Statin-dye conjugates for selective targeting of KRAS mutant cancer cells

Hye-ran Moon; Zhenying Cai; Bo Kyung Cho; Hyeyoun Chang; Seung Taek Hong; Jean J. Zhao; Ick Chan Kwon; Thomas M. Roberts; Bumsoo Han; Ju Hee Ryu

doi:10.1371/journal.pone.0340189

Abstract

Over 90% of pancreatic ductal adenocarcinoma (PDAC) patients involve KRAS mutations (KRAS^MUT), for which current treatment options are limited. Statins, commonly used to lower cholesterol, have demonstrated certain selective toxicity towards KRAS-transformed cells, prompting the question of whether statin-based conjugates could achieve selective uptake specifically in KRAS^MUT cells. To investigate this, we synthesized statin-dye conjugates by attaching a fluorescent dye (Cy5.5) to two statins: simvastatin and pravastatin, aiming to assess whether selective uptake indeed occurs. Our findings revealed that these conjugates exhibited markedly enhanced uptake in KRAS^MUT cells compared to KRAS wild-type (KRAS^WT) cells. We evaluated the uptake of these conjugates in both KRAS^MUT and KRAS^WT cells and examined their potential to selectively target KRAS^MUT pancreatic cancer cells (PCCs) using an engineered PDAC tumor model co-cultured with PCCs and cancer-associated fibroblasts (CAFs). Our findings indicate that KRAS^MUT cancer cells exhibited higher uptake of statin-Cy5.5 conjugates via enhanced macropinocytosis compared to KRAS^WT cancer cells and CAFs. We also found enhanced uptake of the statin-Cy5.5 conjugate in MCF10A cells with PTEN deficiency, a condition known to elevate macropinocytosis, compared to control MCF10A cells with wild-type PTEN. Notably, in the PCC and CAF co-culture model, the pravastatin-Cy5.5 conjugate selectively killed KRAS^MUT PCCs without affecting the KRAS^WT CAFs. These findings highlight the unique synergistic potential of statin-Cy5.5—distinct from either component alone—as targeted delivery vehicles for KRAS^MUT cancer therapy.

Citation: Moon H-r, Cai Z, Cho BK, Chang H, Hong ST, Zhao JJ, et al. (2026) Statin-dye conjugates for selective targeting of KRAS mutant cancer cells. PLoS One 21(1): e0340189. https://doi.org/10.1371/journal.pone.0340189

Editor: Luca Pesce, Università di Pisa: Universita degli Studi di Pisa, ITALY

Received: April 24, 2025; Accepted: December 17, 2025; Published: January 9, 2026

Copyright: © 2026 Moon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data underlying the results are included in the paper and its Supporting Information files. The minimal data set required to replicate the findings is provided.

Funding: This work was supported by the Intramural Research Program of KIST and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT; No. RS-2024-00463774 to JHR). This work was partially supported by grants from the National Institutes of Health (U01 HL143403, R01 CA254110, U01 CA274304 to BH). HM was partially supported by the Purdue University Center for Cancer Research (P30 CA023168) and a Postdoc Challenge Award from Indiana Clinical and Translational Sciences Institute which is funded in part by Award Number UM1TR004402 from the National Institutes of Health.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, with a five-year survival rate of only 12% [1]. PDAC’s aggressive nature and resistance to conventional treatments, such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy, underscore the urgent need for new therapeutic approaches. The tumor microenvironment (TME) of PDAC is particularly complex, consisting of pancreatic cancer cells (PCCs), cancer-associated fibroblasts (CAFs), and various immune cells, further complicating treatment [2]. Consequently, current treatment options are often inadequate, and novel strategies are needed to improve patient outcomes.

A key feature of PDAC is the high prevalence of KRAS mutations, present in over 90% of PDAC patients [3]. These mutations play a crucial role in driving tumorigenesis by maintaining KRAS in a continuously active state, promoting uncontrolled cell growth and division. This persistent signaling contributes to the cancer’s aggressive behavior and poor prognosis, making KRAS a significant target for cancer therapy. Recent breakthroughs have shown promise; for example, the KRAS^G12C inhibitor sotorasib (AMG 510) demonstrated promising results in phase I clinical trial for non-small cell lung cancer, with partial response or stable disease observed in 88.1% of patients with KRAS^G12C mutations [4,5]. Additionally, a recent study suggests that KRAS^G12D inhibitors may offer potential benefits in treating pancreatic cancers [6]. However, the heterogeneity of KRAS mutations, including subtypes such as G12C, G13D, G12D, G12V, and G12R presents a challenge for developing treatments that are effective across all variants [7]. Therefore, there is an urgent need to develop therapies that can effectively target the diverse range of KRAS mutation subtypes.

Interestingly, statins, widely used to lower cholesterol, have shown selective efficacy in killing KRAS mutant (KRAS^MUT) cells in vitro and in tumor models [5,8–10]. Statins inhibit the mevalonate pathway, which is necessary for KRAS prenylation, a post-translational modification required for its localization to the cell membrane where it exerts oncogenic effects [9,11–14]. By inhibiting prenylation, statins disrupt KRAS function, impeding its role in promoting tumor growth [15]. This raises the question of whether statin-based conjugates might also achieve selective uptake specifically in KRAS^MUT cells.

To investigate this, we synthesized statin-dye conjugates by attaching a fluorescent dye (Cy5.5) to two statins, simvastatin and pravastatin, aiming to assess whether selective uptake occurs. To our knowledge, no previous studies have directly examined selective uptake of statin-fluorescent dye conjugates in KRAS^MUT cells, and we sought to investigate the uptake mechanism of these conjugates. Our findings revealed that these conjugates exhibited markedly enhanced uptake in KRAS^MUT cells compared to KRAS wild-type (KRAS^WT) cells.

Furthermore, we investigated their ability to selectively target KRAS^MUT PCCs using an engineered PDAC tumor model with co-cultures of PCCs and CAFs. Our results showed that these statin-dye conjugates were selectively taken up by KRAS^MUT cells compared to isogenic KRAS^WT cells and CAFs. We also found that statin-Cy5.5 conjugates were selectively taken up in PTEN knockout (PTEN^KO) cells via macropinocytosis. Moreover, the pravastatin-Cy5.5 conjugate showed modest selective killing toward PCCs in a 3D co-culture model. This suggests that the conjugation of statins to Cy5.5 creates a unique compound with selective uptake in KRAS^MUT cells—an effect not observed with statin or Cy5.5 alone—demonstrating the therapeutic potential of such conjugates. These findings underscore the functional synergy of the statin-Cy5.5 conjugate and support its potential as a prototype for KRAS^MUT-selective drug delivery.

Materials and methods

Synthesis of statin-dye conjugates

Simvastatin-Cy5.5: Cy5.5 acid (15 mg, 0.026 mmol, Lumiprobe, MD, USA) was added to a mixture of simvastatin (11 mg, 0.026 mmol), EDC (5.5 mg, 0.035 mmol), and DMAP (1.6 mg, 0.012 mmol) in dichloromethane (DCM, 1 mL). The mixture was stirred at room temperature for 1 h, followed by solvent evaporation. The residue was purified by silica gel column chromatography using DCM/methanol (19/1) as the mobile phase. The final product was obtained as a blue solid. The mass and purity of simvastatin-Cy5.5 were confirmed by liquid chromatography-mass spectrometry (1260 Infinity II; Agilent Technologies, CA, USA). The calculated m/z was 983.6, with a measured m/z of 983.7. The purity of simvastatin-Cy5.5 was analyzed by HPLC in solvent gradient conditions of acetonitrile/H₂O from 5:95–100:0 for 30 min, followed by 100:0 for 5 min.

