Comparing the Effect of Multiple Histone Deacetylase Inhibitors on SSTR2 Expression and [111In]In-DOTATATE Uptake in NET Cells

Simple Summary Patients diagnosed with neuroendocrine tumors (NETs) are often treated with peptide receptor radionuclide therapy (PRRT). This therapy targets the somatostatin type-2 receptors (SSTR2) frequently overexpressed on these types of tumors. Although this therapy has proven to be effective, complete responses are rare and therapy improvement is desirable. We aimed to increase SSTR2 expression on NET cells, potentially increasing the number of patients eligible for SSTR2-targeted PRRT and improving clinical outcomes. We used histone deacetylase inhibitors (HDACis) to manipulate the epigenetic machinery and hereby aimed to increase SSTR2 gene transcription. Our results showed that the HDACis increased SSTR2 expression in several NET cell lines. Moreover, the uptake of radiolabeled DOTATATE, the tracer used for PRRT, was enhanced. The observed reversibility profile after HDACi withdrawal of the induced effects suggests that proper timing of HDACi treatment is likely essential. Abstract The aim of this study was to increase somatostatin type-2 receptor (SSTR2) expression on neuroendocrine tumor (NET) cells using histone deacetylase inhibitors (HDACis), potentially increasing the uptake of SSTR2-targeted radiopharmaceuticals and subsequently improving treatment efficacy of peptide receptor radionuclide therapy (PRRT). Human NET cell lines BON-1, NCI-H727, and GOT1 were treated with HDACis (i.e., CI-994, entinostat, LMK-235, mocetinostat, panobinostat, or valproic acid (VPA); entinostat and VPA were the HDACis tested in GOT1 cells) to examine SSTR2 mRNA expression levels and uptake of SSTR2-targeting radiotracer [111In]In-DOTATATE. Reversibility of the induced effects was examined after drug-withdrawal. Finally, the effect of VPA on radiosensitivity was investigated. A strong stimulatory effect in BON-1, NCI-H727, and GOT1 cells was observed after HDACi treatment, both on SSTR2 mRNA expression levels and [111In]In-DOTATATE uptake. The effects of the HDACis were largely reversible over a period of seven days, demonstrating largest reductions within the first day. The reversibility profile of the induced effects suggests that proper timing of HDACi treatment is most likely essential for a beneficial outcome. In addition to increasing SSTR2 expression levels, VPA enhanced the radiosensitivity of all cell lines. In conclusion, HDACi treatment increased SSTR2 expression, and radiosensitivity was also enhanced upon VPA treatment.


Introduction
Neuroendocrine tumors (NETs) form a heterogeneous group of tumors which are often metastasized upon time of diagnosis. Unfortunately, treatment options for NETs are still limited [1]. The frequent overexpression of the somatostatin type-2 receptor (SSTR2) forms a pivotal target for therapy. Treatment with somatostatin analogues (SSAs) and the subsequent development of radiolabeled SSAs, i.e., [ 177 Lu]Lu-[DOTA-Tyr3]octreotate ([ 177 Lu]Lu-DOTATATE) used for peptide receptor radionuclide therapy (PRRT), have both proven their efficacy in the treatment of NETs [2][3][4]. Unfortunately, complete responses after PRRT are still rare [4,5]. Several promising approaches are under investigation to improve the efficacy of PRRT, such as SSTR2 upregulation using epigenetic drugs.
Varying SSTR2 expression levels among patients [6] and the absence of known mutations in the human SSTR2 gene, indicate that its expression may be regulated by epigenetic mechanisms, rather than genetic mutations. Previous studies have described an important role for epigenetic regulation in both NET pathogenesis and SSTR2 expression [7][8][9][10]. Due to the prominent role of epigenetics in this disease, it is hypothesized that epigenetic drugs will mainly target the tumor cells and, to a lesser extent, control tissue. By using synthetic inhibitors targeting the epigenetic machinery, it is possible to modify the epigenetic landscape. For the scope of this study, we focused only on histone acetylation. Histone deacetylase inhibitors (HDACis) specifically target histone deacetylases. Inhibition of these enzymes results in stimulation of the active euchromatin state, the state in which DNA is actively being transcribed. In short, the use of HDACis may modify the epigenetic profile in such a way that protein expression levels are increased [11,12].
In this study, the aim was to thoroughly compare the effect of different HDACis targeting several classes of HDAC enzymes in three NET models: BON-1, NCI-H727, and GOT1 cells derived from pancreatic NET, lung carcinoid tumor, and midgut NET, respectively. We evaluated the effect of the selected HDACis on the different NET cell lines with respect to SSTR2 mRNA expression levels and SSTR2 functionality using [ 111 In]In-DOTATATE uptake studies. Moreover, we analyzed reversibility profiles over time after HDACi withdrawal and investigated the radiosensitivity upon exposure to one of the HDACis.

