analogues as inhibitors of tubulin polymerization

The synthesis and evaluation of small-molecule inhibitors of tubulin polymerization remains a promising approach for the development of new therapeutic agents for cancer treatment. The natural products colchicine and combretastatin A-4 (CA4) inspired significant drug discovery campaigns targeting the colchicine site located on the beta-subunit of the tubulin heterodimer, but so far these efforts have not yielded an approved drug for cancer treatment in human patients. Interest in the colchicine site was enhanced by the discovery that a subset of colchicine site agents demonstrated dual functionality as both potent antiproliferative agents and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery and development of a 2-aryl-3-aroyl-indole analogue (OXi8006) that inhibited tubulin polymerization and demonstrated low nM IC50 values against a variety of human cancer cell lines. A water-soluble phosphate prodrug salt (OXi8007), synthesized from OXi8006, displayed promising vascular disrupting activity in mouse models of cancer. To further extend structure-activity relationship correlations, a series of 6-aryl-3-aroyl-indole analogues was synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines. Several structurally diverse molecules in this small library were strong inhibitors of tubulin polymerization and of MCF-7 and MDA-MB-231 human breast cancer cells. One of the most promising analogues (KGP591) caused significant G2/M arrest of MDA-MB-231 cells, disrupted microtubule structure and cell morphology in MDA-MB-231 cells, and demonstrated significant inhibition of MDA-MB-231 cell migration in a wound healing (scratch) assay. A phosphate prodrug salt, KGP618, synthesized from its parent phenolic precursor, KGP591, demonstrated significant reduction in bioluminescence signal when evaluated in vivo against an orthotopic model of kidney cancer (RENCA-luc) in BALB/c mice, indicative of VDA efficacy. The most active compounds from this series offer promise as anticancer therapeutic agents.

Flavonoids as omnipotent candidates for cancer management

The development of the cancer is a complex process as it proliferates, alters the signaling pathways, and ultimately reduced the patient's survival. The altered cellular events and compromised cellular apoptosis restricts the sensitization of cancer cells to the existing chemotherapeutics. Hence, cancer therapy needs to focus on the development of novel, cost effective, and potent therapeutics approaches which can even overcome the chemo resistance. Plant-based secondary metabolites, such as flavonoids, and their versatile use in health benefits as well as research interest pertaining to their therapeutic benefits are increasing. Flavonoids are used as dietary supplements and are involved in several biological functions and activities. Flavonoids are polyphenolic compounds composed of a 15-carbon skeleton. Due to their diverse pharmacological activities, flavonoids are used for the management of several diseases via oxidative stress and the induction/regression of proteins, enzymes, genes and signaling pathways. The present review aims to extensively discuss the mechanism of flavonoids and their potential utility in inducing programmed cell death. The role of flavonoids as anti-angiogenic, vascular disruption agent and overcoming drug resistance are also discussed. Presently, we have reviewed the various studies on different types of flavonoids, the working mechanism of flavonoids, pharmacological effects, targeting pathways and clinical studies of flavonoids related to multiple cancers. This review offers a prospect for scientists worldwide to understand the potential of flavonoids in the management of cancer.

Light-Driven Self-Recruitment of Biomimetic Semiconducting Polymer Nanoparticles for Precise Tumor Vascular Disruption.

Tumor vascular disrupting therapy has offered promising opportunities to treat cancer in clinical practice, whereas the overall therapeutic efficacy is notably limited due to the off-target effects and repeated dose toxicity of vascular disrupting agents (VDAs). To tackle this problem, a VDA-free biomimetic semiconducting polymer nanoparticle (SPNP ) is herein reported for precise tumor vascular disruption through two-stage light manipulation. SPNP consists of a semiconducting polymer nanoparticle as the photothermal agent camouflaged with platelet membranes that specifically target disrupted vasculature. Upon the first photoirradiation, SPNP administered in vivo generates mild hyperthermia to trigger tumor vascular hemorrhage, which activates the coagulation cascade and recruits more SPNP to injured blood vessels. Such enhanced tumor vascular targeting of photothermal agents enables intense hyperthermia to destroy the tumor vasculature during the second photoirradiation, leading to complete tumor eradication and efficient metastasis inhibition. Intriguingly, the mechanism study reveals that this vascular disruption strategy alleviates splenomegaly and reverses the immunosuppressive tumor microenvironment by reducing myeloid-derived suppressor cells. Therefore, this study not only illustrates a light-driven self-recruitment strategy to enhance tumor vascular disruption via a single dose of biomimetic therapeutics but also deciphers the immunotherapeutic role of vascular disruption therapy that is conducive to clinical studies.

