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. 2025 Aug 1;15:98. doi: 10.1186/s13550-025-01302-x

Chemotherapy alters radiosensitivity and GRPR expression of prostate and breast cancer cells

Tyrillshall ST Damiana 1, Lilian van den Brink 1, Lisette W de Kreij-de Bruin 1, Debra C Stuurman 1, Corrina MA de Ridder 2, Simone U Dalm 1,✉
PMCID: PMC12316622  PMID: 40751101

Abstract

Background

Gastrin releasing peptide receptor (GRPR)-targeting radiotracers have been studied (pre)-clinically with promising results. Patients eligible for this treatment are likely to have undergone prior treatments with other anti-cancer agents, including chemotherapy. Chemotherapies are known to alter cancer cell’s gene expression and radiosensitivity, potentially impacting GRPR expression and the response to radionuclide therapy. We studied the effect of two commonly applied chemotherapies, doxorubicin (DXR) and docetaxel (DTX), on GRPR expression, GRPR radiotracer uptake, and response to external beam radiation therapy (EBRT) and targeted radionuclide treatment, in prostate cancer (PCa) and breast cancer (BC) cells. Additionally, in-vivo uptake of the GRPR-targeting radiotracer “NeoB” in PC-3 and T47D xenograft-bearing mice was assessed using SPECT/CT following chemotherapy treatment.

Results

DTX significantly decreased GRPR expression, radiotracer uptake, and radiosensitivity of PC-3 cells in-vitro. DXR pre-treated T47D cells demonstrated an increased GRPR expression and radiotracer uptake, and were less sensitive to EBRT. In-vivo, DTX pre-treatment increased [177Lu]Lu-NeoB uptake in PC-3 xenografts, but this was not GRPR mediated. DXR pre-treatment did not alter [177Lu]Lu-NeoB uptake in T47D xenografts, but an increase in GRPR mRNA expression was observed.

Conclusion

Our data demonstrated that chemotherapy alters mechanisms relevant for the success of GRPR-mediated radionuclide therapy in PCa and BC cells in-vitro. These finding were less prominent in-vivo and additional studies are needed to unravel this.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13550-025-01302-x.

Keywords: GRPR, NeoB, Chemotherapy, Prostate cancer, Breast cancer

Background

The gastrin-releasing peptide receptor (GRPR) is a G-protein coupled receptor involved in a variety of physiological processes such as signal transmission, smooth muscle contraction and release of gastrointestinal hormones. GRPR is also known to be associated with many pathological indications. In particular GRPR has been shown to promote growth and differentiation of tumor cells. In line herewith, overexpression of the GRPR is described for various tumor types including prostate cancer (PCa) and breast cancer (BC). The overexpression of GRPR in these cancer types, and the low/lack of GRPR expression in healthy organs, makes the receptor a promising target for targeted radionuclide imaging and therapy (TRT) [1, 2]. Accordingly, many GRPR-targeting radiopharmaceuticals have been developed and successfully studied preclinically for radionuclide imaging and treatment. The results of these studies are promising, and indicate the clinical potential of GRPR-targeting radiopharmaceuticals for management of GRPR-expressing cancers [3–6]. Currently, multiple clinical studies evaluating the safety and efficacy of GRPR-targeting radiopharmaceuticals are ongoing, e.g., NCT05870579, NCT05283330, and NCT03872778.

TRT is currently only applied for treatment of advanced disease, i.e. TRT directed at the somatostatin receptor subtype 2 and the prostate specific membrane antigen, the two routinely applied TRTs in the clinic so far, are approved for treatment of neuroendocrine tumors and prostate cancer, respectively, at advanced disease stages [7, 8]. Similarly, the aforementioned clinical trials evaluating GRPR-targeted TRT are all performed in patients with advanced disease. These patients have an extensive treatment history which might impact the success for TRT.

For one, studies have shown that anti-cancer treatments, including chemotherapy, can alter gene expression, and thus prior treatment can affect expression of genes relevant for TRT efficacy, including expression of the targeted biomarker [9–13]. So, preclinical and clinical studies that reported on up and downregulation of genes after chemotherapy, demonstrated that the majority of these genes are involved in cell cycle, apoptosis, and DNA repair mechanisms [14–16]. Moreover, potential downregulation of the targeted biomarker, would negatively influence the uptake of radiopharmaceuticals. In fact, studies have reported changes in the expression of SSTR2 and PSMA after chemotherapy [17–19]. To the best of our knowledge this has not been studied for GRPR yet.