Dose-Response Curves and DNA Quantification
For all HDACis, dose-response studies based on a seven-day treatment schedule were performed. One day before HDACi treatment started, cells were plated in 24-well plates. Drug-supplemented medium was refreshed on day three and medium was removed on day seven. A cell growth assay was then performed by DNA quantification using Hoechst 33256 as previously described [16] with the adjustment of using 0.2% (v/v) Triton X-100 for cell lysis. For GOT1 cells, Quant-iT PicoGreen dsDNA reagent (Invitrogen, Breda, The Netherlands) was used. For this, Quant-iT PicoGreen dsDNA was diluted 120 times and 20 µL was added to each well. The absorbance was subsequently measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

HDACi-Treatment Regimen
To study the effect of HDACis on SSTR2-expression, cells were plated in T75 flasks on day zero. HDACis were added on day one at their IC 50 growth inhibitory concentrations and drug-supplemented medium was refreshed on day three. On day five, cells were trypsinized and plated for further analysis. Exactly four hours after cell plating, HDACis were added again. On day seven, samples were collected for analysis by RT-qPCR analysis (24-well plates) and for internalization studies (12-well plates). For reversibility and radiosensitivity studies (24-well plates), culture periods were prolonged for another week in the absence and presence of HDACis, respectively.

mRNA Analysis
For mRNA analysis, cells were lysed and subsequently incubated with oligo(dT) 25 dynabeads (Invitrogen, Breda, The Netherlands) to isolate poly-A + mRNA. Then, 23 µL H 2 O was added for elution, and 10 µL poly-A + mRNA was used in the next steps. Poly-A + mRNA was converted into cDNA using the commercial RevertAid First Strand cDNA synthesis kit (Thermo Scientific, Breda, The Netherlands). To exclude the possibility of DNA contamination, cDNA was also prepared without the addition of RevertAid Reverse Transcriptase. Samples were diluted by adding 180 µL H 2 O and RT-qPCR was performed. In short, 5 µL sample was mixed with 7.5 µL Taqman Universal PCR mastermix (Applied Biosystems, Breda, The Netherlands) supplemented with primers and probes. SSTR2 expression was determined relative to three housekeeping genes (HKGs). For analysis, the QuantStudio 7 Flex RT-qPCR system with QuantStudio Real-Time PCR software v1.5 was used. The number of copies for SSTR2 and all HKGs was calculated by the efficiency factor to the power of ∆Ct (i.e., 40 minus measured Ct). Subsequently, the relative SSTR2 expression was calculated by dividing the number of SSTR2 copies by the geometric mean of all HKGs. Details on primers are represented in Table S1.

