New Therapy Options for Neuroendocrine Carcinoma of the Pancreas—The Emergent Substance GP-2250 and Gemcitabine Prove to Be Highly Effective without the Development of Secondary Resistances In Vitro and In Vivo

Simple Summary Neuroendocrine carcinoma of the pancreas is a highly aggressive form of neuroendocrine tumor associated with poor survival and increasing occurrence. GP-2250 is an emergent substance showing antineoplastic properties, especially in combination with Gemcitabine. This study was the first to evaluate the antineoplastic effects of GP-2250 on pancreatic neuroendocrine carcinoma. The combination of GP-2250 and Gemcitabine showed highly synergistic effects in a cell culture model, as well as in mice, without the development of secondary resistances. These findings form the basis for further clinical evaluation of a highly promising combination therapy. Abstract Neuroendocrine carcinoma of the pancreas (pNEC) is an aggressive form of neuroendocrine tumor characterized by a rising incidence without an increase in survival rates. GP-2250 is an oxathiazinane derivate possessing antineoplastic effects, especially in combination with Gemcitabine on the pancreatic adenocarcinoma. The cytotoxic effects of the monotherapy of GP-2250 (GP-2250mono) and Gemcitabine (Gemmono), as well as the combination therapy of both, were studied in vitro using an MTT-assay on the QGP-1 and BON-1 cell lines, along with in vivo studies on a murine xenograft model of QGP-1 and a patient-derived xenograft model (PDX) of Bo99. In vitro, Gemmono and GP-2250mono showed a dose-dependent cytotoxicity. The combination of GP-2250 and Gemcitabine exhibited highly synergistic effects. In vivo, the combination therapy obtained a partial response in QGP-1, while GP-2250mono and Gemmono showed progressive disease or stable disease, respectively. In Bo99 PDX, the combination therapy led to a partial response, while the monotherapy resulted in progressive disease. No development of secondary resistances was observed, as opposed to monotherapy. This study was the first to evaluate the effects of the emerging substance GP-2250 on pNEC. The substance showed synergism in combination with Gemcitabine. The combination therapy proved to be effective in vitro and in vivo, without the development of secondary resistances.

The cells were grown as a monolayer and cultured in 10 cm dishes at 37 • C and 5% CO 2 in a humidified atmosphere.
For the in vivo patient-derived xenograft (PDX) experiments, human pancreatic tissue Bo99 was used. This tissue was derived from a neuroendocrine carcinoma of the pancreas head, pT3 pN1 (4/15) M0 L1 V1 Pn1, UICC Stadium IIb, G3. This patient postoperatively received six cycles of Cisplatin and Etoposide and developed tumor relapse 6 months afterwards. The treatment with Carboplatin and Etoposide was initiated and modified. Eventually, chemotherapy was terminated due to the deterioration of the patient's general condition followed by exitus letalis 23 months after the initial diagnosis.

Reagents
GP-2250 ultrapure powder (Geistlich Pharma AG, Wolhusen, Switzerland) was set to physiological pH after being dissolved in double distilled water (ddH 2 O) and subsequently sterile filtered. The preparation was freshly performed once weekly.
A total of 2 h prior to measurement, 10 µL yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) reagent (5 mg/mL) was applied into each well. Viable cells metabolize yellow MTT into violet Formazan crystals; thus, the number of viable cells is directly proportional the amount of violet Formazan. The test media was removed, and 100 µL DMSO (Dimethylsulfoxide) was added. After the complete solution of the crystals, the viability was analyzed via a microplate absorbance reader measuring the optical density at a wavelength of 550 nm and at 720 nm as reference (UVM 340, Biochrom, Cambridge, UK). The assay was repeated in four to six independent experiments in consecutive passages. The MTT assay was performed as previously described by van Meerloo et al. [23].