Abstract 6315: Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature

Abstract VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature, as well as several tumor cells. While anti-VEGF therapy is a key therapeutic in several cancer treatment regimens, none of the current therapeutics (bevacizumab, VEGF-trap, tyrosine kinase inhibitors) are directly cytotoxic against tumor cells. The serine protease Granzyme B (GrB) is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We previously reported the development and characterization of a fusion protein composed of GrB and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein that incorporates a human IgG heavy chain Fc fragment for dimerization which increases the molecular weight and improves in vivo circulation and targeting potential. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the conditioned medium with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. In vitro studies of GrB-Fc-VEGF121 showed cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2. Receptor-negative cells demonstrated IC50 levels in the high micromolar range. Immunofluorescence and confocal microscopy demonstrated that GrB-Fc-VEGF121 readily internalized into VEGFR-2+ tumor cells and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR-2+ endothelial and tumor cells at the IC50 dose resulted in &amp;gt;50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall stability of about 50% over 96 hours. No toxicity was observed in BALB/c mice treated with a total 475 mg/kg given IP every other day over 5 doses, indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. We observed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells in treated tumors compared to controls. Thus, GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature [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 6315.

New 4,5-Diarylimidazol-2-ylidene-iodidogold(I) Complexes with High Activity against Esophageal Adenocarcinoma Cells.

Inspired by the vascular-disrupting agent combretastatin A-4 and recently published anticancer active N-heterocyclic carbene (NHC) complexes of Au(I), a series of new iodidogold(I)-NHC complexes was synthesized and characterized. The iodidogold(I) complexes were synthesized by a route involving van Leusen imidazole formation and N-alkylation, followed by complexation with Ag2O, transmetalation with chloro(dimethylsulfide)gold(I) [Au(DMS)Cl], and anion exchange with KI. The target complexes were characterized by IR spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. The structure of 6c was validated via single-crystal X-ray diffraction. A preliminary anticancer screening of the complexes using two esophageal adenocarcinoma cell lines showed promising nanomolar activities for certain iodidogold(I) complexes accompanied with apoptosis induction, as well as c-Myc and cyclin D1 suppression in esophageal adenocarcinoma cells treated with the most promising derivative 6b.

Feasibility of the novel vascular disrupting agent C118P for facilitating high-intensity focused ultrasound ablation of uterine fibroids

Objective In this study, C118P, a novel vascular disrupting agent (VDA), was evaluated for its ability in improving the ablative effect of high-intensity focused ultrasound (HIFU) on uterine fibroids by reducing blood perfusion. Methods Eighteen female rabbits were infused with isotonic sodium chloride solution (ISCS), C118P or oxytocin for 30 min, and an HIFU ablation of the leg muscles was performed within the last 2 min. Blood pressure, heart rate and laser speckle flow imaging (LSFI) of the auricular blood vessels were recorded during perfusion. Ears with vessels, uterus and muscle ablation sites were collected and sliced for hematoxylin–eosin (HE) staining to compare vascular size, as well as nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) staining to observe necrosis after ablation. Results Analyses revealed that the perfusion of C118P or oxytocin steadily reduced blood perfusion in the ears to approximately half by the end of the perfusion, constricted the blood vessels of the ears and uterus, and improved HIFU ablation in the muscle tissues. C118P increased blood pressure and decreased heart rate. The degree of contraction of the auricular and uterine blood vessels was positively correlated. Conclusion This study confirmed that C118P could reduce blood perfusion in various tissues and had a better synergistic effect with HIFU ablation of muscle (the same tissue type as fibroids) than did oxytocin. C118P could therefore possibly replace oxytocin in facilitating HIFU ablation of uterine fibroids; however, electrocardiographic monitoring is required.

Vascular disruption agent and phototherapeutic assembled nanoparticles for enhanced tumor inhibition.

Vascular disruption agent (combretastatin A-4 phosphate) and phototherapeutic (IEICO-4F) assembled nanoparticles (IFC NPs) were prepared for the first time. The IFC NPs have a high photo energy utilization efficiency of up to 96.1%, and could significantly inhibit tumor growth by photodynamic and photothermal therapy enhanced tumor vascular disruption.

Exploration of Chick Embryo and Chorioallantoic Membrane on Imaging Navigated Platforms for Anticancer Pharmaceutical Evaluations.

Conforming to the current replace-reduce-refine 3Rs' guidelines in animal experiments, a series of explorative efforts have been made to set up operable biomedical imaging-guided platforms for qualitative and quantitative evaluations on pharmacological effects of tumor vascular-disrupting agents (VDAs), based on the chick embryos (CEs) with its chorioallantoic membrane (CAM), in this overview. The techniques and platforms have been hierarchically elaborated, from macroscopic to microscopic and from overall to specific aspects. A protocol of LED lamplight associated with a new deep-learning algorithm was consolidated to screen out weak CEs by using the CAM vasculature imaging. 3D magnetic resonance imaging (MRI) and laser speckle contrast imaging (LSCI) to monitor the evolution of CE and vascular changes in CAM are introduced. A LSCI-CAM platform for studying the effects of VDAs on normal and cancerous vasculature of CAM and possible molecular mechanisms has been demonstrated. Finally, practical challenges and future perspectives are highlighted. The aim of this article is to help peers in biomedical research to familiarize with the CAM platform and to optimize imaging protocols for the evaluation of vasoactive pharmaceuticals, especially anticancer vascular targeted therapy.