Related to the above, chemotherapy treatment has also been associated with a change in cancer cell’s radiosensitivity. Several studies demonstrated that chemotherapy has a radiosensitizing effect. However, this radiosensitizing effect was only observed in combined chemoradiation approaches [20–23]. Radioresistance, however, is acquired in cells that survive chemotherapy. These cells undergo changes which allows them to withstand treatments that cause e.g., cell cycle dysregulation and pathway inhibition, and most important DNA damage, all which are induced by most chemotherapeutic agents [14, 24, 25]. As TRT efficacy is primarily based on radiation induced DNA damage, these changes might influence TRT efficacy as well.

Commonly used chemotherapies for GRPR-expressing PCa and BC are doxorubicin (DXR) and docetaxel (DTX). Both DTX and DXR have demonstrated the ability to alter gene expression levels and potentially radiosensitivity of cancer cells [9, 16]. DXR’s antitumor effect is based on two primary mechanisms of action: (1) DNA intercalation and (2) inhibition of the topoisomerase II enzyme. This mechanism of action of DXR leads to DNA damage and inhibition of both DNA and RNA synthesis, potentially leading to cell death [26, 27]. DTX’s primary mechanism of action is to inhibit microtubule depolymerization by binding to beta-tubulin. By doing so, DTX disrupts the normal mitotic process and arrests the cells in the G2M phase, which can lead to cell death [28]. Figure 1 demonstrates the mechanism of action of DTX and DXR.

Fig. 1.

Fig. 1

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Schematic overview of the mechanism of action of (left) DXR and (right) DTX

As the majority of PCa and BC patients that initially will be eligible for GRPR-mediated TRT have received prior treatment with DTX or DXR, there is a need to determine how these treatments effects the GRPR expression and sensitivity of the cancer cells to TRT to correctly position GRPR-targeting radiopharmaceuticals in the clinical management of these diseases.

Methods

Cell culture

The human PCa cell line PC-3 and the human BC cell line T47D were cultured in Ham’s F-K12 nutrient mix (Gibco) or RPMI (Gibco), respectively, supplemented with 10% fetal bovine serum (Gibco) and penicillin (100 IU/ml)/streptomycin (100 µg/ml). Cells were cultured in T175 flasks (CELLSTAR, Greiner bio-one) at 37˚C in a humidified atmosphere of 5% CO2 of air (NuAire), and passaged routinely.

IC50 doxorubicin and docetaxel

PC-3 cells (3 × 103) and T47D cells (5 × 103) were seeded in 96 well-plates and cultured overnight. Hereafter, cells were treated with increasing concentrations of either DXR (doxorubicin hydrochloride; Sigma Aldrich) or DTX (anhydrous docetaxel; Sigma Aldrich) in the presence of the vehicle (0.4 µM dimethyl sulfoxide, DSMO; Sigma Aldrich) for 72 h at 37 °C in a humidified atmosphere with 5% CO2. Cells were then washed twice with PBS, fresh culture media was added and cells were left to grow for 48 h. Subsequently, cells were fixed with 10% trichloroacetic acid (TCA; Sigma Aldrich) overnight, and stained with 0.5% SRB for 20 min. The excess dye was removed by washing the cells repeatedly with 1% acetic acid (Sigma Aldrich) and plates were left to dry. Subsequently, the protein-bound dye was dissolved in 10 mM Tris base solution, and the optical density (OD) was measured with a spectrometer (SpectraMax) at 560 nm wavelength. To determine cell density, OD of untreated cells was set at 100% and that of chemotherapy treated cells was expressed relative to that of the untreated cells.

Drug pre-treatment

PC-3 cells (1.2 × 106) and T47D cells (4 × 106) were seeded into T175 flask and cultured overnight. Hereafter, cells were treated with the IC50 concentrations of either DXR or DTX in the presence of 0.4% DSMO for 72 h at 37 °C in a humidified atmosphere with 5% CO2. Cells were then washed twice with PBS, fresh culture media was added and cells were left to grow for 24 h. Hereafter cells were used in the following experiments.

Uptake assay

After drug pre-treatment, 2.5 × 105 PC-3 cells, and 6 × 105 T47D cells were seeded into 12 well-plates. The next day, adherent cells were incubated with 10− 9 M [111In]In-NeoB for 1 h at 37 °C. Hereafter, cells were washed twice with cold PBS and 1 M NaOH was added for 20 min at room temperature. The cell lysate was collected and measured in a γ-counter (1480 WIZARD automatic γ-counter; PerkinElmer). To correct for cell density, cells from two separate wells were counted (Countess counter, Invitrogen) and data was expressed as percentage of added dose (%AD) per 2.5 × 105 PC-3 cells or 6 × 105 T47D cells.