Xenograft QGP-1 and Patient Derived Xenograft Bo99
For this study, 6-week old female NMRI Foxn1nu/Foxn1nu mice (Janvier, Saint-Berthevin, France) were used. One week prior to initiation, they were acclimated into a 12 h light cycle-controlled environment. Standard laboratory food and water were allowed ad libitum. Anaesthesia was performed by Isofluran per inhalationem. As described previously, either 5 × 10 6 QGP-1 [20,24] cells, or tumor tissue fragments in the case of Bo99 [25], were implanted subcutaneously into each flank region. Afterwards, the mice were monitored for general health status and the development of subcutaneous tumors. The tumor volume was calculated as V = 1 2 (ab) 2 (a = larger axis, b = smaller axis and a⊥b) via the measurement of the tumor diameter using a caliper. After reaching a tumor volume of 200 mm 3 , the mice were randomized into several groups: Group 1, controls (n = 6); group 2, GP-2250 monotherapy (n = 6); group 3, Gemcitabine monotherapy (n = 6); group 4, combination therapy GP-2250 and Gemcitabine (n = 6). In Bo99, GP-2250 monotherapy was not performed. The controls received a physiological saline solution, group 2 was intraperitoneally treated with 500 mg/kg*BW GP-2250 three times per week, group 3 was intraperitoneally treated with 50 mg/kg*BW Gemcitabine twice weekly and group 4 intraperitoneally received 500 mg/kg*BW 2250 and 50 mg/kg*BW Gemcitabine thrice and twice weekly, respectively, on alternating days. The tumor volume was measured twice a week. The experiment was terminated when the tumor volume reached 1000 mm 3 . The tumors were evaluated after 4 and 6 weeks according to the Response Evaluation Criteria in Solid Tumors (RECIST) [3].
To analyze the development of a secondary resistance against the combination of GP-2250 and Gemcitabine a therapeutical break was performed after 60 days of treatment. The combination therapy was started once again after the initial volume was reached. The above-mentioned scheme was used again. Due to a primary resistance, the Gemcitabine monotherapy was not interrupted.

Ethics Approval and Consent to Participate
The local ethical committee approved the collection of sample tissue from patients with pancreatic cancer as well as the implantation and expansion of cancer tissue in xenograft mouse models. The written, informed consent of all patients was documented according to the local ethics guidelines. The study was conducted according to the Declaration of helsinki. All procedures were performed according to a protocol approved by the Ethics Committee of the Ruhr-University Bochum, Germany (permission no 2392, 10th amendment). All of the animal experiments were performed according to the guidelines of the local Animal Use and Care Committees (permission no. 81-02.04.2018.A169).

Statistical Analysis
The results of the MTT assay (percentage of viable cells), as well as the characteristics of mice (body weight, tumor volume), are presented as mean ± standard derivation. The hypotheses of no difference between the four groups for the continuous variables were tested using ANOVA, and pairwise tests were performed using t tests. p values ≤ 0.05 were considered to be statistically significant and were indicated in the figures as follows: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05. The testing was performed using Graph Pad Prism 9.1.0 by Graph Pad Software, San Diego, CA, USA.

MTT
As previously described, GP-2250 has a dose-dependent effect on the cell viability of pancreatic adenocarcinoma [20]. In order to determine the effects and potential synergism of GP-2250 in combination with Gemcitabine on pancreatic neuroendocrine tumors, MTT assays were performed. In QGP-1, concerning GP-2250, a significant reduction in cell viability was achieved in comparison to the control, starting at concentrations of 100 µM, with an exemption of 150 µM. On average, incubation with 100 µM and 150 µM GP-2250 led to a decrease in cell viability of 7% ± 0.069 (p = 0.038) and 5% ± 0.113 (p = 0.147), and the application of 200 µM, 250 µM and 300 µM reduced cell viability to 35% ± 0.080, 50% ± 0.124 and 5% ± 0.033 (p < 0.001), respectively ( Figure 1).
In Gemcitabine, a significant reduction in the cell viability in the QGP-1 cell line was observed at concentrations of 0.1 µM and higher. The application of 0.001 µM and 0.01 µM did not significantly impact cell viability, whereas at concentrations of 0.1 µM, 1 µM and 10 µM, 46% ± 0.054, 28% ± 0.071 and 13% ± 0.047 (p < 0.001) of cells were viable ( Figure 1).
The cultivation of Bo99 was not successful in a monolayer cell culture.  175 µM GP-2250mono, respectively. This was also observed when combining 200 µM GP-2250 and 0.001 µM or 0.01 µM Gemcitabine, respectively, which proved to decrease cell viability significantly in comparison to 200 µM GP-2250mono.