Pravastatin-Cy5.5: A mixture of pravastatin (20 mg, 0.045 mmol), EDC (14 mg, 0.090 mmol), and NHS (8.6 mg, 0.075 mmol) in dimethylformamide (DMF) was stirred at RT for 30 min. Subsequently, Cy5.5 amine (34 mg, 0.045 mmol, Lumiprobe, MD, USA) was added in one portion to the mixture. The mixture was stirred at RT for 2 h, followed by solvent evaporation. The residue was purified by silica gel column chromatography using DCM/methanol (4/1) as the mobile phase. The final product was obtained as a blue solid. The mass of pravastatin-Cy5.5 was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF, Voyager DE-STR; Applied Biosystems, CA, USA), with a calculated m/z of 1087.7, and a found m/z of 1087.8. The purity was confirmed by HPLC analysis in solvent gradient conditions of acetonitrile/H₂O from 5:95–100:0 for 30 min, followed by 100:0 for 5 min).

Cell culture and cell line information

Panc1 (CRL-1469) and BxPC3 (CRL-1687) cell lines were purchased from the American Type Culture Collection. Panc1 cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, MA, USA) and 1% antibiotic-antimycotic solution (Thermo Fisher, MA, USA). BxPC3 cells were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640; GenDEPOT, TX, USA) medium containing 10% FBS and 1% antibiotic-antimycotic solution.

The KRAS-inducible human pancreatic ductal epithelial cells (HPDE iKRAS) cell line was kindly provided by Dr. Allen-Petersen from Purdue University. HPDE iKRAS cells were genetically modified to allow for the KRAS G12D mutation to be induced by the presence of doxycycline. Details about the modification and characterization of the cell line are described in Tsang et al [16]. HPDE iKRAS cells were maintained in keratinocyte-serum free medium (Invitrogen, MA, USA) supplemented with bovine pituitary extract (0.05 mg/ml), recombinant human epidermal growth factor (5 ng/ml) and L-glutamine. For KRAS induction, HPDE iKRAS cells were treated with 25 ng/ml doxycycline for 48 h (HPDE KRAS^MUT), whereas the HPDE iKRAS cells were treated with a corresponding dimethyl sulfoxide (DMSO) control media for HPDE KRAS^WT in the normal culture conditions. The KRAS induction was processed after the cells were harvested and seeded in the experimental platforms. CAF19 transduced with enhanced GFP was kindly provided by Dr. Melissa Fishel [17] where the CAF19 cells were originally obtained from Dr. Anirban Maitra at Johns Hopkins University [18]. CAF19 cells were maintained in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX™ supplement (Invitrogen, MA, USA), and 100 µg/ml penicillin/streptomycin. KRAS isogenic HCT116 and DLD1 cell lines (KRAS^WT and KRAS^WT/G13D) were kindly provided by Dr. Bert Vogelstein at Johns Hopkins Kimmel Cancer Center to Dr. Jean Zhao’s Lab (S1 Table) [19].

The cells were regularly harvested by 0.05% trypsin and 0.53 mM EDTA (Life Technologies, CA, USA) when grown to ~80% confluency in 25 or 75 cm² T-flasks and incubated at 37°C with 5% CO₂. Harvested cells were used for experiments or sub-cultured while maintaining them within the 15^th passage.

Cellular uptake assay

The cells were seeded on poly-D-lysine coated coverslips (Neuvitro, WA, USA) at a density of 60–70%. For the inhibitor treatment experiment, cells were pre-incubated with DMSO (as mock) or different signaling inhibitors for 1.5 h and treated with 50 nM statin-dye conjugates for 1 h. The signaling inhibitor used for pre-treatment was EIPA (50 μM, 1.5 h) for macropinocytosis inhibition. After treatments, the cells were repeatedly washed with PBS and fixed with 4% paraformaldehyde. The nuclei were stained with Hoechst 33342 (Thermo Fisher, MA, USA), and the coverslips were mounted on a glass slide with mounting medium (90% glycerol/0.2% n-propyl gallate/20 mM Tris, pH 8.0). The fluorescence images were obtained by spinning-disk confocal on an inverted fluorescence microscope.

Cytotoxicity assay

The cytotoxicity of simvastatin-Cy5.5, pravastatin-Cy5.5, and Cy5.5 was determined by 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay which is a colorimetric method to measure cellular proliferation (CellTiter 96® AQueous One Solution, Promega, WI, USA). Briefly, cells were pre-cultured in a 96-well plate for 48 h to achieve ~60% confluency. The cells were then treated with varying concentrations (0, 0.05, 0.1, 1, 5, 10 μM) of simvastatin-Cy5.5, pravastatin-Cy5.5, or Cy5.5 for 24 h. For the drug solutions, the induction medium containing 25 ng/ml doxycycline was used for the HPDE KRAS^MUT cells, while a base medium consisting of 1% DMSO was used for the HPDE KRAS^WT cells. Following this treatment, cell proliferation was assessed using the MTS solution according to the vendor’s instructions. Viability was quantitatively measured by comparing the relative absorbance values to those of the respective control groups (0 μM) for each cell type.

Cellular uptake assay using T-MOC model

The uptake of the statin-dye conjugates was assessed in PCC-CAF co-cultured T-MOC model to investigate differential response in drug accumulation between PCC and CAF cells. The co-cultured T-MOC model was a microfluidic platform to demonstrate dynamic transport as described in our prior studies [20–23]. Briefly, the T-MOC model is composed of capillary, interstitial, and lymphatic channels. The pravastatin-Cy5.5 is accumulated in the cell body through cellular uptake while the compound transports from capillary to interstitial, then lymphatic channels. The drug transport is governed by the perfusion flow condition of the T-MOC which is controlled by differences in hydrostatic pressure of the capillary, interstitial, and lymphatic channels as described in our prior publications [21,22]. In the present study, a hydrostatic pressure difference of 20 mm H₂O was used to mimic the average interstitial flow rates typically observed in TME [24]. We measured the accumulation by using a live-cell imaging technique with time-lapse microscopy for fluorescent statin-dye conjugates. An inverted microscope (Olympus IX71, Japan) was equipped with a stagetop incubator as described in [25], which allowed maintaining the microfluidic platform at 37°C with 5% CO₂ environment during imaging. After the 2 μM of each statin-dye conjugate (simvastatin-Cy5.5 and pravastatin-Cy5.5) and free Cy5.5 was introduced into the capillary channel, Cy5.5 fluorescence in the T-MOC platform was captured every 2 h for 24 h-duration. Temporal drug accumulation in the cells was measured by fluorescence intensity at each cell type where the intensity was calibrated with fluorescence of 2 μM of the corresponding compound. The accumulation was measured for each cell type separately. Since the CAF19 cell line is transfected with GFP, CAF19 cell area was defined with the FITC-fluorescence. On the other hand, Panc1 cell boundary was defined by using the bright-field images. The contrast differences between cells and background were processed to convert the images to monochrome by using ImageJ. The control experiment with doxorubicin also followed the above procedure to compare the differential drug accumulation. All experiments were repeated at least 3 times for each treatment group. The data was reported in the form of mean ± standard deviation.