[ 111 In]In-DOTATATE Internalization Studies
Internalization studies were performed as previously described [18]. Cells were incubated with internalization medium (DMEM (1x)-GlutaMAX-I, 1% (wt/v) BSA, and 20 mM HEPES (pH 7.4)) supplemented with 10 −9 M [ 111 In]In-DOTATATE (50 MBq/1 nmol), with or without 10 −6 M unlabeled DOTATATE, for four hours. Following incubation, the excess unbound radiotracer was removed and the membrane-bound and internalized radioactivity were determined. For GOT1 cells, the protocol was slightly adjusted due to insufficient cell adherence. In each step, non-adherent cells were pelleted by centrifugation (3.5× g, 5 min) and combined with the attached cells. In addition, the total uptake was determined for GOT1 cells. To correct for possible differences in cell numbers, cell pellets of additional wells were collected and DNA content was measured with Hoechst 33258 using the same protocol described previously.

Reversibility
To determine the reversibility of HDACi-induced effects, drug-supplemented medium was removed on day seven. Cells were subsequently maintained in normal growth medium up to an additional period of seven days, with medium renewal after three days. Samples were collected one, three, and seven days after drug withdrawal and analyzed by RT-qPCR using the previously described method.

Radiosensitivity
HDACi-supplemented growth medium was refreshed on day seven and cells were irradiated up to 8 grays (Gy) using the RS320 (Xstrahl Live Sciences; 1.6554 Gy/min, 195 kV, 10 mA). On day eleven, HDACi-supplemented growth medium was refreshed again. Cells were fixated on day 14 using 10% (wt/v) trichloroacetic acid. Subsequently, plates were incubated with 0.5% (wt/v) sulforodamine B (SRB) solution. After incubation, the excess SRB solution was removed by washing plates with 1% (v/v) acetic acid and protein-bound dye was solubilized with 10 mM tris-base solution. Using a SpectraMax iD3 plate reader (Molecular Devices), the optical density was measured at 560 nm. For each plate, a background measurement was included.

Statistical Analysis
To estimate IC 50 values, the obtained percentages were plotted using spline/LOWESS analysis using the point-to-point curve. For detecting correlations, the Pearson r 2 was determined. For all other analysis, results were calculated as percentage increase or decrease compared to the control situation. The resulting percentages were log-transformed. One-way ANOVA analysis using the Tukey post-hoc test was performed to detect significant differences between HDACi-treated cells. To detect differences in radiosensitivity and obtain IC 50 values, a dose-response curve was plotted with variable slope. For all experiments, both biological and technical replicates were included. All results represent the mean ± SD of at least two independent biological replicates and at least three technical replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, NS; non-significant. All statistical analyses were performed using GraphPad Prism 5 software.

Effects of HDACis in BON-1 and NCI-H727 Cells
BON-1 cells showed to be slightly more sensitive for HDACi treatment than NCI-H727 cells. The IC 50 values on growth of BON-1 and NCI-H727 cells were, respectively, 1.85 µM and 3.05 µM for CI-994, 218 nM and 315 nM for ENT, 154 nM and 348 nM for LMK-235, 84.3 nM and 171 nM for MOC, 3.11 nM and 8.53 nM for PAN, and 1.12 mM and 1.31 mM for VPA ( Figure 1A-F, Table S2). Treatment at these IC 50 values demonstrated an expected inhibition of approximately 50% for all HDACis ( Figure 1G).  In-DOTATATE and radiosensitivity were evaluated, a proper reduction in DNA amount (i.e., approximately 50%) was confirmed as the internal control for each experiment. As HDACis were either dissolved in 40% DMSO or sterile aquadest, two vehicle-controls were included.
In BON-1 cells, all HDACis significantly increased SSTR2 mRNA expression levels (p < 0.001). CI-994, ENT, and PAN induced the strongest effects, i.e., a 3.07-, 3.13-, and 2.87fold increase, respectively (Figure 2A). In line with this, uptake of [ 111 In]In-DOTATATE was significantly enhanced, i.e., 8.14-, 8.30-, and 7.54-fold, respectively ( Figure 2B). Surprisingly, even though VPA had a relatively modest effect on the SSTR2 mRNA expression level, the uptake of [ 111 In]In-DOTATATE after VPA was most pronounced, i.e., an 8.63-fold increase in uptake. In-DOTATATE and radiosensitivity were evaluated, a proper reduction in DNA amount (i.e., approximately 50%) was confirmed as the internal control for each experiment. As HDACis were either dissolved in 40% DMSO or sterile aquadest, two vehicle-controls were included.

