Combination of 131I-trastuzumab and lanatoside C enhanced therapeutic efficacy in HER2 positive tumor model

Lanatoside C has a promising anti-tumor activity and is a potential candidate for radiosensitizers. In this study, we have investigated the therapeutic efficacy of the combination of 131I-trastuzumab and lanatoside C for inhibition of human epidermal growth factor receptor 2 (HER2) positive tumor progression in NCI-N87 xenograft model. The combination treatment (131I-trastuzumab and lanatoside C) showed highest cytotoxicity when compared to non-treated control or trastuzumab alone or 131I alone or 131I-trastuzumab alone in vitro. Biodistribution studies using 131I-trastuzumab or combination of 131I-trastuzumab and lanatoside C showed tumor uptake in BALB/c nude mice bearing HER2 positive NCI-N87 tumor xenograft model. The higher tumor uptake was observed in 131I-trastuzumab (19.40 ± 0.04% ID/g) than in the combination of 131I-trastuzumab and lanatoside C (14.02 ± 0.02% ID/g) at 24 h post-injection. Most importantly, an antitumor effect was observed in mice that received the combination of 131I-trastuzumab and lanatoside C (p = 0.009) when compared to control. In addition, mice received lanatoside C alone (p = 0.085) or 131I-trastuzumab alone (p = 0.160) did not significantly inhibit tumor progression compared with control. Taken together, our data suggest that combination of 131I-trastuzumab and lanatoside C might be a potential synergistic treatment for radioimmunotherapy to control the HER2 positive tumor.


Scientific Reports
| (2021) 11:12871 | https://doi.org/10.1038/s41598-021-92460-0 www.nature.com/scientificreports/ In the present study, we investigated the effect of cell proliferation of lanatoside C on two cancer cells (NCI-N87 and MDA-MB231). In addition, the cytotoxicity and therapeutic effects of combined treatment with 131 I-trastuzumab and lanatoside C were evaluated in HER2 positive cancer cells in vitro and in vivo.

Effect of lanatoside C on cell proliferation of cancer cells. Before investigating the 131 I-trastuzumab
in combination with lanatoside C, we determined the cytotoxic effects of lanatoside C in NCI-N87 (HER2 positive) and MDA-MB231 (HER2 negative) cancer cells. Both the cells were treated with various concentrations of lanatoside C and assesses for cell viability using Ez-Cytox cell viability assay. All the doses of lanatoside C shows strong decreases of cell proliferation in both cancer cells when compared to non-treated control cells (p < 0.001), suggesting efficient cellular uptake of those lanatoside C concentrations (Fig. 1). Significant decrease of cell viability relative to untreated control was apparent, and most evident following treatment of NCI-N87 with 0.125 nM/well lanatoside C. A notable difference in cell viability was observed between 0.125 µM and 1 µM of lanatoside C. However, no significant difference was found between 0.25 µM and 0.5 µM in both cancer cells.
Lanatoside C increases the sensitivity of NCI-N87 cells to 131 I-trastuzumab radioimmunotherapy in-vitro. The cytotoxic effects of lanatoside C on treatment of 131 I-trastuzumab in NCI-N87 cells was determined using the Ez-Cytox cell viability assay following 96 h incubation.
Lanatoside C increases the sensitivity of NCI-N87 xenografts to 131 I-trastuzumab radioimmunotherapy in-vivo. The maximum dose of 131 I for therapeutic efficacy is well known through several studies 17 . Therefore, we performed experiments at a concentration of 400 μCi based on the results of previous studies. We observed that tumor growth rapidly occurred in the vehicle control group administered 0.01% DMSO in saline. Most importantly, it was confirmed that the growth of the tumor was significantly decreased in the group treated with combination of 131 I-trastuzumab and lanatoside C (p < 0.01) and 131 I-trastuzumab alone (p < 0.05) compared to the non-treated control group (Fig. 3). Moreover, there is no significant differences between lanatoside C treated group and the non-treated control group.
Biodistribution of 131 I-trastuzumab or combination of 131 I-trastuzumab and lanatoside C in vivo. The results of the biodistribution data of 131 I-trastuzumab or combination of 131 I-trastuzumab and lanatoside C in NCI-N87 xenografted BALB/c nude mice obtained at 4, 24, 48 h post injection (h.p.i) are shown in Fig. 4. Higher uptake of 131 I was clearly observed in blood, spleen, liver,heart, lung, kidney, and NCI-N87 tumor in 131 I-trastuzumab alone and combination of 131 I-trastuzumab and lanatoside C groups at 4 h (Fig. 4A). Notably, 131 I uptake in the lung was slightly higher in combination of 131 I-trastuzumab and lanatoside C group (12.14 ± 8.39% ID/g) compared to 131 I-trastuzumab group (6.70 ± 2.62% ID/g) at 4 h. However, there were no significant differences between groups. The high accumulation of 131 I in 131 I-trastuzumab is consistent with extraction of the activity from the blood at 4 h (24.9 ± 0.09% ID/g), 24 h (16.5 ± 0.02% ID/g), and 48 h (11.4 ± 0.04% ID/g). Moreover, the data reveal that NCI-N87 tumor uptake was shown 4 h.p.i in 131 I-trastuzumab (11.1 ± 0.01% ID/g) with a steady increase through 24 h (19.4 ± 0.04% ID/g) and 48 h (16.8 ± 0.04% ID/g) ( Fig. 4A-C). How-   www.nature.com/scientificreports/ C group showed significantly higher inhibition of tumor growth compared to 131 I-trastuzumab alone group (Fig. 3).

