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. 2025 Jan:202:110646.
doi: 10.1016/j.radonc.2024.110646. Epub 2024 Nov 21.

Altering fractionation during radiation overcomes radio-resistance in patient-derived glioblastoma cells assessed using a novel longitudinal radiation cytotoxicity assay

Lauren C Nassour-Caswell  1 , Manoj Kumar  2 , Christian T Stackhouse  3 , Hasan Alrefai  4 , Taylor L Schanel  5 , Benjamin M Honan  6 , Andee M Beierle  7 , Patricia H Hicks  8 , Joshua C Anderson  9 , Christopher D Willey  10 , Jeffrey S Peacock  11
Affiliations

Altering fractionation during radiation overcomes radio-resistance in patient-derived glioblastoma cells assessed using a novel longitudinal radiation cytotoxicity assay

Lauren C Nassour-Caswell et al. Radiother Oncol. 2025 Jan.

Abstract

Purpose: Current radiotherapy (RT) in glioblastoma (GBM) is delivered as constant dose fractions (CDF), which do not account for intratumoral-heterogeneity and radio-selection in GBM. These factors contribute to differential treatment response complicating the therapeutic efficacy of this principle. Our study aims to investigate an alternative dosing strategy to overcome radio-resistance using a novel longitudinal radiation cytotoxicity assay.

Methods: Theoretical In-silico mathematical assumptions were combined with an in-vitro experimental strategy to investigate alternative radiation regimens. Patient-derived xenograft (PDX) brain tumor-initiating cells (BTICs) with differential radiation-sensitivities were tested individually with sham control and three regimens of the same nominal and average dose of 16 Gy (over four fractions), but with altered doses per fraction. Fractions were delivered conventionally (CDF: 4, 4, 4, 4 Gy), or as dynamic dose fractions (DDF) "ramped down" (RD: 7, 5, 3, 1 Gy), or DDF "ramped up" (RU: 1, 3, 5, 7 Gy), every 4 days. Interfraction-longitudinal data were collected by imaging cells every 5 days, and endpoint viability was taken on day 20.

Results: The proposed method of radiosensitivity assessment allows for longitudinal-interfraction investigation in addition to endpoint analysis. Delivering four-fraction doses in an RD manner proves to be most effective at overcoming acquired radiation resistance in BTICs (Relative cell viability: CDF vs. RD: P < 0.0001; Surviving fraction: CDF: vs. RD: P < 0.0001).

Conclusions: Using in-silico cytotoxicity prediction modeling and an altered radiosensitivity assessment, we show DDF-RD is effective at inducing cytotoxicity in three BTIC lines with differential radiosensitivity.