Reversibility Profile of SSTR2 Expression in BON-1 and NCI-H727 Cells
Reversibility profiles of the effects of HDACis in BON-1 cells showed that control expression levels were not reached after treatment with CI-994, ENT, and LMK-235, i.e., 2.19-, 2.51-, and 1.57-fold increase seven days after drug withdrawal, respectively ( Figure  3A). For these inhibitors, strong reductions within the first day after HDACi removal were observed. For PAN-treated cells, control levels were also not reached (1.81-fold increase). In these cells, a gradual downregulation of SSTR2 expression over time was observed,  Figure 2B). SSTR2 mRNA expression levels followed an identical pattern (Figure 2A), resulting in a significant positive correlation between SSTR2 mRNA expression levels and [ 111 In]In-DOTATATE uptake levels (r 2 = 0.9005; p = 0.0038) ( Figure S2B). A statistically significant correlation was not reached in BON-1 cells (r 2 = 0.4534) ( Figure S2C).

Reversibility Profile of SSTR2 Expression in BON-1 and NCI-H727 Cells
Reversibility profiles of the effects of HDACis in BON-1 cells showed that control expression levels were not reached after treatment with CI-994, ENT, and LMK-235, i.e., 2.19-, 2.51-, and 1.57-fold increase seven days after drug withdrawal, respectively ( Figure 3A). For these inhibitors, strong reductions within the first day after HDACi removal were observed. For PAN-treated cells, control levels were also not reached (1.81-fold increase). In these cells, a gradual downregulation of SSTR2 expression over time was observed, suggesting a slow wash out of the effect of PAN on these cells. For MOC-and VPA-treated BON-1 cells, control levels were already reached after one day. At day three and day seven, a slight increase in SSTR2 mRNA expression levels was observed in MOC treated cells, reaching significance for both time points.  In NCI-H727 cells, one day after VPA withdrawal control SSTR2 mRNA expression levels were already observed ( Figure 3B). Surprisingly, after three days, a small but significant upregulation was observed, i.e., 1.37-fold. For all other conditions, another reversibility pattern was observed. SSTR2 expression levels were significantly reduced already one day after drug withdrawal, i.e., 0.53-, 0.55-, 0.63, 0.71-, and 0.43-fold for CI-994, ENT, LMK-235, MOC, and PAN, respectively. Generally, SSTR2 mRNA expression levels reached control expression levels three days after drug removal. For MOC treatment, the To determine reversibility of SSTR2 upregulation upon HDACi withdrawal, SSTR2 mRNA levels were determined by RT-qPCR directly after treatment with HDACi at the IC 50 dose (D0), and one (D1), three (D3), and seven days (D7) after HDACi withdrawal for BON-1 (A), and at D0, D1, and D3 for NCI-H727 (B). Results were normalized to vehicle-treated cells. * p < 0.05, ** p < 0.01, *** p < 0.001, NS; non-significant.
In NCI-H727 cells, one day after VPA withdrawal control SSTR2 mRNA expression levels were already observed ( Figure 3B). Surprisingly, after three days, a small but significant upregulation was observed, i.e., 1.37-fold. For all other conditions, another reversibility pattern was observed. SSTR2 expression levels were significantly reduced already one day after drug withdrawal, i.e., 0.53-, 0.55-, 0.63, 0.71-, and 0.43-fold for CI-994, ENT, LMK-235, MOC, and PAN, respectively. Generally, SSTR2 mRNA expression levels reached control expression levels three days after drug removal. For MOC treatment, the only HDACi inducing SSTR2 mRNA downregulation directly after treatment, control levels were not reached within this time frame.

