Poster 70 - Evaluating Cytotoxicity and Vascular Disrupting Activity of Novel Agents Against Renal Cell Carcinoma

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Steven Z. Sun

Student
J. J. Pearce High School & The University of Texas Southwestern Medical Center
Dallas, Texas, United States

Co-Author(s)

TW

Tianyuan Wang

Postdoctoral Researcher
The University of Texas Southwestern Medical Center, Texas, United States
RV

Rebecca Vairin

Research Assistant
Baylor University, United States
KP

Kevin G. Pinney

Professor of Chemistry
Baylor University, Texas, United States
Ralph Mason, PhD, FRSC, FISMRM (he/him/his)

Professor
The University of Texas Southwestern Medical Center, Texas, United States

Primary Investigator(s)

Li Liu, PhD (she/her/hers)

Associate Professor
The University of Texas Southwestern Medical Center
Dallas, Texas, United States

Introduction: Tumor viability requires a functioning vascular network. Renal cell carcinoma (RCC) is an angiogenesis-dependent and hypoxia-driven malignancy [1]. RCC is characterized by extensive vascular development, and the tumor vascular endothelium is distinct from normal blood vessels in terms of abnormal morphology, permeability, and lack of pericyte coverage [2]. Most vascular disrupting agents (VDAs) function via targeting the actively proliferating endothelial cells. We are assessing KGP265, a water-soluble phosphate prodrug of a benzosuberene-based tubulin-binding agent [3] and KGP415, a prodrug salt prepared from indole-based agent KGP369. The VDAs were tested against three RCC cell lines: ACHN (a papillary metastatic human cell line), 786-O (a clear cell adenocarcinoma human cell line), and RENCA (a mouse-derived renal cortical adenocarcinoma cell line) [4].

Materials and

Methods: MTT colorimetric assays were used to assess the effects of the VDAs on cell viability of three RCC cell lines ACHN, 786-O, and RENCA. Cells were seeded in a 96 well plate at a density of 4x10^4 cells/mL, incubated, then treated with the VDAs and medium solvents. Following additional incubation, the MTT labeling reagent was added, and after 3 h, the medium was removed and replaced with100 µl of DMSO. For quantification, the optical density was measured at an absorbance of 570 nM. RENCA cells were injected subcutaneously into the right flanks of female and male BALB/c mice, and tumor growth was monitored weekly by bioluminescence imaging (BLI) [5]. All 3 RCC cell lines contained the luciferase expression (luc) gene, allowing tumor progression and vascular effects to be tracked using luciferin and BLI. ACHN-luc cells were injected into NOD SCID mice with treatment initiated when tumors reached ~8 mm in diameter and BLI signals were ~5x10^6 photons/sd. Acute effects of VDA KGP415 were assessed at 4, 24, 48, and 72 h post-treatment for RENCA-luc tumors, and up to 48 h for ACHN-luc tumors. The effects associated with KGP265 treatment were evaluated 2 and 24 h post-treatment for 786-O-luc orthotopic tumors.

Results, Conclusions, and Discussions: RCC cell viability in vitro via the MTT assay assessment (Figure 1 A-C) indicated that the half maximal inhibitory concentration (IC50) of KGP265 was consistently lower than KGP415 (Figure 1D). The RENCA cells were more sensitive to treatment with KGP265 and KGP415 as compared to human RCC cell lines. For RENCA-luc subcutaneous tumor-bearing mice, KGP415 (150 mg/kg) caused significantly reduced luminescence within 4 h, which remained low for 72 h, while control tumors showed consistent signals. ACHN subcutaneous tumors responded similarly. Quantification of the tumors following imaging revealed a treatment tumor with complete vascular shutdown (Figure 1F). A similar study was carried out on 786-O orthotopic tumors treated with 7.5 mg/kg KGP265 (Figure 2A &B). The tumor vasculature was disrupted 24 h after KGP265 treatment as evidenced by BLI. These findings suggest that KGP265 and KGP415 are effective VDAs, with potential therapeutic benefits in RCC treatment through targeted disruption of tumor vasculature. KGP265 and KGP415 effectively decreased the viability of ACHN-luc, 786-O-luc, and RENCA-luc cell lines in a dose-dependent manner (Figure 1 A-D and Figure 2 A&B). KGP415 caused a vascular shutdown in RENCA subcutaneous tumor in BALB/c mice (Figure 1 E &F), KGP265 caused the vascular shutdown in human RCC 786-O-luc orthotopic tumor in NOD SCID mice (Figure 2 A & B). This demonstrates the potential of KGP415 and KGP265 in the treatment of RCC. This study suggests that VDAs may inhibit RCC cell viability and tumor growth. BLI is an effective method for monitoring tumor growth and the acute effects of VDAs. Further research must be conducted to determine the long-term effects of KGP265 and KGP415 on mice and assess the possibility of a combination of therapeutic treatments to combat possible tumor regrowth of the periphery. Future histological analysis of tumors should provide mechanistic insights.

Acknowledgements (Optional): Imaging was facilitated by UT Southwestern Small Animal Imaging Resource (P30 CA142543, CPRIT RP210099 and R01 CA244579-01). KGP265 and KGP415 were generously supplied by Dr. Kevin G. Pinney at Baylor University with partial financial support from NIH R01 CA238624.

References (Optional): [1] Ellis, L., Shah, P., Hammers, H., Lehet, K., Sotomayor, P., Azabdaftari, G., Seshadri, M., & Pili, R. (2012). Vascular disruption in combination with mTOR inhibition in renal cell carcinoma. Molecular cancer therapeutics, 11(2), 383–392. https://doi.org/10.1158/1535-7163.MCT-11-0748
[2] Murphy, K. A., James, B. R., Wilber, A., & Griffith, T. S. (2017). A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies. Journal of visualized experiments: JoVE, (122), 55080. https://doi.org/10.3791/55080
[3] Guo Y, Wang H, Gerberich JL, Odutola SO, Charlton-Sevcik AK, Li M, Tanpure RP, Tidmore JK, Trawick ML, Pinney KG, et al. Imaging-Guided Evaluation of the Novel Small-Molecule Benzosuberene Tubulin-Binding Agent KGP265 as a Potential Therapeutic Agent for Cancer Treatment. Cancers. 2021; 13(19):4769. https://doi.org/10.3390/cancers13194769
[4] Sobczuk P, Brodziak A, Khan MI, et al. Choosing The Right Animal Model for Renal Cancer Research. Transl Oncol. 2020;13(3):100745. doi: 10.1016/j.tranon.2020.100745
[5] Liu, L.; O’Kelly, D.; Schuetze, R.; Carlson, G.; Zhou, H.; Trawick, M.L.; Pinney, K.G.; Mason, R.P. Non-Invasive Evaluation of Acute Effects of Tubulin Binding Agents: A Review of Imaging Vascular Disruption in Tumors. Molecules 2021, 26, 2551. https://doi.org/10.3390/molecules26092551