Background and study design
CXCR4-targeted radioligand therapy (RLT) with [
177Lu]Lu/[
90Y]Y-PentixaTher (Yttrium (
90Y) anditixafortide) has recently evolved as a promising therapeutic option for patients with advanced hematological cancers such as multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL) [
1‐
3]. Generally, a prerequisite for patient eligibility for [
177Lu]Lu/[
90Y]Y-PentixaTher RLT is high tumoral CXCR4 expression, confirmed by high uptake of the companion diagnostic, [
68Ga]Ga-PentixaFor (Gallium (
68Ga) boclatixafortide), in the respective tumor lesions by pre-therapeutic PET/CT. However, as most patients scheduled for PentixaTher RLT suffer from advanced stages of their disease, concomitant or bridging chemotherapy to prevent tumor progression between diagnostic imaging and RLT is often required. That these intermittent therapeutic regimens may have significant impact on tumoral CXCR4 expression has recently been demonstrated. For three patients with different hematological malignancies, substantial downregulation of CXCR4 expression in response to bridging chemotherapy was observed [
4], rendering [
177Lu]Lu/[
90Y]Y-PentixaTher RLT unsuitable or possibly much less effective.
To date, the mechanisms of this CXCR4 downregulation are unclear, and the observed effects are all the more surprising since two of the reported patients received Dexamethasone (albeit in conjunction with cyclophosphamide or other chemotherapeutic agents [
4]). Dexamethasone was shown to substantially increase surface expression of CXCR4 in MM cells [
4,
5], in murine B-cells [
6] and in human T-cells [
7]. In view of the implementation of an early phase clinical study on [
177Lu]Lu/[
90Y]PentixaTher RLT in patients with DLBCL, it is of particular importance to understand the role of corticosteroid treatment on the regulation of CXCR4 expression in this malignancy. Such information could possibly guide the safe use of bridging chemotherapies prior to considering the patients for CXCR4-targeted [
177Lu]Lu/[
90Y]Y-PentixaTher RLT.
Thus, this study aimed at investigating the influence of corticosteroid (Dexamethasone, Prednisolone) treatment on CXCR4 expression in a panel of DLBCL cell lines (Daudi, OCI-LY1, SUDHL-4, SUDHL-5, SUDHL-6, SUDHL-8) with different baseline CXCR4 expression levels. The human T-cell leukemia cell line Jurkat with high CXCR4 expression was also included.
Clinically, in the management of DLBCL, the combination of CHOP (Cyclophosphamide, Doxorubicin, Vincristine and Prednisone
a) and rituximab is considered a standard first-line treatment [
8], whereas modified/extended DHAP (Dexamethasone, Cytarabine, Cisplatin) protocols are used as second-line chemotherapies [
9]. Since patients with DLBCL eligible for [
177Lu]Lu/[
90Y]Y-PentixaTher RLT are very likely to undergo/have undergone one of these treatments, Prednisolone
1 and Dexamethasone were both included into this investigation.
To ensure the reliability of the in vitro data, the concentrations for Dexamethasone and Prednisolone were chosen such as to resemble as closely as possible to the maximum plasma concentrations observed in humans receiving standard treatments (i.e., 40 mg Dexamethasone/day [
9] and 100 mg Prednisone/day [
8]). On the basis of this dosing, the corresponding maximum plasma concentrations for Dexamethasone and Prednisolone were found to be approximately 0.5 µM [
10] and 5 µM [
11], respectively. Thus, these concentrations were used consistently throughout the study. In some experiments, however, to assess a potential concentration dependence of the (up)regulation of CXCR4 and of cell viability, a tenfold concentration of the chosen corticosteroids was also investigated.
Data from the literature indicate variable kinetics of CXCR4 upregulation in different cell types, with the first detection of receptor upregulation ranging from 1 to 3 h [
6,
7] to 24 h of incubation [
5]. We therefore performed initial pilot studies to establish the most suitable incubation time for detecting potential effects of corticosteroid treatment on CXCR4 expression in the different DLBCL cell lines. Ultimately, after having established appropriate experimental conditions, a second set of experiments was performed, in which the changes in CXCR4 expression observed in flow cytometry were correlated with changes in radioligand binding induced by corticosteroid therapy. Given the particularly high sensitivity of [
125I]CPCR4.3 for quantifying different CXCR4 expression levels in vitro [
12], this ligand was used instead of [
177Lu]Lu-PentixaTher for the in vitro studies.