Effects of HDACis in GOT1 Cells
Based on effects induced in BON-1 and NCI-H727 cells, the effects of ENT and VPA were examined in GOT1 cells. Measured IC 50 values were 384 nM for ENT and 1.36 mM for VPA treatment ( Figure 4A,B, Table S2). Both HDACis significantly increased SSTR2 mRNA expression levels: 2.27-and 2.37-fold, respectively ( Figure 4C). In line with this, also the total uptake of [ 111 In]In-DOTATATE was significantly enhanced, i.e., 1.34-and 2.06-fold, respectively ( Figure 4D). only HDACi inducing SSTR2 mRNA downregulation directly after treatment, control levels were not reached within this time frame.

Effects of HDACis in GOT1 Cells
Based on effects induced in BON-1 and NCI-H727 cells, the effects of ENT and VPA were examined in GOT1 cells. Measured IC50 values were 384 nM for ENT and 1.36 mM for VPA treatment ( Figure 4A -B, Table S2). Both HDACis significantly increased SSTR2 mRNA expression levels: 2.27-and 2.37-fold, respectively ( Figure 4C). In line with this, also the total uptake of [ 111 In]In-DOTATATE was significantly enhanced, i.e., 1.34-and 2.06-fold, respectively ( Figure 4D).

Reversibility Profile of SSTR2 Expression in GOT1 Cells
Concordant with the observations in BON-1 and NCI-H727 cells, the effects of ENT and VPA gradually decreased over the seven-day period. However, control SSTR2 mRNA expression levels were not reached within the examined time period (Figure 4D -E).

Reversibility Profile of SSTR2 Expression in GOT1 Cells
Concordant with the observations in BON-1 and NCI-H727 cells, the effects of ENT and VPA gradually decreased over the seven-day period. However, control SSTR2 mRNA expression levels were not reached within the examined time period (Figure 4D,E).

Radiosensitizing Effects upon VPA Treatment
As VPA treatment induced strong and significant stimulatory effects on SSTR2 mRNA expression and [ 111 In]In-DOTATATE uptake in all three NET cell lines, the effect of this HDACi was also examined in terms of radiosensitivity ( Figure 5). Irradiated VPA-treated NET cells proliferated less than irradiated control cells. IC 50 values were 4.39 Gy, 3.22 Gy, and 1.34 Gy for untreated BON-1, NCI-H727, and GOT-1 cells, whereas VPA-treated cells had IC 50 values of 2.82 Gy, 1.88 Gy, and 0.72 Gy, respectively, demonstrating a statistically significant increased sensitivity to external beam irradiation induced by VPA.

Radiosensitizing Effects upon VPA Treatment
As VPA treatment induced strong and significant stimulatory effects on SSTR2 mRNA expression and [ 111 In]In-DOTATATE uptake in all three NET cell lines, the effect of this HDACi was also examined in terms of radiosensitivity ( Figure 5). Irradiated VPAtreated NET cells proliferated less than irradiated control cells. IC50 values were 4.39 Gy, 3.22 Gy, and 1.34 Gy for untreated BON-1, NCI-H727, and GOT-1 cells, whereas VPAtreated cells had IC50 values of 2.82 Gy, 1.88 Gy, and 0.72 Gy, respectively, demonstrating a statistically significant increased sensitivity to external beam irradiation induced by VPA. Figure 5. After a seven-day treatment with the IC50 dose, VPA-treated and untreated BON-1 (A), NCI-H727 (B), and GOT1 (C) cells were exposed to different dosages of irradiation using external beam radiation to investigate increased radiosensitivity.

Discussion
As the number of complete responses in NET patients is limited after PRRT, therapy improvement is highly needed. For this reason, we focused on HDACi-induced SSTR2 Figure 5. After a seven-day treatment with the IC 50 dose, VPA-treated and untreated BON-1 (A), NCI-H727 (B), and GOT1 (C) cells were exposed to different dosages of irradiation using external beam radiation to investigate increased radiosensitivity.