To quantitatively address the cell-type specific accumulation of the statin-dye conjugates, we modeled the drug intracellular transport model based on mass conservation (Fig 5d) [23].

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Fig 1. Synthesis and characterization of the statin-dye conjugates.

(a) Schematic diagram illustrating the synthesis of the simvastatin-Cy5.5 and pravastatin-Cy5.5 conjugates. EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, DMAP: 4-dimethylaminopyridine, DCM: dichloromethane, NHS: N-hydroxysuccinimide, DMF: dimethylformamide. (b) High-performance liquid chromatography (HPLC) chromatograms and (c) mass spectrometry analysis of simvastatin-Cy5.5 and pravastatin-Cy5.5.

https://doi.org/10.1371/journal.pone.0340189.g001

where C_cell is an intracellular drug concentration and V_cell is an estimated volume of each cellular compartment. Transient drug accumulation is governed by a balance between drug influx (J_in) and efflux (J_out) at the cell surface. The cell’s drug uptake affinity (k_on) at the cell surface and drug concentration at the extracellular region near the cells (C_ex) determine the drug influx, while the drug efflux is dependent on the drug dissociation affinity (k_off) and intracellular drug concentration (C_cell). Assuming that k_on and k_off are constant parameters, we quantitatively estimated the cellular capability of drug uptake and efflux.

Cytotoxicity assay using T-MOC model

The efficacy of statin-dye conjugates was assessed in the T-MOC to investigate the differential cell response between PCC and CAF cells. Cells cultured in the T-MOC were pre-cultured for 2 days after loading. Then, simvastatin-Cy5.5 of 0 (control), 0.2, 2, and 5 μM and pravastatin-Cy5.5 of 0, 2, 5 μM was perfused through for 24 h. After the drug treatment, cells were washed with the normal culture medium and cultured in the normal condition for an additional 2 days to have sufficient time to capture the latent effects of drug action. At the final stage of the experiment, cells’ nucleic acid was stained with Hoechst 33342. Although nucleic acid staining marks both live and dead cells, viable cells were assessed in the T-MOC platform on the observation that nuclei of dead cells diminish after 48 hours of post-culture, as verified using Propidium Iodide, dead cell staining in our precious work (Moon, Ozcelikkale et al., 2020).Cell survival was defined by normalizing nuclear area of the treatment group to control group (0 μM) as follow:

The cell survival metric indicated drug response to the drug by comparing viable cell growth of the treatment group with respect to the growth of control. In the co-culture model, PCCs (Panc1) and CAFs (CAF19) were distinguished by using green-fluorescent CAF19. Specifically, the stained nuclear area included Panc1 and CAF19, while CAF19 nuclear area was identified based on overlap with the green fluorescent signal. While the stained nuclear area includes both Panc1 and CAF19, the CAF19 nuclear area overlaps with the green fluorescent signal. By selectively measuring the nuclear area that overlaps with green fluorescence, we quantified CAF19 nuclei, while the remaining nuclear area was attributed to Panc1 cells (Fig 6a). All experiments were repeated at least 3 times for each treatment group. The data was reported in the form of mean ± standard estimated error (S.E.). Data points in the drug cytotoxicity assay with T-MOC were statistically analyzed by using student t-test. The comparison was done with cell survivals between PCC and CAFs. The differences were recognized as statistically significant when p-value < 0.05.

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Fig 2. Cellular uptake of statin-dye conjugates by KRAS^MUT and KRAS^WT cells.

Simvastatin-Cy5.5, pravastatin-Cy5.5, and free Cy5.5 uptake was measured by fluorescence (red) in (a) Panc1 (KRAS^MUT) and BxPC3 (KRAS^WT) and (b) HPDE KRAS^MUT (HPDE iKRAS with doxycycline) and HPDE KRAS^WT (HPDE iKRAS without doxycycline). The scale bars indicate 10 μm. The cell nuclei were stained with DAPI (blue). Quantitative measurements of the cellular uptake of the statin-dye conjugates are presented relative fluorescence intensity per cell count. Bars indicate Mean ± S.E. (n ≥ 3). Statistically significant differences are represented as * for p < 0.05, *** for p < 0.001, and **** for p < 0.0001.

https://doi.org/10.1371/journal.pone.0340189.g002

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Fig 3. Regulation of cellular uptake of statin-Cy5.5 conjugates by inhibitors and pathways associated with macropinocytosis.

(a) Treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) blocks the selective uptake of statin-dye conjugates in KRAS^MUT cells, specifically Panc1 and HPDE KRAS^MUT. Ctrl (Control) indicates cells with no EIPA treatment. (b) MCF10A cells with PTEN^KO show enhanced uptake of statin-dye conjugates (red) compared to PTEN^WT MCF10A control cells. The cell nuclei were stained with DAPI (blue). The scale bars indicate 10 μm. Bars represent Mean ± S.E. (n ≥ 3). Statistically significant differences are represented as * for p < 0.05 and *** for p < 0.001.

https://doi.org/10.1371/journal.pone.0340189.g003

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Fig 4. The cytotoxic effects of statin-dye conjugates.

Cell viability (%) was measured after treating the 2D cell monolayers with simvastatin-Cy5.5, pravastatin-Cy5.5, and free Cy5.5 in (a) HPDE KRAS^MUT and KRAS^WT cells and (b) MCF10A with PTEN^KO and MCF10A with PTEN^WT. Bars represent Mean ± S.E. (n ≥ 3). Statistically significant differences with p < 0.01 are indicated by **.

https://doi.org/10.1371/journal.pone.0340189.g004

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Fig 5. Selective uptake of statin-Cy5.5 conjugates into KRAS^MUT Panc1 cells over KRAS^WT cancer-associated fibroblasts (CAFs) in an in vitro cancer-stroma tumor environment-on-a-chip model (T-MOC).

(a) Schematic configuration of the cancer-stroma T-MOC model operation, illustrating the setup and flow dynamics within the model. (b) Micrograph showing drug accumulation of simvastatin-Cy5.5, pravastatin-Cy5.5, and doxorubicin (used as a control). In the image, red indicates accumulated drugs, and green indicates transfected CAFs. The scale bars represent 100 μm. (c) Quantified drug accumulation measured in cell type-specific areas for simvastatin-Cy5.5, pravastatin-Cy5.5, and doxorubicin. Bars indicate Mean ± S.E. (n ≥ 3). (d) Drug accumulation model based on mass conservation principles, illustrating the quantified rate constants of drug influx and efflux within the T-MOC system.

https://doi.org/10.1371/journal.pone.0340189.g005

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Fig 6. The tumor-selective cytotoxicity of statin-dye conjugates in in vitro T-MOC platforms.