Radiosensitivity

Following drug pre-treatment, 4 × 104 PC-3 cells, and 3.5 × 104 T47D cells were seeded into 24 well-plates. The following day, cells were irradiated with 0, 2, 4, 6, 8, 10, 12, and 14 Gy (Gy) using an external beam (external beam radiation treatment; EBRT) RS320 X-Ray irradiator at a dose rate of 0.5 mm Cu Gy/min. Cells were then incubated for 5 days at 37˚C in a humidified atmosphere with 5% CO2. Cell density was determined using an SRB assay as described above.

Sensitivity to GRPR-mediated radionuclide therapy

DXR and DTX pretreated PC-3 cells (2 × 106) and T47D cells (3 × 106) were seeded into T25 flasks. The following day, cells were washed twice with PBS and incubated with 5 mL of 50 MBq/ml [177Lu]Lu-NeoB, [177Lu]Lu-DTPA (the latter to determine GRPR specificity of treatment), or medium for 1 h at 37˚C in a humidified atmosphere with 5% CO2. Hereafter, cells were washed with PBS, and 3 mL Ham’s F-K12 or RPMI medium was added to the PC-3 and T47D cells, respectively. Cells were then scraped and collected into 15 mL tubes. The collected cells fractions were centrifuged 5 min at 1500 rpm, and resuspended in 3 mL culture medium. 2 × 103 PC-3 cells and 4 × 103 T47D cells were seeded into 96 well plates and cultured for 6 days at 37˚C in a humidified atmosphere with 5% CO2. Subsequently, cells were fixed with TCA and cell density was determined using the SRB assay as previously described. The results are expressed as % cell density relative to non-irritated cells.

Animals and drug treatment

All animal experiments were approved by the Animal Welfare Committee of the Erasmus MC, and conducted in accordance to institutional guidelines. Male and female NMRI-Foxn1 nu/nu mice (6 weeks old) were subcutaneously inoculated with 5 × 106 PC-3 or 8 × 106 T47D cells, respectively, on the right shoulder (100 µL: 1/3 Matrigel (Corning) and 2/3 Hank’s Balanced Salt Solution (Gibco)). Tumors were grown for 2 weeks resulting in an average volume of 116 ± 30 mm3 and 75 ± 25 mm3 for PC-3 and T47D cells, respectively. Hereafter, the mice bearing PC-3 xenografts were treated with an injection of 5 mg/kg DTX in polysorbate-80 (1:1) diluted in 5% glucose (N = 4) or placebo (polysorbate-80 (1:1) diluted in 5% glucose) (N = 4) once a week for 3 consecutive weeks [29]. T47D tumor bearing female mice received an injection of 5 mg/kg DXR in polysorbate-80 (1:1) diluted in 5% glucose (N = 4) or placebo (N = 4) once a week for 2 weeks [30]. As T47D BC cells are estrogen receptor positive, T47D tumor bearing female animals were supplemented with β-estradiol (4 mg/L; Sigma-Aldrich) in drinking water during the entire length of the study. Figure 2 shows an overview of the study design.

Fig. 2.

Fig. 2

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Schematic overview of the time schedule for the in-vivo studies performed with NMRI-nude animals. *Female T47D tumor bearing animals were euthanized after de second SPECT/CT cycle for biodistribution purposes

The mean body weight of male animals was 34.35 ± 1.39 g (N = 8), while that of female animals was 26.02 ± 0.95 g (N = 7), based on measurements obtained from the first through the final treatment cycle.

In-vivo tumor growth and biodistribution

Four days after each injection of DTX, DXR, or placebo (days 4, 11, and 18), animals received 4 MBq/200 pmol [177Lu]Lu-NeoB, with an average injected dose of 4.01 ± 0.15 and 3.80 ± 0.40 for male (N = 8) and female (N = 7) animals, respectively. After 24 h whole-body single-photon emission computed tomography and computed tomography (SPECT/CT; VECTor/CT, MILabs) images were acquired. Following the final scan, organs were collected for biodistribution analysis. Tumors were divided into two portions: one half for biodistribution studies, and the other half was processed for GRPR mRNA expression analysis. Detailed methods for SPECT/CT, biodistribution, and RT-qPCR analysis of GRPR mRNA expression are provided in the supplementary material (Supplementary method 1 & 2, and supplementary Table 1).

Statistics

All in-vitro studies were performed in triplicate, and data are expressed as mean ± standard deviation (SD). Statistical analysis were performed using GraphPad Prism 9.0.0 Software (La Jolla, California). IC50 curves were performed by nonlinear fit using the [Inhibitor] vs. response-Variable slope (four parameters) with a confidence interval level of 0.05 and 95%. Significance in in-vitro and in-vivo uptake, and GRPR mRNA expression level were determined using One-Way ANOVA followed by Dunnett’s multiple comparisons test with a confidence interval level of 0.05 and 95%.