Discussion
As the number of complete responses in NET patients is limited after PRRT, therapy improvement is highly needed. For this reason, we focused on HDACi-induced SSTR2 upregulation in three NET cell-line models as a way of improving uptake of radiolabeled SSAs, and thereby possibly increasing treatment efficacy.
In line with our results, previous studies also reported on the effects of HDACi treatment, showing SSTR2 upregulation and/or increased uptake of radiolabeled SSAs in different NET models [16,[19][20][21][22][23][24][25][26]. However, the strength of our study was the use of HDACi-specific and cell line-based IC 50 values allowing for a comparison of effects induced by HDACis targeting different classes of HDAC enzymes. Moreover, we investigated the induced effects in three frequently used NET cell-line models derived from different origins. Importantly, the established IC 50 values of the HDACis were below or within the same order of magnitude as the therapeutic dose, indicating the clinical relevance of the used concentrations.
The effects induced by the HDACis clearly enhanced uptake of the radiolabeled SSA [ 111 In]In-DOTATATE, as well as SSTR2 mRNA expression levels in both BON-1 and NCI-H727 cells. VPA and ENT induced the strongest effects in both cell lines. CI-994, targeting the same HDAC enzymes as VPA and ENT, also induced strong upregulation in BON-1 cells, suggesting that epigenetic modifiers targeting HDAC class I enzymes are strongly involved in SSTR2 transcription. Therefore, we expected that MOC, targeting both HDAC class I and class IV, would also enhance SSTR2 expression. However, only weak SSTR2 upregulation and even downregulation was observed, suggesting that HDACis may have induced cell-specific responses.
The extent of receptor upregulation upon HDACi treatment on SSTR2 mRNA expression level and uptake of [ 111 In]In-DOTATATE correlated significantly in NCI-H727 cells (r 2 = 0.9005). For BON-1 cells, an r 2 of 0.4534 was obtained. This correlation was not statistically significant due to the potent effects of VPA treatment on the uptake of [ 111 In]In-DOTATATE, i.e., imbalance between mRNA and uptake levels. With the exception of this condition, all other conditions indicated that the enhanced uptake was caused by SSTR2 upregulation, instead of other mechanisms-of-actions, e.g., faster recycling of SSTR2 to the cell membrane after internalization. Next to the correlation between SSTR2 mRNA expression levels and uptake of [ 111 In]In-DOTATATE, it has been demonstrated in literature that the uptake of radiolabeled SSTR2 analogue is associated with SSTR2 protein expression levels [27,28]. We therefore hypothesized that HDACi treatment increased the uptake of [ 111 In]In-DOTATATE by upregulation of SSTR2 protein expression levels.
In our study, we also evaluated whether SSTR2 baseline expression levels were associated with the extent of HDACi-induced SSTR2 upregulation. In general, our results showed that the strongest effects were induced in BON-1 cells, e.g., 8.63-, 4.16-, and 2.06-fold increased uptake of [ 111 In]In-DOTATATE after VPA treatment in BON-1, NCI-H727, and GOT1 cells, respectively. This pattern was observed for the majority of HDACis, suggesting that there may have been a relationship between extent of receptor upregulation and SSTR2 baseline expression levels. In the study of Exner et al. [29], it was demonstrated that SSTR2 mRNA expression levels of BON-1 and NCI-H727 cells are lower than or comparable to control pancreatic and ileum tissue, respectively. Since GOT1 has higher SSTR2 mRNA expression, we hypothesized that these cells had more expression than control tissue. We thereby demonstrated that HDACis could upregulate SSTR2 expression in NET cell lines characterized by a broad range of baseline expression levels, with SSTR2 expression levels lower and higher than control tissue.
To the best of our knowledge, we are the first reporting on the reversibility of SSTR2 upregulation after HDACi withdrawal. As epigenetic histone modifications are part of a dynamic process and the resulting marks are therefore reversible, we hypothesized that SSTR2 expression levels would go back to baseline. In line with this, our results showed that effects induced by HDACi treatment were largely and rapidly reversible. Generally, the largest reductions were observed in the first day. One day after drug withdrawal in NCI-H727 cells, a significant reduction was observed in all conditions, which frequently resulted in SSTR2 downregulation compared to control cells. We hypothesized that upon drug withdrawal, HDACi enzymes were over-activated, resulting in strong histone deacetylation and thus reduced SSTR2 expression levels. However, over a time course of three days, control expression levels were reached again. This quick reversibility upon HDACi withdrawal can provide guidance for the timing between HDACi administration and [ 177 Lu]Lu-DOTATATE injection in preclinical studies in order to obtain beneficial effects.