Materials and methods
Cell culture
Jurkat human T-cell leukemia cells were cultured in RPMI-1640 medium, supplemented with 10% FCS. All DLBCL cell lines, namely Daudi, OCI-LY1, SUDHL-4, -5, -6 and -8, were kindly supplied by Prof. Ulrich Keller, Department of Hematology and Oncology, Charité, Berlin, Germany, and were grown in RPMI-1640 medium, supplemented with 20% FCS. All cell lines were maintained at 37 °C in a humidified 5% CO2 atmosphere. Media and supplements were obtained from Biochrom (Berlin, Germany) or Gibco (life technologies, Darmstadt, Germany). For cell counting, an automated CytoSMART Lux cell counter (Axion BioSystems, Atlanta, USA) was used.
For treatment with Dexamethasone and Prednisolone (obtained as suspensions/solutions for oral application from the clinical pharmacy at CHUV), the respective cell suspensions were centrifuged (3 min, 1300 rcf, Megafuge 1.0, Heraeus Thermo Scientific). The culture medium was removed and the cell pellet was resuspended in assay medium (DMEM/F-12 medium with Glutamax-I (1:1) supplemented with 5% BSA) to yield a cell suspension with a concentration of app. 5–7·106 cells/ml. For treatment, either 140 μL of assay medium (untreated control cells) or 140 μL of tenfold concentrated solutions of Dexamethasone and Prednisolone (5 μM and 50 μM as well as 50 μM and 500 μM, respectively) was added to 1.26 mL of cell suspension. After incubation of the cells at 37° for 24 h in an incubator, the cells were centrifuged, washed once with assay medium, and resuspended in assay medium to a concentration of 5·106 cells/mL. This suspension was either used directly for the radioligand binding assay or processed further for flow cytometry analysis.
Flow cytometry
The treated and untreated cells were washed twice with cold flow cytometry buffer (5% fetal bovine serum in PBS). For the staining, triplicates of 1·106 cells were prepared and incubated 45 min on ice with a concentration of 1 µg/mL PE anti-human CD184 CXCR4 antibody (BioLegend) or PE mouse IgG2a isotype control (BioLegend) in 100 µL FACS buffer. Next, the cells were spun down at 300 × g and the staining agent was discarded. The cells were washed twice and were resuspended in 500 µL of cold FACS buffer. In addition, DAPI was added to each sample shortly before the analysis to yield a final concentration of 0.5 µg/mL. The flow cytometry analyses were conducted immediately on a Beckman CoulterGallios flow cytometer. The acquired data were analyzed with FlowJo v10.7.1.
Radioligand binding assay
Radioiodination of CXCR4.3 was carried out using the IodoGen® method as described previously [
12].
For the binding assay, samples containing app. 1·106 cells in assay medium were incubated with [125I]CPCR4.3 (0.2 nM) at RT for 60 min in the presence (non-specific binding) or absence (control) of 100 µM unlabeled CPCR4.3 (n = 3 per condition, total sample volume: 250 µL). After incubation, the tubes were centrifuged (3 min, 1300 rcf, Megafuge 1.0, Heraeus Thermo Scientific) and the supernatant was carefully removed. The cells were washed once with 200 µL of cold HBSS, and the supernatant of the washing step was pooled with the supernatant from the previous step (free ligand). Then, the amount of bound radioligand in the cell pellet as well as the amount of free radioligand in the combined supernatants was quantified using a γ-counter (WALLAC; 1480 WIZARD™ 3″). For each sample, the cellular uptake in % of total added dose was calculated and then used for further data processing.
Tumor model and in vivo biodistribution studies
For induction of tumor growth, female NSG mice (6–8 weeks) were subcutaneously injected with 5 × 106 OCI-LY1 cells in HBSS/Matrigel (1:1). After 25 days, small palpable tumors had grown in all animals, and animals were divided into a control group (no treatment, n = 5) and a treatment group (n = 5). Treated animals received 50 μg Dexamethasone in 100 μL PBS as an i.p. injection for 6 consecutive days (day 25-day 30 post tumor implant). The following day, all animals were injected intravenously with 3–4 MBq (0.16–0.18 nmol) [68Ga]Ga-PentixaTher, and a biodistribution study was carried out. The animals were sacrificed at 1 h post injection (p.i.), and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-counter. Data are expressed in % ID/g tissue (mean ± SD).
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