Cancer stem cell populations are resistant to 5-aminolevulinic acid- photodynamic therapy (5-ALA-PDT)

Cancer stem cell populations are resistant to 5-aminolevulinic acid- photodynamic therapy (5-ALA-PDT) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cancer stem cell populations are resistant to 5-aminolevulinic acid- photodynamic therapy (5-ALA-PDT) Chantel PJ Rice, Vipin Shankar Chelakkot, Noah Conohan, Kensuke Hirasawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5039795/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Photodynamic therapy (PDT) is a minimally invasive treatment approved for many types of cancers. PDT involves the administration of photoactive substances called photosensitizers (PS) that selectively accumulate in cancer cells and are subsequently excited/activated by irradiation with specific wavelengths of light. Activated PS leads to the generation of singlet oxygen and reactive oxygen species (ROS), promoting cancer cell death. 5-aminolevulinic acid (5-ALA) is a naturally occurring PS precursor, which is metabolically converted to the PS, protoporphyrin IX (PPIX). Although 5-ALA-PDT is effective at killing cancer cells, we normally observed in in vitro experiments that approximately 5–10% of cells survive 5-ALA-PDT. Identifying the mechanisms of resistance to 5-ALA-PDT-mediated cell death is important to prevent tumor recurrence following 5-ALA-PDT. Previously, we reported that oncogenic activation of Ras/MEK promotes PPIX efflux and reduces cellular sensitivity to 5-ALA-PDT through increased expression of ABCB1 transporter. As cancer stem cells (CSCs) are known to drive resistance to other cancer treatments and have high efflux of chemotherapeutic agents via ABC-family transporters, we hypothesize that CSCs underlie 5-ALA-PDT resistance. In this study, we determined 1) if CSCs are resistant to 5-ALA-PDT and 2) if CSCs play roles in establishing resistant populations of 5-ALA-PDT. When we compared CSC populations before and after 5-ALA-PDT, we found that CSCs were less susceptible to 5-ALA-PDT. Moreover, we found that the CSC population was enriched in 5-ALA-PDT-resistant cell lines compared to the parental cell line. Our results indicate that CSCs are not sensitive to 5-ALA-PDT, which may contribute to establishment of 5-ALA-PDT resistance. 5-aminolevulinic acid (5-ALA) photodynamic therapy (PDT) cancer stem cells (CSCs) CD133 5-ALA-PDT resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The World Health Organization’s Global Cancer Observatory (GCO) estimated an occurrence of ~ 20 million new cases of cancer and ~ 9.7 million cancer deaths during 2022 (Global Cancer Observatory, 2022). Conventional cancer treatments such as radiotherapy, surgery, and chemotherapy, alongside contemporary advancements in immunotherapy and hormonal therapy, have collectively led to substantial improvements in cancer prognosis [ 1 ]. However, the development and refinement of therapeutic strategies remains paramount in the effort to improve patient outcomes. Photodynamic therapy (PDT) is a minimally invasive cancer treatment that has been approved by the FDA for use against many cancers [ 2 ]. Fundamentally, PDT involves the administration of a photosensitive drug known as a photosensitizer (PS) that preferentially accumulates within tumors, which are subsequently irradiated with a specific wavelength of visible light. Once subjected to light, PSs are excited from a ground state and achieve a triplet state through intersystem crossing, resulting in the formation of singlet oxygen and reactive oxygen species (ROS) that damage cellular components and initiate cell death [ 3 ]. 5-aminolevulinic acid (5-ALA) is a non-proteogenic amino acid and a key precursor in the biosynthesis of heme. The generation of 5-ALA is mediated by aminolevulinic acid synthase (ALAS), which catalyzes the condensation of glycine and succinyl-CoA at the mitochondrial membrane. Through a series of metabolic reactions in the cytosol and mitochondria, 5-ALA is converted to protoporphyrin IX (PPIX), a fluorescent PS and immediate precursor of heme. Importantly, the generation of 5-ALA by ALAS is the rate limiting step in the pathway, a key element that is exploited by 5-ALA-PDT [ 4 ]. Under homeostatic conditions, ALAS is regulated by negative feedback from free heme, preventing excessive heme production that would otherwise result in oxidative stress. However, the addition of exogenous water soluble 5-ALA bypasses homeostatic control of 5-ALA synthesis, leading to an accumulation of intracellular photosensitive PPIX. Many cancers display a preferential accumulation of PPIX compared to normal cells following 5-ALA administration, which has been attributed to metabolic reprogramming that upregulates components of the heme biosynthesis pathway during oncogenesis [ 5 ]. The preferential accumulation of PPIX in cancer cells is another exploitable element of 5-ALA-PDT that allows for the selective elimination of tumors with minimal damage to normal cells. Importantly, the subcellular accumulation of PPIX occurs primarily in mitochondria, which can become damaged by ROS generated during PDT and cause mitochondrial dysfunction that results in the initiation of programmed cell death [ 6 ] Although 5-ALA-PDT is effective at killing cancer cells, we have observed that 5–10% of cells survive in vitro experiments. The resistant population that remains post-treatment could lead to tumor resurgence with a phenotype that is less-responsive to subsequent 5-ALA-PDT. As such, identifying the mechanisms of 5-ALA-PDT resistance is crucial to improve treatment outcomes. Previous work in our lab demonstrated that RasV12-transformed NIH3T3 cells have upregulated PPIX biosynthesis, but also upregulate ATP-binding cassette (ABC) transporters and ferrochelatase (FECH) via MEK signalling-axes that reduce PPIX accumulation [ 7 ]. ABC transporters are responsible for PPIX efflux outside of the cell by exporting PPIX through the mitochondrial and plasma membranes [ 8 , 9 ]. Inhibition of MEK and downstream targets significantly enhanced the accumulation of PPIX in cancer cells, demonstrating a key mechanism of resistance to 5-ALA-PDT. Cancer stem cells (CSCs) are a minor population of cancer cells that possess high proliferative capability and produce cancer cells with heterogenous phenotypes in a similar fashion to the differentiation of normal stem cells. Both tissue resident stem cells and differentiated cells have the potential to become CSCs because stem cells have a low genetic barrier to transform into cancer cells, and partially differentiated progenitor cells can reacquire stem cell characteristics during transformation [ 10 ]. CSCs are known to drive resistance to cancer treatments and express high levels of ABC transporters that mediate the export of therapeutic drugs, preventing elimination and promoting tumor recurrence [ 11 – 14 ]. As a result, development of strategies to enhance the ability of PDT to target CSCs is an active area of research, with an emphasis on drug delivery systems that overcome mechanisms of resistance [ 15 – 17 ]. Therefore, it is pertinent to ascertain the sensitivity of CSCs to 5-ALA-PDT, given the potential resistant phenotype that could serve as founding population for future tumors. In this study, our goal is to determine if CSCs are resistant to 5-ALA-PDT, and if CSCs establish 5-ALA-PDT resistant populations post-treatment. Methods Cell culture The human lung cancer cell line H1299, human breast cancer cell line Hs578T and human colon cancer cell lines DLD-1, were purchased from the American Type Culture Collection (ATCC). All cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning, MA) supplemented with 10% fetal bovine serum (HyClone, Cytiva), 1 mM sodium pyruvate (Life Technologies) and antibiotic-antimycotic (Thermo Scientific). Cells were maintained in 10 cm culture dishes at 37°C with 5% CO 2 . For generation of 5-ALA-PDT resistant cells, DLD-1 cells (5000 cells/well) were plated in 96-well plates and subjected to 5-ALA-PDT 24 hours later. At 72 to 96 hours following 5-ALA-PDT, live cells were trypsinized, pooled and re-plated in a 10 cm dish to generate a cell line with a PDT-conditioned population (DLD-1 PDTx1). DLD-1 PDTx1 cells were plated in 96-well plates and subjected to another round of 5-ALA-PDT to generate DLD-1 PDTx2. This cycle was repeated until we obtained cell lines with population of DLD-1 that had been conditioned by PDT to varying degrees, with a maximum of four treatments (DLD-1 PDTx4). 5-ALA-PDT Human cancer cells plated in 96-well plates were treated with 5-aminolevulinic acid (5-ALA) (5 mM) (Sigma, A3785, USA) diluted in culture media (as described in previous section) and incubated at 37°C for 4 hours. Following the incubation period, culture media was replaced, and the cells were subjected to PDT using a Theralase TLC 3000A modular light source (Theralase Technologies Inc., Toronto, Canada; λ = 618–630 nm, fluence rate = 150 mW/cm 2 , energy density (ED) = 27 J/cm 2 ). Cell viability assay Cell viability was measured using the Colorimetric Cell Viability Kit I (WST-8) following manufacturer’s instructions (PromoCell GmbH, Germany). Ten (10) µl of WST-8 reagent was added to each well of 96-well plates containing cells and incubated in the dark at 37°C for 35 minutes. Absorbance was measured at 450 nm using a Bio-Rad Model 3550 microplate reader and cell viability was determined based on standard curves for each cell line. Flow cytometry Cells were fixed with Inside Stain Kit (Miltenyl Biotec, Teterow, Germany) and subsequently stained with anti-CD133 antibody (Miltenyl Biotec) and Zombie Violet™ Fixable Viability Kit (BioLegend, CA, USA) following manufacturer’s instructions. Flow cytometry analysis was conducted using a CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA). The data was analyzed using FlowJo (FlowJo LLC, OR). Soft agar assay UltraPure TM agarose (0.7%) (Invitrogen, CA, USA) and purified agar (1%) (Oxoid LTD, Hampshire UK) were mixed and incubated in a hot water bath at 37°C. First, the mixtures of agarose and 2X DMEM were plated in a 6-well plate (1.5ml/well) to generate the bottom layer. After 15 minutes of cooling to solidify the bottom layer, it was overlaid with mixtures of cell suspension and agarose