Although X atoms possess very high ionization energies, these resulting X@36adz complexes possess low adiabatic ionization energies (AIEs) of 0.78–5.28 eV. In particular, the adiabatic ionization energies (AIEs) of X@36adz  are even lower than the ionization energy (3.89 eV) of Cs atoms, and thus, can be classified as novel nonmetallic superalkalis. Moreover, due to the existence of diffuse excess electrons in B@36adz, this complex not only possesses pretty low AIE of 2.16 eV but also exhibits a remarkably large first hyperpolarizability (β0) of 1.35 × 106 au, indicating that it can also be considered as a new kind of nonlinear optical molecule. As a result, this study provides an effective approach to achieve new metal-free species with an excellent reducing capability by utilizing the cage-like organic complexants as building blocks.In this study, to examine the possibility of using cage-like complexants to design nonmetallic superalkalis, a series of X@3 In MLk+1, one more alkali metal atom will bring an extra valence electron for the electronic shell of M according to the octet rule. Consequently, such an MLk+1 complex has a great tendency to lose the extra valence electron and thus possess strong reducibility (Reducing agents with low ionization energies (IEs) play a crucial role in chemical synthesis. As is well-known, alkali metal atoms possess the lowest ionization energies (5.39–3.89 eV)  among alcibility .2 and N2 molecules  superalkali cations by using hydrogen atoms as ligands. Following a similar rule, the other two series of nonmetallic superalkali cations, namely, FnHn+1+ (n = 1–10) and CxHx+14+ (x = 1–5), have been proposed by In view of the great importance of superalkalis in chemistry, various superalkalis have been theoretically  and expeinuclear  and polyinuclear  superalkinuclear , Zintl-iinuclear , hyperalinuclear , alkali-inuclear , and so inuclear , particuinuclear , have beinuclear  designedMore recently, 6adamanzane (36adz) has been chosen as a representative to design a series of X@36adz  by encapsulating nonmetallic atoms into the cavity of this cage-like complexant in this work  of 8.15–17.42 eV  molecule with a remarkably large first hyperppolarizability of 1.35 × 106 au because the valence electron of boron atom is pushed out of cage to form diffuse excess electrons. We hope that this work will not only provide new nonmetallic members for the superatom family, but will also open the door to design strong reducing matters by embedding nonmetallic atoms into the various cage-like complexants.To verify this hypothesis, the 3work see . The 36ac chains , which hadz]+Na− . In thisadz]+Na− . Under t17.42 eV . In part6adz  level. Based on the optimized structures, the single-point energies, nature population analysis (NPA) charges, and static electric properties were calculated at the CAM-B3LYP/6-311++G  level.In this work, all the calculations were carried out by using the coulomb-attenuated hybrid exchange-correlation functional (CAM-B3LYP) , which h6adz see  to verif6adz  were calculated as the energy difference between the optimized neutral complex and the cation in the geometry of the neutral complex, while their adiabatic ionization energies (AIEs) are defined as the energy difference between the neutral and cationic complex at their respective optimized structures. In addition, the TD-M06-2X calculations were performed to obtain the transition energies and oscillator strengths of the crucial excited states as well as the difference of the dipole moments between the ground state and crucial excited state of X@36adz by using the 6-311++G  basis set. Herein, the dipole moments (µ0), polarizabilities (α0), and first hyperpolarizabilities (β0) are defined as follows,i, j = {x, y, z}.In this work, the vertical ionization energies (VIEs) of X@3All the above calculations were performed by using the GAUSSIAN 16 program package . The dim6adz  compounds have been constructed by encapsulating one X atom into a 36adz cage. After optimization, the geometric structures of X@36adz are illustrated in 6adz compounds are summarized in Initially, seven X@36adz is a cage-like complexant with S4 symmetry. From 6adz cage is well-preserved in these X@36adz compounds. However, the geometric symmetries of these compounds are lowered to C1 and C2, except for H@36adz, which maintains the S4 symmetry of 36adz. To be specific, the encapsulated hydrogen atom located at the central position of 36adz in H@36adz, yields the newly formed N-H bonds of 2.11 Å and ∠N1-H-N2 of 113.5°. As for B@36adz, the boron