Significance in sensitivity to EBRT and [177Lu]Lu-NeoB, as well as tumor volume were determined using Two-Way ANOVA followed by Dunnett’s multiple comparisons test with a confidence interval level of 0.05 and 95%.

Results

IC50 DXR and DTX

The DXR IC50 value was 22.68 ± 4.17 nM and 22.33 ± 7.04 nM for PC-3 and T47D cells, respectively (Fig. 3a and b). PC-3 cells were less sensitive to DTX compared to T47D cells, with IC50 values of 14.05 ± 3.72 nM and 0.94 ± 0.39 nM, respectively (Fig. 3c and d).

Fig. 3.

Fig. 3

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IC50 concentration of DXR and DTX for PC-3 cells (a, c) and T47D cells (b, d). Data is expressed as cell density relative to untreated cells. N = 3 individual experiments, with technical triplicates

GRPR mRNA expression and radiotracer uptake

GRPR mRNA expression level in DXR treated PC-3 cells did not alter compared to untreated cells (93.20 ± 7.11%), while a significant increase in GRPR mRNA expression level was observed in DXR treated T47D cells (122.17 ± 6.82% relative to untreated cells set at 100% (P < 0.001)). In PC-3 cells, DTX treatment decreased GRPR mRNA expression significantly (42.30 ± 4.55%, P < 0.001), but DTX did not alter GRPR mRNA expression in T47D cells (99.53 ± 2.95%) (Fig. 4a and b). The observed change in GRPR mRNA expression following treatment was also evident in the uptake of the GRPR radiopharmaceutical [111In]In-NeoB. [111In]In-NeoB uptake decreased to 47.50 ± 4.74% AD (P = 0.05) in DTX pretreated PC-3 cells and increased to 242.44 ± 65.41% AD (P = 0.02) in DXR pretreated T47D cells (Fig. 4c and d).

Fig. 4.

Fig. 4

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GRPR mRNA expression levels of treated relative to vehilce treated (a) PC-3 and (b) T47D cells after DXR and DTX treatement. GRPR radiotracer uptake of [111In]In-NeoB in DXR and DTX pretreated (c) PC-3 and (d) T47D cells. Uptake is expressed as precentage added dose (% AD) per 2.5 × 105 PC-3 cells or 6 × 105 T47D cells. N = 3 individual experiments, with technical triplicates

Radiosensitivity

Vehicle and DXR pretreated PC3-cells showed a decrease in cell density after EBRT in a dose dependent manner (Fig. 5a). Remarkably, EBRT did not affect the cell density of the DTX pretreated PC-3 cells; cell density of the irradiated (2–14 Gy) DTX pretreated PC-3 cells were in the same range as of the non-irradiated (0 Gy) DTX treaded cells. Concerning T47D cells, vehicle and DTX pretreated cells showed a dose dependent response to EBRT, but the cell density of the irradiated DXR treated T47D cells remained in the same range as that of non-irradiated (0 Gy) DXR pre-treaded T47D cells (Fig. 5b).

Fig. 5.

Fig. 5

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Cell density of vehicle, DXR and DTX pretreated (a) PC-3 and (b) T47D cells 5 days after EBRT. Data is expressed % of untreated (0 Gy) cells. N = 3 individual experiments, with technical triplicates

Sensitivity to GRPR-mediated radionuclide therapy

The cell density of non-pretreated, and DTX pretreated PC-3 cells, treated with [177Lu]Lu-NeoB was lower compared to medium and [177Lu]Lu-DTPA treated cells (Fig. 6a). However, this difference was only significant between the medium and [177Lu]Lu-NeoB treated PC-3 cells of the non-chemotherapy pretreated PC-3 cells. Interestingly, the vehicle pretreated PC-3 cells showed no clear difference in cell density between the medium, [177Lu]Lu-DTPA or [177Lu]Lu-NeoB treated conditions. Furthermore no significant difference in cell density was observed between the non-chemotherapy pretreated, vehicle and DTX cells treated with 50 MBq [177Lu]Lu-NeoB. Non-chemotherapy pretreated PC-3 cells that were treated with [177Lu]Lu-NeoB had the lowest cell density (76.86 ± 18.30%), followed by DTX treated cells with (81.24 ± 12.61%) and vehicle treated cells (98.91 ± 9.01%) (Fig. 6a).

Fig. 6.