to generate the top layer (2500 cells/well). Once the top layer solidified, the plate was incubated at 37°C for 21 days, after which colonies were counted under microscope. Western blot analysis Anti-ALDH1 antibody was purchased from Abcam (Boston, MA, USA) and HRP-conjugated secondary antibody from Santa Cruz Biotechnology (Dallas, TX, USA). Protein samples were prepared, and western blot was conducted as previously described [ 18 ]. Quantification of intracellular PPIX Parental and PDT-resistant (PDTx1-4) DLD-1 cells were plated in 24-well plates (5 x 10 4 cells/well) and incubated for 24 hours to attain confluency. Following this, cells were treated with 5-ALA (5 mM) and incubated in the dark for 8 or 24 hours. At each time point, samples were obtained by washing cells with PBS before lysis with supplemented RIPA buffer (RIPA, PMSF, aprotinin, and Halt™ phosphatase and protease inhibitor cocktail (Thermo Scientific)). Lysate was collected in opaque amber tubes and stored at -80°C. In minimally lit conditions, lysate was diluted in PBS (2.5% v/v), added to solid black 96-well plates (Greiner Bio-one), and covered with foil. Fluorescence of PPIX was measured with a Biotek Synergy MX plate reader with an excitation wavelength of 405 nm and emission wavelength of 635 nm. Statistical analyses Statistical analyses were performed using Prism 7.0 (GraphPad). Student’s t -test was used for inter-group comparison, and one-way ANOVA with Tukey’s posthoc test was used to compare between multiple groups. P < 0.01 were considered statistically significant. Data availability: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Results CD133 + cells are not sensitive to 5-ALA-PDT. To determine if the CD133 + stem cell (CSC) population is sensitive to 5-ALA-PDT, we quantified the percentage of CD133 + cell populations in three different cell lines [human colon cancer (DLD-1), human lung cancer (H1299) and human breast cancer (Hs578T)] (Fig. 1 ). The average percentage of CD133 + stem cell population in DLD-1, H1299 and Hs578T were 1.0%, 1.9% and 1.6% respectively. We next sought to determine if the CD133 + cells are sensitive to 5-ALA-PDT. The concentration of 5-ALA (5mM) we used in the study did not cause cell death without light exposure (Supplementary Fig. 1). Each cell line was treated with 5-ALA for 4 hours and then irradiated for 3 minutes using a Theralase TLC 3000A modular light source (27 J/cm 2 ) (5-ALA-PDT). At 24 hours after 5-ALA-PDT, the cells were incubated with Zombie Violet, fixed with inside stain kit (Miltenyl Biotec), and then incubated with an anti-CD133 antibody for 10 minutes. Cell viability [dead cells (red) and live cells (green)] and CD133 expression was evaluated by flow cytometry (Fig. 2 ). In the scatter plots of DLD-1 cells, 0.77% of live cells were CD133 + before 5-ALA-PDT which increased to 2.03% after 5-ALA-PDT (Fig. 2 A), indicating that this population is enriched in surviving cells. In the total population of DLD-1 cells, live cells decreased from 83–46% after 5-ALA-PDT, but the reduction was not statically significant. In contrast, the live cell populations of CD133 + DLD-1 cells were 77% and 69% before and after 5-ALA-PDT respectively, suggesting that 5-ALA-PDT is not effective on CD133 + DLD-1 cells. For H1299, a lung cancer cell line, the percentage of CD133 + cells in the total population was increased to 3.67% from 2.32% after 5-ALA-PDT (Fig. 2 B). Moreover, there was a significant difference in the percentage of live cells in the total cell population before and after 5-ALA-PDT, with an approximate 44% decrease of living cells following 5-ALA-PDT (Fig. 2 B). In contrast, CD133 + H1299 cells were resistant to 5-ALA-PDT as almost 80% of CD133 + cells were alive even after 5-ALA-PDT and statistically insignificant compared to the control. Similar results were found in Hs578T cells (Fig. 2 C). The CD133 + population was increased after 5-ALA-PDT and cell death was effectively induced in the total cell population of Hs578T cells as the live cell population significantly reduced from 79–30%. However, the live percentages of CD133 + Hs578T cells were not significantly reduced by 5-ALA-PDT. These results demonstrate that the CD133 + populations of each cell line were not sensitive to cell death induced by 5-ALA-PDT. Cancer stem cells are enriched in 5-ALA-PDT resistant populations. We hypothesize that cancer stem cells play a critical role in forming populations with resistance to 5-ALA-PDT. If this is the case, cancer stem cells could be enriched in 5-ALA-PDT resistant populations. To this end, we first established 5-ALA-PDT resistant cell lines from DLD-1 cells (Fig. 3 ). We plated parental DLD-1 cells in a 96-well plate and subjected them to 5-ALA-PDT (5 mM 5-ALA, 27 J/cm 2 ). At 3–4 days after 5-ALA-PDT, the surviving cells were pooled and further subjected to 5-ALA-PDT. This process was repeated up to 4 times (PDTx1: one cycle, PDTx2: two cycles, PDTx3: three cycles and PDTx4: four cycles) (Fig. 3 A). To confirm if the established cell lines were resistant to 5-ALA-PDT, the viability of the parental DLD-1 and the 5-ALA-PDT resistant cells (PDTx1, PDTx2, PDTx3 and PDTx4) were measured 24 hours after 5-ALA-PDT by a WST-8 assay. The cell viabilities of parental DLD-1 and PDTx1 cell lines were significantly reduced after 5-ALA-PDT, suggesting that they are sensitive to 5-ALA-PDT (Fig. 3 B). In contrast, most of the PDTx2, PDTx3 or PDTx4 cells survived after 5-ALA-PDT, indicating that they are 5-ALA-PDT resistant cell lines. To determine if CSCs were enriched in the DLD-1 PDTx2, PDTx3 and PDTx4 resistant cell lines, we first performed a soft agar analysis of anchorage-independent growth. The parental DLD-1, PDTx2, PDTx3 and PDTx4 resistant cells were plated in soft agar. After 21 days, colonies were counted under the microscope. PDTx3 and PDTx4 resistant cells showed a significant increase in the average number of colonies compared to the parental DLD-1 cell line, whereas a significant change was not observed in PDTx2 resistant cells (Fig. 4 A). To further confirm that CSCs are enriched in the 5-ALA-PDT resistant populations, we conducted western blot analysis of ALDH1 on cell lysates prepared from parental DLD-1 cells and 5-ALA-PDT resistant DLD-1 cells (PDTx2, PDTx3 and PDTx4) (Fig. 4 B and Supplementary Fig. 2). ALDH1 is another marker for CSCs, which was expressed on DLD-1 CSCs, but not in other cell lines. While we did not observe changes in ALDH1 expression between PDTx2 cells and parental DLD-1 cells, ALDH1 expression was increased in PDTx3 and PDTx4 cells. Finally, we determined the expression levels of the CSC marker CD133 + expression in the 5-ALA-PDT resistant cells (PDTx4) by flow cytometry. The mean fluorescent intensity of the CD133 + population of cells was increased in the PDTx4 compared to the parental DLD-1 cell line (Fig. 4 C). We also found that the percentage of CD133 + cells was significantly increased in the PDTx4 cell line compared to DLD-1 parent cells. These results further support that CSCs are resistant to 5-ALA-PDT and are enriched in 5-ALA-PDT resistant cell populations. We further determined if decreased PPIX accumulation could be one of causes of 5-ALA-PDT resistance (Fig. 4 D). We found that 5-ALA-PDT resistant DLD-1 cells (PDTx1, PDTx2, PDTx3 and PDTx4) accumulated significantly lower amounts of PPIX than parental control DLD-1 cells when stimulated with 5-ALA. The results suggest that low PPIX accumulation is one of the underlying mechanisms for 5-ALA-PDT resistance. Discussion Although 5-ALA-PDT has been approved for treating different types of cancers, cancer recurrence remains as a problem [ 19 – 21 ]. One of the primary factors of cancer recurrence is resistance to treatment, leading to survival of a small number of cancer cells that potentially reform tumors. Therefore, addressing the mechanisms of cancer resistance against 5-ALA-PDT is essential to improve its efficacy. In this study, we investigated the possible role of CSCs as a driver of 5-ALA-PDT resistance because CSCs have been reported to resist chemotherapy partially due to upregulation of ABC transporters [ 22 – 24 ]. ABC transporters efflux chemotherapeutics from cancer cells, which decreases its intracellular accumulation and thus prevents the cells from dying [ 25 , 26 ]. Our previous studies also demonstrated that PPIX efflux via ABCB1 transporter reduces cancer cell sensitivity to 5-ALA-PDT [ 7 , 9 ]. Therefore, we hypothesized that CSCs are less sensitive to 5-ALA-PDT and subsequently contribute to the establishment of resistant populations to 5-ALA-PDT due to their ability to efflux 5-ALA via ABC transporters. Through our efforts, we confirmed that 5-ALA-PDT is not effective in killing CSCs and that CSC populations are increased in the 5-ALA-PDT resistant cell lines. These results clearly indicate that CSCs play an essential role in establishing cancer resistance to 5-ALA-PDT in the cells lines evaluated (Fig. 5). Interestingly, our results contrast previous reports of 5-ALA-PDT efficacy against CSCs [ 27 – 29 ]. CSCs from oral squamous cell carcinoma cell lines were effectively differentiated by 5-ALA-PDT and susceptibility to treatment was due to increased PPIX accumulation [ 27 ]. Similarly, glioma CSC lines accumulate more PPIX compared to their differentiated counterparts and therefore more sensitive to treatment [ 28 ]. When evaluating 5-ALA-PDT against head and neck cancer CSC lines, 5-ALA-PDT reduced the expression of CSC markers and sensitized cells to chemotherapy, despite modest cell killing [ 29 ]. In comparison, our study showed that CSCs became enriched in PDT-resistant populations, which accumulated significantly less PPIX (Fig. 4 D). These results highlight the importance of PPIX accumulation on efficacy of treatment and the consideration of CSC heterogeneity between cancers. CD133 was used as a CSC marker in this study as we found that the levels of CD133 + cells in all three cell lines were sufficient (1-1.9%, Fig. 1 ) to conduct our proposed experiments. Other CSC markers such as CD24, CD32, CD44 and ALDH1A1 were not commonly expressed in the cell lines. As CD133 has been widely used as a CSC marker for different types of cancer, we utilized CD133 as a CSC marker of DLD-1 cells to determine CSC functions throughout