atom tends to bind with 3 N atoms of the complexant, forming 3 N-B bonds of 1.63 Å ∼ 1.66 Å, while the distance between the uncombined N and B atoms is as long as 3.02 Å. The C@36adz complex possesses a C2-symmetric structure, where the introduced carbon atom prefers to bind with 2 N atoms of 36adz by forming two N-C bonds of 1.52 Å. Differently, the more electronegative N, O, and F atoms are linked to only 1 N atom of the cage complexant via N-N, N-O, and N-F bonds of 1.41, 1.34, and 1.87 Å, respectively, generating the very similar structures of X@36adz . Similar to B@36adz, the introduced silicon atom tends to bind with 3 N atoms of complexant via 3 N-Si bonds of 2.06–2.35 Å in Si@36adz.As shown in 6adz, it is found that only the optimized structure of [B@36adz]+ cation almost coincides with the geometry of the corresponding neutral one , quite different geometries of cationic and neutral complexes were found. For instance, the H+ is attached to 1 N atom of the complexant in the resulting [H@36adz]+, while the doped N atom turns to combine with 2 N atoms of 36adz in [N@36adz]+ and Si atom almost moves to the center of the cage in [Si@36adz]+. The geometry of C@36adz is distorted from C2 symmetry to C1 with the changes of 0.29 Å for the C-N2 bond and 7.3° for ∠N1-C-N2. As for [F@36adz]+, the N-F bond is shortened from 1.87 Å to 1.38 Å because the introduced F atom further loses 0.333e  species.By turning to the cations of X@3 one see . For ins333e see  and thus6adz  complexes, although X atoms possess very high ionization energies (IEs) of 8.15–17.42 eV , B@36adz (2.16 eV), C@36adz (2.72 eV), N@36adz (3.15 eV), and Si@36adz (1.79 eV) are even lower than the IE of 3.89 eV  than that (6.80 eV) of 36adz. In particular, B@36adz exhibits the highest HOMO level of −1.81 eV, and thus possesses the lowest VIE of 2.18 eV among these X@36adz complexes. This is because that the valence electron of embedded boron atom is pushed out of the cage by the lone pairs of N atoms of the complexant, forming a electride-like molecule [B+@36adz](e‒) with obvious diffuse electrons in the HOMO of B@36adz , which show obvious antibonding character with respect to the central atom-complexant interaction. Such antibonding HOMOs destabilize the neutral structures of X@36adz  and result in their low VIE values  are larger than that of 5.39 eV for Li atom, although their HOMOs also possess obvious antibonding character. This is attributed to the larger elertonegativities of N, O, and F atoms than H, C, and Si atoms, which hinders the ionization of the valence electrons on their np orbitals in the HOMOs of X@36adz .Differently, as shown in E values . Hence, 6adz. To be specific, the geometric structure of B@36adz is hardly changed after its diffuse excess electron of HOMO is lost, resulting in its nearly equal VIE (2.18 eV) and AIE (2.16 eV) values. However, the destabilization of antibonding HOMOs for the neutral X@36adz  complexes drives the embedded X atom to lose nearly one valence electron . Note that the AIE of H@36adz is as low as 0.78 eV because the formed [H@36adz]+ is very stable and has been identified in various synthesized ionic compounds, such as [H@36adz]+X‒   .6adz, it is highly expected that this superalkali also exhibits considerable nonlinear optical (NLO) response. Thus, the static electric properties of these studied X@36adz compounds and 36adz complexant were calculated and listed in 6adz has the largest dipole moment (3.326 au) and polarizability  among these X@36adz complexes because of the existence of diffuse electrons in the HOMO of this superalkali. In particular, the first hyperpolarizability (β0) of B@36adz is as large as 1.35 × 106 au, which is significantly larger than those of the reported superalkalis and superalkali-based NLO materials, such as the aromatic organometallic superalkali Au3(Py)3 (3.74 × 104 au) M‒  (1.18 × 104–3.33 × 104 au) (3O@Al12N12 (8.73 × 105 au)  , superal 104 au) , and sup 105 au) , indicatβ0 value of B@36adz, we focus our attention on the simple two-level model  compounds, which rationalizes its largest β0 value among these studied X@36adz species. In addition, the proposed C@36adz and Si@36adz superalkalis also show considerable β0 values of 4.05 × 103 au and 1.95 × 104 au, respectively, because of their relatively smaller ΔE values and larger Δµ values.To understand the eminently large el model ,β0∝Δμ⋅fzability ,c. Hence6adamanzane (36adz) as a complexant, a series of X@36adz  compounds were constructed and studied based on the density functional theory. It is interesting