PDX and Xenograft
In order to elucidate the therapeutic effects in vivo, the treatment was tested in the PDX model Bo99 and in xenografts derived from the established cancer cell line QGP-1.
In Bo99, the treatment with Gemcitabine and the combination of GP-2250 and Gemcitabine both led to a significant decrease in tumor growth volume in comparison to the controls ( Figure 4). All of the controls showed progressive disease according to the RECIST (mean increase in tumor volume: 8.6 fold ± 4.094) and reached the abortion criteria within 6 weeks and therefore had to be terminated. The treatment with Gemcitabine mono reduced tumor growth in comparison to the control group; however, the tumor volume increased 1.77 fold ± 0.385 within 8 weeks and therefore showed progressive disease according to the RECIST. The combination therapy of GP-2250 and Gemcitabine, on the other hand, resulted in a partial response after 8 weeks, with a mean reduction to 50.5% ± 0.145 of the initial tumor volume. The combination therapy therefore proved to be highly effective in comparison to Gemcitabine mono (p < 0.001).  In order to further elucidate the development of secondary resistances against therapy, a therapeutic break was introduced in the combination therapy cohort afte days of treatment but not in the Gemcitabine mono cohort, due to the developmen primary resistance in the latter ( Figure 5). The treatment of the combination cohort resumed when the tumor reached the initial volume after two weeks. The rene  In order to further elucidate the development of secondary resistances against the therapy, a therapeutic break was introduced in the combination therapy cohort after 60 days of treatment but not in the Gemcitabine mono cohort, due to the development of primary resistance in the latter ( Figure 5). The treatment of the combination cohort was resumed when the tumor reached the initial volume after two weeks. The renewed application of Gemcitabine and GP-2250 led to a decrease in tumor volume, resulting in a partial response again. The observed reduction under combination therapy was significant when compared to the tumor volume increase and therefore the progressive disease under Gemcitabine mono (p = 0.007).
Similarly, in QGP-1 (Figure 6), the controls exhibited a pronounced increase in tumor volume, which nearly doubled in size over the observation period (increase by 188% ± 0.413). The monotherapy with GP-2250 resulted in an average increase in tumor volume by 25.4% ± 0.334 in progressive disease as well. The treatment with Gemcitabine alone decreased tumor volume significantly compared to the untreated control (relative reduction in tumor volume: 22.4% ± 0.265, p = 0.007) and led to a stable disease according to the RECIST. however, only the combination of GP-2250 and Gemcitabine reduced the tumor volume to an extent that met the criteria for partial response according to the RECIST [3] (77.9% ± 0.091, p = 0.002, respectively). This presents a significant decrease in the tumor volume compared to Gemcitabine mono (p ≤ 0.001). A follow-up period with a therapeutical break was not feasible due to the tumor's tendency to form necrosis and thus present abortion criteria. Similarly, in QGP-1 (Figure 6), the controls exhibited a pronounced increase in tumor volume, which nearly doubled in size over the observation period (increase by 188% ± 0.413). The monotherapy with GP-2250 resulted in an average increase in tumor volume by 25.4% ± 0.334 in progressive disease as well. The treatment with Gemcitabine alone decreased tumor volume significantly compared to the untreated control (relative reduction in tumor volume: 22.4% ± 0.265, p = 0.007) and led to a stable disease according to the RECIST. However, only the combination of GP-2250 and Gemcitabine reduced the tumor volume to an extent that met the criteria for partial response according to the RECIST [3] (77.9% ± 0.091, p = 0.002, respectively). This presents a significant decrease in the tumor volume compared to Gemcitabine mono (p ≤ 0.001). A follow-up period with a