(a) Fluorescence micrograph showing the co-cultured Panc1 (KRAS^MUT) and green fluorescent-labeled CAF19 (KRAS^WT) cell nuclei in the T-MOC device, both with and without treatment of simvastatin-Cy5.5 (0.2 µM) and pravastatin-Cy5.5 (2 µM). CAF19 cells are represented in green, while the nuclear areas of both Panc1 and CAF19 cells appear in blue. The overlapping regions of the nuclear area with the green fluorescent signal are attributed to CAF19 nuclei (cyan), while the remaining nuclear areas correspond to Panc1 nuclei (red). The scale bars represent 100 μm. (b) Cell survival (%) measured after treating the co-cultured Panc1 and CAF19 cells in the T-MOC with simvastatin-Cy5.5 and pravastatin-Cy5.5. Bars represent mean ± S.E. (n ≥ 3). Statistically significant differences are indicated by * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 (student t-test).

https://doi.org/10.1371/journal.pone.0340189.g006

Statistical analysis

To compare results, groups were evaluated using the Student’s t-test, with each group containing at least three biological replicates (n ≥ 3). Statistical significance was indicated by p-values, with a threshold of p < 0.05 for significance. We presented the statistical significance of p < 0.05, 0.01, 0.001 and 0.0001 as *, **, *** and **** respectively.

Results

Synthesis of statin-dye conjugates

We synthesized statin-dye conjugates by attaching the fluorescent dye Cy5.5 to simvastatin and pravastatin. Simvastatin was conjugated to Cy5.5 using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP), resulting in the formation of simvastatin-Cy5.5. Pravastatin was similarly conjugated to Cy5.5 using EDC and N-hydroxysuccinimide (NHS), producing pravastatin-Cy5.5 (Fig 1a). The synthesized conjugates, simvastatin-Cy5.5 and pravastatin-Cy5.5, were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry (Fig 1b,c). Mass spectrometry confirmed the expected molecular weights of the conjugates, validating successful synthesis. HPLC analysis further confirmed the purity of both conjugates, achieving over 95% purity. NMR analysis was also performed to support the structural characterization of the conjugates (S1,S2 Fig), and the NMR spectrum of the raw material used for conjugation is additionally provided (S3 Fig).

Selective uptake of statin-dye conjugates in KRAS^MUT cancer cells

To investigate the preferential uptake of statin-dye conjugates in KRAS^MUT cancer cells, we evaluated the cellular uptake of simvastatin-Cy5.5, pravastatin-Cy5.5, and Cy5.5 across several cell lines. We first compared the uptake of these statin-dye conjugates in two distinct pancreatic cancer cell types: Panc1, featuring a KRAS G12D mutation (KRAS^G12D), and BxPC3 with KRAS^WT using confocal microscopy. Notably, the uptake of simvastatin-Cy5.5 was significantly higher in Panc1 cells than in BxPC3 cells (Fig 2a). Similarly, the uptake of pravastatin-Cy5.5 was also significantly higher in Panc1 cells compared to in BxPC3 cells, although the level of uptake was lower than that of simvastatin-Cy5.5 in Panc1 cells. In this study, all instances of Cy5.5 refer specifically to Cy5.5 acid, as shown in Fig 1a. Free Cy5.5 also showed selective uptake by KRAS^MUT cancer cells in vitro. To further assess the concentration dependency of statin-dye conjugate uptake via macropinocytosis, we examined the uptake of simvastatin-Cy5.5 and Cy5.5 alone in KRAS^MUT Panc1 cells at two different concentrations: 50 nM and a lower concentration of 17 nM (1/3 of 50 nM). Our results demonstrate that simvastatin-Cy5.5 exhibits concentration-dependent cellular uptake in Panc1 cells, with a higher intracellular accumulation at 50 nM compared to 17 nM. In contrast, Cy5.5 alone showed minimal uptake at both concentrations, suggesting that the statin moiety plays a crucial role in facilitating uptake in KRAS^MUT cells (S4 Fig). Flow cytometric data quantitatively assessing the uptake of the statin-dye conjugates in KRAS^MUT Panc1 cells indicated significantly enhanced uptake of both simvastatin-Cy5.5 and pravastatin-Cy5.5 compared to Cy5.5 alone (S5a Fig).

To clarify the contribution of the Cy5.5 moiety, additional control experiments were performed. We synthesized PEG-Cy5.5 (structurally unrelated to statins) and included Cy5.5 acid (neutral) and Cy5.5 amine (+2) as controls (S6 Fig). Cellular uptake analysis showed that simvastatin-Cy5.5 exhibited the highest uptake, PEG-Cy5.5 showed moderate uptake, and Cy5.5 acid and Cy5.5 amine had the lowest and comparable uptake (S7 Fig). These results indicate that net charge alone does not explain internalization, and that the statin contributes to the enhanced cellular uptake.

To further evaluate the potential preferential uptake of statin-dye conjugates in KRAS^MUT cells, we assessed their uptake in genetically modified human pancreatic ductal epithelial (HPDE) cells engineered to inducibly express activated KRAS^G12D following doxycycline treatment (HPDE iKRAS) [26]. Notably, we observed significantly higher fluorescence intensity from the statin-dye conjugates in HPDE iKRAS cells treated with doxycycline induction (HPDE KRAS^MUT) than in control cells without doxycycline induction (HPDE KRAS^WT) (Fig 2b). For each experimental group, multiple representative fields were acquired by confocal microscopy, and fluorescence intensity was quantified. The mean fluorescence per cell was statistically analyzed and presented as bar graphs to ensure quantitative interpretation of uptake differences.

Quantitative assessment of the relative Cy5.5 intensity per cell revealed a significantly augmented accumulation of all compounds, simvastatin-Cy5.5, pravastatin-Cy5.5 and Cy5.5 in KRAS^MUT cells as opposed to KRAS^WT cells. Additionally, to determine whether the selective uptake of statin-Cy5.5 conjugates observed in KRAS^G12D cells extends to other KRAS mutation subtypes, we tested two isogenic colorectal cell line pairs, HCT116 and DLD1, which carry the KRAS G13D mutation alongside their KRAS^WT counterparts (S1 Table). Consistent with the results from KRAS^G12D cells, we observed significantly higher uptake of the statin-Cy5.5 conjugates in KRAS^WT/G13D cells compared to their KRAS^WT controls (S5b, c Fig). These findings suggest that statin-Cy5.5 conjugates undergo specific uptake in KRAS^MUT cells, indicating a potentially distinct transport mechanism for these statin conjugates in KRAS^MUT cells.

Macropinocytosis as a key mechanism in statin-dye conjugate uptake

Given that macropinocytosis is known to be activated in KRAS^MUT cells to facilitate nutrient uptake [27], we next tested whether this process might contribute to the enhanced uptake of statin-Cy5.5 conjugates. Macropinocytosis is a form of endocytosis that allows cells to engulf extracellular fluid and molecules, which is often upregulated in cancer cells with certain mutations, including KRAS mutations. To test this hypothesis, we employed the pharmacological inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA), which specifically targets macropinocytosis. Before assessing the effects of EIPA on statin-dye conjugate uptake, we first validated its efficacy in inhibiting macropinocytosis using fluorescein isothiocyanate-labeled BSA (FITC-BSA), a widely recognized marker for macropinocytosis (S8 Fig). KRAS^MUT Panc1 cells were pre-treated with 50 μM EIPA for 1.5 hours, followed by a 1-hour incubation with FITC-BSA (2 mg/mL). As expected, EIPA treatment significantly reduced FITC-BSA uptake, confirming its effectiveness as a macropinocytosis inhibitor. Additionally, we also confirmed that KRAS^MUT Panc1 cells exhibited higher macropinocytotic activity compared to KRAS^WT BxPC3 cells, further supporting the selective engagement of this pathway in KRAS^MUT cells as assessed by FITC-BSA uptake.