to find that the X@36adz  complexes possess lower AIE values than the IE of Cs atoms though the X atoms and 36adz possess very high IE values. Thereby, they can be regarded as a new kind of nonmetallic superalkalis. In particular, different from other complexes, the low IE of B@36adz is derived from the diffuse excess electron formed by the repulsion between the valence electron of the embedded boron atom and lone pairs of N atoms of the complexant. Due to the existence of diffuse electrons, this superalkali also possesses a remarkably large β0 of 1.35 × 106 au, which can serve as a new kind of NLO molecule. Hence, it is highly hoped that the theoretical design and characterization of these nonmetallic superalkali species could provide meaningful references to further design novel reducing matters or NLO materials by using such cage-like molecules as complexants.By using 3


Urinary bladder cancer is a common cancer worldwide. Currently, the modality of treating and monitoring bladder cancer is wide. Nonetheless, the high recurrence rate of non-muscle-invasive bladder cancer after surgical resection is still unsatisfactory. Hereby, our study demonstrated whether the intra-operative and post-operative environments will affect bladder cancer recurrence utilizing in vitro cell line model. Bladder cancer cell lines were submerged in four different irrigating fluids for assessing their tumorigenic properties. Our results showed that sterile water performed the best in terms of the magnitude of cytotoxicity to cell lines. Besides, we also investigated cytotoxic effects of the four irrigating agents as well as mitomycin C (MMC) in normothermic and hyperthermic conditions. We observed that sterile water and MMC had an increased cytotoxic effect to bladder cancer cell lines in hyperthermic conditions. Altogether, our results could be translated into clinical practice in the future by manipulating the intra-operative and post-operative conditions in order to lower the chance of residual cancer cells reimplant onto the bladder, which in turns, reducing the recurrence rate of bladder cancers. With the advancement in diagnostic techniques and nonsurgical therapies over the past decades, the treatment and surveillance of bladder cancer have been greatly improved. However, non-muscle-invasive bladder cancer (NMIBC) is still accompanied with high recurrence rate and the prognosis of muscle-invasive bladder cancer (MIBC) has also remained poor5 which in turns burdening the healthcare system.Urinary bladder cancer is the ninth most common cancer in the world. There are more than 550,000 new cases diagnosed and 220,000 deaths every year6. After TURBT, intravesical instillations of chemotherapy such as Mitomycin-C (MMC) can be given to impose a cytotoxic effect on the floating tumour cells. Combining TURBT and an immediate post-operative instillation of chemotherapy can reduce the risk of recurrence by 35%7. In addition, synergism of hyperthermia and chemotherapy is suggested to impose a greater cytotoxic effect to residual cancer cells, therefore, further lower the chance of cancer recurrence after resection.Transurethral resection of bladder tumour (TURBT) is the current standard in the initial diagnosis and treatment of NMIBC. Conventionally, monopolar resection in non-conducting intra operative irrigation solutions such as 1.5% glycine and water, or bipolar resection in isotonic (0.9% NaCl) normal saline is commonly performed. Although TURBT is safe and relatively simple to perform, cancer cells can be released during the resection procedure. The floating tumour cells may reimplant to the bladder wall resulting in early disease recurrence8. We believed the condition in vivo maybe similar as in vitro, therefore, it becomes extremely important to understand if the surrounding fluid environment has any beneficial or harmful effects on the floating tumour cells. A fluid environment which can avoid tumour reimplantation may be the key in avoiding tumour recurrence after a complete resection.After resection, the tumour cells will be deprived of blood supply and cell death is an inevitable outcome unless early tumour reimplantation occurs. It was found that human bladder cancer cells form firm attachment onto the culture dish take place within one hour in vitro and reached maximum within 24 h, followed by proliferation of bladder cancer cellsIn this study, we investigated the effects of common irrigating fluids  and mitomycin C, with or without hyperthermia, in terms of tumorigenic properties on four bladder cancer (CaB) cell lines. We believe the results may provide valuable insights on how we can manipulate the fluid environment in order to optimise the oncological outcomes.6 T24, UMUC3, RT4 and HTB9 cells were prepared in suspension with sterile water, normal saline (Baxter), 1.5% glycine (Baxter), 10 µg/mL MMC , 100 µg/mL MMC, 1000 µg/mL MMC or culture media (as controls). Suspension cells in different reagents were incubated at 37 °C or 43 °C for 1 h. Cells were then centrifuged, and cell viability was assessed by 0.4% Trypan Blue dye exclusion test. Living cells were further tested by four different assays. All the assays were performed in triplicate and at least three independent experiments.Four human urinary bladder cancer cell lines  were used in this study. T24 and RT4 cell lines were maintained in McCoy’s 5A Medium with high-glucose and L-glutamine (ThermoFisher Scientific Inc.). UMUC3 and HTB9 were grown in Minimum Essential Medium (MEM) (ThermoFisher Scientific Inc.) and in Roswell Park Memorial Institute (RPMI) 1640 Medium with L-glutamine (ThermoFisher Scientific Inc.) respectively. All the cells were stocked in liquid nitrogen in dimethyl sulfoxide (DMSO) for permanent storage until use. The cell experiments were performed within 40 passages of the cells. After trypsinization, 1 × 104 cells/mL. After seeding and incubation for 24 and 48 h, cells were treated with 10 µl 3--2,5-diphenyltetrazolium bromide (MTT) (ThermoFisher Scientific Inc.) and incubated for 4 h. MTT-mixture was removed and DMSO was added to dissolve the purple formazan produced by reduction of MTT by living cells and the absorbance was measured at 590 nm using a microplate reader.The growth rates of T24, UMUC3, RT4 and HTB9 cells were tested by the proliferation assay. Living cells were first seeded in 96-well plates (100 µl/well) at a density of 1 × 104 cells/mL on Gibco® Collagen I-coated plate (ThermoFisher Scientific Inc.). Cells were centrifuged at 71g for 5 min at room temperature (RT), followed by incubation for 15 min at 37 °C in a humidified incubator with 5% CO2. After incubation, media in each well containing cells was then removed and the whole plate underwent centrifugation upside down at 7g for 5 min at RT. A MTT assay was then performed. After incubation for 4 h, MTT-mixture was removed and DMSO was added, the absorbance at 590 nm was measured by a microplate reader.Adhesion assay was performed by plating 2 × 104 cells/mL living cells were seeded in each transwell containing 0.5% serum medium. The transwell insert was plated onto the 24-well plated containing 5% serum medium as a lower compartment, serving as an attractant for the cells to invade from the upper compartment towards to lower compartment through the microporous Matrigel® matrix-reconstituted membrane on the basement of each transwell insert. After incubation at 37 °C in a humidified incubator with 5% CO2 for 48 h, the transwell insert was removed and crystal violet stain was performed, accumulating the cell nuclei of the invasive cells which could enzymatically degrade the Matrigel® matrix and invade through membrane pores to the lower compartment. Stained cells were photographed by an inverted microscope and counted by the Image J software in three randomly selected fields.Cell invasion assays were carried out using BD BioCoat Matrigel Invasion chambers  according to manufacturer’s protocol. Briefly, 5 × 102 for 9 days. After the incubation, crystal violet stain was performed, and the living cells were indicated by the total number of stained cell clones on the 100 mm plates. At least 50 cells were required to define a colony.To assess the cell’s ability to grow into a colony, 1000 living cells were seeded onto a 100 mm plate in their respective culture media. The plates were then incubated at 37 °C in a humidified incubator with 5% CO6 cells/mL suspended living cells of the 4 CaB cell lines were incubated at different irrigation fluids and MMC of different concentrations or culture media for 1 h at 37 °C and 43 °C. After incubation, cells were washed twice with PBS , followed by Annexin V-FITC and PI staining. Fluorescent signal was immediately measured by flow cytometry  and data was further analysed by FlowJo software (version 10.4).To evaluate the effect of apoptosis induced by irrigation fluids and MMC at normothermic and hyperthermic conditions to CaB cells, Annexin V-FITC and propidium iodide (PI)  double stain was employed. In this assay, 1 × 10Data are plotted as mean ± standard error of the mean (SEM) at least three independent experiments. