Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance

Samir V Jenkins; Shruti Shah; Azemat Jamshidi-Parsian; Amir Mortazavi; Hailey Kristian; Gunnar Boysen; Kieng B Vang; Robert J Griffin; Narasimhan Rajaram; Ruud PM Dings

doi:10.1667/RADE-23-00005.1

. Author manuscript; available in PMC: 2025 Feb 1.

Published in final edited form as: Radiat Res. 2024 Feb 1;201(2):174–187. doi: 10.1667/RADE-23-00005.1

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance

Samir V Jenkins ^a, Shruti Shah ^a, Azemat Jamshidi-Parsian ^a, Amir Mortazavi ^a, Hailey Kristian ^a, Gunnar Boysen ^b, Kieng B Vang ^a, Robert J Griffin ^a, Narasimhan Rajaram ^c, Ruud PM Dings ^a,¹

PMCID: PMC10993299 NIHMSID: NIHMS1973344 PMID: 38329819

Abstract

Jenkins SV, Shah S, Jamshidi-Parsian A, Mortazavi A, Kristian H, Boysen G, Vang KB, Griffin RJ, Rajaram N, Dings RPM. Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance. Radiat Res. 201, 174–187 (2024).

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5–2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed in vivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

INTRODUCTION

Approximately 50% of patients diagnosed with cancer annually, lung cancer included, are treated with radiotherapy either alone or in combination with chemotherapy (1, 2). Due to intrinsic or acquired radiation resistance (RR), treatment failure remains a significant problem. Thus, delineating molecular and cellular adaptations of treatment resistance aids in identifying prognosticators and/or selective avenues for drug targeting to prevent or overcome resistance. In the past, assessment of radiation resistance was often based on a comparative analysis of patient-derived cell lines grouped generally per tumor type (3, 4). Unfortunately, as a result of the distinct genetic backgrounds and complex medical and treatment histories, a clear understanding of radiation resistance has been challenging and may explain why the subsequent translation into the clinic has not lived up to its promise.

Conventional radiation is given to most primary tumors in fractions over a period of 5–7 weeks (2 Gy/fraction) (5). Dose fractionation is given as it may improve the tumor oxygenation and consequently the efficacy of the additional radiation treatments. However, the protracted treatment schedule also allows for the induction of acquired resistance. Comparing genetically unrelated patient-derived specimens has limited value as the distinction between intrinsic and acquired resistance is difficult to discern. Therefore, to understand, and possibly overcome acquired resistance, we used established NSCLC models, i.e., the murine Lewis lung carcinoma (LLC) and the human A549 cell line and exposed them to a conventional clinical regimen, i.e., 25 fractions of an accumulated dose of 55 Gy to generate matched models of ionizing radiation resistance in non-small cell lung carcinoma (NSCLC). These two models are potential valuable examples of recurrent disease, because the parental and daughter lines were passage-matched and are isogenic in origin. Moreover, the resistance to radiation was acquired from known radiation exposures only, rather than using distinct cell lines with different intrinsic resistance, wherein the origin of their treatment resistance may be operating through an orthogonal and unrelated mechanism(s) as compared to acquired resistance to ionizing radiation.

Here we show that clonogenic and cell viability assays confirmed the acquired radiation resistance as compared to their isogenically-matched parental counterpart. Mechanistically, the radiation resistance was reflected in higher levels of DNA damage and repair, reduced apoptosis, and reduced radiation-induced cell killing. Morphologically, we found that the cell size and nucleus-to-cytosol (N/C) ratio changes as radiation resistance is acquired. Regarding possible changes in the epithelial-to-mesenchymal transition (EMT) phenotype, we found that A549 cells have a greater plasticity as compared to LLC, as measured by E-cadherin, vimentin, and double positive (DP) modulation. Functionally, this was reflected in suppressed A549-RR migration, as compared to its radiation naïve matched cells. Subsequently, we confirmed in vivo that LLC-RR and A549-RR cells are also more resistant to radiation than their isogenic matched counterpart.

Platinum-based chemotherapy is the standard-of-care for patients with advanced NSCLC, and it is frequently combined with radiation in the clinic (6). We next assessed whether cross resistance was induced in the radiation-resistant cells. We found that the acquired radiation resistance also induced a cross resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. To delineate the mechanism of this cross-resistance we investigated the redox system. The dysregulation of the redox system, which includes reactive oxygen species (ROS), has been associated with radiation resistance and impairment of radiation efficacy (7). Reduced thiols or sulfhydryl groups are a significant component of the intracellular redox system. We found that the acquired resistance was attributed to a gross upregulation of protein thiols, which are known to scavenge and inactivate ROS. Moreover, that buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, reduced thiol formation and sensitized the radioresistant cells to cisplatin, to similar levels as its naive counterpart. Thus, we postulate that, at least in part, radiation-induced cross-resistance to cisplatin is thiol dependent and that this can be overcome with glutathione pathway inhibitors.

MATERIALS AND METHODS

Cell Lines and Chemicals

At the start of the project authenticated NSCLC murine LLC and human lung carcinoma A549 cells were newly purchased from American Type Culture Collection (ATCC; CCL185) and cultured according to the company’s instructions. Cisplatin, carboplatin, and oxaliplatin were purchased from SelleckChem (Houston, TX), and γ-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) from Sigma-Aldrich (St. Louis, MO).

Irradiation

Cells were irradiated every three days for a cumulative dose of 55 Gy (25 fractions) to generate the radiation-resistant cells. All cells were passaged biweekly and at least 24 h was allowed both before and after the intervention. Non-irradiated cells were passaged along-side to have passaged-matched cells. Irradiation was carried out in a CP 160 X-ray system (Faxitron X-ray Corporation Tucson, AZ). Here, for all experiments shelf 6 (SSD 0 43.2 cm covering, ~39-cm diameter field, and 0.8 mm Be/0.5 mm copper filtration) was used with 150 kVp and 6.6 mA beam. Dosimetry was carried out using a pinpoint ion chamber (PTW N301013, ADCL calibrated for 225 kV) following the AAPM TG-61 protocol. The radiation dose rate in our setup was 1.018 ± 0.10 Gy/min at 150 kV and 6.6 mA.