Fig. 6

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Cell density of non-pretreated (grey), vehicle-pretreated (blue), and DTX-pretreated (green) PC-3 cells (a), and non-pretreated (grey), vehicle-pretreated (blue), and DXR-pretreated (red) T47D cells (b). Following pretreatment, cells were incubated with medium, 50 MBq [177Lu]Lu-DTPA, or 50 MBq [177Lu]Lu-NeoB, and cell density was determined after 6 days using an SRB assay. Data are expressed as percentage relative to medium-treated cells within each pretreatment group. Results represent the mean of three independent experiments performed in triplicate

The cell density of the non-chemotherapy pretreated T47D, treated with 50 MBq [177Lu]Lu-NeoB was lower than medium treated cell. However, this difference was not statistically significant (Fig. 6b). Compared to [177Lu]Lu-DTPA similar cell densities were found as for [177Lu]Lu-NeoB treated cells in the non-pretreated T47D cells. For the vehicle and DXR pretreated T47D cells no effect was observed between the medium, 50 MBq [177Lu]Lu-DTPA, and [177Lu]Lu-NeoB treated cells. However, the cell density of [177Lu]Lu-DTPA treated DXR-pretreated cells was slightly higher compared to the other conditions.

In-vivo GRPR uptake and Ex-vivo GRPR mRNA expression

Uptake in PC-3 xenografts was measured at 11.87 ± 2.93% injected dose/gram) (D/g) in the placebo group and 19.81 ± 1.01% ID/g in the DTX treated group. In T47D xenografts, a tumor uptake of 5.90 ± 1.22% ID/g in the placebo group and 6.61 ± 6.08% ID/g in the DXR treated group was measured. Background organ uptake was consistent with our previous study using radiolabeled NeoB [4], with particularly high uptake noted in the physiological GRPR-expressing pancreas (Supplementary Fig. 1).

Throughout the study, no significant differences in body mass were observed between the treatment groups (Supplementary Fig. 2c, d). During the first injection of [¹⁷⁷Lu]Lu-NeoB, there was little to no difference in PC-3 xenograft volume between the placebo and DTX-treated groups (Table 1, Day 18). However, a reduction in PC-3 xenograft growth rate was observed during the second and third treatment cycles, with the placebo group exhibiting tumors nearly twice the size of those in the DTX-treated group (Table 1, Day 32). While an increase in PC-3 xenograft volume was observed over time in the placebo group (Supplementary Fig. 2a), uptake (kBq/ml) remained constant across all cycles (Fig. 7a). In contrast, an increase in uptake was noted over time in DTX-treated animals. During the second and third SPECT/CT scans after [¹⁷⁷Lu]Lu-NeoB injection, uptake per tumor volume was higher in DTX-treated animals compared to the placebo group. This difference in tumor size and uptake between the placebo and DTX groups is clearly visible on the SPECT/CT scans (Fig. 7c). Notably, despite the difference in [¹⁷⁷Lu]Lu-NeoB uptake observed during the third scan, GRPR mRNA levels were similar between the placebo and DTX-treated animals (Fig. 7e).

Table 1.

Tumor volume (mm3) during the course of the experiment for PC-3 and T47D tumor bearing animals

Days after tumor inoculation Volume (mm3) n Volume (mm3) n P value (placebo vs. treatment)
PC-3 bearing mice
Placebo DTX
11 72.75 ± 20.32 4 78.75 ± 15.97 4 > 0.99
14 117.75 ± 34.93 4 113.50 ± 31.10 4 > 0.99
18 148.50 ± 48.56 4 140.75 ± 53.58 4 > 0.99
21 314.25 ± 113.66 4 169.75 ± 22.47 4 0.18
25 310.25 ± 88.98 4 197.00 ± 44.82 4 0.45
28 429.00 ± 180.94 4 207.25 ± 65.97 4 0.008
32 558.25 ± 202.39 4 219.75 ± 65.25 4 < 0.001
T47D bearing mice
Placebo DXR*
10 66.25 ± 25.10 4 57.00 ± 18.73 3 0.99
13 83.00 ± 35.14 4 67.67 ± 9.07 3 0.9
17 78.50 ± 17.99 4 63.33 ± 16.92 3 0.91
20 69.50 ± 23.78 4 53.00 ± 23.39 3 0.87
24 70.25 ± 17.75 4 45.33 ± 14.19 3 0.56
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*one animal was excluded because there was no xenograft formation

Fig. 7.