Keywords: BTIC; Cell viability; Glioblastoma; Radiosensitivity; Xenograft.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1:
Figure 1:. Schematic summary of constant dose fraction and proposed altered fractionation based
. (A) Standard CDF (top row, pink RT bolts) delivery to radiosensitive cells (blue) are preferentially killed early on, whereas radioresistant subpopulations emerge (red) or persist (yellow). Eventually, resistant phenotypes dominate the population. DDF-RD (purple RT bolts) during radiation could compensate for the evolving radioresistance, leading to a higher chance for tumor eradication (middle row), whereas DDF-RU (teal RT bolts) could potentially select for it (bottom row). (B) To model radiotherapy effects, at each treatment fraction delivery (tRT), a proportion of (1 - S) of the viable tumor (Vl) is transferred to a dying compartment, Vd. S is the surviving fraction. Here, t−RT denotes the time immediately before delivery of a radiation fraction, t+RT the time immediately after treatment delivery. (C) Volume change as an exponential reduction of Vd(t) at rate λ, the growth rate. (D) Methodology of alternative radiosensitivity assessment. Cells obtained from an anonymous patient donor with classical GBM were grown as xenografts in athymic nude mice and harvested as radiation-sensitive or selected to be acquired radiation-resistant. Cells were plated and irradiated according to respective RT dosage and fraction. Interfraction data was collected every 5 days in addition to endpoint viability. All schematics were created with Biorender.
Figure 2:
Figure 2:. Longitudinal Assessment of Patient-derived Brain Tumor Initiating Cells with Differential RT-Sensitivity.
(A) Cell division rate in JX14P and JX14P-RT when stained with Carboxyfluorescein, succinimidyl ester (CFSE) dye (24h: P<0.0001, 48h: P<0.0017). (B) Cell division rate in JX14P-RT in FBS-containing PDX media compared to stem-promoting PDX media (24h: P<0.0001, 48h: P<0.0001). (C) Sphere formation comparison between JX14P and JX14P-RT demonstrating a larger quantity and average size in JX14P-RT compared to JX14P (Sphere counts: P<0.0001; Average sphere size: P<0.0001). Representative image of neurosphere formation in JX14P and JX14P-RT stained with Calcein-AM after 6 days are shown. (D) Representative images of adherent cell growth on day 1 vs. day 20 mCherry stably expressing cells. (E) Longitudinal growth assessment based on surviving cell ratio in JX14P vs. JX14P-RT (Day 20: P>0.0001, N=4). (F) (E) Longitudinal growth assessment based on relative fluorescence in JX14P and JX14P-RT (Day 20: P>0.0001, N=4). (G) Dose-response of JX14P and JX14P-RT relative cell viability when treated with 3 or 5 Gy (3Gy: P<0.0001; 5Gy: P<0.0001). Error bars represent standard error of mean for all graphs.
Figure 3:
Figure 3:. DDF-RD overcomes radio-resistance in acquired RT-resistant BTICs.
(A) Longitudinal surviving cell ratio in JX14P (Day 20: CDF: 0.29 ± 0.03 vs. RD: 0.28 ± 0.01, P>0.9999; RU: 0.47 ± 0.03, RU vs. CDF: P=0.0253; RU vs. RD: P=0.0205, N=4). (B) Longitudinal relative fluorescence in JX14P (Day 20: CDF: 0.90 ± 0.07 vs. RD: 0.82 ± 0.05, P=0.7364; RU: 1.2 ± 0.01, RU vs. CDF: P=0.0264; RU vs. CDF: P=0.0056, N=4). (C) Relative cell viability in JX14P (CDF vs. RD: P=0.8546; CDF vs. RU: P=0.5996; RD vs. RU: P=0.8941). (D) Longitudinal surviving cell ratio in JX14P-RT (Day 20: Day 20: CDF: 1.22 ± 0.05 vs. RD: 0.92 ± 0.02, P=0.0294; RU: 2.10 ± 0.05, RU vs. CDF: P=0.0006; RU vs. RD: P=0.0002, N=4). (E) Longitudinal relative fluorescence in JX14P-RT (CDF: 3.42 ± 0.15 vs. RD: 2.03 ± 0.13, P=0.0050; RU: 9.01 ± 0.04, RU vs. CDF: P<0.0001; RU vs. RD: P<0.0001, N=4). (F) Relative cell viability in JX14P-RT (CDF vs. RD: P<0.0001; CDF vs. RU: P<0.0001; RD vs. RU: P<0.0001). (G) Correlation between relative cell viability and surviving cell ratio in JX14P (R2 = 0.7026, P=0.3672, Y=0.2525X+0.2609). (H) Correlation between relative cell viability and relative fluorescence in JX14P (R2 = 0.5506, P=0.4678, Y=0.1098X+0.2387). (I) Correlation between relative cell viability and surviving cell ratio in JX14P-RT (R2 = 1.000, P=0.0007, Y = 4.480X – 2.465). (H) Correlation between relative cell viability and relative fluorescence in JX14P-RT (R2 = 0.9967, P=0.0364, Y = 0.7325X + 0.3206). (K) Representative images of surviving cell ratio and relative fluorescence in JX14P and JX14P-RT CT, CDF, RD, and RU groups on day 20. (L) Representative images of colony formation assay of JX14P and JX14P-RT CT, CDF, RD, and RU groups on day 20. (M) Surviving fraction in JX14P (CDF vs. RD: P>0.9999; CDF vs. RU: P=0.0003; RD vs. RU: P=0.0003). (N) Surviving fraction in JX14P-RT (CDF vs. RD: P=0.0308; CDF vs. RU: P=0.0008; RD vs. RU: P<0.0001). Error bars represent standard error of mean for all graphs.
Figure 4:
Figure 4:. CDF and DDF-RD induce cytotoxicity in inherently resistant BTICs.
(A) Longitudinal surviving cell ratio in X1441 (Day 20: CDF: 0.02 ± 0.003 vs. RD: 0.03 ± 0.12, P=0.9614; RU: 0.59 ± 0.02; RU vs. CDF: P=0.0998; RU vs. RD: P=0.0988, N=3). (B) Longitudinal relative fluorescence in X1441 (Day 20: CDF: 1.10 ± 0.03 vs. RD: 1.09 ± 0.08, P=0.9851; RU: 2.39 ± 0.01; RU vs. CDF: P=0.0040; RU vs. RD: P=0.0082, N=3). (C) Relative cell viability in X1441 (CDF vs. RD: P=0.9871; CDF vs. RU: P=0.0009; RD vs. RU: P=0.0008). (D) Correlation between relative cell viability and surviving cell ratio on day 20 (R2 = 0.9775, P=0.0960, Y = 0.8372X + 0.01874). (E) Correlation between relative cell viability and relative fluorescence on day 20 (R2 = 0.9549, P=0.1363, Y = 0.2194X + 0.2042). (F) Representative images of surviving cell ratio and relative fluorescence on day 20. (G) Quantification and representative images of X1441 colony formation assay when treated with CT, CDF, RD, and RU (CDF vs. RD: P=0.9903; CDF vs. RU: P<0.0001; RD vs. RU: P<0.0001). Error bars represent standard error of mean for all graphs.
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