Discussion
The present study demonstrated that the combination of 131 I-trastuzumab RIT and lanatoside C can improve the therapeutic effects in HER2 positive tumor. We demonstrated that lanatoside C increases the sensitivity of NCI-N87 cells and xenograft models to 131 I-trastuzumab RIT in vitro and in vivo (Figs. 2 and 3). The results of the present study are consistent with those of earlier studies, in which treatment with lanatoside C led to dosedependent cytostatic or cytotoxic responses of radiosensitization in two colorectal cancer cell line 16 . Previous studies have shown the effect of lanatoside C as a radiosensitizer at external radiotherapy. However, no study has been conducted in case of RIT. We demonstrated the effect of lanatoside C as a radiosensitizer of 131 I-trastuzumab RIT in HER2 positive cells (Fig. 2). According to literature, tumor cells are most radiosensitive in the M and G2 phases 18 . Moreover, lanatoside C induces cell cycle arrest in the G2/M phase could be responsible for the difference in radiosensitization 19 . Another study also shown that lanatoside C increased cell sensitivity to radiation by inhibiting DNA damage repair 16 . Lanatoside C is known to inhibit cell proliferation and induces cell apoposis in tumor cells involving various cellular signaling pathways [19][20][21] . Additional killing of NCI-N87 cells by 131 I-trastuzumab could be due to associated high energy beta radiation (0.2 MeV of 131 I). The enhanced magnitudes of damage are due to the localization of radioisotope very close to cellular targets at membrane and cytoplasm level. The present study also showed that the higher level of cell death measured by Ez-Cytox cell viability, proliferation, and cytotoxicity assay kit was observed after treatment of lanatoside C at various concentrations. Moreover, lanatoside C enhanced 131 I-trastuzumab RIT in vitro. Combination of 131 I-trastuzumab and lanatoside C showed highest tumor cell growth inhibition when compared to other groups such as 131 I-trastuzumab RIT alone, trastuzumab alone and 131 I alone in NCI-N87 cells. The combinatorial treatment of lanatoside C with 131 I -trastuzumab RIT would result in higher tumor cell growth inhibition and thus, there is an increase in the number of cell death.
Radionuclide 131 I emits both β-emission and γ-emission which could be used for radiotherapy. In this work, BALB/c nude mice bearing NCI-N87 tumors were intravenously injected with 400 μCi 131 I-trastuzumab. It was found that 131 I-trastuzumab after intravenous injection exhibited obvious tumor accumulation. Moreover, the biodistribution of 131 I-trastuzumab in mice bearing HER2 positive tumors showed maximum higher tumor uptake at 24 h, but there is no significant difference from combination of 131 I-trastuzumab and lanatoside C. Biodistribution studies in nude mice showed that 131 I-trastuzumab targeted the tumors overexpressing the Human HER2 receptor in vivo. 131 I-trastuzumab accumulated to a significant extent in tumors with % ID/g of 19.4 ± 0.04 in the tumor tissues at 24 h.p.i. which decreased to 16.8 ± 0.045 at 48 h.p.i. Combination of 131 I-trastuzumab and lanatoside C also showed similar pattern of tumor uptake in mice bearing HER2 positive tumors. Although the combination of 131 I-trastuzumab and lanatoside C group showed less tumor uptake than the 131 I-trastuzumab alone group at 24 h.p.i and 48 h.p.i, the combination of 131 I-trastuzumab and lanatoside C group revealed better treatment response than the 131 I-trastuzumab alone group. This result might imply that lanatoside C can be considered as a radiosensitizer (Fig. 3). Moreover, there is no significant difference between 131 I-trastuzumab alone and combination of 131 I-trastuzumab and lanatoside C at all the time points (4 h, 24 h and 48 h). Steady blood clearance of 131 I-trastuzumab and 131 I-trastuzumab combined with lanatoside C demonstrated the stability of the complex under in vivo conditions. The high uptake of 131 I-trastuzumab by the liver, lungs and spleen may be due to the rich blood flow and its effective metabolism in the reticuloendothelial system of these organs. The tumor uptake and biodistribution ratio of 131 I-trastuzumab was found to be almost same when compared to combination of 131 I-trastuzumab and lanatoside C at 4, 24, and 48 h.p.i. However, lung and muscle showed slightly higher uptake in combination of 131 I-trastuzumab and lanatoside C than 131 I-trastuzumab at 4 h.p.i. Most importantly, those tissues and other non-targeted tissuses did not show significant differences of uptake between 131 I-trastuzumab alone and 131 I-trastuzumab combined with lanatoside C at 4, 24, and 48 h.p.i. The expectation of high tumor uptake of the radiolabeled 131 I-trastuzumab in HER2 positive tumors was confirmed by the present biodistribution study. Most importantly, combination of 131 I-trastuzumab and lanatoside C showed better therapeutic effect than 131 I-trastuzumab or lanatoside C alone in this HER2 positive tumor model. However, there is a limitation because we performed our study only in one HER2 positive tumor model and this might warrant further diverse experiments in other HER2 positive tumor models.
We have performed the clinical trial of RIT for NHL patients using 131 I-rituximab for 17 years [22][23][24][25][26] . RIT demonstrated excellent outcomes, but there are some refractory patients who revealed resistance to RIT. We expected that it is necessary to treat these refractory patients using more enhanced RIT protocols. We hope that 131 I-rituximab with lanatoside C can be applied for refractory NHL patients, because we found out that addition of lanatoside C can enhance the RIT of 131 I-trastuzumab.