Following this validation, we conducted EIPA experiments on statin-dye conjugates to determine whether their uptake was mediated by macropinocytosis. As shown in Fig 3a, the intracellular fluorescence from both simvastatin-Cy5.5 and pravastatin-Cy5.5 was significantly decreased when EIPA was added to the KRAS^MUT cells in both Panc1 and HPDE KRAS^MUT-induced cells. The selective uptake of statin-dye conjugates in KRAS^MUT cells, along with the significant reduction in intracellular fluorescence following EIPA treatment, indicates that macropinocytosis plays a significant role in their uptake in KRAS^MUT cells.

We also measured the uptake of statin-dye conjugates in cells with PTEN deficiency, a condition known to elevate macropinocytosis due to the activation of phosphoinositide 3-kinase (PI3K) signaling. PTEN is a tumor suppressor gene that negatively regulates the PI3K/AKT signaling pathway. Loss of PTEN function leads to increased activation of the PI3K pathway, which can promote macropinocytosis, a process often upregulated in cancer cells to meet their increased nutrient demands. By using CRISPR-Cas9 technology, we created isogenic cell lines with either sgRNA targeting PTEN (PTEN^KO) or sgRNA targeting a non-coding sequence (PTEN^WT) in MCF10A cells. As can be seen in Fig 3b, MCF10A cells with PTEN^KO exhibited stronger fluorescence compared to the PTEN^WT isogenic control, indicating enhanced uptake of simvastatin-Cy5.5 and pravastatin-Cy5.5. Upon treatment with either EIPA or a pan-PI3K inhibitor (BKM120), the fluorescence signal of simvastatin-Cy5.5 in MCF10A PTEN^KO cells decreased significantly (S9 Fig). These results suggest that PTEN loss can increase the cellular uptake of statin-Cy5.5 conjugates in a PI3K pathway-dependent manner, further highlighting the role of macropinocytosis in this process.

Selective cytotoxicity of statin-dye conjugates in KRAS^MUT cells in vitro

To investigate the selective cytotoxic effects of statin-Cy5.5 conjugates on KRAS^MUT cancer cells, we performed cytotoxicity assays using HPDE iKRAS cell monolayers. Among the conjugates tested, pravastatin-Cy5.5 displayed some selective effectiveness against HPDE KRAS^MUT cells compared to HPDE KRAS^WT cells at concentrations of 0.05 µM and 0.1 µM (Fig 4a). However, neither simvastatin-Cy5.5 nor Cy5.5 alone demonstrated selective killing effects on HPDE KRAS^MUT cells compared to HPDE KRAS^WT cells. Additionally, in MCF10A PTEN^KO cells that exhibited selective uptake of statin-Cy5.5 conjugates, no statistically significant differences in cytotoxic effects were observed compared to their isogenic PTEN^WT counterparts (Fig 4b).

Previous studies have reported that statins can kill KRAS^MUT cancer cells more effectively than KRAS^WT cells. [8,9] To further clarify the contribution of the statin scaffold itself, we compared the cytotoxicity of unconjugated statins and their Cy5.5 conjugates in KRAS^MUT and KRAS^WT cells. As shown in S10 and S11 Fig, both simvastatin and pravastatin alone showed slight cytotoxicity across a range of concentrations in Panc1, CT26, and CAF19 cells. In contrast, simvastatin-Cy5.5 and pravastatin-Cy5.5 exhibited dose-dependent cytotoxicity in KRAS^MUT (Panc1 and CT26), while showing little to no effect in CAF19 cells.

This selective cytotoxicity, confined to specific KRAS^MUT cell lines, suggests that the statin-Cy5.5 conjugates possess unique properties distinct from either statins or Cy5.5 alone, thereby warranting further investigation into the underlying mechanisms.

KRAS^MUT cancer cells selectively uptake statin-Cy5.5 conjugates in the engineered TME

To further evaluate the transport and accumulation of statin-Cy5.5 conjugates in targeting KRAS^MUT cancer cells, we expanded our assessment using a microfluidic TME-on-a-chip model (T-MOC). This biomimetic tumor model recapitulates the stroma tissue in PDAC, where PCCs and CAFs are embedded in a 3D matrix. The fluid flow passes through an endothelium-mimicking membrane interfaced with a capillary channel (Fig 5a), simulating the hydrodynamic conditions of drug transport. The T-MOC model is designed to regulate the transport of fluid, nutrients, and drugs by adjusting hydrostatic pressure variations across channels, mimicking the dynamic transport conditions within the TME [20–23]. Additionally, this system reconstitutes pharmacokinetic processes including extravasation from a capillary vessel, interstitial diffusion and convection, cellular uptake, and lymphatic drainage.

Using the T-MOC model, we perfused either simvastatin-Cy5.5 or pravastatin-Cy5.5 along the capillary channel and monitored transient drug accumulation with time-lapse microscopy. All T-MOC experiments were conducted using living cells without fixation, allowing us to assess cellular uptake under physiologically relevant conditions. The PDAC tumor model with T-MOC included non-transfected cancer cells (Panc1) and green fluorescent protein (GFP)-transfected CAFs (CAF19), allowing for clear differentiation of drug accumulation in each cell type. Results showed that the fluorescence intensity of simvastatin-Cy5.5 and pravastatin-Cy5.5 was significantly higher in the Panc1 cell area compared to the CAF19 area. In contrast, doxorubicin, used as a control drug, showed no significant difference in fluorescence intensity between Panc1 and CAF19 cells (Fig 5b). This comparison highlights the preferential uptake of statin-Cy5.5 conjugates in KRAS^MUT cancer cells over CAFs, unlike doxorubicin.

Drug accumulation was measured by calibrating fluorescence intensity to 2 μM concentrations of simvastatin-Cy5.5, pravastatin-Cy5.5, and doxorubicin within the capillary channel. Quantitative results showed that KRAS^MUT Panc1 cells exhibited significantly higher uptake of both simvastatin-Cy5.5 and pravastatin-Cy5.5 compared to CAF19 cells (Fig 5c). Specifically, simvastatin-Cy5.5 accumulated approximately 2.5 times more, and pravastatin-Cy5.5 around 5 times more, in Panc1 cells compared to CAF19 cells during a 24 h perfusion. In contrast, doxorubicin showed no significant difference in accumulation between the two cell types (dash lines in Fig 5c), suggesting that statin-Cy5.5 conjugates selectively target cancer cells with minimal impact on surrounding stromal cells in PDAC tissue.

To further investigate drug transport mechanisms, we applied a simple drug accumulation model based on mass conservation (Fig 5d) [23]. In this model, the parameter k_on represented the cellular capability for drug uptake affinity at the cell surface, while k_off represented drug efflux as the drug dissociation affinity. Assuming that k_on and k_off are constant, quantitative analysis showed that k_on values for Panc1 cells were significantly higher than those for CAF19 cells for both statin-Cy5.5 conjugates. Although Panc1 cells also exhibited a higher k_on for doxorubicin than CAF19, this difference was less pronounced compared to statin-Cy5.5 conjugates. The high k_on to k_off ratio for statin-dye conjugates in KRAS^MUT Panc1 cells suggests that selective uptake may be linked to cancer cell-specific endocytic activity, particularly macropinocytosis, which is known for facilitating large-scale uptake.

Tumor-selective cytotoxicity of statin-dye conjugates using the engineered TME

Building on previous findings that demonstrated the KRAS^MUT targeting potential of statin-dye conjugates, we hypothesized that these conjugates could selectively kill KRAS^MUT tumor cells over KRAS^WT stroma cells in tumor tissue. To maximize screening effectiveness, we applied the drug solution in a stationary manner, eliminating flow dynamics. Devices were pre-cultured for 48 h, followed by a 24-h drug treatment. After treatment, the model was perfused with drug-free media and cultured for an additional 48 h to capture any latent drug effects. Cell viability was assessed by measuring the stained nuclear area of the non-transfected PCCs (Panc1) and GFP-transfected CAFs (CAF19) separately (Fig 6). Notably, GFP-labeled CAFs retained viability at up to 0.2 µM for simvastatin-Cy5.5 conjugates and 2 µM for pravastatin-Cy5.5 conjugates (Fig 6a), while these concentrations inhibited the growth of Panc1 cells. The calculated IC₅₀ values further support the observed selective cytotoxicity. For the simvastatin-Cy5.5 conjugate, the IC₅₀ was 1.13 µM for Panc1 cells and 1.51 µM for CAF19 cells. Remarkably, the pravastatin-Cy5.5 conjugate showed an IC₅₀ of 6.52 µM for Panc1, while CAF19 cells exhibited no significant viability reduction up to 5 µM. Post-treatment viability results confirmed that statin-dye conjugates selectively killed Panc1 cancer cells at 0.2 μM for simvastatin-Cy5.5 and 2 and 5 μM for pravastatin-Cy5.5 (Fig 6b), demonstrating cancer cell-targeted cytotoxicity with minimal impact on stromal cells. This suggests a promising and novel approach for treating PDAC tumors, which frequently harbor KRAS mutations.

Discussion

In this study, we synthesized statin-dye conjugates by attaching the fluorescent dye Cy5.5 to two commercially available statins, simvastatin and pravastatin, to evaluate their potential for selectively targeting KRAS^MUT cancer cells. Statins, commonly prescribed for cholesterol reduction, have also shown selective cytotoxicity against KRAS^MUT cells, leading us to explore whether statin-based conjugates might similarly exhibit selective uptake in these cells. With Cy5.5 labeling, we aimed to track the internalization of statin-dye conjugates specifically in KRAS^MUT cells and examine their uptake mechanism. To our knowledge, this study represents the first to directly examine the uptake of statin-dye conjugates in KRAS^MUT cells using fluorescent labeling, offering new insights on statin-based drug delivery and selective targeting potential.

The conjugates demonstrated preferential uptake by KRAS^MUT cells over KRAS^WT cells, including various isogenic cell pairs with KRAS G12D and G13D mutations. Our findings indicate that macropinocytosis significantly contributes to the uptake of statin-dye conjugates in KRAS^MUT cells. Although other mechanisms may be involved, our findings confirm that macropinocytosis plays a major role in this selective uptake of statin-dye conjugates in KRAS^MUT cells. The use of EIPA, a macropinocytosis inhibitor, substantially reduced the internalization of both statin-Cy5.5 conjugates in KRAS^MUT cells, strongly suggesting that macropinocytosis is a key mechanism facilitating the enhanced uptake of these conjugates in KRAS^MUT cells [28].

To determine whether the enhanced uptake arises from specific structural features of the statin-Cy5.5 conjugate, we synthesized simvastatin-Rhodamine B, a conjugate incorporating a fluorophore that is spectrally and structurally distinct from Cy5.5. Unlike statin-Cy5.5, the Rhodamine B conjugate showed negligible uptake and no KRAS- or PTEN-selective accumulation (S12 Fig). These findings support the notion that the observed behavior is not due to the statin alone, but rather a synergistic interaction between the statin and Cy5.5 that facilitates uptake. This conclusion is further supported by additional control experiments using PEG-Cy5.5 (structurally unrelated to statins) and charged/neutral Cy5.5 variants (S7 Fig). These results indicate that neither conjugation to a neutral polymer, net charge, nor Cy5.5 alone accounts for the enhanced internalization, highlighting the specific contribution of the statin moiety to the enhanced cellular uptake.

This study highlights a unique property of the statin-Cy5.5 conjugates, suggesting that Cy5.5 may serve as a functional delivery motif that enhances cellular uptake and therapeutic effects of conjugated small molecules like statins. The lack of significant cytotoxicity with Cy5.5 or statin alone further underscores the significance of their combination (Fig 4, S10, S11 Fig). This strategy may be extended to other therapeutic agents, where conjugation to Cy5.5-like scaffolds enhances macropinocytic entry into genetically defined cancer cells such as those with KRAS mutations.

Additionally, we demonstrated that PTEN deficiency, known to elevate macropinocytosis through activation of the PI3K/RAC pathway, further increased the uptake of statin-Cy5.5 conjugates. In MCF10A cells with PTEN^KO, the enhanced uptake of these conjugates was significantly reduced upon treatment with either EIPA or a pan-PI3K inhibitor (S9 Fig). Taken together, our data support the potential of exploiting the macropinocytosis pathway in targeting tumors with certain specific genetic lesions.

In addition to the macropinocytosis pathway, variations in membrane lipid composition—potentially altered by KRAS or PTEN mutations—may also influence endocytic pathways [29,30]. Lipid modeling can modulate membrane curvature, fluidity, or protein recruitment, all of which are critical for macropinocytic vesicle formation [31]. This concept is supported by previous reports showing that oncogenic KRAS signaling alters membrane lipid dynamics and promotes macropinocytosis and nutrient scavenging [32]. Although we did not directly measure lipid composition in this study, we regard such analysis as indispensable for a complete understanding of the uptake mechanism of our statin-dye conjugates, and we therefore plan to address this question in future work.

While our data clearly demonstrate enhanced uptake of statin-dye conjugates in KRAS^MUT cells (Fig 2), the observed cytotoxicity differences between KRAS^MUT and KRAS^WT cells were relatively modest. This discrepancy can be attributed to several factors. Cellular uptake does not always directly translate to cytotoxicity, as the intracellular fate of the internalized compounds such as metabolism, degradation, and efflux can significantly impact their biological activity and overall effect on cell viability. Although KRAS^MUT cells exhibit increased macropinocytotic uptake, their dependency on the mevalonate pathway may differ from that of KRAS^WT cells. As a result, even with differential uptake, the downstream cytotoxic effects may be relatively modest between the two cell types.

We initially hypothesized that our statin-dye conjugates might be internalized via albumin binding, as statins are known to bind albumin strongly, and albumin is often taken up through macropinocytosis. However, our findings indicated that the uptake of simvastatin-Cy5.5 was albumin-independent, occurring even in the absence of albumin (S13 Fig). Furthermore, the presence of physiological levels of bovine serum albumin did not significantly increase the uptake. These observations suggest an alternative mechanism, potentially involving nanoparticle formation. Given the physicochemical properties of statin-dye conjugates, they may self-assemble into nanoparticles, facilitating their macropinocytotic uptake into KRAS^MUT cells. Further studies are warranted to characterize the nanoparticle formation of these conjugates under physiological conditions and its impact on their selective accumulation in KRAS^MUT cells.