Clonogenic Assay

To determine clonogenic survival in response to radiation, cells in exponential growth phase were trypsinized, washed, counted, and seeded at a concentration of 100 cells per well into 6-well plates. All conditions were done in triplicate. The plates were incubated overnight at 37°C, 5% CO₂. Approximately 16 h after seeding, plates were treated with radiation and incubated for 8 days to form colonies. To quantify colony formation, plates were washed with normal saline, and then fixed in 90:10 methanol: acetic acid for 30 min. The fixative was removed and the colonies were stained for 2 h with 1% crystal violet in the methanol/acetic acid mixture. The stain was removed and residual crystal violet was washed off with tap water. The number of resulting colonies (defined as >50 cells per colony) per well was counted using a stereomicroscope. The number of identified colonies at 0 Gy divided by the seeding density was used to normalize the number of colonies formed per cell line at the various doses, as described elsewhere (8). The dose-modifying factor (DMF₃₀) was calculated as the ratio between two cell line at the dose with 30% survival (9).

Cell Viability

The cell viability was assessed using the CCK-8 assay (Dojindo, Japan), as described previously (10, 11). In brief, cells were seeded in a 96-well plate at 1,000 cells/well and allowed to adhere overnight. The cells were treated with different radiation or chemotherapeutics doses. After 72 h of incubation the cell viability was assessed using the CCK-8 assay (Dojindo, Japan), according to the manufacturer instructions.

Gamma H2AX Assay

To determine DNA damage and repair in response to radiation, cells in exponential growth phase were trypsinized, washed, counted, and seeded at a concentration of 100,000 cells per well into 12-well plates. LLC, LLC-RR, A549, and A549-RR cells were grown overnight and then irradiated with 2 Gy or left untreated. Samples were then incubated for 1 h at 37°C and collected, washed, and stained with Fixable Viability Dye 780 for 1 h. Cells were subsequently washed with phosphate buffered saline (PBS) and fixed with 2% paraformaldehyde for 20 min. Cells were washed with PBS then permeabilized with 2% Triton-X-100 for five min, then washed with PBS and blocked with 5% bovine calf serum and Fc Block for 30 min. Anti-mouse/human γH2AX monoclonal antibody (Clone:CR55T33) and rat anti-mouse IgG conjugated with Dylight488 were used to stain DNA damage sites. Samples were processed using BD LSRFortessa (BD Biosciences) and data were analyzed using FlowJo^™ (BD Biosciences), as described earlier (12).

Apoptosis

To determine baseline and induced apoptosis index after 2 Gy, LLC, LLC-RR, A549, and A549-RR cells were grown overnight (100,000 cells per well in a 12-well plate) and then irradiated with 2 Gy or left untreated. According to the manufacturer’s instructions (Biolegend, San Diego), Annexin V (AnxV)-FITC was used to assess early apoptosis, and propidium iodide (PI) was used to assess late apoptosis and/or necrosis detection. Samples were analyzed 24 h later using BD LSRFortessa^™ (BD Biosciences) and data were analyzed using FlowJo as described earlier (13–15).

Cell Cycle Analysis

Cell cycle analysis was performed by flow cytometry. In brief, 100,000 cells were grown overnight onto 24-wells plates. After approximately 18 h the cells were irradiated with 2 Gy. At 24 h post-irradiation, adherent and non-adherent cells were recovered, washed with PBS, fixed in 70% ethanol and stored at 4°C overnight. Cells were rehydrated in PBS, incubated for 20 min at room temperature with 250 μg/mL RNAse A, and for 20 min at 4°C with 50 μg/mL propidium iodide in the dark. Cell cycle distribution was recorded using a BD LSRFortessa and data were analyzed using FlowJo, as described earlier (16).

Nuclear/Cytoplasmic Ratio Determination

To assess possible morphological changes after acquired resistance the nuclear/cytoplasmic (N/C) ratio was determined, as previously described (3). In brief, 10,000 cells per well were seeded in a 24-well plate and allowed to adhere overnight in complete medium. Cells were then washed with PBS and incubated for 20 min with 1X PBS containing Ca²⁺, Mg²⁺, 0.5% bovine serum albumin, 1 mM fluorescein diacetate, and 2 drops/mL of NucBlue live cell stain (Hoechst 33342). Cells were then imaged directly at 10X using phase contrast, FITC filter cube, and DAPI filter cube on a ImageXpress Pico Automated Cell imaging system (Molecular Devices, LLC, San Jose, CA). Quantification was based on the fluorescein diacetate signal representing the cytoplasm and the signal in the DAPI channel representing the nucleus to determine relative areas per cell on at least ten sets of images per cell line.

Migration

The migration or wound healing assay was performed as described previously, although with some modifications (8, 17). Cells were grown overnight until confluence in a 6-well plate. When confluent, 5 parallel scratches were made in the well, using a blunt, sterile, plastic 200 μl pipette tip. The well was carefully washed with PBS and the medium was replaced with medium containing 0.5% fetal bovine serum (FBS). The wound width was measured at four different pre-defined places. Photographs were made using an inverted photomicroscope (Olympus IX71) and measured and digitally quantified, as described elsewhere (8, 17).