the study. However our research objective was not to specifically characterize the roles of CD133 and its downstream signalling pathways in 5-ALA-PDT resistance. The CSC populations (CD133 + cells) were low in the cancer cell lines (1–3%) (Fig. 1 ) and even in PDTx4 DLD-1 cells (8%) in Fig. 4 C. Generally, the CSC population in tumors range from less than 0.02 to 25% depending on the tumor types (30). Our current study falls short to identify the precise mechanisms underlying 5-ALA-PDT resistance in CSCs. There are several possible mechanisms that could lead to CSCs resistant against 5-ALA-PDT. First, as mentioned above, CSCs could be resistant to 5-ALA-PDT due to high expression of the ABCtransporters as they are commonly overexpressed by CSCs [ 11 , 31 ]. High expression of the ABC transporters may decrease PPIX accumulation in CSCs induced by 5-ALA treatment and subsequently the sensitivity of CSCs to 5-ALA-PDT. In a study examining the efficacy of photodiagnosis of cancers using 5-ALA (5-ALA PDD), Kawai et al., reported that the CSC populations of PANC-1 cells expressing high levels of ABCG2 were responsible for decreased PPIX accumulation [ 32 ]. We observed that the 5-ALA-PDT resistant cells accumulated lower amounts of PPIX (Fig. 4 D), suggesting that this mechanism underlies the CSC resistance to 5-ALA-PDT. Secondly, high expression of stress response genes in CSCs could be one of the resistant mechanisms [ 33 , 34 ]. CSCs have increased genetic diversity to adapt and survive under stress conditions such as lack of oxygen, starvation, and exposure to DNA-damaging compounds [ 35 – 38 ]. Moreover, CSCs have increased activity of autophagy, which promotes cellular survival by enabling the ability to overcome stress conditions through nutrient recycling and preventing the accumulation of damaged cellular components [ 39 – 42 ]. These survival mechanisms of CSCs may reduce cell death caused by 5-ALA-PDT. Fourth, CSCs have lower levels of reactive oxygen species (ROS) compared to their differentiated counterparts, which is one of the essential effectors for cancer cell death induced by 5-ALA-PDT [ 43 ]. Although our current study did not identify the cellular mechanisms that underlie CSC resistance to 5-ALA-PDT, it is essential to improve the efficacy of 5-ALA-PDT and reduce cancer recurrence after 5-ALA-PDT. Combined treatment with 5-ALA-PDT and inhibitors targeting ABC transporters has been shown to increase anticancer efficacy in vitro and in vivo , which may be an effective way to eliminate CSCs by 5-ALA-PDT [ 44 – 46 ]. As the development of ABC transporter inhibitors for clinical trials is an active area of research, it would be a feasible idea to evaluate inhibitors in combination with 5-ALA-PDT in clinical settings in the near future [ 47 ]. To clarify the mechanism of CSC resistance to 5-ALA-PDT, this study warrants further investigation. It would be essential to determine cellular localization and quantification of PPIX, activities of enzymes involved in the heme pathway, cellular ROS levels, activation of cell death pathways, expression of ABC transporters and expression of stress response genes. Declarations Author Contribution Conceptualization/design: CPJR, VSC, KH; Data collection: CPJR, VSC, NC; Data analysis: CPJR, VSC, NC, KH; Writing original draft: CPJR, NC, KH; Review & editing: VSC, NC, KH. References Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA: A Cancer Journal for Clinicians. 2023 Jan 12;73(1):17–48. doi:10.3322/caac.21763 Baskaran R, Lee J, Yang S-G. Clinical development of photodynamic agents and therapeutic applications. Biomaterials Research. 2018 Sept 26;22(25). doi:10.1186/s40824-018-0140-z Mishchenko T, Balalaeva I, Gorokhova A, Vedunova M, Krysko DV. Which cell death modality wins the contest for photodynamic therapy of cancer? Cell Death & Disease. 2022 May 13;13(455). doi:10.1038/s41419-022-04851-4 Zheng J, Shan Y, Lambrecht RW, Donohue SE, Bonkovsky HL. Differential regulation of human ALAS1 mrna and protein levels by heme and cobalt protoporphyrin. Molecular and Cellular Biochemistry. 2008 Aug 22;319(1–2):153–61. Doi:10.1007/s11010-008-9888-0 Yang X, Palasuberniam P, Kraus D, Chen B. Aminolevulinic acid-based tumor detection and therapy: Molecular mechanisms and strategies for enhancement. International Journal of Molecular Sciences. 2015;16(10):25865–80. doi:10.3390/ijms161025865 Zhang Z-J, Wang K-P, Mo J-G, Xiong L, Wen Y. Photodynamic therapy regulates fate of cancer stem cells through reactive oxygen species. World Journal of Stem Cells. 2020 Jul 26;12(7):562-84. doi: 10.4252/wjsc.v12.i7.562 Chelakkot VS, Liu K, Yoshioka E, Saha S, Xu D, Licursi M, et al. MEK reduces cancer-specific PPIX accumulation through the RSK-ABCB1 and HIF-1α-Fech Axes. Scientific Reports. 2020 Dec 17;10(22124). doi:10.1038/s41598-020-79144-x Kobuchi H, Moriya K, Ogino T, Fujita H, Inoue K, Shuin T, et al. Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation. PLOS ONE. 2012 Nov 26;7(11). doi:10.1371/journal.pone.0050082 Yoshioka E, Chelakkot VS, Licursi M, Rutihinda SG, Som J, Derwish L, et al. Enhancement of cancer-specific protoporphyrin IX fluorescence by targeting oncogenic Ras/Mek Pathway. Theranostics. 2018 Mar 8;8(8):2134–46. doi:10.7150/thno.22641 Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011 Mar 4;144(5):646–74. doi:10.1016/j.cell.2011.02.013 Moitra K. Overcoming multidrug resistance in cancer stem cells. BioMed Research International. 2015 Nov 16;2015. doi:10.1155/2015/635745 Zinzi L, Contino M, Cantore M, Capparelli E, Leopoldo M, Colabufo NA. ABC transporters in CSCs membranes as a novel target for treating tumor relapse. Frontiers in Pharmacology. 2014 Jul 10;5(163). doi:10.3389/fphar.2014.00163 Cui J, Christin JR, Reisz JA, Cendali FI, Sanawar R, Coutinho De Miranda M, et al. Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance. Science Advances. 2023 Dec 1;9(48). doi:10.1126/sciadv.adh1891 Fuchs D, Daniel V, Sadeghi M, Opelz G, Naujokat C. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1A cells. Biochemical and Biophysical Research Communications. 2010 Mar 27;394(4):1098–104. doi:10.1016/j.bbrc.2010.03.138 Li L, Ni R, Zheng D, Chen L. Eradicating the tumor “seeds”: Nanomedicines-based therapies against Cancer Stem Cells. DARU Journal of Pharmaceutical Sciences. 2023 Mar 27;31:83–94. doi:10.1007/s40199-023-00456-0 Duan H, Liu Y, Gao Z, Huang W. Recent advances in drug delivery systems for targeting cancer stem cells. Acta Pharmaceutica Sinica B. 2021 Jan;11(1):55-70. doi:10.1016/j.apsb.2020.09.016 Yang Y, Peng Y, Du Y, Lin M, Li J, Gao D, et al. Hierarchical self-recognition and response in CSC and Non-CSC micro-niches for cancer therapy. Biomaterials. 2024 Jul;308. doi: 10.2139/ssrn.4635630 Duncan JK, Xu D, Licursi M, Joyce MA, Saffran HA, Liu K, et al. Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection. Frontiers in Immunology. 2023 Apr 30;14. doi:10.3389/fimmu.2023.930086 Railkar R, Agarwal PK. Photodynamic therapy in the treatment of bladder cancer: Past challenges and current innovations. European Urology Focus. 2018 Aug 22;4(4):509–11. doi:10.1016/j.euf.2018.08.005 Van den Broeck T, van den Bergh RCN, Arfi N, Gross T, Moris L, Briers E, et al. Prognostic value of biochemical recurrence following treatment with curative intent for prostate cancer: A systematic review. European Urology. 2019 Jun;75(6):967–87. doi:10.1016/j.eururo.2018.10.011 Kim TE, Chang J-E. Recent studies in photodynamic therapy for cancer treatment: From basic research to clinical trials. Pharmaceutics. 2023 Aug 31;15(9):2257. doi:10.3390/pharmaceutics15092257 Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene. 2003 Oct 23;22:7340–58. doi:10.1038/sj.onc.1206938 Cho Y, Kim YK. Cancer stem cells as a potential target to overcome multidrug resistance. Frontiers in Oncology. 2020 Jun 1;10. doi:10.3389/fonc.2020.00764 Begicevic R-R, Falasca M. ABC transporters in cancer stem cells: Beyond Chemoresistance. International Journal of Molecular Sciences. 2017 Nov 8;18(11):2362. doi:10.3390/ijms18112362 Lou H, Dean M. Targeted therapy for cancer stem cells: The patched pathway and ABC Transporters. Oncogene. 2007 Feb 26;26:1357–60. doi:10.1038/sj.onc.1210200 Wu C-P, Hsiao S-H, Huang Y-H, Hung L-C, Yu Y-J, Chang Y-T, et al. Sitravatinib sensitizes ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs. Cancers. 2020 Jan 13;12(1):195. doi:10.3390/cancers12010195 Pinto MA, Ferreira CB, Lima BE, Molon AC, Coppa Ibarra AM, Cecatto RB, et al. Effects of 5-ALA mediated photodynamic therapy in Oral cancer stem cells. Journal of Photochemistry and Photobiology B: Biology. 2022 Oct;235(112552). doi:10.2139/ssrn.4146140 Omura N, Nonoguchi N, Fujishiro T, Park Y, Ikeda N, Kajimoto Y, et al. Ablation efficacy of 5-aminolevulinic acid-mediated photodynamic therapy on human glioma stem cells. Photodiagnosis and Photodynamic Therapy. 2023 Mar;41:103119. doi:10.1016/j.pdpdt.2022.103119 Yu C-H, Yu C-C. Photodynamic therapy with 5-aminolevulinic acid (ALA) impairs tumor initiating and chemo-resistance property in head and neck cancer-derived cancer stem cells. PLoS ONE. 2014 Jan 24;9(1). doi:10.1371/journal.pone.0087129 Toledo-Guzmán ME, Bigoni-Ordóñez GD, Hernández MI, Ortiz-Sánchez E. Cancer stem cell impact on clinical oncology. World Journal of Stem Cells. 2018 Dec 26;10(12):183–95. doi:10.4252/wjsc.v10.i12.183 Eyre R, Harvey I, Stemke-Hale K, Lennard TW, Tyson-Capper A, Meeson AP. Reversing paclitaxel resistance in ovarian cancer cells via inhibition of the ABCB1 expressing side population. Tumor Biology. 2014 Jul 4;35(10):9879–92. doi:10.1007/s13277-014-2277-2 Kawai N, Hirohasi Y, Ebihara Y, Saito T, Murai A, Saito T, et al. ABCG2 expression is related to low 5-ala photodynamic diagnosis (PDD) efficacy and cancer stem cell phenotype, and suppression of ABCG2 improves the efficacy of PDD. PLoS ONE. 2019 May 13;14(5). doi:10.1371/journal.pone.0216503 Casas A, Perotti C, Di Venosa G, Batlle A. Mechanisms of resistance to photodynamic therapy: An update. Resistance to Targeted Anti-Cancer Therapeutics. 2011;18(16):2486–515. doi:10.1007/978-3-319-12730-9_2 Torigoe T, Hirohashi Y, Yasuda K, Sato N. Constitutive expression and activation of stress response genes in cancer stem-like cells/tumour initiating cells: Potent targets for cancer stem cell therapy. International Journal of Hyperthermia. 