The T-MOC model, which closely mimics the complex TME of PDAC, provided a realistic platform to evaluate the efficacy and selectivity of these conjugates. Within this model, KRAS^MUT PCCs co-cultured with CAFs exhibited selective uptake of statin-Cy5.5 conjugates, with simvastatin-Cy5.5 and pravastatin-Cy5.5 accumulating 2.5 and 5 times more, respectively, in KRAS^MUT Panc1 cells than in CAFs. This differential accumulation underscores their potential for precise targeting within the complex environment of PDAC. Notably, in the same T-MOC model, doxorubicin did not exhibit selective uptake in KRAS^MUT Panc1 cells compared to CAF19 stromal cells, whereas statin-dye conjugates preferentially accumulated in KRAS^MUT cells. This contrast suggests that the physicochemical properties of statin-dye conjugates, potentially including nanoparticle formation and macropinocytosis-driven uptake, differentiate them from conventional small-molecule chemotherapeutics. While doxorubicin relies on passive diffusion and transporter-mediated uptake, statin-dye conjugates may be actively internalized through macropinocytosis, enabling selective tumor targeting. Furthermore, the higher drug uptake affinity (k_on) observed in Panc1 cells further supports the idea that KRAS^MUT cells may have enhanced endocytic activity, particularly macropinocytosis. These findings highlight the potential advantages of leveraging macropinocytosis in designing targeted drug delivery strategies for KRAS^MUT cancers.

Importantly, our results highlight that neither Cy5.5 nor statins alone induces selective cytotoxicity or uptake, whereas the statin-Cy5.5 conjugate shows a unique synergy, suggesting that their combination imparts new functional properties not observed with either component alone.

Conclusions

Our study shows that synthesizing statin-Cy5.5 conjugates, which are selectively internalized into KRAS-transformed tumor cells via macropinocytosis, can significantly enhance the selectivity of drug delivery. This finding demonstrates strong potential for targeting KRAS^MUT and PTEN-deficient tumors, where macropinocytosis is upregulated. By leveraging this pathway, statin-based drug conjugates could broaden the application range of existing drugs, creating a novel class of potential treatments for KRAS-driven tumors. Future research should focus on optimizing these statin-drug conjugates to achieve selective and effective treatments, which will be crucial for maximizing their potential and possibly extending their use to other cancers with similar characteristics.

Supporting information

S1 Fig. Chemical structure of simvastatin-Cy5.5 was confirmed via (a) ¹H-NMR and (b) ¹³C-NMR, respectively.

Conjugation was verified by the appearance of ester-specific methylene (¹H: δ 5.20-5.06 ppm) and carbonyl (¹³C: δ 176.4-168.6 ppm) signals.

https://doi.org/10.1371/journal.pone.0340189.s001

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S2 Fig. Chemical structure of pravastatin-Cy5.5 was confirmed via (a) ¹H-NMR and (b) ¹³C-NMR, respectively.

Conjugation was verified by the appearance of amide-specific methylene (¹H: δ 4.79, 4.45 ppm) and carbonyl (¹³C: δ 175.3-170.6 ppm) signals.

https://doi.org/10.1371/journal.pone.0340189.s002

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S3 Fig. Chemical structures of Simvastatin, Pravastatin and Cy 5.5 acid were confirmed by (a, c, e) ¹H-NMR and (b, d, f) ¹³C-NMR, respectively.

https://doi.org/10.1371/journal.pone.0340189.s003

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S4 Fig. Concentration-dependent uptake of simavastatin-Cy5.5 in KRAS-mutant (KRAS^MUT) Panc1 cells.

Confocal microscopy images show the cellular uptake of simvastatin-Cy5.5 (a) and Cy5.5 alone (b) in KRAS^MUT Panc1 cells at two different concentrations: 50 nM and 17 nM. Nuclei were stained with DAPI (blue), and Cy5.5 fluorescence is shown in red. Simvastatin-Cy5.5 exhibited concentration-dependent uptake, with higher fluorescence intensity observed at 50 nM compared to 17 nM. In contrast, Cy5.5 alone showed minimal uptake at both concentrations, suggesting that the statin moiety facilitates selective internalization in KRAS^MUT cells. The scale bars indicate 500 μm.

https://doi.org/10.1371/journal.pone.0340189.s004

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S5 Fig. Enhanced cellular uptake of simvastatin-Cy5.5 in KRAS^MUT cancer cells.

(a) Cellular uptake of simvastatin-Cy5.5, pravastatin-Cy5.5, and Cy5.5 in Panc1 cells measured by flow cytometry. Bars indicate Mean ± S.E. (n ≥ 3). Statistically significant differences are represented as **** for p < 0.0001. Cellular uptake of simvastatin-Cy5.5 (red) in isogenic (b) HCT116 and (c) DLD1 cells. The scale bars indicate 100 μm. The cell nuclei were stained with DAPI (blue).

https://doi.org/10.1371/journal.pone.0340189.s005

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S6 Fig. Synthesis and characterization of PEG-Cy5.5.

(a) Schematic diagram illustrating the synthesis of the PEG-Cy5.5 conjugate. SPA: Succinimidyl propionate; DMSO: Dimethylsulfoxyde. (b) High-performance liquid chromatography (HPLC) chromatograms of Cy5.5 amine and PEG-Cy5.5.

https://doi.org/10.1371/journal.pone.0340189.s006

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S7 Fig. Cellular uptake of Cy5.5 conjugates in KRAS^MUT cancer cells.

(a) Confocal images showing the cellular uptake of Cy5.5 acid, Cy5.5 amine, PEG-Cy5.5 and simvastatin-Cy5.5 in Panc1 cells. Cells were incubated with 50 nM of each molecule for 1 hour. Red fluorescence represents internalized Cy5.5 conjugates, and blue fluorescence (DAPI) indicates cell nuclei. The scale bars indicate 20 μm. (b) Corresponding quantification of mean fluorescence intensity (MFI) per cell. Data are presented as mean ± S.E. (n = 5 randomly selected images per group). Statistical significance was analyzed by one-way ANOVA; *p < 0.05, ****p < 0.0001.

https://doi.org/10.1371/journal.pone.0340189.s007

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S8 Fig. Validation of macropinocytosis inhibition by (5-(N-ethyl-N-isopropyl)amiloridein (EIPA; macropinocytosis inhibitor) in KRAS-mutant (KRAS^MUT) Panc1 cells.

EIPA (50 μM) was pre-treated into cells for 1.5 h, followed by a 1 h incubation with fluorescein isothiocyanate-labeled BSA (FITC-BSA; 2 mg/mL), a widely used marker for macropinocytosis. Representative fluorescence images show FITC-BSA uptake (green) and nuclear staining with DAPI (blue). EIPA treatment significantly reduced FITC-BSA uptake in KRAS^MUT Panc1 cells, confirming its efficacy in inhibiting macropinocytosis. Additionally, Panc1 cells exhibited higher macropinocytosis activity compared to KRAS wild-type (KRAS^WT) BxPC3 cells. The scale bar indicates 100 μm.

https://doi.org/10.1371/journal.pone.0340189.s008

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S9 Fig. Cellular uptake of statin-Cy5.5 conjugates (red) by MCF10A cells with PTEN knockout (PTEN^KO) and wild-type (PTEN^WT).

BKM120 (pan-PI3K inhibitor; 10 μM) was pre-treated into cells for 1 h and EIPA (macropinocytosis inhibitor; 50 μM) was pre-treated into cells for 1.5 h. The cell nuclei were stained with DAPI (blue).

https://doi.org/10.1371/journal.pone.0340189.s009

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S10 Fig. Selective cytotoxicity of simvastatin-Cy5.5 in KRAS^MUT cell lines compared to simvastatin.