Discussion
The data given are the first to evaluate the effects of the emerging substance GP-225 on pNEC. The substance showed strong synergism in combination with Gemcitabine, an the combination therapy proved to be highly effective in vitro on two cell lines of pNE and in vivo in a cell line model and a PDX model. The latter is an important model translational medicine, bridging the gap between animal and human studies [26].
pNECs are a highly aggressive subgroup of neuroendocrine tumors tied to a extremely unfavorable prognosis [9,10]. The current first-line therapy, consisting Cisplatin/Carboplatin in combination with Etoposide, is accompanied by poor tolerabili and severe side effects [16] and requires a good performance status for application [15]. second-line therapy has yet to be fully established. The German guidelines express a open preference for Capecitabine plus Temozolomid, FOLFOX (folinic acid, Fluorouracil, Oxaliplatin) or FOLFIRI (folinic acid, 5-Fluorouracil, Irinotecan) [15], whic in turn, depends on the sufficient performance status of the patients as well [27]. The on targeted therapy that has been FDA approved in the last 30 years is the mTor inhibito Everolimus, which has shown a median progression-free survival rate of 11 months in th Everolimus group compared to 4.6 months in the placebo group [28][29][30].
This underlines the crucial need to establish new therapy regimens and concepts wi better tolerability, especially for frailer or more comorbid patients.
In the selection of the therapeutic agents for this study, we focused on agen applicable as a second-line therapy. The clinical course of our PDX model Bo99 indicat a necessary second-line therapy with moderate side effects. The first experiments pancreatic adenocarcinoma suggest a synergistic effect of the novel agents GP-2250 an Gemcitabine [21].