2013 Jul 31;29(5):436–41. doi:10.3109/02656736.2013.814809 Chen M, Xie S. Therapeutic targeting of cellular stress responses in cancer. Thoracic Cancer. 2018 Oct 12;9(12):1575–82. doi:10.1111/1759-7714.12890 Qureshi-Baig K, Kuhn D, Viry E, Pozdeev VI, Schmitz M, Rodriguez F, et al. Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-Ezr (ezrin) pathway. Autophagy. 2019 Nov 27;16(8):1436–52. doi:10.1080/15548627.2019.1687213 Srivastava AK, Han C, Zhao R, Cui T, Dai Y, Mao C, et al. Enhanced expression of DNA polymerase ETA contributes to cisplatin resistance of ovarian cancer stem cells. Proceedings of the National Academy of Sciences. 2015 Mar 23;112(14):4411–6. doi:10.1073/pnas.1421365112 Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006 Oct 18;444:756–60. doi:10.1038/nature05236 Ferrand A, Sandrin MS, Shulkes A, Baldwin GS. Expression of gastrin precursors by CD133-positive colorectal cancer cells is crucial for tumour growth. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2009 Jan 15;1793(3):477–88. doi:10.1016/j.bbamcr.2009.01.004 Wang Y, Zhou L, Qing Q, Li Y, Li L, Dong X, et al. Gene expression profile of cancer stem‑like cells in the SW480 colon adenocarcinoma cell line. Oncology Reports. 2019 May 2;42(1):386–98. doi:10.3892/or.2019.7146 Sato K, Tsuchihara K, Fujii S, Sugiyama M, Goya T, Atomi Y, et al. Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Research. 2007 Oct 15;67(20):9677–84. doi:10.1158/0008-5472.can-07-1462 Togano S, Yashiro M, Masuda G, Sugimoto A, Miki Y, Yamamoto Y, et al. Gastric cancer stem cells survive in stress environments via their autophagy system. Scientific Reports. 2021 Oct 19;11(20664). doi:10.1038/s41598-021-00155-3 Li Y-R, Fang Y, Lyu Z, Zhu Y, Yang L. Exploring the dynamic interplay between cancer stem cells and the tumor microenvironment: Implications for novel therapeutic strategies. Journal of Translational Medicine. 2023 Oct 2;21(683). doi:10.1186/s12967-023-04575-9 Robey RW, Steadman K, Polgar O, Bates SE. ABCG2-mediated transport of photosensitizers: Potential impact on photodynamic therapy. Cancer Biology & Therapy. 2005 Feb 2;4(2):187–94. doi:10.4161/cbt.4.2.1440 Ishikawa T, Kajimoto Y, Inoue Y, Ikegami Y, Kuroiwa T. Critical role of ABCG2 in ala-photodynamic diagnosis and therapy of human brain tumor. Advances in Cancer Research. 2015 Jan 8;125:197–216. doi:10.1016/bs.acr.2014.11.008 Chandratre S, Olsen J, Howley R, Chen B. Targeting ABCG2 transporter to enhance 5-aminolevulinic acid for tumor visualization and photodynamic therapy. Biochemical Pharmacology. 2023 Nov 1;217:115851. doi:10.1016/j.bcp.2023.115851 Toyoda Y, Takada T, Suzuki H. Inhibitors of human ABCG2: From technical background to recent updates with clinical implications. Frontiers in Pharmacology. 2019 Mar 4;10. doi:10.3389/fphar.2019.00208 Additional Declarations No competing interests reported. Supplementary Files supplfiguresSept2024.pdf Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Sep, 2024 Reviews received at journal 18 Sep, 2024 Reviews received at journal 18 Sep, 2024 Reviewers agreed at journal 18 Sep, 2024 Reviewers agreed at journal 18 Sep, 2024 Reviewers invited by journal 17 Sep, 2024 Editor assigned by journal 17 Sep, 2024 Editor invited by journal 17 Sep, 2024 Submission checks completed at journal 17 Sep, 2024 First submitted to journal 05 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5039795","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":356609776,"identity":"24a13d59-8309-4ce8-aa4b-d26a0e94b81e","order_by":0,"name":"Chantel PJ Rice","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Chantel","middleName":"PJ","lastName":"Rice","suffix":""},{"id":356609777,"identity":"34c61a56-b47e-421b-a8f9-117ee1a5f2d0","order_by":1,"name":"Vipin Shankar Chelakkot","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Vipin","middleName":"Shankar","lastName":"Chelakkot","suffix":""},{"id":356609778,"identity":"35194401-359c-43b8-819f-2865f5b366ee","order_by":2,"name":"Noah Conohan","email":"","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":false,"prefix":"","firstName":"Noah","middleName":"","lastName":"Conohan","suffix":""},{"id":356609779,"identity":"5185563d-f4b4-48bf-9ea6-1d17cefd3b4d","order_by":3,"name":"Kensuke Hirasawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYBACCTB5gEGOdC3GpGtJbCBai2R77+HPPGds0tfOyD3A8KOGCC3SPOfSpHlupOVuu5GXwNhzjAgtchI5Zsw8Hw4DteQYMDOwEaNF/o3xZ54P/9PNwFr+EeMwCR4DoMMOJIC1MLYRoUWyJ8dMcs6ZZMNtZ94YHOztI0KLxPEzxh/eHLOTNzueY/jgxzcitKCAA6RqGAWjYBSMglGAAwAA4m42DjgO4LsAAAAASUVORK5CYII=","orcid":"","institution":"Memorial University of Newfoundland","correspondingAuthor":true,"prefix":"","firstName":"Kensuke","middleName":"","lastName":"Hirasawa","suffix":""}],"badges":[],"createdAt":"2024-09-05 17:09:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5039795/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5039795/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-88173-3","type":"published","date":"2025-02-05T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69336941,"identity":"49ca0be2-dd47-4627-9a12-4d263db2cb2a","added_by":"auto","created_at":"2024-11-19 10:08:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD133+ stem cell population in DLD-1, H1299 and Hs578T cells\u003c/strong\u003e. DLD-1, H1299 and Hs578T cells were stained with anti-CD133 antibody. Flow cytometry analysis was conducted using the CytoFLEX Flow Cytometer and analyzed using FlowJo.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/1a14f5ff42a16ae4bdaf5acc.jpg"},{"id":69336956,"identity":"55a536af-ea97-46a2-9f4a-b429c3bb3f56","added_by":"auto","created_at":"2024-11-19 10:08:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD133+ stem cells are not sensitive to 5-ALA-PDT.\u003c/strong\u003e (A) DLD-1, (B) H1299 and (C) Hs578T cells were treated with 5-ALA (5mM) (5-ALA-PDT) or with control vehicle (control) for 4 hours and then irradiated for 3 minutes using a Theralase TLC 3000A modular light source (27 J/cm\u003csup\u003e2\u003c/sup\u003e). At 24 hours post-irradiation, Cell viability (dead cells (red) and live cells (green)) by Zombie Violet staining and numbers of CD133 positive cells was measured by flow cytometry analysis. *p\u0026lt;0.01 by one-way ANOVA with Tukey's post-hoc test.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/10a9abb163ab7b0917bf8108.jpg"},{"id":69336046,"identity":"71cd3489-0d8b-447f-9646-b24e8bd1f5de","added_by":"auto","created_at":"2024-11-19 10:00:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of 5-ALA-PDT resistant DLD-1 cells.\u003c/strong\u003e (A) Illustration of the workflow of generation of 5-ALA-PDT resistant cells. DLD-1 cells plated in 96-well plates were subjected to 5-ALA-PDT. At 72 to 96 hours after 5-ALA-PDT, live cells were pooled and re-plated in a 10 cm dish (DLD-1 PDTx1). The cycle was repeated until we obtained DLD-1 PDTx4. (B) Parental DLD-1, PDTx1, PDTx2, PDTx3 and PDTx4 cells were treated with 5-ALA (5mM) (5-ALA-PDT) or with vehicle (control) for 4 hours and then irradiated for 3 minutes using a Theralase TLC 3000A modular light source (27 J/cm\u003csup\u003e2\u003c/sup\u003e). At 24 hours post-irradiation, Cell viability was measured by WST-8 assay.\u0026nbsp; *p\u0026lt;0.01 by one-way ANOVA with Tukey's post-hoc test.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/358a3ee3c4242950895c3553.jpg"},{"id":69336946,"identity":"27dbbf7b-551b-480a-95ac-6dba721dc3d5","added_by":"auto","created_at":"2024-11-19 10:08:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121251,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCSCs are enriched in 5-ALA-PDT resistant DLD-1 cells.\u003c/strong\u003e (A) Parental DLD-1 cells and 5-ALA-PDT resistant DLD-1 cells (PDTx2, PDTx3 and PDTx4) were plated for assaying anchorage-independent growth assay in soft agar. Mean ± standard error of mean (SEM) in average colonies numbers per microscopic field are presented. *p\u0026lt;0.01 by one-way ANOVA with Tukey's post-hoc test. (B) The expression levels of ALDH1 and β-actin in parental DLD-1 cells and the 5-ALA-PDT resistant DLD-1 cells was determined by western blot analysis. (C) The expression levels of CD133 on parental DLD-1 cells and 5-ALA-PDT resistant DLD-1 cells (PDTx4) was measured by flow cytometry. Representative histogram of fluorescent intensity of CD133 on parental DLD-1 and PDTx4 cells (left) and quantitative analysis of CD133 expression based on 3 independent experiments (right). *p\u0026lt;0.01 by one-way ANOVA with Turkey's post-hoc test. (D) Intracellular accumulation of PPIX was measured by fluorometric analysis of total lysate of DLD-1 cells (Parental, PDTx1, PDTx2, PDTx3, and PDTx4) treated with 5 mM of 5-ALA for 8h and 24h in the dark. Excitation λ = 405 nm, emission λ = 635 nm. *p\u0026lt;0.01 by one-way ANOVA with Tukey's post-hoc test.\u003c/p\u003e","description":"","filename":"Figure4revised.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/d70fea2c54d8adebbc394417.jpg"},{"id":69336048,"identity":"b8c0f11b-d506-42aa-9c3d-07e2dfb0d68e","added_by":"auto","created_at":"2024-11-19 10:00:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCSCs are resistant to 5-ALA-PDT which play critical roles in tumor recurrence. \u003c/strong\u003e5-ALA-PDT does not kill CSCs efficiently due to 1) low accumulation of PPIX caused by high expression levels of the ABC transporter, 2) promoted cell survivability caused by expression of stress response genes and constitutively active autophagy and 3) resistance to cell death due to low ROS levels. Surviving CSCs serve as a founding population for future tumors, resulting in cancer recurrence.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/756c287a88eb1ec595a9476a.jpg"},{"id":75930412,"identity":"f0852894-8daf-4a7d-b1ab-51195f7f00cf","added_by":"auto","created_at":"2025-02-10 16:11:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1083541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/150e2a7c-0c62-4223-97c3-dd222bd01d11.pdf"},{"id":69336050,"identity":"615b69b7-403b-4f38-8f67-7fcad4a7004c","added_by":"auto","created_at":"2024-11-19 10:00:50","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":150325,"visible":true,"origin":"","legend":"","description":"","filename":"supplfiguresSept2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5039795/v1/47299519322ee0b25fa4a699.