In vivo NSCLC Models

Animals (5–6 weeks old) were provided water and standard chow ad libitum, allowed to acclimatize to local conditions for at least 1 week, and were maintained on a 12 h light/dark cycle. For tumor cell inoculation of the syngeneic murine LLC, a 100 μL solution containing 1 × 10⁶ LLC cells was injected subcutaneously (s.c.) in C57BL/6J mice (strain #0664; The Jackson Laboratory), as described elsewhere (18). For the human lung epithelial A549 carcinoma cells, initially exponentially growing A549 cells were cultured, harvested, suspended in serum-free Ham’s F12K medium (4.0 × 10⁷ cells/ml) and 100 μL of the solution was inoculated s.c. in Foxn1^−/− mice (strain #002019; The Jackson Laboratory), similarly as previously described (19). Tumors were subsequently established by transplanting 0.85 g viable tissues s.c. on new recipient mice. When tumors reached approximately 100 mm³ on average, mice were randomized and divided in groups. In the radiation-cohort tumors were locally irradiated with 2 Gy once every 3 days (q3dx3 – 6 Gy total dose for the LLC and q3dx4 – 8 Gy total dose for the A549), and for the cohort receiving platinum-based chemotherapy 3 mg/kg i.p. cisplatin in sterile saline was administered (q3dx3) at the indicated time points. Tumor volume was determined by measuring the diameters of tumors with calipers and calculated by the equation for volume of a spheroid: (a² × b × π)/6, where a is the short axis and b is the long axis of the tumor. Tumor mass was assessed at the end of the study after humanely sacrificing the mice and excising the tumors and weighing them on a Sartorius A200S analytical balance (18). As an indirect measurement of general health and possible toxicity, body weights were monitored using a digital balance (Ohaus Florham, NJ) (20). All experiments were approved by the University of Arkansas for Medical Sciences institutional animal care and use committee.

Flow Cytometry

To quantify cellular membrane protein expression on viable cells we employed fluorescence-activated cell sorting (FACS) analysis. Anti-mouse and anti-human antibodies E-cadherin (CD324; clone DECMA-1) and vimentin (clone V9) were used and the analysis was based on pre-gating on single cells and fixable viability dye (FVD-780; eBioscience) negative cells. Positivity for the marker of interest was determined by using the fluorescence minus one (FMO) strategy. Samples were acquired by multiparameter flow cytometry on a LSR II flow cytometer (BD Biosciences), analyzed by FlowJo software and plotted using Prism 9.3.1 (Graphpad), as previously described (15).

Thiol Determination

The dysregulation of the redox system, which includes reactive oxygen species (ROS), has been associated with radioresistance and impairment of radiotherapy efficacy. Thiols can scavenge and inactivate ROS, thus, to assess thiol levels, maleimide staining was done by seeding cells at 100,000 cells per well in a 6-well plate. After being allowed to adhere, cells were treated with radiation and allowed to incubate overnight. Medium was removed and the cells were washed once with PBS followed by a 45 min incubation of 1 μM fluorescein-maleimide. Cells were harvested with a rubber policeman, washed by centrifugation 3 times, then fixed with paraformaldehyde (2%, 20 min, 4°C) prior to flow cytometry analysis.

Statistical Analysis

Data are reported as mean ± SEM unless otherwise stated and were analyzed by an unpaired two-tailed t-test. P values <0.05 were considered statistically significant.

RESULTS

Generating Acquired Radiation Resistance in NSCLC

Acquired radiation resistance in murine LLC and human A549 NSCLC was generated by exposing them to a clinically relevant fractionated irradiation, namely 25 fractions for a cumulative total of 55 Gy (Fig. 1). As a result, LLC-RR (Fig. 1A, C, E and G) and A549-RR (Fig. 1B, D, F and H) cells increased their radiation resistance by up to 1.5–1.7 times, as compared to their original intrinsic resistance, as measured by clonogenicity and cell viability. This acquired resistance could not be explained by changes in seeding efficiency, regarding clonogenicity, nor doubling time with respect to cell viability, as both did not change (Supplementary Fig. S1A–D;² https://doi.org/10.1667/RADE-23-00005.1.S1).

FIG. 1. — LLC-RR and A549-RR exhibit increased radiation resistance as compared to their intrinsic origins. Panel A: LLC-RR and (panel B) A549-RR are radiation resistant as compared to their parental counterparts, displaying a DMF₃₀ of 1.5 for the LLC-RR and a DMF₃₀ of 2.5 for the A549-RR, as measured by clonogenicity. (panel C) LLC-RR are up to 1.6× and (panel D) A549-RR up to 1.7× more resistant to radiation as determined by clonogenicity than parental LCC and A549. Panel E: LLC-RR and (panel F) A549-RR are radiation resistant as compared to their parental counterparts, as measured by cell viability. Panel G: LLC-RR are up to 1.6× and (panel H) A549-RR are up to 1.5× more resistant to radiation as determined by cell viability than parental LLC and A549. Data presented as mean ± SEM, pooled from 3 experiments with triplicates. *P < 0.05; two-tailed t-test. DMF = dose-modifying factor at 30% survival.

Phenotypic Characterization

To assess if the resistant cells acquired any changes in amount of DNA damage and/or repair capacity, we quantified γH2AX, an early cellular response to the induction of DNA double-strand breaks (Fig. 2). Radiation-resistant cells had more intrinsic γH2AX foci as compared to their parental counterparts. However, the resistant cells did not show an increase in number of γH2AX foci after 2 Gy (Fig. 2A–G). Similarly, the cell cycle distribution was also not altered with or without radiation exposure between LLC and LLC-RR, or A549 and A549-RR cells (Supplementary Fig. S1E and F; https://doi.org/10.1667/RADE-23-00005.1.S1). In contrast, the resistant cells did show a suppressed induction of apoptosis and necrosis after receiving 2 Gy (Fig. 2H–J).

Morphologically the LLC-RR and the A549-RR cells showed a change in N/C ratio as compared to their respective matched origins (Fig. 2K–N). By using the outlines of the cells in brightfield and fluorescein diacetate, as compared to the signal of the Hoechst DNA stain, it was noted that the acquired radiation resistant resulted in decreased N/C ratio for the LLC-RR as compared to the LLC (Fig. 2K and L). This resulted in approximate 40% reduction N/C ratio on average: LLC (0.68 ± 0.04) as compared to LLC-RR (0.41 ± 0.03; Fig. 2L). In contrast, the N/C ratio for A549 changed in the opposite direction by an increase of 33% on average: A549 (0.30 ± 0.02) as compared to A549-RR (0.40 ± 0.03; Fig. 2M and L). Cell size assessment as detected by forward scattering, showed that both LLC-RR and A549-RR decreased in overall cell size as compared to their matched counterparts (Fig. 2N).