Discussion
The data given are the first to evaluate the effects of the emerging substance GP-2250 on pNEC. The substance showed strong synergism in combination with Gemcitabine, and the combination therapy proved to be highly effective in vitro on two cell lines of pNEC and in vivo in a cell line model and a PDX model. The latter is an important model in translational medicine, bridging the gap between animal and human studies [26].
pNECs are a highly aggressive subgroup of neuroendocrine tumors tied to an extremely unfavorable prognosis [9,10]. The current first-line therapy, consisting of Cisplatin/Carboplatin in combination with Etoposide, is accompanied by poor tolerability and severe side effects [16] and requires a good performance status for application [15]. A second-line therapy has yet to be fully established. The German guidelines express an open preference for Capecitabine plus Temozolomid, FOLFOX (folinic acid, 5-Fluorouracil, Oxaliplatin) or FOLFIRI (folinic acid, 5-Fluorouracil, Irinotecan) [15], which, in turn, depends on the sufficient performance status of the patients as well [27]. The only targeted therapy that has been FDA approved in the last 30 years is the mTor inhibitor Everolimus, which has shown a median progression-free survival rate of 11 months in the Everolimus group compared to 4.6 months in the placebo group [28][29][30].
This underlines the crucial need to establish new therapy regimens and concepts with better tolerability, especially for frailer or more comorbid patients.
In the selection of the therapeutic agents for this study, we focused on agents applicable as a second-line therapy. The clinical course of our PDX model Bo99 indicates a necessary second-line therapy with moderate side effects. The first experiments in pancreatic adenocarcinoma suggest a synergistic effect of the novel agents GP-2250 and Gemcitabine [21].
Gemcitabine is a reputable chemotherapeutic agent that is approved for, among other things, locally advanced and/or metastasized ductal adenocarcinoma of the pancreas [31]. It is mainly applied in patients whose performance status does not allow for treatment with other agents [32]. Concerning neuroendocrine tumors, Gemcitabine has been systematically evaluated nearly exclusively in small collectives with small-cell lung cancer (SCLC). Cormier et al. observed a response rate of 29% in 29 patients with previously untreated SCLC [33], while van der Lee et al. found an overall response rate of 13% in a collective of 41 patients with pre-treated, limited-or extensive-stage SCLC [34]. In 2004, Kulke and colleagues examined a collective of 18 patients with metastatic neuroendocrine tumors, of which seven presented pancreatic primaries, and found disease stabilization in 65% of them following monotherapy with Gemcitabine, although no radiological or biochemical response was achieved [35]. All of the studies concurringly found Gemcitabine to be well tolerated, with a favorable side-effects profile [33][34][35]. Concerning pNEC in particular, data are currently lacking; however, several case-reports observed a response and satisfactory tolerability to treatment with Gemcitabine as a salvage therapy [36], third-line therapy [37] or in combination with S-1 [38,39].
The oxathiazinane derivate GP-2250, on the other hand, has been proven to possess antiproliferative and antineoplastic properties on pancreatic cancer in vitro and in vivo [20]. The tolerability of the combination therapy with Gemcitabine has been established as favorable; correspondingly, no test animal had to be terminated due to any adverse effects of the treatment [20]. Currently, the combination therapy is being tested in a Phase I/II trial in subjects with advanced pancreatic cancer [40].
This study in pNEC supports the major relevance of this combination. While the monotherapy with Gemcitabine achieved stable disease in a PDX model and progressive disease in QGP-1 cells, the combination therapy of GP-2250 and Gemcitabine even led to partial response according to the RECIST and thereby led to a significant decrease in tumor volume in comparison to the monotherapy. The number of PDX studies, especially for pNET, is quite small. Chetser et al. showed just a stable disease in a pNET PDX model using the FDA approved agent Everolimus and Sapanisertib, another mTor inhibitor [41]. In another study, the traditional Chinese drug baicalein induced apoptosis and protein changes in vitro and inhibited the migration and tumor growth of BON-1 in vivo [42].
Furthermore, after the therapeutic break, the treatment with the combination therapy resulted in a renewed response, while the monotherapy with Gemcitabine led to the development of resistances after 30 days of treatment. Chemoresistance to Gemcitabine is a well-described problem, developing within weeks after initiation [43]. The mechanisms promoting resistances against Gemcitabine are multifaceted; mediation via pathways including p53 [44], NF-κB, Akt/PI3K [45], reactive oxygen species [46], heat shock proteins [47] and micro RNAs [48] was identified. Overcoming this hurdle presents an enormous opportunity towards the improvement of both the survival and quality of life of this highly vulnerable collective of patients.
This study has several limitations. As in every PDX model, the direct extrapolation of the results to humans is not possible. Furthermore, the investigation is limited due to the absent control with the first-line therapy. In the clinical course, however, the patient showed recurrence and progress during this treatment, which ultimately had to be terminated due to poor tolerability. Moreover, based on the rare tumor entity, there is a limited number of cell lines accessible.
The combination of GP-2250 and Gemcitabine proved to be effective for the treatment of an established cell line and a PDX model of pNEC in vitro and in vivo, without the development of secondary resistances. Further studies are needed to comprehensively evaluate the effects of GP-2250 and Gemcitabine in neuroendocrine carcinoma, especially in auxiliary cell lines.
The present study forms the basis for future clinical trials of a promising combination therapy.

Conclusions
The present study was the first to evaluate the effects of the emerging substance GP-2250 on pNEC. The substance showed strong synergism in combination with Gemcitabine, and the combination therapy proved to be highly effective in vitro and in vivo on both an established cell line and a PDX model of pNEC. Additionally, no development of secondary resistances was observed for the combination treatment. These results are consistent with the findings of prior studies involving PDAC cells and concerning the combination therapy with Gemcitabine and GP-2250 [22]. The additional analysis of GP-2250 may be valuable not only in PDAC but also in pNEC. Although the transfer of animal study results to humans is limited, this study forms the basis for further clinical evaluation of a highly promising combination therapy. Funding: The consumables used in this work were partially funded by Geistlich Pharma AG, Wolhusen, Switzerland. Geistlich Pharma AG played no role in the design of the study or in the collection, analysis or interpretation of the data.

Institutional Review Board Statement:
The local ethical committee approved the collection of sample tissue from patients with pancreatic cancer as well as the implantation and expansion of cancer tissue in xenograft mouse models. The written, informed consent of all patients was documented according to the local ethics guidelines. The study was conducted according to the Declaration of helsinki. All procedures were performed according to a protocol approved by the Ethics Committee of the Ruhr-University Bochum (permission no. 2392, 10th amendment). All animal experiments were performed according to the guidelines of the local Animal Use and Care Committees (permission no. 81-02.04.2018.A169).