Fig. 7

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Tumor uptake of [177Lu]Lu-NeoB in (a) PC-3 and (b) T47D xenograft bearing mouse measured in-vivo after different chemotherapy treatment cycles. Representative SPECT/CT scans of (c) PC-3 and a (d) T47D xenograft bearing animals 24 p.i. of 4 MBq/200 pmol of [177Lu]Lu-NeoB, 4 days after the last DTX and DXR injection, respectively. Tumor xenografts are indicated with yellow arrows. Bladder and pancreas uptake were exclude from the SPECT/CT images. GRPR mRNA expression in (e) PC-3 xenograft and (f) T47D xenografts treated in-vivo with DTX or DXR, respectively. Data is expressed as mean + SD of N = 4 (or N3 for DXR treated female animals)

In T47D tumor-bearing mice, the xenograft volume in DXR-treated animals was slightly lower than that in the placebo group at the time of the first [¹⁷⁷Lu]Lu-NeoB injection (Table 1, Day 17). The difference in xenograft size became more pronounced by the second injection (Day 24), with the placebo and DXR-treated animals showing mean tumor sizes of 70.25 ± 17.75 mm³ and 45.33 ± 14.19 mm³, respectively (Supplementary Fig. 2b). DXR treatment did not significantly affect [¹⁷⁷Lu]Lu-NeoB uptake, as the mean uptake per xenograft volume remained similar between the DXR and placebo groups (Fig. 7b and d). However, RT-qPCR analysis revealed increased GRPR mRNA expression in T47D tumors from DXR-treated animals (Fig. 7f).

SUV values calculated for male and female animals at the different dosing cycles showed a similar pattern to the calculated kBq/mL values, confirming consistency in uptake trends across both quantification methods (Supplementary Fig. 3).

Discussion

GRPR-mediated TRT is emerging with significant potential, particularly for PCa and BC management. Preclinical studies have shown that GRPR-targeting radiopharmaceuticals can effectively and safely be used to target tumors [31–33]. Currently, clinical studies are ongoing focused on assessing the efficacy and safety of these radiopharmaceuticals. Apart from these studies, that are crucial studies to establish their potential, for successful and optimal clinical application, research into identification of specific patient populations that will optimally benefit from the treatment, among others taking in to account medical history, should also be considered. Here, we describe the effect of two commonly applied chemotherapeutic drugs, DTX and DXR, on the success of GRPR-mediated TRT of PCa and BC with the aim of guiding clinical positioning of GRPR-targeting radiopharmaceuticals in the disease management course.

First, we determined the effect of DTX and DXR treatment on GRPR mRNA expression levels and GRPR-targeted radiopharmaceutical uptake. Ideally, GRPR protein expression levels would have been determined. This was not possible as a validated GRPR antibody for performing the respective analysis is not available. However, in prior studies we demonstrated a positive correlation between GRPR mRNA expression and GRPR-targeted radiopharmaceutical uptake, indicating that mRNA expression levels provide an indirect indication of the amount of functional GRPR protein present [34].

Exposure of PC-3 cells to DTX resulted in downregulation of GRPR mRNA expression and a corresponding decrease of [177Lu]Lu-NeoB uptake. A previous study by Kuroda. K et al., 2009 demonstrated downregulation of the androgen receptor (AR) in two PCa cell lines (LNCaP and MDA-PCa-2b cells) after DTX exposure [35]. Moreover, several groups have shown an interplay between AR and GRPR expression. Additionally, Schroeder et al. showed that castration of three androgen-dependent human PCa xenografts (PC295, PC-310 and PC82), resulted in a significant reduction of GRPR mRNA expression and binding of the GRPR-targeted radiopharmaceutical [125I]I-universal-BN. A decrease in AR due to DTX exposure might have an effect similar to castration resulting in downregulation of GRPR expression. However, the majority of studies reported that PC-3 cells lack AR expression and are thus androgen independent, while some have provided evidence that PC-3 cells do express low levels of AR mRNA and protein [36–38]. It should be determined whether the limited AR levels reported in the afore-mentioned studies can potentially have an effect on GRPR expression if AR is (further) downregulated. Interestingly, DTX did not cause any alteration in GRPR mRNA expression nor [177Lu]Lu-NeoB uptake in T47D cells, a AR positive human BC cell line. This discrepancy might be due to difference in cellular kinetics and AR functions in male and female cells, and/or sensitivity to DTX between the two cell lines. Nevertheless, further investigations are necessary to elucidate the underlying mechanism that led to the decrease of GRPR in PC-3 cells after DTX treatment.

The increased GRPR mRNA expression and [177Lu]Lu-NeoB uptake observed in T47D cells following DXR treatment might be part of a survival response. Cells often response to stress or damage by upregulating various signaling pathways. Hallasch, S. et al., for example, demonstrated an increase in various cell proliferation markers including GRPR after hyperthermia induced cell stress [39]. Furthermore, other studies have shown that DXR promotes mitotic activity, migration and invasion of cancer cells [40, 41]. GRPR might be involved in all of these processes. These mechanisms could be used as compensation by the cancer cells to counteract the cytotoxic effect of DXR. Additionally, the ability of DXR to modulate estrogen-receptor (ER) signaling pathways could indirectly upregulate GRPR expression. GRPR expression is highly correlated with ER, thus an increase in ER might lead to an increase in GRPR. A study by Pritchard. J., demonstrated DXR to lead to increase in ER [42]. Since T47D cells are ER positive, an increase in ER induced by DXR could potentially also lead to an increase in GRPR expression. Based on the latter mentioned, DXR in combination with GRPR-meditated TRT might lead to an improved treatment outcome in ER positive tumor cells. However, it should be noted that these finding can differ between cell lines and might be dose dependent.