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cancer stem cell populations are resistant to 5-aminolevulinic acid- photodynamic therapy (5-ALA-PDT)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe World Health Organization\u0026rsquo;s Global Cancer Observatory (GCO) estimated an occurrence of ~\u0026thinsp;20\u0026nbsp;million new cases of cancer and ~\u0026thinsp;9.7\u0026nbsp;million cancer deaths during 2022 (Global Cancer Observatory, 2022). Conventional cancer treatments such as radiotherapy, surgery, and chemotherapy, alongside contemporary advancements in immunotherapy and hormonal therapy, have collectively led to substantial improvements in cancer prognosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the development and refinement of therapeutic strategies remains paramount in the effort to improve patient outcomes. Photodynamic therapy (PDT) is a minimally invasive cancer treatment that has been approved by the FDA for use against many cancers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Fundamentally, PDT involves the administration of a photosensitive drug known as a photosensitizer (PS) that preferentially accumulates within tumors, which are subsequently irradiated with a specific wavelength of visible light. Once subjected to light, PSs are excited from a ground state and achieve a triplet state through intersystem crossing, resulting in the formation of singlet oxygen and reactive oxygen species (ROS) that damage cellular components and initiate cell death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5-aminolevulinic acid (5-ALA) is a non-proteogenic amino acid and a key precursor in the biosynthesis of heme. The generation of 5-ALA is mediated by aminolevulinic acid synthase (ALAS), which catalyzes the condensation of glycine and succinyl-CoA at the mitochondrial membrane. Through a series of metabolic reactions in the cytosol and mitochondria, 5-ALA is converted to protoporphyrin IX (PPIX), a fluorescent PS and immediate precursor of heme. Importantly, the generation of 5-ALA by ALAS is the rate limiting step in the pathway, a key element that is exploited by 5-ALA-PDT [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Under homeostatic conditions, ALAS is regulated by negative feedback from free heme, preventing excessive heme production that would otherwise result in oxidative stress. However, the addition of exogenous water soluble 5-ALA bypasses homeostatic control of 5-ALA synthesis, leading to an accumulation of intracellular photosensitive PPIX. Many cancers display a preferential accumulation of PPIX compared to normal cells following 5-ALA administration, which has been attributed to metabolic reprogramming that upregulates components of the heme biosynthesis pathway during oncogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The preferential accumulation of PPIX in cancer cells is another exploitable element of 5-ALA-PDT that allows for the selective elimination of tumors with minimal damage to normal cells. Importantly, the subcellular accumulation of PPIX occurs primarily in mitochondria, which can become damaged by ROS generated during PDT and cause mitochondrial dysfunction that results in the initiation of programmed cell death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAlthough 5-ALA-PDT is effective at killing cancer cells, we have observed that 5\u0026ndash;10% of cells survive \u003cem\u003ein vitro\u003c/em\u003e experiments. The resistant population that remains post-treatment could lead to tumor resurgence with a phenotype that is less-responsive to subsequent 5-ALA-PDT. As such, identifying the mechanisms of 5-ALA-PDT resistance is crucial to improve treatment outcomes. Previous work in our lab demonstrated that RasV12-transformed NIH3T3 cells have upregulated PPIX biosynthesis, but also upregulate ATP-binding cassette (ABC) transporters and ferrochelatase (FECH) via MEK signalling-axes that reduce PPIX accumulation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. ABC transporters are responsible for PPIX efflux outside of the cell by exporting PPIX through the mitochondrial and plasma membranes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Inhibition of MEK and downstream targets significantly enhanced the accumulation of PPIX in cancer cells, demonstrating a key mechanism of resistance to 5-ALA-PDT.\u003c/p\u003e \u003cp\u003eCancer stem cells (CSCs) are a minor population of cancer cells that possess high proliferative capability and produce cancer cells with heterogenous phenotypes in a similar fashion to the differentiation of normal stem cells. Both tissue resident stem cells and differentiated cells have the potential to become CSCs because stem cells have a low genetic barrier to transform into cancer cells, and partially differentiated progenitor cells can reacquire stem cell characteristics during transformation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CSCs are known to drive resistance to cancer treatments and express high levels of ABC transporters that mediate the export of therapeutic drugs, preventing elimination and promoting tumor recurrence [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As a result, development of strategies to enhance the ability of PDT to target CSCs is an active area of research, with an emphasis on drug delivery systems that overcome mechanisms of resistance [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, it is pertinent to ascertain the sensitivity of CSCs to 5-ALA-PDT, given the potential resistant phenotype that could serve as founding population for future tumors. In this study, our goal is to determine if CSCs are resistant to 5-ALA-PDT, and if CSCs establish 5-ALA-PDT resistant populations post-treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cstrong\u003eCell culture\u003c/strong\u003e \u003cp\u003eThe human lung cancer cell line H1299, human breast cancer cell line Hs578T and human colon cancer cell lines DLD-1, were purchased from the American Type Culture Collection (ATCC). All cells were cultured in high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Corning, MA) supplemented with 10% fetal bovine serum (HyClone, Cytiva), 1 mM sodium pyruvate (Life Technologies) and antibiotic-antimycotic (Thermo Scientific). Cells were maintained in 10 cm culture dishes at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. For generation of 5-ALA-PDT resistant cells, DLD-1 cells (5000 cells/well) were plated in 96-well plates and subjected to 5-ALA-PDT 24 hours later. At 72 to 96 hours following 5-ALA-PDT, live cells were trypsinized, pooled and re-plated in a 10 cm dish to generate a cell line with a PDT-conditioned population (DLD-1 PDTx1). DLD-1 PDTx1 cells were plated in 96-well plates and subjected to another round of 5-ALA-PDT to generate DLD-1 PDTx2. This cycle was repeated until we obtained cell lines with population of DLD-1 that had been conditioned by PDT to varying degrees, with a maximum of four treatments (DLD-1 PDTx4).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e5-ALA-PDT\u003c/strong\u003e \u003cp\u003eHuman cancer cells plated in 96-well plates were treated with 5-aminolevulinic acid (5-ALA) (5 mM) (Sigma, A3785, USA) diluted in culture media (as described in previous section) and incubated at 37\u0026deg;C for 4 hours. Following the incubation period, culture media was replaced, and the cells were subjected to PDT using a Theralase TLC 3000A modular light source (Theralase Technologies Inc., Toronto, Canada; λ\u0026thinsp;=\u0026thinsp;618\u0026ndash;630 nm, fluence rate\u0026thinsp;=\u0026thinsp;150 mW/cm\u003csup\u003e2\u003c/sup\u003e, energy density (ED)\u0026thinsp;=\u0026thinsp;27 J/cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell viability assay\u003c/strong\u003e \u003cp\u003eCell viability was measured using the Colorimetric Cell Viability Kit I (WST-8) following manufacturer\u0026rsquo;s instructions (PromoCell GmbH, Germany). Ten (10) \u0026micro;l of WST-8 reagent was added to each well of 96-well plates containing cells and incubated in the dark at 37\u0026deg;C for 35 minutes. Absorbance was measured at 450 nm using a Bio-Rad Model 3550 microplate reader and cell viability was determined based on standard curves for each cell line.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFlow cytometry\u003c/strong\u003e \u003cp\u003eCells were fixed with Inside Stain Kit (Miltenyl Biotec, Teterow, Germany) and subsequently stained with anti-CD133 antibody (Miltenyl Biotec) and Zombie Violet\u0026trade; Fixable Viability Kit (BioLegend, CA, USA) following manufacturer\u0026rsquo;s instructions. Flow cytometry analysis was conducted using a CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA). The data was analyzed using FlowJo (FlowJo LLC, OR).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSoft agar assay\u003c/strong\u003e \u003cp\u003eUltraPure TM agarose (0.7%) (Invitrogen, CA, USA) and purified agar (1%) (Oxoid LTD, Hampshire UK) were mixed and incubated in a hot water bath at 37\u0026deg;C. First, the mixtures of agarose and 2X DMEM were plated in a 6-well plate (1.5ml/well) to generate the bottom layer. After 15 minutes of cooling to solidify the bottom layer, it was overlaid with mixtures of cell suspension and agarose to generate the top layer (2500 cells/well). Once the top layer solidified, the plate was incubated at 37\u0026deg;C for 21 days, after which colonies were counted under microscope.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWestern blot analysis\u003c/strong\u003e \u003cp\u003eAnti-ALDH1 antibody was purchased from Abcam (Boston, MA, USA) and HRP-conjugated secondary antibody from Santa Cruz Biotechnology (Dallas, TX, USA). Protein samples were prepared, and western blot was conducted as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eQuantification of intracellular PPIX\u003c/strong\u003e \u003cp\u003eParental and PDT-resistant (PDTx1-4) DLD-1 cells were plated in 24-well plates (5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well) and incubated for 24 hours to attain confluency. Following this, cells were treated with 5-ALA (5 mM) and incubated in the dark for 8 or 24 hours. At each time point, samples were obtained by washing cells with PBS before lysis with supplemented RIPA buffer (RIPA, PMSF, aprotinin, and Halt\u0026trade; phosphatase and protease inhibitor cocktail (Thermo Scientific)). Lysate was collected in opaque amber tubes and stored at -80\u0026deg;C. In minimally lit conditions, lysate was diluted in PBS (2.5% v/v), added to solid black 96-well plates (Greiner Bio-one), and covered with foil. Fluorescence of PPIX was measured with a Biotek Synergy MX plate reader with an excitation wavelength of 405 nm and emission wavelength of 635 nm.