Influence of corticosteroid treatment on CXCR4 expression in DLBCL

Pilot studies—time dependence and concentration dependence

In a first set of experiments, the time dependence of CXCR4 (up)regulation by corticosteroid treatment was investigated. As opposed to results from the literature [6, 7], an incubation time of 2 h at 37 °C with both the high dose (5 µM Dexamethasone, 50 µM Prednisolone) and the respective low-dose mimicking plasma concentration (0.5 µM Dexamethasone, 5 µM Prednisolone) did not induce any notable change in CXCR4 expression in any of the cell lines investigated (data not shown). However, after 24 h of incubation, flow cytometry analysis revealed increased CXCR4 surface expression levels for Daudi, OCI-LY1, SUDHL-4, SUDHL-5 and SUDHL-6 cells at both drug concentrations (Fig. 1). Consequently, an incubation time of 24 h was selected for all subsequent experiments.

Fig. 1figure 1

Dependence of CXCR4 upregulation on drug concentration. Data are shown as the mean fluorescence intensity observed by flow cytometry for each of the cell lines at different concentrations (low and high dose) of Dexamethasone and Prednisolone, respectively, after 24 h of incubation in percent of untreated controls (n = 1)

Importantly, the effect of corticosteroid treatment on CXCR4 surface expression was highly variable between cell lines (Fig. 1). For SUDHL-4, SUDHL-5 and SUDHL-6 cells, substantial CXCR4 upregulation by treatment with Dexamethasone and Prednisolone was observed, which was also found to be dependent on drug concentration in the case of SUDHL-4 and SUDHL-6 cells. Only moderate, concentration-independent CXCR4 upregulation was observed for Daudi and OCI-LY1 cells, whereas the treatment effect was negligible for Jurkat and SUDHL-8 cells. Overall, there was a slight trend toward a more notable CXCR4 upregulation by Dexamethasone than by Prednisolone in the responding cell lines (Daudi, OCI-LY1, SUDHL-4, -5 and -6). However, since these pilot experiments were performed only once for estimation of effects (n = 1), their significance is limited and does not allow conclusive interpretation. Despite the observed variability between different cell lines in response to corticosteroid treatment, however, no negative effect of Dexamethasone and Prednisolone on CXCR4 surface expression was detected.

Correlation of treatment effects observed by flow cytometry with radioligand binding data

Based on the above pilot experiments, OCI-LY1, SUDHL-4 and SUDHL-5 cells were selected for more in-depth evaluation of the association of steroid pretreatment, CXCR4 expression and CXCR4 radioligand uptake, based on their gradual response to corticosteroid treatment (SUDHL-4 > SUDHL-5 > OCI-LY1). Despite pronounced CXCR4 upregulation by corticosteroid treatment, SUDHL-6 cells were not included due to practical considerations.

To be able to assess the influence of the CXCR4 upregulation observed via flow cytometry on radioligand uptake an additional set of experiments was performed. Aliquots of the same treated cells (24 h, 37 °C) were analyzed in parallel via flow cytometry and via incubation with [125I]CPCR4.3 to quantify CXCR4 expression. Results are summarized in Fig. 2.

Fig. 2figure 2

CXCR4 upregulation by corticosteroid treatment as assessed in parallel by flow cytometry and a radioligand binding study using [125I]CPCR4.3. All data are shown in percent of the values obtained for untreated control cells. Flow cytometry data are shown as relative mean fluorescence intensity (MFI) values and are means ± SD from 3–4 separate determinations with n = 3, respectively. Radioligand binding data are shown as relative normalized uptake values (% of added dose bound per 1 Mio live cells) and are means ± SD from 2 separate determinations with n = 3, respectively

Interestingly, the extent of CXCR4 upregulation observed by flow cytometry for the three selected cell lines was quite different from the pilot experiments, with the responsiveness to therapy being now in the order of OCI-LY1 > SUDHL-4 > SUDHL-5. Another unexpected finding is the fact that the increase in cellular uptake of [125I]CPCR4.3 was much less pronounced than the change in CXCR4 surface expression observed by flow cytometry. However, as shown in Fig. 3, there is a linear correlation between the relative changes in CXCR4 expression determined by flow cytometry and via [125I]CPCR4.3 binding. This correlation on the one hand corroborates the initial observation (see pilot experiments), that corticosteroid treatment does increase CXCR4 expression in DLBCL cell lines. However, the extent of this effect is strongly cell line dependent. The observed increase in radioligand uptake in cell lines with a strong CXCR4 upregulation upon corticosteroid treatment may even prove beneficial in the context of RLT with [177Lu]Lu/[90Y]Y-PentixaTher.