Based on our analysis, VPA induced the strongest effects on uptake of [ 111 In]In-DOTATATE in all three examined NET cell lines. Therefore, the effect of VPA on radiosensitivity was examined using external beam irradiation. Using external beam irradiation, the radiosensitizing effect was distinguished from other possible mechanisms, e.g., increased therapeutic effect as a consequence of increased [ 177 Lu]Lu-DOTATATE uptake or a combination of these two. We observed that the radiosensitivity of all NET cell lines was significantly increased after VPA treatment. This is in line with earlier published data by Jin et al. [19] who showed a slightly increased radiosensitivity upon treatment of BON-1 and QGP-1 cells with CI-994. Similar results were demonstrated for several other cancer types [30][31][32][33]. This suggests that VPA may have a dual function; it increases both SSTR2 mRNA expression levels and uptake of [ 111 In]In-DOTATATE, and it increases radiosensitivity towards PRRT. Another major advantage is that VPA is already used in a clinical setting, e.g., for treatment of epilepsy and psychiatric disorders [34,35]. This HDACi can therefore be of great interest for its potency to upregulate SSTR2.
Although upregulation of the target receptor has already been demonstrated to be successful for improving radioligand therapy for NETs in vitro [36] and increasing uptake of radiolabeled somatostatin analogues in vivo [20,22], the improved therapeutic effect on tumor growth in vivo is not established yet for upregulation of the target receptor in combination with [ 177 Lu]Lu-DOTATATE treatment. In prostate cancer, several approaches are under investigation to increase target expression levels, i.e., treatment with antiandrogen MDV3100 resulting in an increased uptake of radiolabeled PSMA-targeting antibody 64 Cu-J591 [37], and, more importantly, treatment with enzalutamide enhancing PSMA expression and thereby improving survival in xenograft models upon combination with PSMA antibody drug conjugates [38]. In contrast to the above-described study by DiPippo et al. [38], Lückerath et al. [39] did not show an increased therapeutic response after treatment with [ 177 Lu]Lu-PSMA617 in combination with enzalutamide which caused an increased PSMA expression. In the study of McDevitt et al. [40] a feed-forward loop was described in prostate cancer xenografts irradiated with [ 225 Ac]hu11B6, causing upregulation of the human kallikrein peptidase 2, which is targeted by hu11B6 itself. Therefore, the potential of a combinational therapy consisting of HDACi and [ 177 Lu]Lu-DOTATATE in NET models should be addressed in future preclinical studies. Moreover, in vivo studies are required to examine the effect of HDACi treatment on SSTR2 expression level itself. There are several variables which should be taken into account, e.g., HDACi dose, treatment duration, and frequency and route of administration. Moreover, the quick reversibility after HDACi withdrawal in vitro, as was observed in our study, indicates that timing between HDACi and DOTATATE injection may be an important factor in preclinical studies. With these in vivo studies, it is also important to determine tumor-to-background ratios, thereby taking into account the effect of HDACis on physiological tissues.

Conclusions
In conclusion, we demonstrated that SSTR2 upregulation by HDACi treatment was possible in NET cell lines of different origins, especially using HDACi specifically targeting class I HDACs, and with strongest effects observed in cells characterized by low SSTR2 baseline expression levels. Generally, the effects were rapidly and largely reversible within one day after HDACi withdrawal. This suggests that proper timing of HDACi treatment could be an important factor in both preclinical and clinical settings. Future studies will provide definite answers about the potential for this combinational therapy in order to improve NET patient outcomes.