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analyses\u003c/strong\u003e \u003cp\u003eStatistical analyses were performed using Prism 7.0 (GraphPad). Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for inter-group comparison, and one-way ANOVA with Tukey\u0026rsquo;s posthoc test was used to compare between multiple groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were considered statistically significant.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u0026nbsp; The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCD133\u0026thinsp;+\u0026thinsp;cells are not sensitive to 5-ALA-PDT.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine if the CD133\u0026thinsp;+\u0026thinsp;stem cell (CSC) population is sensitive to 5-ALA-PDT, we quantified the percentage of CD133\u0026thinsp;+\u0026thinsp;cell populations in three different cell lines [human colon cancer (DLD-1), human lung cancer (H1299) and human breast cancer (Hs578T)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average percentage of CD133\u0026thinsp;+\u0026thinsp;stem cell population in DLD-1, H1299 and Hs578T were 1.0%, 1.9% and 1.6% respectively. We next sought to determine if the CD133\u0026thinsp;+\u0026thinsp;cells are sensitive to 5-ALA-PDT. The concentration of 5-ALA (5mM) we used in the study did not cause cell death without light exposure (Supplementary Fig.\u0026nbsp;1). Each cell line was treated with 5-ALA for 4 hours and then irradiated for 3 minutes using a Theralase TLC 3000A modular light source (27 J/cm\u003csup\u003e2\u003c/sup\u003e) (5-ALA-PDT). At 24 hours after 5-ALA-PDT, the cells were incubated with Zombie Violet, fixed with inside stain kit (Miltenyl Biotec), and then incubated with an anti-CD133 antibody for 10 minutes. Cell viability [dead cells (red) and live cells (green)] and CD133 expression was evaluated by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the scatter plots of DLD-1 cells, 0.77% of live cells were CD133\u0026thinsp;+\u0026thinsp;before 5-ALA-PDT which increased to 2.03% after 5-ALA-PDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that this population is enriched in surviving cells. In the total population of DLD-1 cells, live cells decreased from 83\u0026ndash;46% after 5-ALA-PDT, but the reduction was not statically significant. In contrast, the live cell populations of CD133\u0026thinsp;+\u0026thinsp;DLD-1 cells were 77% and 69% before and after 5-ALA-PDT respectively, suggesting that 5-ALA-PDT is not effective on CD133\u0026thinsp;+\u0026thinsp;DLD-1 cells. For H1299, a lung cancer cell line, the percentage of CD133\u0026thinsp;+\u0026thinsp;cells in the total population was increased to 3.67% from 2.32% after 5-ALA-PDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, there was a significant difference in the percentage of live cells in the total cell population before and after 5-ALA-PDT, with an approximate 44% decrease of living cells following 5-ALA-PDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In contrast, CD133\u0026thinsp;+\u0026thinsp;H1299 cells were resistant to 5-ALA-PDT as almost 80% of CD133\u0026thinsp;+\u0026thinsp;cells were alive even after 5-ALA-PDT and statistically insignificant compared to the control. Similar results were found in Hs578T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The CD133\u0026thinsp;+\u0026thinsp;population was increased after 5-ALA-PDT and cell death was effectively induced in the total cell population of Hs578T cells as the live cell population significantly reduced from 79\u0026ndash;30%. However, the live percentages of CD133\u0026thinsp;+\u0026thinsp;Hs578T cells were not significantly reduced by 5-ALA-PDT. These results demonstrate that the CD133\u0026thinsp;+\u0026thinsp;populations of each cell line were not sensitive to cell death induced by 5-ALA-PDT.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCancer stem cells are enriched in 5-ALA-PDT resistant populations.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe hypothesize that cancer stem cells play a critical role in forming populations with resistance to 5-ALA-PDT. If this is the case, cancer stem cells could be enriched in 5-ALA-PDT resistant populations. To this end, we first established 5-ALA-PDT resistant cell lines from DLD-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We plated parental DLD-1 cells in a 96-well plate and subjected them to 5-ALA-PDT (5 mM 5-ALA, 27 J/cm\u003csup\u003e2\u003c/sup\u003e). At 3\u0026ndash;4 days after 5-ALA-PDT, the surviving cells were pooled and further subjected to 5-ALA-PDT. This process was repeated up to 4 times (PDTx1: one cycle, PDTx2: two cycles, PDTx3: three cycles and PDTx4: four cycles) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To confirm if the established cell lines were resistant to 5-ALA-PDT, the viability of the parental DLD-1 and the 5-ALA-PDT resistant cells (PDTx1, PDTx2, PDTx3 and PDTx4) were measured 24 hours after 5-ALA-PDT by a WST-8 assay. The cell viabilities of parental DLD-1 and PDTx1 cell lines were significantly reduced after 5-ALA-PDT, suggesting that they are sensitive to 5-ALA-PDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, most of the PDTx2, PDTx3 or PDTx4 cells survived after 5-ALA-PDT, indicating that they are 5-ALA-PDT resistant cell lines.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo determine if CSCs were enriched in the DLD-1 PDTx2, PDTx3 and PDTx4 resistant cell lines, we first performed a soft agar analysis of anchorage-independent growth. The parental DLD-1, PDTx2, PDTx3 and PDTx4 resistant cells were plated in soft agar. After 21 days, colonies were counted under the microscope. PDTx3 and PDTx4 resistant cells showed a significant increase in the average number of colonies compared to the parental DLD-1 cell line, whereas a significant change was not observed in PDTx2 resistant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further confirm that CSCs are enriched in the 5-ALA-PDT resistant populations, we conducted western blot analysis of ALDH1 on cell lysates prepared from parental DLD-1 cells and 5-ALA-PDT resistant DLD-1 cells (PDTx2, PDTx3 and PDTx4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;2). ALDH1 is another marker for CSCs, which was expressed on DLD-1 CSCs, but not in other cell lines. While we did not observe changes in ALDH1 expression between PDTx2 cells and parental DLD-1 cells, ALDH1 expression was increased in PDTx3 and PDTx4 cells. Finally, we determined the expression levels of the CSC marker CD133\u0026thinsp;+\u0026thinsp;expression in the 5-ALA-PDT resistant cells (PDTx4) by flow cytometry. The mean fluorescent intensity of the CD133\u0026thinsp;+\u0026thinsp;population of cells was increased in the PDTx4 compared to the parental DLD-1 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also found that the percentage of CD133\u0026thinsp;+\u0026thinsp;cells was significantly increased in the PDTx4 cell line compared to DLD-1 parent cells. These results further support that CSCs are resistant to 5-ALA-PDT and are enriched in 5-ALA-PDT resistant cell populations. We further determined if decreased PPIX accumulation could be one of causes of 5-ALA-PDT resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We found that 5-ALA-PDT resistant DLD-1 cells (PDTx1, PDTx2, PDTx3 and PDTx4) accumulated significantly lower amounts of PPIX than parental control DLD-1 cells when stimulated with 5-ALA. The results suggest that low PPIX accumulation is one of the underlying mechanisms for 5-ALA-PDT resistance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough 5-ALA-PDT has been approved for treating different types of cancers, cancer recurrence remains as a problem [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. One of the primary factors of cancer recurrence is resistance to treatment, leading to survival of a small number of cancer cells that potentially reform tumors. Therefore, addressing the mechanisms of cancer resistance against 5-ALA-PDT is essential to improve its efficacy.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the possible role of CSCs as a driver of 5-ALA-PDT resistance because CSCs have been reported to resist chemotherapy partially due to upregulation of ABC transporters [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ABC transporters efflux chemotherapeutics from cancer cells, which decreases its intracellular accumulation and thus prevents the cells from dying [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our previous studies also demonstrated that PPIX efflux via ABCB1 transporter reduces cancer cell sensitivity to 5-ALA-PDT [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, we hypothesized that CSCs are less sensitive to 5-ALA-PDT and subsequently contribute to the establishment of resistant populations to 5-ALA-PDT due to their ability to efflux 5-ALA via ABC transporters. Through our efforts, we confirmed that 5-ALA-PDT is not effective in killing CSCs and that CSC populations are increased in the 5-ALA-PDT resistant cell lines. These results clearly indicate that CSCs play an essential role in establishing cancer resistance to 5-ALA-PDT in the cells lines evaluated (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eInterestingly, our results contrast previous reports of 5-ALA-PDT efficacy against CSCs [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. CSCs from oral squamous cell carcinoma cell lines were effectively differentiated by 5-ALA-PDT and susceptibility to treatment was due to increased PPIX accumulation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Similarly, glioma CSC lines accumulate more PPIX compared to their differentiated counterparts and therefore more sensitive to treatment [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. When evaluating 5-ALA-PDT against head and neck cancer CSC lines, 5-ALA-PDT reduced the expression of CSC markers and sensitized cells to chemotherapy, despite modest cell killing [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In comparison, our study showed that CSCs became enriched in PDT-resistant populations, which accumulated significantly less PPIX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results highlight the importance of PPIX accumulation on efficacy of treatment and the consideration of CSC heterogeneity between cancers.