Epithelial and Mesenchymal Markers are Elevated due to Acquired Resistance

Since the N/C ratio has been linked to the maturity and/or differentiated state of the cell, we next investigated the epithelial to mesenchymal transition (EMT) proteomic phenotype and the plasticity thereof after radiation exposure (Supplementary Fig. S2; https://doi.org/10.1667/RADE-23-00005.1.S1 ). We found that E-cadherin, indicative of an epithelial phenotype, was significantly higher on LLC (23.1 × 0.9%) as compared to A549 (14.5 ± 0.5%; Supplementary Fig. S2A). Whereas vimentin, a mesenchymal indicator is significantly lower on LLC (40.7 ± 1.4%), as compared to A549 (49.1 ± 0.5%; Supplementary Fig. S2B). The population of cells positive for both markers simultaneously or double positives (DP), was significantly higher on A549 (6.9 ± 0.02%), as compared to LLC (4.7 ± 0.2%; Supplementary Fig. S2C).

Radiation (2 Gy) increased expression levels of E-cadherin from 14.5 ± 0.5% to 39.0 ± 2.0%, vimentin from 49.1 ± 0.5% to 71.6 ± 2.9%, and DP from 6.9 ± 0.02% to 20.6 ± 2.1%, on A549. However, the intrinsic relatively higher expression levels of the EMT markers on A549-RR did not additionally increase after 2 Gy irradiation (Supplementary Fig. S2D–F).

We found that expression levels of E-cadherin, vimentin and DP were not statistically different between LLC and LLC-RR. Radiation only significantly increased E-cadherin on LLC (from 23.1 ± 0.9% to 30.1 ± 1.7%; P < 0.05) and LLC-RR (from 22.2 ± 2.3% to 33.7 ± 0.8%; P < 0.01), and it increased vimentin levels on LLC-RR (47.5 ± 1.1%; P < 0.05), as compared to LLC (35.3 ± 1.5%) after 2 Gy (Supplementary Fig. S3).

Since EMT is involved in migration, we measured whether the acquired resistance had an influence on the ability to migrate (Supplementary Fig. S2G–J; https://doi.org/10.1667/RADE-23-00005.1.S1). We found that migration is suppressed in resistant cells, as compared to the parental cell lines. This is in line with the greater fold-increase in intrinsic levels of E-cadherin (2.8-fold; from 14.5% to 40.3%), as compared to vimentin (1.4-fold; from 49.1% to 68.2%) in resistant cells (Supplementary Fig. S2D and E). However, the migration rate after 2 Gy irradiation is similar between the respective sensitive and resistant cell lines (Supplementary Fig. S2H), like their EMT expression levels (Supplementary Fig. S2D–F). Intriguingly, radiation suppressed migration of radiation naïve cells, whereas the resistant cells migrated faster after radiation (Supplementary Fig. S2G–J).

Acquired Radiation Resistance is Maintained In Vivo

In vivo, the tumor growth kinetics of LLC-RR were similar to the original tumor growth kinetics of LLC (Fig. 3). Namely, as measured by tumor volume in time, both tumor models grew approximately 50 mm³ per day (Fig. 3A). This was also reflected in similar tumor weights after sacrifice and excision: 0.32 ± 0.04 grams for LLC and 0.34 ± 0.05 grams for LLC-RR on average (Fig. 3B). However, LLC tumor growth and mass was inhibited approximately 60% by (3 × 2 Gy) radiation exposure, i.e. 555 ± 33.8 mm³ vs. 212 ± 37.6 mm³ and 0.32 ± 0.04 grams vs. 0.18 ± 0.05 grams in tumor mass on average (Fig. 3C and D). In contrast, LLC-RR tumor progression was not significantly affected by radiation exposure. Namely, approximately 600 mm³ final tumor volumes and 0.34–0.39 grams in tumor mass on average with or without radiation (Fig. 3E and F). No obvious signs of toxicity were noted as evidenced by maintaining body weight during the study (Fig. 3G and H).

Similar trends were seen comparing A549 vs. A549-RR (Supplementary Fig. S4; https://doi.org/10.1667/RADE-23-00005.1.S1). Namely, both tumor models grew approximately 12 mm³ per day (Supplementary Fig. S4A). This was also reflected in similar tumor weights after sacrifice and excision: on average 0.31 ± 0.17 grams for A549 and 0.32 ± 0.2 grams for A549-RR (Supplementary Fig. S4B). A549 tumor growth and mass was inhibited approximately 60% after (4 × 2 Gy) irradiation, i.e., 615 ± 339 mm³ vs. 255 ± 99 mm³ and on average 70% in final tumor mass (0.31 ± 0.17 grams vs. 0.09 ± 0.04 grams; Supplementary Fig. S4C and D). However, A549-RR tumor growth and their tumor mass were not significantly affected by radiation. Namely, approximately 700 mm³ final tumor volumes and 0.30 grams in tumor mass on average with or without radiation (Supplementary Fig. S4E and F). No obvious signs of toxicity or overall health loss were noted as evidenced by maintaining body weight during the study (Supplementary Fig. S4G and H).

Acquired Radiation Resistance causes Cisplatin Cross-Resistance

Since cross-resistance is a clinical challenge, we next tested the sensitivity of the acquired radiation resistant lines against platinum-based chemotherapy, the standard-of-care for patients with advanced NSCLC. We noted a cisplatin cross-resistance, whereas the sensitivity to carboplatin or oxaliplatin was maintained (Fig. 4). Namely, acquired radiation resistance increased the half-maximum effective concentration (EC₅₀) in inhibiting cell viability from 0.5 μM in LLC to 1.5 μM in LLC-RR, whereas the sensitivity was maintained for both carboplatin (EC₅₀ = 10 μM) and oxaliplatin (EC₅₀ = 0.7 μM; Fig. 4A–C). Similarly, A549-RR showed cisplatin resistance induction (EC₅₀ = 3.5 μM in A549 to 8 μM in A549-RR), whereas carboplatin (EC₅₀ = 60 μM) and oxaliplatin (EC₅₀ = 2 μM) sensitivity was maintained (Fig. 4D–F).