Fig. 3figure 3

Correlation of CXCR4 upregulation by corticosteroid treatment quantified by flow cytometry and via a radioligand binding study, respectively. All data are shown in percent of the values obtained for untreated control cells. Flow cytometry data are shown as relative MFI values and are means ± SD from 3–4 separate determinations with n = 3, respectively. Radioligand binding data using [125I]CPCR4.3 are shown as relative normalized uptake values (% of added dose bound per 1 Mio live cells) and are means ± SD from 2 separate determinations with n = 3, respectively

In vivo assessment of the effect of Dexamethasone treatment on tumoral CXCR4 expression

To verify this hypothesis, mice bearing subcutaneous OCI-LY1 DLBCL xenografts were randomized into a control group (no treatment) and a treatment group (50 μg Dexamethasone i.p. for 6 consecutive days), and a comparative biodistribution study using [68Ga]Ga-PentixaTher was carried out at the end of the treatment period. Of note, since at the time of the experiment, 177LuCl3 was not available from the manufacturer due to production shortages, 68Ga-labeled PentixaTher was chosen as a substitute for the therapeutic agent [177Lu]Lu-PentixaTher.

As summarized in Fig. 4, Dexamethasone treatment (dosing as in [6]) had a pronounced effect on the overall biodistribution of [68Ga]Ga-PentixaTher. The observed increased concentration of the tracer in blood (increase by 58% in treated vs untreated animals) was reflected by a 53–68% higher absolute tracer uptake in all organs in the treated animals. The only exception from this consistent general effect was the OCI-LY1 xenograft, with an increase in [68Ga]Ga-PentixaTher uptake by 128% compared to untreated animals. As of now, the reasons for the tendency toward an increased blood concentration of [68Ga]Ga-PentixaTher are unclear; it may either be the result of a delayed blood clearance as a side effect of corticosteroid treatment, or be related to an upregulation of mCXCR4 expression on circulating mouse immune cells (T-lymphocytes, B-cells [6]). [177Lu]Lu-PentixaTher is known to display moderate affinity toward mCXCR4 [13], and it is highly probable that the mCXCR4 affinity of [68Ga]Ga-PentixaTher lies in the same range, and thus, the increased [68Ga]Ga-PentixaTher concentration in blood in treated animals may thus be related to specific tracer binding. However, further experiments are needed to confirm this hypothesis.

Fig. 4figure 4

Biodistribution (A) and Tumor-to-organ ratios (B) of [68Ga]Ga-PentixaTher in OCI-LY1 DLBCL xenograft bearing NSG mice at 1 h p.i.. The treatment group (n = 5) received 50 μg Dexamethasone i.p. for 6 consecutive days before tracer injection. The biodistribution data are given in % injected dose per gram tissue (%iD/g) and are means ± SD (n = 5 animals/group)

In contrast, however, the over-proportional increase in tracer accumulation in the OCI-LY1 xenograft is in line with our in vitro findings, i.e., a dexamethasone-treatment induced CXCR4 upregulation on the tumor cells. This is further underlined by the consistently higher tumor/organ ratios observed for the treated animals (Fig. 4). Although the differences in tumor/background ratios between the treated and the untreated animals are not statistically significant (P = 0.4–0.8 for all organs) due to the relatively high standard deviation of the absolute tumor uptake value for the treatment group, our data nevertheless indicate a clear trend, which is in accordance with our in vitro observations.

In summary, we observed that corticosteroid treatment (Dexamethasone, Prednisolone) consistently induced an upregulation of CXCR4 expression DBLCL cells in vitro. Of note, the effect varied significantly between cell lines, the increase ranging from 20 to 300% of baseline CXCR4 expression. For the cell line with the most pronounced response to Dexamethasone treatment, OCI-LY1, the in vitro findings could also be recapitulated in the corresponding in vivo xenograft model. This confirms that at least the corticosteroid component of stabilizing chemotherapy regimens in DLBCL patients [8, 9] prior to CXCR4-targeted RLT with [177Lu]Lu-PentixaTher does not lead to downregulation of the molecular target CXCR4 and may even have a contrary, beneficiary effect. However, it needs to be investigated in more detail to which extent rituximab or the other chemotherapeutic agents used in CHOP or DHAP treatment protocols affect CXCR4 expression, since these effects may limit the use of CXCR4-targeted diagnostics and/or CXCR4-targeted therapies [4]. A better understanding of CXCR4 (de)regulation by DLBCL lymphoma directed chemotherapies may help to ensure the choice of an appropriate treatment regimen prior to [177Lu]Lu/[90Y]Y-PentixaTher RLT in these diseases.

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