\u003c/p\u003e \u003cp\u003eCD133 was used as a CSC marker in this study as we found that the levels of CD133\u0026thinsp;+\u0026thinsp;cells in all three cell lines were sufficient (1-1.9%, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to conduct our proposed experiments. Other CSC markers such as CD24, CD32, CD44 and ALDH1A1 were not commonly expressed in the cell lines. As CD133 has been widely used as a CSC marker for different types of cancer, we utilized CD133 as a CSC marker of DLD-1 cells to determine CSC functions throughout the study. However our research objective was not to specifically characterize the roles of CD133 and its downstream signalling pathways in 5-ALA-PDT resistance. The CSC populations (CD133\u0026thinsp;+\u0026thinsp;cells) were low in the cancer cell lines (1\u0026ndash;3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and even in PDTx4 DLD-1 cells (8%) in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Generally, the CSC population in tumors range from less than 0.02 to 25% depending on the tumor types (30).\u003c/p\u003e \u003cp\u003eOur current study falls short to identify the precise mechanisms underlying 5-ALA-PDT resistance in CSCs. There are several possible mechanisms that could lead to CSCs resistant against 5-ALA-PDT. First, as mentioned above, CSCs could be resistant to 5-ALA-PDT due to high expression of the ABCtransporters as they are commonly overexpressed by CSCs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. High expression of the ABC transporters may decrease PPIX accumulation in CSCs induced by 5-ALA treatment and subsequently the sensitivity of CSCs to 5-ALA-PDT. In a study examining the efficacy of photodiagnosis of cancers using 5-ALA (5-ALA PDD), Kawai et al., reported that the CSC populations of PANC-1 cells expressing high levels of ABCG2 were responsible for decreased PPIX accumulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We observed that the 5-ALA-PDT resistant cells accumulated lower amounts of PPIX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting that this mechanism underlies the CSC resistance to 5-ALA-PDT. Secondly, high expression of stress response genes in CSCs could be one of the resistant mechanisms [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CSCs have increased genetic diversity to adapt and survive under stress conditions such as lack of oxygen, starvation, and exposure to DNA-damaging compounds [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, CSCs have increased activity of autophagy, which promotes cellular survival by enabling the ability to overcome stress conditions through nutrient recycling and preventing the accumulation of damaged cellular components [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These survival mechanisms of CSCs may reduce cell death caused by 5-ALA-PDT. Fourth, CSCs have lower levels of reactive oxygen species (ROS) compared to their differentiated counterparts, which is one of the essential effectors for cancer cell death induced by 5-ALA-PDT [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although our current study did not identify the cellular mechanisms that underlie CSC resistance to 5-ALA-PDT, it is essential to improve the efficacy of 5-ALA-PDT and reduce cancer recurrence after 5-ALA-PDT. Combined treatment with 5-ALA-PDT and inhibitors targeting ABC transporters has been shown to increase anticancer efficacy \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, which may be an effective way to eliminate CSCs by 5-ALA-PDT [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. As the development of ABC transporter inhibitors for clinical trials is an active area of research, it would be a feasible idea to evaluate inhibitors in combination with 5-ALA-PDT in clinical settings in the near future [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. To clarify the mechanism of CSC resistance to 5-ALA-PDT, this study warrants further investigation. It would be essential to determine cellular localization and quantification of PPIX, activities of enzymes involved in the heme pathway, cellular ROS levels, activation of cell death pathways, expression of ABC transporters and expression of stress response genes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eConceptualization/design: CPJR, VSC, KH; Data collection: CPJR, VSC, NC; Data analysis: CPJR, VSC, NC, KH; Writing original draft: CPJR, NC, KH; Review \u0026amp; editing: VSC, NC, KH.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA: A Cancer Journal for Clinicians. 2023 Jan 12;73(1):17\u0026ndash;48. doi:10.3322/caac.21763\u003c/li\u003e\n \u003cli\u003eBaskaran R, Lee J, Yang S-G. Clinical development of photodynamic agents and therapeutic applications. Biomaterials Research. 2018 Sept 26;22(25). doi:10.1186/s40824-018-0140-z\u003c/li\u003e\n \u003cli\u003eMishchenko T, Balalaeva I, Gorokhova A, Vedunova M, Krysko DV. Which cell death modality wins the contest for photodynamic therapy of cancer? Cell Death \u0026amp; Disease. 2022 May 13;13(455). doi:10.1038/s41419-022-04851-4\u003c/li\u003e\n \u003cli\u003eZheng J, Shan Y, Lambrecht RW, Donohue SE, Bonkovsky HL. Differential regulation of human ALAS1 mrna and protein levels by heme and cobalt protoporphyrin. Molecular and Cellular Biochemistry. 2008 Aug 22;319(1\u0026ndash;2):153\u0026ndash;61. Doi:10.1007/s11010-008-9888-0\u003c/li\u003e\n \u003cli\u003eYang X, Palasuberniam P, Kraus D, Chen B. Aminolevulinic acid-based tumor detection and therapy: Molecular mechanisms and strategies for enhancement. International Journal of Molecular Sciences. 2015;16(10):25865\u0026ndash;80. doi:10.3390/ijms161025865 \u003c/li\u003e\n \u003cli\u003eZhang Z-J, Wang K-P, Mo J-G, Xiong L, Wen Y. Photodynamic therapy regulates fate of cancer stem cells through reactive oxygen species. World Journal of Stem Cells. 2020 Jul 26;12(7):562-84. doi: 10.4252/wjsc.v12.i7.562\u003c/li\u003e\n \u003cli\u003eChelakkot VS, Liu K, Yoshioka E, Saha S, Xu D, Licursi M, et al. MEK reduces cancer-specific PPIX accumulation through the RSK-ABCB1 and HIF-1\u0026alpha;-Fech Axes. Scientific Reports. 2020 Dec 17;10(22124). doi:10.1038/s41598-020-79144-x\u003c/li\u003e\n \u003cli\u003eKobuchi H, Moriya K, Ogino T, Fujita H, Inoue K, Shuin T, et al. Mitochondrial localization of ABC transporter ABCG2 and its function in 5-aminolevulinic acid-mediated protoporphyrin IX accumulation. PLOS ONE. 2012 Nov 26;7(11). doi:10.1371/journal.pone.0050082\u003c/li\u003e\n \u003cli\u003eYoshioka E, Chelakkot VS, Licursi M, Rutihinda SG, Som J, Derwish L, et al. Enhancement of cancer-specific protoporphyrin IX fluorescence by targeting oncogenic Ras/Mek Pathway. Theranostics. 2018 Mar 8;8(8):2134\u0026ndash;46. doi:10.7150/thno.22641\u003c/li\u003e\n \u003cli\u003eHanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011 Mar 4;144(5):646\u0026ndash;74. doi:10.1016/j.cell.2011.02.013\u003c/li\u003e\n \u003cli\u003eMoitra K. Overcoming multidrug resistance in cancer stem cells. BioMed Research International. 2015 Nov 16;2015. doi:10.1155/2015/635745\u003c/li\u003e\n \u003cli\u003eZinzi L, Contino M, Cantore M, Capparelli E, Leopoldo M, Colabufo NA. ABC transporters in CSCs membranes as a novel target for treating tumor relapse. Frontiers in Pharmacology. 2014 Jul 10;5(163). doi:10.3389/fphar.2014.00163\u003c/li\u003e\n \u003cli\u003eCui J, Christin JR, Reisz JA, Cendali FI, Sanawar R, Coutinho De Miranda M, et al. Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance. Science Advances. 2023 Dec 1;9(48). doi:10.1126/sciadv.adh1891\u003c/li\u003e\n \u003cli\u003eFuchs D, Daniel V, Sadeghi M, Opelz G, Naujokat C. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1A cells. Biochemical and Biophysical Research Communications. 2010 Mar 27;394(4):1098\u0026ndash;104. doi:10.1016/j.bbrc.2010.03.138\u003c/li\u003e\n \u003cli\u003eLi L, Ni R, Zheng D, Chen L. Eradicating the tumor \u0026ldquo;seeds\u0026rdquo;: Nanomedicines-based therapies against Cancer Stem Cells. DARU Journal of Pharmaceutical Sciences. 2023 Mar 27;31:83\u0026ndash;94. doi:10.1007/s40199-023-00456-0\u003c/li\u003e\n \u003cli\u003eDuan H, Liu Y, Gao Z, Huang W. Recent advances in drug delivery systems for targeting cancer stem cells. Acta Pharmaceutica Sinica B. 2021 Jan;11(1):55-70. doi:10.1016/j.apsb.2020.09.016\u003c/li\u003e\n \u003cli\u003eYang Y, Peng Y, Du Y, Lin M, Li J, Gao D, et al. Hierarchical self-recognition and response in CSC and Non-CSC micro-niches for cancer therapy. Biomaterials. 2024 Jul;308. doi: 10.2139/ssrn.4635630\u003c/li\u003e\n \u003cli\u003eDuncan JK, Xu D, Licursi M, Joyce MA, Saffran HA, Liu K, et al. Interferon regulatory factor 3 mediates effective antiviral responses to human coronavirus 229E and OC43 infection. Frontiers in Immunology. 2023 Apr 30;14. doi:10.3389/fimmu.2023.930086 \u003c/li\u003e\n \u003cli\u003eRailkar R, Agarwal PK. Photodynamic therapy in the treatment of bladder cancer: Past challenges and current innovations. European Urology Focus. 2018 Aug 22;4(4):509\u0026ndash;11. doi:10.1016/j.euf.2018.08.005 \u003c/li\u003e\n \u003cli\u003eVan den Broeck T, van den Bergh RCN, Arfi N, Gross T, Moris L, Briers E, et al. Prognostic value of biochemical recurrence following treatment with curative intent for prostate cancer: A systematic review. European Urology. 2019 Jun;75(6):967\u0026ndash;87. doi:10.1016/j.eururo.2018.10.011 \u003c/li\u003e\n \u003cli\u003eKim TE, Chang J-E. Recent studies in photodynamic therapy for cancer treatment: From basic research to clinical trials. Pharmaceutics. 