Additionally, we found that resistance to radiation during chemoradiation (dual treatment) was maintained during cisplatin co-treatment (Fig. 4G). Cisplatin inhibited the surviving fraction of A549 by approximately 28 ± 9%, whereas the A549-RR had a slight insignificant increase in surviving fraction to an average of 118 ± 9%, as compared to untreated. Cisplatin combined with either 2 Gy or 6 Gy of radiation resulted in a significant (P < 0.05) greater survival in the acquired resistant cells, as compared to the parental cell line. In contrast, chemoradiation with carboplatin did not show a differential between sensitive and resistant cells (Fig. 4H).

Next, we assessed this cisplatin cross-resistance in vivo (Fig. 5). First, we found that that LLC and LLC-RR did not significantly differ in tumor growth rate, as assessed by tumor volume and tumor mass on the last day of the study (Fig. 5A and B). However, LLC tumor growth and final tumor mass was inhibited by cisplatin (3 mg/kg; q3dx3) by approximately 65%, i.e., 800 ± 166 mm³ vs. 290 ± 29 mm³ and 55%, i.e., 0.69 ± 0.1 grams vs. 0.31 ± 0.05 grams in tumor mass on average (Fig. 5C and D). Whereas the radiation resistant tumor progression was not significantly affected by cisplatin. Namely, approximately 500 mm³ ± 40 mm³ final tumor volumes and 0.5 ± 0.05 grams in tumor mass on average with or without cisplatin (Fig. 5E and F). No obvious signs of toxicity or loss in overall health were noted in any of the tumor models or interventions as evidenced by maintaining body weight during the study (Fig. 5G and H).

Acquired Radiation Resistance is Thiol Dependent

Mechanistically we found that buthionine sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase and analog of the glutamine synthetase (glutamate-ammonia ligase) inhibitor methionine sulfoximine, restored cisplatin sensitivity after the induced resistance (Fig. 6 and Supplementary Fig. S5; https://doi.org/10.1667/RADE-23-00005.1.S1). First, by using maleimide as a thiol reporter we observed that radiation caused an increase in reduced thiol binding sites, i.e., radiation nearly doubled the free thiols in LLC from 20,920 ± 4,105 MFI to 41,733 ± 4,944 MFI at 2 Gy and 51,956 ± 1,739 MFI at 6 Gy (Fig. 6A). Although less pronounced, the same trends were seen for LLC-RR. Radiation increased thiols from 31,463 ± 1,415 MFI to 38,715 ± 929 MFI at 2 Gy and 39,912 ± 2,924 MFI at 6 Gy (Fig. 6B). Similar trends were seen in the A549 and A549-RR cells (Fig. S5).

FIG. 6. — Induced cisplatin cross-resistance is thiol dependent. Panel A: Radiation-induced thiol increase in LLC, and (panel B) LLC-RR. Panel C: BSO reduced free thiols in LLC-RR. Panel D: Acquired cross-resistance to cisplatin was overcome by BSO in LLC-RR. Data presented as mean ± SEM, representative experiment shown from 3 experiments with triplicates. BSO = buthionine sulfoximine [5 mM]. *P < 0.05; two-tailed t-test

BSO was able to prevent thiol generation and consequently overcame the acquired cross-resistance to cisplatin in both LLC and A549 matched cell lines (Fig. 6C and S5C). Whereas the intrinsic thiol levels of resistant cells are higher than the relative sensitive counterparts, 31,463 ± 1,415 MFI vs. 20,920 ± 4,105 MFI, BSO was able to reduce the amount of thiols to 19,987 ± 3,221 MFI (Fig. 6C). Functionally, BSO was able to increase the cisplatin-sensitivity of LLC-RR approximately threefold to similar levels as LLC, namely from an EC₅₀ of 1.5 μM (Fig. 4A) to approximately 0.5 μM (Fig. 6D). Similar trends were seen in the A549 and A549-RR cells: the EC₅₀ of 8 μM improved to 3.5 μM in the presence of BSO, without affecting cell viability in any of the cell lines (Supplementary Fig. S5; https://doi.org/10.1667/RADE-23-00005.1.S1).

DISCUSSION

Recent technical advances in radiotherapy and chemotherapy, as well as the increase in immunotherapy options have helped substantially improve the treatment outcome and quality of life for cancer patients (21, 22). Nonetheless, successful cancer therapy remains a major challenge for many patients, particularly for those with tumors resistant to chemoradiation. It is therefore important to identify the underlying molecular and cellular features involved in this resistance, which would then facilitate the discovery and validation of clinical prognosticators of treatment response and/or treatment selection to prevent or overcome resistance and increase clinical outcome.