In our study PC-3 and T47D cell treated with DTX and DXR, respectively, appear to be less sensitive to EBRT. However, these cells were rather non-responsive to EBRT than not sensitive, since DTX and DXR are known to induce cell cycle arrest [43, 44]. In response to damage e.g., DNA damage, cells can be arrested from growth at different checkpoints of the cell cycle in order to provide time for repair, a state where cells are resistant to stress and toxicity also known as quiescence. When the damage is irreversible, cells can trigger cell death programs or permanently arrest from the cell cycle (cellular senescence) [45]. We believe that cellular quiescence or senescence due to DTX and DXR treatment might be the reason for the lack of response to EBRT. In contrast to EBRT, [177Lu]Lu-NeoB treatment resulted in similar levels of growth inhibition between non-pretreated and chemotherapy-pretreated PC-3 cells. In T47D cells, the DXR-pretreated cells appeared to be less responsive to [177Lu]Lu-NeoB compared to non-pretreated cells, however, this difference was not statistically significant. These findings might suggest that [177Lu]Lu-NeoB treatment may bypass the chemoradiation-induced cellular quiescence/senescence observed with EBRT. However, more studies are needed to support this hypothesis.

The primary mechanism of both EBRT and [177Lu]Lu-NeoB involves the induction of irreparable DNA strand breaks through ionizing radiation. Despite this commonality, the biological effects of these two treatments are different, due to distinct ways of administration, distinct dose distributions, dose rates, and cytotoxic profiles [46]. This may explain the different outcomes observed between EBRT and TRT after chemotherapy.

Furthermore, the cytotoxic effects of [177Lu]Lu-NeoB did not correlate with the uptake and GRPR expression data. This discrepancy could be attributed to the complex biology underlying receptor-mediated therapies, wherein factors such as receptor internalization and ligand dissociation over time may contribute to variable outcomes [47–49]. Additionally, in-vitro TRT cytotoxicity is highly influenced by background radiation exposure, as observed under [177Lu]Lu-DTPA conditions. To minimize this effect, we reduced cell incubation time to just one hour.

Remarkably, DMSO-pretreated cells exhibited reduced responsiveness to TRT. DMSO has been reported in several studies to exhibit radioprotective properties, primarily through its ability to scavenge hydroxyl radicals. This protective effect is particularly significant in the context of low-LET radiation, such as beta particles [50, 51].

In-vivo, [177Lu]Lu-NeoB xenograft uptake in PC-3 xenografted animals pretreated with DTX increased over the course of the study. In contrast, uptake in the non-pretreated animals remained consistent across different cycles. The increased [177Lu]Lu-NeoB uptake in the PC-3 xenografts of DTX-pretreated animals during the second and third cycles, although not statistically significant, was likely due to increased xenograft perfusion or permeability, rather than an increase in GRPR expression. This hypothesis is supported by the RT-qPCR data performed after the third cycle, which showed no increase in GRPR mRNA levels compared to placebo group. Furthermore, the SPECT/CT scans revealed a more homogeneous distribution of the radiotracer in DTX-pretreated xenografts, along with a more intense signal compared to non-pretreated PC-3 xenografts, which were twice as large during the last treatment cycle. Based on these findings, it can be concluded that DTX might enhance radiotracer uptake by increasing tumor perfusion. Previous studies have shown that DTX decreases interstitial fluid pressure (IFP), a factor that often acts as a barrier, reducing the efficiency of therapeutic agent uptake. Lowering tumor IFP has been demonstrated to improve drug uptake and, consequently, treatment efficacy [52, 53]. Additionally, disruption of the tumor microenvironment and extracellular matrix, reduces the physical barriers that normally restrict drug penetration. This makes the tumor more permeable to therapeutic agents, such as radiopharmaceuticals, as observed in this study. In line with the aforementioned, future studies should address changes in perfusion e.g. by imaging (e.g., dynamic contrast-enhanced MRI or PET (Rubidium-82 and Copper-64-PTSM)) or Western blot analysis (e.g., VE-Cadherin and ICAM-1 and VCAM-1). However, next to perfusion, the significantly smaller size of the xenografts might also (partly) be the reason for the increased uptake observed, and should also be taken into account.