Conclusions
In conclusion, our findings suggest that lanatoside C has the potential to sensitize 131 I-trastuzumab induced cytotoxicity in NCI-N87 cells in vitro and enhanced strong antitumor effect in a HER2 positive xenograft model. As the results, the combined therapy with 131 I-trastuzumab and lanatoside C achieved excellent synergistic in vivo therapeutic effects in HER2 positive tumor bearing mice. Therefore, our in vitro and in vivo results provide potentially important and promising therapeutic strategies for future clinical translations in radioimmunotherapy.

Materials and methods
Cells and reagents. NCI-N87 and MDA-MB231 cell lines were purchased from the American Type Culture Collection (New York, USA). All these cells were cultured in RPMI-1640 (WELGENE Inc., Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Omega Scientific, Inc., Tarzana, CA, USA), 2 mmol/L L-glutamine, 5% Penicillin/Streptomycin in a humidified atmosphere of 5% CO 2 at 37° C. Lanatoside C was purchased from Sigma-Aldrich (St. Louis, MO, USA). Herceptin (Trastuzumab), A therapeutic agent that targets HER2 (Human Epidermal growth factor Receptor 2) was purchased from Roche. 131 I was purchased in New Korea Industrial Co., Ltd. Ez-Cytox cell viability, proliferation, and cytotoxicity assay kit was purchased from DoGenBio (Seoul, Korea).
Radiolabeling. Radiolabeling of trastuzumab with 131 I was achieved using the Pierce Pre-coated Iodination Tubes (Thermo scientific, U.S.A.) and carried out in accordance with the protocol provided by Thermo scientific 27 . Briefly, the pierce pre-coated iodination tube was wetted with 1 ml of Tris iodination buffer and decanted. 500 µCi of 131 I was added to the Pierce pre-coated iodination tube and activated for 5 min at room temperature. Subsequently, 100 µg of trastuzumab was added to the tubes and the reaction mixture was incubated for 10 min at room temperature. Radiolabeling purity was determined by instant thin-layer chromatography (Agilent Technologies) using saline. Incorporation purity was always exceeded 95%.

Determination of the effect of lanatoside C on cancer cells.
For the in vitro cell viability assay was carried out according to protocol described by Ez-Cytox cell viability, proliferation, and cytotoxicity assay kit 28  Xenograft model. To make a xenograft mouse model, NCI-N87 cells (5 × 10 6 /mouse/0.1 ml) were injected subcutaneously into the dorsal right flank of 6-week-old BALB/c nude mice. When the tumor volume reached approximately 150 mm 3 , the mice were randomly assigned to four groups (7 mice/group): (1) non-treated control group (untreated control: NCI-N87 tumor), (2) vehicle control group (0.01% DMSO in saline, 100 μl, the first 3 days), (3) lanatoside C (6 mg/kg body weight, the first 3 days intraperitoneal injection), (4) 131 I-trastuzumab group (400 μCi, once tail vein injection), and (5) 131 I-trastuzumab and lanatoside C combination (lanatoside C, 6 mg/kg body weight, the first 3 days intraperitoneal injection, and 131 I-trastuzumab, 400 μCi, once tail vein injection). Tumor size and body weight were measured once a week, and the tumor volume (V) was calculated using the following formula: V = L × W 2 /2 (L, long diameter of the tumor; W, short diameter of the tumor).
Biodistribution Study. The biodistribution of the 131 I radiolabeled trastuzumab was assessed in BALB/c nude mice bearing established NCI-N87 xenografts. Mice were injected with 131 I-trastuzumab (400 μCi) or combination of 131 I-trastuzumab and lanatoside C by tail vein injection. At 4, 24 and 48 h after post-injection (h.p.i), groups of 4 mice were euthanized by isofluorane anesthesia and then immediately bled via cardiac puncture. Tumors and normal tissues (muscle, bone, lipid, spleen, pancreas, intestine, liver, heart, lung, kidney and tail) were then resected and placed in individual γ-counter tubes. The activity of all samples were then counted on a gamma counter (2480 Wizard 2 , PerkinElmer, Waltham, MA, USA), and the percent injected dose per gram (% ID/g) calculated. Results were expressed as Mean ± SD for each time point.