Our previous work and the work of others have highlighted the multifaceted components of radiation resistance in a variety of solid tumors (3, 4, 23, 24). Thus far, molecular and cellular mechanisms of radiation and chemo-resistance include enhanced DNA repair activity, apoptotic pathway defects, metabolic and reactive oxygen species adaptations, altered target and membrane transporter molecules, enzymatic deactivation, and changes in cell plasticity or tumor tissue physiology (24–27). This resistance to anti-neoplastic therapy is generally classified by either intrinsic genetic characteristics or acquired resistance after repeated radiation and/or drug exposure. Here, our objective was to generate genetically-matched pairs of NSCLC to define cellular adaptations of acquired, rather than intrinsic radiation resistance, through repeated conventional X-ray irradiations to aid and optimize clinical strategies. Moreover, the A549 model allows for studying the response of human NSCLC in an in vivo setting. The observed responses in the A549 model are likely tumor cell intrinsic as parts of the tumor microenvironment, most notably the adaptive immune system, are largely absent to allow interspecies transplantation. In contrast, the syngeneic LLC model allows for an immunocompetent setting with interactions and responses within the variety of (non)cellular tumor microenviroment elements. Thus, the combination of both the A549 and the LLC models are a valuable and unique set of NSCLC tumor models complementing each other to compare and contrast treatment resistance mechanisms based on tumor cell intrinsic only responses, and in an immunocompetent setting. Additionally, our studies showed a complex mechanistic connection between morphology (N/C ratio and cell size), phenotype (epithelial/mesenchymal markers), and cross-resistance to cisplatin. Since the discovery of cisplatin as an anti-tumor agent in the late seventies, it has become a primary and essential treatment modality in the clinic for a variety of solid tumor types, including lung, brain, breast, bladder, prostate, and head and neck cancer. Despite cisplatin’s current, frequent, and broad use, and thus clinical reliance, acquired resistance is often observed in patients (28–30).

An often-highlighted mechanism of resistance is the upregulation of molecules that inactivate ROS. Classically, glutathione has been highlighted extensively, but there is emerging evidence that there is a uniform induction of proteins, particularly proteins with reduced cysteine residues. The thiol side chain in cysteines inactivates various ROS due to its intrinsic ability to scavenge ROS. Namely, sulfur occupies a unique position in biology due to its ability to adopt a wide range of oxidation states (−2 to +6), and chemically unique forms (31). Mercapto (i.e., thiol or -SH), is the most reduced form and acts like a sponge to attract and inactivate numerous ROS. Consequently, cysteine plays an integral role in biology as a cellular target and sensor of reactive intermediates. Thus, based on the biological chemistry involved we suggest that the cisplatin resistance is due to increased prevalence of reduced thiol groups. Specifically, the inorganic cisplatin contains two relatively loosely bound chloro (Cl⁻) ligands and the salinity of extracellular fluid maintains an equilibrium at around ~100 mM Cl⁻, wherein the Cl⁻ are bound to the platinum (Pt²⁺) cation (32). However, the intracellular chloride level is 10–20 mM, shifting the equilibrium to favor dissociation of the Cl⁻ towards aquation, which is subsequently exchanged for DNA nitrogen bases in the nucleus. The attachment of the complex to DNA interferes with replication and leads to cisplatin’s alkylating mechanism of action. Thiols are well known to bind to metals and sulfur poisoning of platinum catalysts is particularly well-described (33). There is evidence that Pt²⁺ ions can migrate from thioethers to N7 in guanine, but transport of many proteins into the nucleus creates a geographical barrier to that transfer. Increasing the level of available thiols in the intracellular space allows more opportunities for binding of the aquated Pt²⁺ ion within the cytoplasm. However, the bidentate ligands used in the synthesis of carboplatin and oxaliplatin provides significantly greater bond strength and a degree of steric protection from thiols in the cytoplasm, which can explain the absence of resistance to carboplatin and oxaliplatin in cells that have developed cisplatin resistance and demonstrate constitutively elevated expression of reduced thiols. Thus, the extreme lability of the two chloro ligands in cisplatin make it more susceptible to sequestration by proteinaceous thiols, leading to cellular resistance, while this development is less likely for carboplatin or oxaliplatin.

Therapeutically, we offer a potential solution to this acquired chemoradiation resistance as γ-glutamylcysteine synthetase inhibitor BSO, was able to overcome this resistance. In addition to glutathione, BSO also decreases the intracellular concentration of cysteine. As such, the reductive potential of the cell is significantly diminished. Others have shown that BSO increases the degree of DNA damage from alkylating agents, and that increases in reduced cysteine levels significantly lowers cisplatin-based nephrotoxicity (34). Our data indicate that there is a constitutive rise in reduced thiol levels as a result of prior exposure to radiation, which imparts a cross-resistance to cisplatin. It is likely that other components in the glutathione pathway are modulated, and a cross-resistance to other therapies that are reliant on elements in this pathway is similarly developed. As such, the treatment regimen for recurrent disease, as a result of acquired resistance to radiation may need to be changed (i.e., use of later generation platinum drugs) or augmented (i.e., combined with BSO or similar therapies) to achieve the desired clinical outcome. Currently, glutamine synthetase inhibitors, as well as associated glutamine pathway disruptors are at various stages in clinical trials and hold great promise (31, 35–38). Namely, glutamine synthetase is highly expressed in the tumor microenvironment as compared to normal tissues. This supports the observed cancer cell dependence of glutamine (39). The metabolism and proliferation of cancer cells is heavily reliant on glutamine, and glutamine synthetase is the only enzyme responsible for de novo synthesis (39, 40). It was shown in various human cancers that the overexpression and increased glutamine pathway signaling appears to be mediated through oncogene Myc (41). Here, we propose an additional feature of elevated levels of glutamine synthetase cancer cells benefit from: increased thiol production to scavenge thiolreactive molecules such as cisplatin.

CONCLUSIONS

This study shows that acquired radiation resistance also induces cisplatin cross-resistance via an elevation of thiol expression. Moreover, this cisplatin cross-resistance can be overcome by preventing thiol generation with a glutathione pathway inhibitor. Further investigations are needed to define whether the adoption of carboplatin or oxaliplatin-based regimens in lung cancer or other solid tumor treatments in patients would indeed improve outcomes by circumventing possible cross-resistance. Finally, although clinical studies are warranted to validate these findings in patients, our results additionally suggest that combinatorial treatment strategies of glutamine pathway inhibitors, with chemoradiation will improve the treatment outcome of cancer patients by overcoming potential acquired resistance.

