Poster G10 - Screening for ATP Binding Cassette Transporter-Mediated Efflux of Photosensitizers for Photodynamic Therapy Applications in Multidrug Resistant Cancer

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Renee A. Jaranson

Undergraduate Bioengineering Student at the University of Maryland, College Park
Optical Therapeutics & Nanotechnology Laboratory, University of Maryland, College Park
Elkridge, Maryland, United States

Co-Author(s)

SV

Shruti Vig, PhD (she/her/hers)

PhD Candidate in the Optical Therapeutics & Nanotechnology Laboratory
Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States
PS

Payal Srivastava, PhD

Postdoctoral Researcher in the Optical Therapeutics & Nanotechnology Laboratory
Fischell Department of Bioengineering, University of Maryland, College Park, United States
IR

Idrisa Rahman (she/her/hers)

PhD Candidate in the Optical Therapeutics & Nanotechnology Laboratory
Fischell Department of Bioengineering at the University of Maryland, College Park
ACCOKEEK, Maryland, United States
Anika Dasgupta (she/her/hers)

Undergraduate Research Assistant
University of Maryland, College Park
Olney, Maryland, United States
Robert Perttilä, MS (he/him/his)

Biomedical Research and Development Engineer
Modulight Corporation
Tampere, Finland
PU

Petteri Uusimaa

Founder and CTO
Modulight Corporation, United States

Primary Investigator(s)

HH

Huang Chiao Huang

Associate Professor of Bioengineering
Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA, Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, 21201, USA, United States

Introduction: Resistance to anti-cancer drugs, otherwise known as multidrug resistance (MDR), accounts for up to 90% of cancer treatment failures clinically. One well-studied mechanism of drug resistance is drug efflux via membrane proteins called ATP binding cassette (ABC) transporters. Three ABC transporters (ABCB1, ABCG2, and ABCC1) are often overexpressed in various cancer cell types. Many chemotherapeutic drugs have been studied and classified as substrates for these ABC transporters, meaning that they are actively effluxed from the cell, limiting the efficacy of the drug treatment. This underscores the importance of ABC drug transporters as targets for improving the efficacy of cancer treatment by overcoming MDR as a limitation. Light based therapies have shown the potential to overcome MDR in cancer. Photodynamic therapy (PDT) is a clinically used treatment modality that combines light with agents called photosensitizers (PS) to generate reactive oxygen species, which can trigger cell death with limited side effects. Similarly to other anticancer agents, photosensitizers can act as substrates for specific ABC transporters, which could potentially reduce their intracellular accumulation and limit their therapeutic efficacy. Knowing which photosensitizers are substrates for ABC transporters is critical for developing effective PDT-based therapies for MDR. However, the ABC transporter substrate status for many clinically used photosensitizers is unknown. Thus, two in-vitro methods were used to screen for ABC transporter-mediated efflux of seven clinically used photosensitizers. Expanding on this study, photosensitizer and ABC transporter inhibitor combination treatments are being studied to determine if increased accumulation of substrate photosensitizers does indeed enhance PDT-mediated cell killing.

Materials and

Methods: Three breast cancer cell lines, derived from the parental MCF7 cell line, were maintained in media with chemotherapy drugs to select for ABC transporter overexpression: MCF7-MX100 - overexpressing ABCG2, MCF7-TX400 - overexpressing ABCB1, MCF7-VP - overexpressing ABCC1. ABC transporter expression of each cell line was verified via western blot. ABC transporter-mediated efflux of seven photosensitizers (benzoporphyrin derivative, temoporfin, redaporfin, talaporfin sodium, rose bengal, indocyanine green, methylene blue) was measured in each cell line via two methods, intracellular photosensitizer extraction and intracellular photosensitizer fluorescence. For both methods, cell lines were treated with media (no treatment), photosensitizer (PS), or the photosensitizer and an ABC transporter inhibitor (PS+I) and subsequently incubated for an hour to allow the PS and inhibitors to accumulate in cells. After the first incubation, cells were washed twice with PBS before a second hour of incubation to allow PS efflux. The PS+I group was maintained in media with the transporter inhibitor. After the second incubation, cells were washed again. For the PS extraction method, cells were lysed with RIPA buffer and the intracellular PS and protein concentration was determined via fluorescence and a BCA assay respectively to normalize PS fluorescence to protein concentration. For the intracellular PS fluorescence method, cells were collected and intracellular PS content was measured via flow cytometry. PDT-mediated cell killing for the PS+I combination was investigated by following the same incubation/wash procedure described above before treating cells with PDT at a range of radiant exposures. Cell viability was measured 24 hours later with CellTiter-Glo.

Results, Conclusions, and Discussions: Rose bengal and benzoporphyrin derivative showed increased accumulation in MX100 and TX400 cells with their respective ABC transporter inhibitors. With fumitremorgin C, methylene blue accumulation decreased in MX100 cells. With KO143, an alternative ABCG2 inhibitor, there was no difference in methylene blue accumulation. FACS data for redaporfin exhibited higher TX400 cellular uptake with valspodar, however, intracellular photosensitizer fluorescence did not change significantly with this inhibitor. The remaining photosensitizers did not demonstrate a change in cellular accumulation upon ABC transporter inhibition. Previous literature has shown that benzoporphyrin derivative is a likely substrate for ABCG2 and ABCB1 while temoporfin is not a substrate for the three ABC transporters tested. These studies correspond with the fluorescence and FACS data gathered, validating the methodologies used. The data suggest that rose bengal is a substrate for all three ABC transporters. Fumitremorgin C facilitated a surprising decrease in methylene blue accumulation in MX100 cells, suggesting that the inhibitor may have off-target interactions with other components in the cell that affect methylene blue uptake. With the KO143 inhibitor, however, methylene blue accumulation did not change, indicating that methylene blue is not a substrate for ABCG2. In order to investigate the discrepancy between the redaporfin accumulation data, the redaporfin and ABCB1 inhibitor PDT cell killing curve will be compared to the curve for redaporfin alone. The remaining photosensitizers are not substrates for any of the ABC transporters that were tested. Using three characterized photosensitizers, including substrate and non-substrates, the PDT cell killing curves will be compared between the PS+I treatment group and the PS alone to determine if increased photosensitizer accumulation facilitates enhanced cell-killing. So far, benzoporphyrin derivative in combination with ABCG2 and ABCB1 inhibitors has led to improved cell-killing compared to benzoporphyrin derivative alone. Previous studies have shown that photosensitizers that are not ABC transporter substrates can resensitize tumor cells to treatment while photosensitizers that are substrates can temporarily reduce transporter expression and improve chemotherapy drug uptake. Because PDT relies on the intracellular localization of photosensitizers, identifying which photosensitizers are substrates for ABC transporters can inform future PDT regimens for improving PDT efficacy and overcoming MDR.

Acknowledgements (Optional): This project was supported by Modulight.