Cell viability of CT26 and Panc1 cells after 24 h treatment with simvastatin-Cy5.5 (left, red) or unconjugated simvastatin (right, green) at concentrations of 0.1, 1, and 10 µM. While simvastatin-Cy5.5 induced dose-dependent cytotoxicity in KRAS^MUT cells, simvastatin alone showed minimal toxicity across the same dose range. Bars indicate mean ± S.E. (n ≥ 3). Statistical comparisons were made against the control group using Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

https://doi.org/10.1371/journal.pone.0340189.s010

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S11 Fig. Selective cytotoxicity of pravastatin-Cy5.5 in KRAS^MUT cell line compared to pravastatin.

Cell viability of Panc1 and CAF19 cells after 24 h treatment with pravastatin-Cy5.5 (left, red) and unconjugated pravastatin (right, gray) at concentrations of 0, 2, and 5 µM. Pravastatin-Cy5.5 exhibited dose-dependent cytotoxicity in Panc1, while showing little to no toxicity in CAF19 cells. In contrast, pravastatin alone had minimal effects across all conditions Bars indicate mean ± S.E. (n ≥ 3). Statistically significant differences are indicated by ** for p < 0.01 and *** for p < 0.001 (student t-test).

https://doi.org/10.1371/journal.pone.0340189.s011

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S12 Fig. Cellular uptake of simvastatin-Rhodamine B in various cell lines.

Confocal fluorescence images showing intracellular localization of simvastatin-Rhodamine B (yellow) after 1 h incubation at 50 nM in DLD1 KRAS^WT and KRAS^MUT cells (top), HCT116 KRAS^WT and KRAS^MUT cells (middle), and MCF10A PTEN^WT and PTEN^KO cells (bottom). Nuclei were counterstained with DAPI (blue). Minimal intracellular fluorescence was observed across all cell types, indicating poor uptake of simvastatin-Rhodamine B and no apparent KRAS- or PTEN-dependent selectivity.

https://doi.org/10.1371/journal.pone.0340189.s012

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S13 Fig. Uptake of simvastatin-Cy5.5 (red) and pravastatin-Cy5.5 (red) in Panc1 under no bovine serum albumin (BSA) and 5% BSA condition.

The cell nuclei were stained with DAPI (blue). The scale bars indicate 100 μm.

https://doi.org/10.1371/journal.pone.0340189.s013

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S1 Table. Characteristics of the cells used in this study.

https://doi.org/10.1371/journal.pone.0340189.s014

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Acknowledgments

We thank Dr. In-San Kim and Dr. Gi-Hoon Nam for their helpful comments and input.

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Figures

Abstract

Introduction

Materials and methods

Synthesis of statin-dye conjugates

Cell culture and cell line information

Cellular uptake assay

Cytotoxicity assay

Cellular uptake assay using T-MOC model

Cytotoxicity assay using T-MOC model

Statistical analysis

Results

Synthesis of statin-dye conjugates

Selective uptake of statin-dye conjugates in KRASMUT cancer cells

Macropinocytosis as a key mechanism in statin-dye conjugate uptake

Selective cytotoxicity of statin-dye conjugates in KRASMUT cells in vitro

KRASMUT cancer cells selectively uptake statin-Cy5.5 conjugates in the engineered TME

Tumor-selective cytotoxicity of statin-dye conjugates using the engineered TME

Discussion

Conclusions

Supporting information

S1 Fig. Chemical structure of simvastatin-Cy5.5 was confirmed via (a) 1H-NMR and (b) 13C-NMR, respectively.

S2 Fig. Chemical structure of pravastatin-Cy5.5 was confirmed via (a) 1H-NMR and (b) 13C-NMR, respectively.

S3 Fig. Chemical structures of Simvastatin, Pravastatin and Cy 5.5 acid were confirmed by (a, c, e) 1H-NMR and (b, d, f) 13C-NMR, respectively.

S4 Fig. Concentration-dependent uptake of simavastatin-Cy5.5 in KRAS-mutant (KRASMUT) Panc1 cells.

S5 Fig. Enhanced cellular uptake of simvastatin-Cy5.5 in KRASMUT cancer cells.

S6 Fig. Synthesis and characterization of PEG-Cy5.5.

S7 Fig. Cellular uptake of Cy5.5 conjugates in KRASMUT cancer cells.

S8 Fig. Validation of macropinocytosis inhibition by (5-(N-ethyl-N-isopropyl)amiloridein (EIPA; macropinocytosis inhibitor) in KRAS-mutant (KRASMUT) Panc1 cells.

S9 Fig. Cellular uptake of statin-Cy5.5 conjugates (red) by MCF10A cells with PTEN knockout (PTENKO) and wild-type (PTENWT).

S10 Fig. Selective cytotoxicity of simvastatin-Cy5.5 in KRASMUT cell lines compared to simvastatin.

S11 Fig. Selective cytotoxicity of pravastatin-Cy5.5 in KRASMUT cell line compared to pravastatin.

S12 Fig. Cellular uptake of simvastatin-Rhodamine B in various cell lines.

S13 Fig. Uptake of simvastatin-Cy5.5 (red) and pravastatin-Cy5.5 (red) in Panc1 under no bovine serum albumin (BSA) and 5% BSA condition.

S1 Table. Characteristics of the cells used in this study.

Acknowledgments

References

Selective uptake of statin-dye conjugates in KRAS^MUT cancer cells

Selective cytotoxicity of statin-dye conjugates in KRAS^MUT cells in vitro

KRAS^MUT cancer cells selectively uptake statin-Cy5.5 conjugates in the engineered TME

S1 Fig. Chemical structure of simvastatin-Cy5.5 was confirmed via (a) ¹H-NMR and (b) ¹³C-NMR, respectively.

S2 Fig. Chemical structure of pravastatin-Cy5.5 was confirmed via (a) ¹H-NMR and (b) ¹³C-NMR, respectively.

S3 Fig. Chemical structures of Simvastatin, Pravastatin and Cy 5.5 acid were confirmed by (a, c, e) ¹H-NMR and (b, d, f) ¹³C-NMR, respectively.

S4 Fig. Concentration-dependent uptake of simavastatin-Cy5.5 in KRAS-mutant (KRAS^MUT) Panc1 cells.

S5 Fig. Enhanced cellular uptake of simvastatin-Cy5.5 in KRAS^MUT cancer cells.

S7 Fig. Cellular uptake of Cy5.5 conjugates in KRAS^MUT cancer cells.

S8 Fig. Validation of macropinocytosis inhibition by (5-(N-ethyl-N-isopropyl)amiloridein (EIPA; macropinocytosis inhibitor) in KRAS-mutant (KRAS^MUT) Panc1 cells.

S9 Fig. Cellular uptake of statin-Cy5.5 conjugates (red) by MCF10A cells with PTEN knockout (PTEN^KO) and wild-type (PTEN^WT).

S10 Fig. Selective cytotoxicity of simvastatin-Cy5.5 in KRAS^MUT cell lines compared to simvastatin.

S11 Fig. Selective cytotoxicity of pravastatin-Cy5.5 in KRAS^MUT cell line compared to pravastatin.