Supplementary Material

Supplementary date 1

Supplementary Fig. S1. Acquired resistance does not alter seeding efficiency or doubling time. Panel A: The seeding efficiency and (panel B) doubling time of LLC and LLC-RR. Panel C: The seeding efficiency and (panel D) doubling time of A549 and A549-RR. Panel E: Cell cycle distribution of LLC and LLC-RR with or without 2 Gy. Panel F: Cell cycle distribution of A549 and A549-RR with or without 2 Gy. All determinations are based on passage-matched counterparts. ns = not significant.

Supplementary Fig. S2. Acquired radiation resistance induces EMT markers on A549, whereas the EMT markers on LLC do not statistically change. Panel A: E-cadherin, panel B: vimentin, and panel C: double positive cells (DP) profiles of LLC and A549 as determined by flow cytometry. Panel D: E-cadherin, panel E: vimentin, and panel F: double positive cells (DP) profiles of A549 and A549-RR with and without radiation. A549-RR have intrinsic higher expression levels of E-cadherin, vimentin and DP as compared to A549, and 2 Gy radiation increased expression levels of E-cadherin, vimentin and DP on A549. Whereas radiation (2 Gy) increased expression levels of E-cadherin, vimentin and DP on A549, it does not further increase expression levels of then EMT markers on A549-RR (panels D–F). Panel G: Migration is suppressed in A549-RR, as compared to A549. Panel H: Migration after 2 Gy of radiation is similar in A549 and A549-RR. Panel I: Radiation (2 Gy) suppressed migration of A549, whereas (panel J) A549-RR migrate faster. Data presented as mean ± SEM, representative experiment shown from 3 experiments with triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test. ns = not significant.

Supplementary Fig. S3. Influence of acquired radiation resistance on EMT markers on LLC. Panel A: E-cadherin, (panel B) vimentin, and (panel C) double positive cells (DP) profiles of LLC and LLC-RR. Expression levels of E-cadherin, vimentin and DP are not statistically different between LLC and LLC-RR. Radiation (2 Gy) increases E-cadherin on LLC and LLC-RR, and vimentin is differentially more expressed on LLC-RR than LLC after irradiation. DP population is not affected. Data presented as mean ± SEM, representative experiment shown from 3 experiments with triplicates. *P < 0.05; **P < 0.01 two-tailed t-test. ns = not significant.

Supplementary Fig. S4. A549-RR tumors are resistant to radiation. Panel A: A549 and A549-RR have similar tumor growth kinetics and (panel B) tumor mass after excision. Panel C: A549 tumor growth is inhibited by 4 × 2 Gy irradiation. Panel D: The tumor mass of A549 is significantly less after irradiation, whereas (panel E) A549-RR tumor growth and their (panel F) mass is not affected by 4 × 2 Gy irradiation. Panel G: A549 and A549-RR body weights are not affected during the study, and (panel H) at the end of the study shown as individual mice. Arrows indicate time of 2 Gy irradiation on days 24, 27, 30 and 33. *P < 0.05; two-tailed t-test. ns = not significant.

Supplementary Fig. S5. Induced-cisplatin cross-resistance is thiol dependent. Panel A: Radiation-induced free thiols in A549, and (panel B) A549-RR. Panel C: BSO reduced free thiols in A549-RR. Panel D: Acquired cross-resistance against cisplatin was overcome by BSO in A549-RR. Panel E: BSO does not have an effect on the cell viability of LLC, LLC-RR, A549, A549-RR. Data presented as mean ± SEM, representative experiment shown from 3 experiments with triplicates. BSO = Buthionine sulfoximine [5 mM]. *P < 0.05; two-tailed t-test. ns = not significant.

NIHMS1973344-supplement-Supplementary_date_1.pdf^{(625.4KB, pdf)}

ACKNOWLEDGMENTS

The study was supported by in part by the National Institutes of Health/National Cancer Institute and National Institute of General Medical Sciences (R01 CA245083, P20 GM103429, P20 GM103625 and P30 GM145393), Department of Defense W81XWH-21–1-0577 through the Congressionally Directed Medical Research Programs to RPMD. The study was also supported in part National Institutes of Health (R01 CA238025) to NR and P20 GM109005 to SVJ through the Center for Host Response to Cancer Therapy. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Footnotes

Editor’s note. The online version of this article (DOI: https://doi.org/10.1667/RADE-23-00005.1) contains supplementary information that is available to all authorized users.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary date 1

NIHMS1973344-supplement-Supplementary_date_1.pdf^{(625.4KB, pdf)}

[R1] 1.Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 2011; 11:239–53. [DOI] [PubMed] [Google Scholar]

[R2] 2.Delaney G, Jacob S, Featherstone C, Barton M. The role of radiotherapy in cancer treatment: Estimating optimal utilization from a review of evidence-based clinical guidelines. Cancer. 2005; 104:1129–37. [DOI] [PubMed] [Google Scholar]

[R3] 3.Bansal N, Mims J, Kuremsky JG, Olex AL, Zhao W, Yin L, et al. Broad phenotypic changes associated with gain of radiation resistance in head and neck squamous cell cancer. Antioxid Redox Signal. 2014; 21:221–36. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Reisz JA, Bansal N, Qian J, Zhao W, Furdui CM. Effects of ionizing radiation on biological molecules - mechanisms of damage and emerging methods of detection. Antioxid Redox Signal. 2014; 21:260–92. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Fowler JF. 21 Years of biologically effective dose. Br J Radiol. 2010; 83:554–68. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Rossi A, Di Maio M. Platinum-based chemotherapy in advanced non-small-cell lung cancer: optimal number of treatment cycles. Expert Rev Anticancer Ther. 2016; 16:653–60. [DOI] [PubMed] [Google Scholar]

[R7] 7.Zou Z, Chang H, Li H, Wang S. Induction of reactive oxygen species: an emerging approach for cancer therapy. Apoptosis. 2017; 22:1321–35. [DOI] [PubMed] [Google Scholar]