2023 Aug 31;15(9):2257. doi:10.3390/pharmaceutics15092257\u003c/li\u003e\n \u003cli\u003eDoyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene. 2003 Oct 23;22:7340\u0026ndash;58. doi:10.1038/sj.onc.1206938 \u003c/li\u003e\n \u003cli\u003eCho Y, Kim YK. Cancer stem cells as a potential target to overcome multidrug resistance. Frontiers in Oncology. 2020 Jun 1;10. doi:10.3389/fonc.2020.00764 \u003c/li\u003e\n \u003cli\u003eBegicevic R-R, Falasca M. ABC transporters in cancer stem cells: Beyond Chemoresistance. International Journal of Molecular Sciences. 2017 Nov 8;18(11):2362. doi:10.3390/ijms18112362 \u003c/li\u003e\n \u003cli\u003eLou H, Dean M. Targeted therapy for cancer stem cells: The patched pathway and ABC Transporters. Oncogene. 2007 Feb 26;26:1357\u0026ndash;60. doi:10.1038/sj.onc.1210200\u003c/li\u003e\n \u003cli\u003eWu C-P, Hsiao S-H, Huang Y-H, Hung L-C, Yu Y-J, Chang Y-T, et al. Sitravatinib sensitizes ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs. Cancers. 2020 Jan 13;12(1):195. doi:10.3390/cancers12010195\u003c/li\u003e\n \u003cli\u003ePinto MA, Ferreira CB, Lima BE, Molon AC, Coppa Ibarra AM, Cecatto RB, et al. Effects of 5-ALA mediated photodynamic therapy in Oral cancer stem cells. Journal of Photochemistry and Photobiology B: Biology. 2022 Oct;235(112552). doi:10.2139/ssrn.4146140 \u003c/li\u003e\n \u003cli\u003eOmura N, Nonoguchi N, Fujishiro T, Park Y, Ikeda N, Kajimoto Y, et al. Ablation efficacy of 5-aminolevulinic acid-mediated photodynamic therapy on human glioma stem cells. Photodiagnosis and Photodynamic Therapy. 2023 Mar;41:103119. doi:10.1016/j.pdpdt.2022.103119 \u003c/li\u003e\n \u003cli\u003eYu C-H, Yu C-C. Photodynamic therapy with 5-aminolevulinic acid (ALA) impairs tumor initiating and chemo-resistance property in head and neck cancer-derived cancer stem cells. PLoS ONE. 2014 Jan 24;9(1). doi:10.1371/journal.pone.0087129 \u003c/li\u003e\n \u003cli\u003eToledo-Guzm\u0026aacute;n ME, Bigoni-Ord\u0026oacute;\u0026ntilde;ez GD, Hern\u0026aacute;ndez MI, Ortiz-S\u0026aacute;nchez E. Cancer stem cell impact on clinical oncology. World Journal of Stem Cells. 2018 Dec 26;10(12):183\u0026ndash;95. doi:10.4252/wjsc.v10.i12.183 \u003c/li\u003e\n \u003cli\u003eEyre R, Harvey I, Stemke-Hale K, Lennard TW, Tyson-Capper A, Meeson AP. Reversing paclitaxel resistance in ovarian cancer cells via inhibition of the ABCB1 expressing side population. Tumor Biology. 2014 Jul 4;35(10):9879\u0026ndash;92. doi:10.1007/s13277-014-2277-2\u003c/li\u003e\n \u003cli\u003eKawai N, Hirohasi Y, Ebihara Y, Saito T, Murai A, Saito T, et al. ABCG2 expression is related to low 5-ala photodynamic diagnosis (PDD) efficacy and cancer stem cell phenotype, and suppression of ABCG2 improves the efficacy of PDD. PLoS ONE. 2019 May 13;14(5). doi:10.1371/journal.pone.0216503\u003c/li\u003e\n \u003cli\u003eCasas A, Perotti C, Di Venosa G, Batlle A. Mechanisms of resistance to photodynamic therapy: An update. Resistance to Targeted Anti-Cancer Therapeutics. 2011;18(16):2486\u0026ndash;515. doi:10.1007/978-3-319-12730-9_2\u003c/li\u003e\n \u003cli\u003eTorigoe T, Hirohashi Y, Yasuda K, Sato N. Constitutive expression and activation of stress response genes in cancer stem-like cells/tumour initiating cells: Potent targets for cancer stem cell therapy. International Journal of Hyperthermia. 2013 Jul 31;29(5):436\u0026ndash;41. doi:10.3109/02656736.2013.814809\u003c/li\u003e\n \u003cli\u003eChen M, Xie S. Therapeutic targeting of cellular stress responses in cancer. Thoracic Cancer. 2018 Oct 12;9(12):1575\u0026ndash;82. doi:10.1111/1759-7714.12890\u003c/li\u003e\n \u003cli\u003eQureshi-Baig K, Kuhn D, Viry E, Pozdeev VI, Schmitz M, Rodriguez F, et al. Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-Ezr (ezrin) pathway. Autophagy. 2019 Nov 27;16(8):1436\u0026ndash;52. doi:10.1080/15548627.2019.1687213\u003c/li\u003e\n \u003cli\u003eSrivastava AK, Han C, Zhao R, Cui T, Dai Y, Mao C, et al. Enhanced expression of DNA polymerase ETA contributes to cisplatin resistance of ovarian cancer stem cells. Proceedings of the National Academy of Sciences. 2015 Mar 23;112(14):4411\u0026ndash;6. doi:10.1073/pnas.1421365112\u003c/li\u003e\n \u003cli\u003eBao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006 Oct 18;444:756\u0026ndash;60. doi:10.1038/nature05236\u003c/li\u003e\n \u003cli\u003eFerrand A, Sandrin MS, Shulkes A, Baldwin GS. Expression of gastrin precursors by CD133-positive colorectal cancer cells is crucial for tumour growth. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2009 Jan 15;1793(3):477\u0026ndash;88. doi:10.1016/j.bbamcr.2009.01.004\u003c/li\u003e\n \u003cli\u003eWang Y, Zhou L, Qing Q, Li Y, Li L, Dong X, et al. Gene expression profile of cancer stem‑like cells in the SW480 colon adenocarcinoma cell line. Oncology Reports. 2019 May 2;42(1):386\u0026ndash;98. doi:10.3892/or.2019.7146\u003c/li\u003e\n \u003cli\u003eSato K, Tsuchihara K, Fujii S, Sugiyama M, Goya T, Atomi Y, et al. Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Research. 2007 Oct 15;67(20):9677\u0026ndash;84. doi:10.1158/0008-5472.can-07-1462\u003c/li\u003e\n \u003cli\u003eTogano S, Yashiro M, Masuda G, Sugimoto A, Miki Y, Yamamoto Y, et al. Gastric cancer stem cells survive in stress environments via their autophagy system. Scientific Reports. 2021 Oct 19;11(20664). doi:10.1038/s41598-021-00155-3\u003c/li\u003e\n \u003cli\u003eLi Y-R, Fang Y, Lyu Z, Zhu Y, Yang L. Exploring the dynamic interplay between cancer stem cells and the tumor microenvironment: Implications for novel therapeutic strategies. Journal of Translational Medicine. 2023 Oct 2;21(683). doi:10.1186/s12967-023-04575-9\u003c/li\u003e\n \u003cli\u003eRobey RW, Steadman K, Polgar O, Bates SE. ABCG2-mediated transport of photosensitizers: Potential impact on photodynamic therapy. Cancer Biology \u0026amp; Therapy. 2005 Feb 2;4(2):187\u0026ndash;94. doi:10.4161/cbt.4.2.1440\u003c/li\u003e\n \u003cli\u003eIshikawa T, Kajimoto Y, Inoue Y, Ikegami Y, Kuroiwa T. Critical role of ABCG2 in ala-photodynamic diagnosis and therapy of human brain tumor. Advances in Cancer Research. 2015 Jan 8;125:197\u0026ndash;216. doi:10.1016/bs.acr.2014.11.008 \u003c/li\u003e\n \u003cli\u003eChandratre S, Olsen J, Howley R, Chen B. Targeting ABCG2 transporter to enhance 5-aminolevulinic acid for tumor visualization and photodynamic therapy. Biochemical Pharmacology. 2023 Nov 1;217:115851. doi:10.1016/j.bcp.2023.115851 \u003c/li\u003e\n \u003cli\u003eToyoda Y, Takada T, Suzuki H. Inhibitors of human ABCG2: From technical background to recent updates with clinical implications. Frontiers in Pharmacology. 2019 Mar 4;10. doi:10.3389/fphar.2019.00208 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"5-aminolevulinic acid (5-ALA), photodynamic therapy (PDT), cancer stem cells (CSCs), CD133, 5-ALA-PDT resistance","lastPublishedDoi":"10.21203/rs.3.rs-5039795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5039795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhotodynamic therapy (PDT) is a minimally invasive treatment approved for many types of cancers. PDT involves the administration of photoactive substances called photosensitizers (PS) that selectively accumulate in cancer cells and are subsequently excited/activated by irradiation with specific wavelengths of light. Activated PS leads to the generation of singlet oxygen and reactive oxygen species (ROS), promoting cancer cell death. 5-aminolevulinic acid (5-ALA) is a naturally occurring PS precursor, which is metabolically converted to the PS, protoporphyrin IX (PPIX). Although 5-ALA-PDT is effective at killing cancer cells, we normally observed in \u003cem\u003ein vitro\u003c/em\u003e experiments that approximately 5\u0026ndash;10% of cells survive 5-ALA-PDT. Identifying the mechanisms of resistance to 5-ALA-PDT-mediated cell death is important to prevent tumor recurrence following 5-ALA-PDT. Previously, we reported that oncogenic activation of Ras/MEK promotes PPIX efflux and reduces cellular sensitivity to 5-ALA-PDT through increased expression of ABCB1 transporter. As cancer stem cells (CSCs) are known to drive resistance to other cancer treatments and have high efflux of chemotherapeutic agents via ABC-family transporters, we hypothesize that CSCs underlie 5-ALA-PDT resistance. In this study, we determined 1) if CSCs are resistant to 5-ALA-PDT and 2) if CSCs play roles in establishing resistant populations of 5-ALA-PDT. When we compared CSC populations before and after 5-ALA-PDT, we found that CSCs were less susceptible to 5-ALA-PDT. Moreover, we found that the CSC population was enriched in 5-ALA-PDT-resistant cell lines compared to the parental cell line. Our results indicate that CSCs are not sensitive to 5-ALA-PDT, which may contribute to establishment of 5-ALA-PDT resistance.\u003c/p\u003e","manuscriptTitle":"Cancer stem cell populations are resistant to 5-aminolevulinic acid- photodynamic therapy (5-ALA-PDT)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 10:00:45","doi":"10.21203/rs.3.rs-5039795/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-20T05:19:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T03:40:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-18T15:12:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62874109431267684607974834152546022136","date":"2024-09-18T14:00:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261368920830155076916282475595631988903","date":"2024-09-18T08:20:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-18T03:14:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-18T03:05:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-18T01:14:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-17T06:49:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-05T17:08:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b926efe0-4020-4ad4-8a25-65193a2afbc0","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:02:38+00:00","versionOfRecord":{"articleIdentity":"rs-5039795","link":"https://doi.org/10.1038/s41598-025-88173-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-05 15:57:38","publishedOnDateReadable":"February 5th, 2025"},"versionCreatedAt":"2024-11-19 10:00:45","video":"","vorDoi":"10.1038/s41598-025-88173-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-88173-3","workflowStages":[]},"version":"v1","identity":"rs-5039795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5039795","identity":"rs-5039795","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}