In T47D tumor-bearing female animals, [177Lu]Lu-NeoB uptake remained similar between the placebo and DXR-pretreated groups across different cycles. However, an increase in GRPR mRNA levels was observed in the DXR-pretreated tumors, which was consistent with our in-vitro data. Apart from this, the in-vivo [177Lu]Lu-NeoB uptake in DTX and DXR-pretreated animals bearing PC-3 and T47D xenografts, respectively, did not align with our in-vitro findings. These discrepancies likely arise from the complexity of living organisms, including factors such as drug delivery, pharmacokinetics, tumor microenvironment, and tumor cell heterogeneity, which differ significantly from the controlled conditions of cell cultures.

Conclusion

This study demonstrated a pronounced effect of chemotherapy on GRPR expression and consequently radiotracer uptake in-vitro. However, in-vivo the observed increased [177Lu]Lu-NeoB uptake in PC-3 xenografted animals treated with DTX appears to be influenced by changes in tumor perfusion, permeability, and potentially tumor size, rather than GRPR expression. Nonetheless, our findings indicate that pretreatment with DTX might alter the therapeutic efficacy of [177Lu]Lu-NeoB by facilitating enhanced drug delivery. However, this strategy might not be universally applicable across all tumor types, particularly those exhibiting resistance to DTX. Additionally, for imaging and diagnostic purposes, an increase in tracer uptake following DTX treatment should be interpreted cautiously, as it may reflect changes in perfusion or permeability rather than tumor growth or alterations in GRPR expression. In contrast, our findings suggest that DXR could enhance radiotracer uptake, potentially by increasing GRPR expression in ER-positive tumors, thereby improving TRT efficacy; however, these effects are likely influenced by tumor-specific factors. Future studies should investigate the underlying mechanisms and the discrepancies between the in-vitro and in-vivo findings, as well as whether DTX or DXR pretreatment before [177Lu]Lu-NeoB therapy is beneficial in a clinical setting.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material (269.5KB, docx)

Acknowledgements

We would like to thank the Radiopharmaceutical Chemistry group (Radiology & Nuclear Medicine, Erasmus MC, The Netherlands) for radiolabeling of NeoB.

Abbreviations

ANOVA

Analysis of variance

BC

Breast cancer

DNA

Deoxyribonucleic acid

DMSO

Dimethyl sulfoxide

DTPA

Diethylenetriaminepentaacetic acid

DTX

Docetaxel

DXR

Doxorubicin

EBRT

External beam radiation treatment

FBS

Fetal bovine serum

GRPR

Gastrin-releasing peptide receptor

Gy

Gray (unit of radiation dose)

IC50

Half maximal inhibitory concentration

ID/g

Injected dose per gram

IFP

Interstitial fluid pressure

kBq

Kilobecquerel (unit of radioactivity)

LET

Linear energy transfer

M

Molar (unit of concentration)

MBq

Megabecquerel (unit of radioactivity)

mL

Milliliter

mm³

Cubic millimeter

mRNA

Messenger ribonucleic acid

nM

Nanomolar (unit of concentration)

OD

Optical density

PCa

Prostate cancer

PBS

Phosphate-buffered saline

PSMA

Prostate specific membrane antigen

RPMI

Roswell park memorial institute (medium for cell culture)

RT

qPCR-reverse transcription quantitative polymerase chain reaction

SPECT/CT

Single-photon emission computed tomography/computed tomography

SRB

Sulforhodamine B

SSTR2

Somatostatin receptor subtype 2

TCA

Trichloroacetic acid

TRT

Targeted radionuclide therapy

Author contributions

This study was coordinated by T.S.T. Damiana with the supervision of S.U. Dalm. Material preparation, data collection and analysis were performed by T.S.T. Damiana, L. van den Brink, L.W. de Kreij-de Bruin, C. de Ridder, and D. Stuurman. The first draft of the manuscript was written by T.S.T. Damiana and S.U. Dalm. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was partly supported by NWO ZonMw Veni #09150161810061.

Data availability

All available data are described in the manuscript and available through the supplementary material. Additional information can be obtained by contacting the authors upon reasonable request.

Declarations

Ethics approval and consent to participate

He study was conducted according to the guidelines of the Declaration of Helsinki. Approval was granted by the Animal Welfare Committee of the Erasmus MC.

Consent for publication

Not applicable.

Competing interests

The authors have no potential conflicts of interest to disclose.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary Material (269.5KB, docx)

Data Availability Statement

All available data are described in the manuscript and available through the supplementary material. Additional information can be obtained by contacting the authors upon reasonable request.

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