[R8] 8.Jenkins SV, Alimohammadi M, Terry AS, Griffin RJ, Tackett AJ, Leung JW, et al. Dysbiotic stress increases the sensitivity of the tumor vasculature to radiotherapy and c-Met inhibitors. Angiogenesis. 2021; 24(3):597–611. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Prevc A, Kranjc S, Cemazar M, Todorovic V, Zegura B, Novak M, et al. Dose-Modifying Factor of Radiation Therapy with Concurrent Cisplatin Treatment in HPV-Positive Squamous Cell Carcinoma: A Preclinical Study. Radiat Res. 2018; 189(6):644–51. [DOI] [PubMed] [Google Scholar]

[R10] 10.Jenkins SV, Nima ZA, Vang KB, Kannarpady G, Nedosekin DA, Zharov VP, et al. Triple-negative breast cancer targeting and killing by EpCAM-directed, plasmonically active nanodrug systems. NPJ Precis Oncol. 2017. Dec; 1(1). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Läppchen T, Dings RPM, Rossin R, Simon JF, Visser TJ, Bakker M, et al. Novel analogs of antitumor agent calixarene 0118: Synthesis, cytotoxicity, click labeling with 2-[18F]fluoroethylazide, and in vivo evaluation. Eur J Med Chem. 2015; 89:279–95. [DOI] [PubMed] [Google Scholar]

[R12] 12.Kelliher JL, West KL, Gong Q, Leung JWC. Histone H2A variants alpha1-extension helix directs RNF168-mediated ubiquitination. Nat Commun. 2020; 11(1). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Dings RPM, Van Der Schaft DWJ, Hargittai B, Haseman J, Griffioen AW, Mayo KH. Anti-tumor activity of the novel angiogenesis inhibitor anginex. Cancer Lett. 2003; 194(1):55–66. [DOI] [PubMed] [Google Scholar]

[R14] 14.van der Schaft DW, Dings RP, de Lussanet QG, van Eijk LI, Nap AW, Beets-Tan RG, et al. The designer anti-angiogenic peptide anginex targets tumor endothelial cells and inhibits tumor growth in animal models. Faseb J. 2002; 16(14):1991–3. [DOI] [PubMed] [Google Scholar]

[R15] 15.Dings RPM, Kumar N, Mikkelson S, Gabius HJ, Mayo KH. Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity. Biochem Biophys Rep. 2021. Dec; 28:101116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Dings RPM, Levine JI, Brown SG, Astorgues-Xerri L, MacDonald JR, Hoye TR, et al. Polycationic calixarene PTX013, a potent cytotoxic agent against tumors and drug resistant cancer. Invest New Drugs. 2013. Oct; 31(5):1142–50. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Nowak-Sliwinska P, Alitalo K, Allen E, Anisimov A, Aplin AC, Auerbach R, et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis. 2018; 21:425–532. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Jenkins SV, Robeson MS, Griffin RJ, Quick CM, Siegel ER, Cannon MJ, et al. Gastrointestinal tract dysbiosis enhances distal tumor progression through suppression of leukocyte trafficking. Cancer Res. 2019; 79(23):5999–6009. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Dings RPM, Vang KB, Castermans K, Popescu F, Zhang Y, Oude Egbrink MGA, et al. Enhancement of T-cell-mediated antitumor response: Angiostatic adjuvant to immunotherapy against cancer. Clinl Cancer Res. 2011; 17(10):3134–45. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Dings RPM, Williams BW, Song CW, Griffioen AW, Mayo KH, Griffin RJ. Anginex synergizes with radiation therapy to inhibit tumor growth by radiosensitizing endothelial cells. Int J Cancer. 2005. Jun 10; 115(2):312–9. [DOI] [PubMed] [Google Scholar]

[R21] 21.Garon EB, Hellmann MD, Rizvi NA, Carcereny E, Leighl NB, Ahn MJ, et al. Five-Year Overall Survival for Patients with Advanced Non-Small-Cell Lung Cancer Treated with Pembrolizumab: Results From the Phase I KEYNOTE-001 Study. J Clin Oncol. 2019; 37:2518–27. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Csőszi T, Andrea F. et al. Five-Year Outcomes With Pembrolizumab Versus Chemotherapy for Metastatic Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score ≥50. J Clin Oncol. 2021; 39:2339–49. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Lewis JE, Forshaw TE, Boothman DA, Furdui CM, Kemp ML. Personalized Genome-Scale Metabolic Models Identify Targets of Redox Metabolism in Radiation-Resistant Tumors. Cell Syst. 2021. Jan 20; 12(1):68–81.e11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Dadgar S, Troncoso JR, Siegel ER, Curry NM, Griffin RJ, Dings RPM, et al. Spectroscopic investigation of radiation-induced reoxygenation in radiation-resistant tumors. Neoplasia (United States). 2021. Jan 1; 23(1):49–57. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Lin X, Kong D, Chen ZS. Editorial: Chemo-Radiation-Resistance in Cancer Therapy. Front Pharmacol. 2022. May 18; 13. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance

Samir V Jenkins

Shruti Shah

Azemat Jamshidi-Parsian

Amir Mortazavi

Hailey Kristian

Gunnar Boysen

Kieng B Vang

Robert J Griffin

Narasimhan Rajaram

Ruud PM Dings

Abstract

INTRODUCTION

MATERIALS AND METHODS

Cell Lines and Chemicals

Irradiation

Clonogenic Assay

Cell Viability

Gamma H2AX Assay

Apoptosis

Cell Cycle Analysis

Nuclear/Cytoplasmic Ratio Determination

Migration

In vivo NSCLC Models

Flow Cytometry

Thiol Determination

Statistical Analysis

RESULTS

Generating Acquired Radiation Resistance in NSCLC

FIG. 1.

Phenotypic Characterization

FIG. 2.

Epithelial and Mesenchymal Markers are Elevated due to Acquired Resistance

Acquired Radiation Resistance is Maintained In Vivo

FIG. 3.

Acquired Radiation Resistance causes Cisplatin Cross-Resistance

FIG. 4.

FIG. 5.

Acquired Radiation Resistance is Thiol Dependent

FIG. 6.

DISCUSSION

CONCLUSIONS

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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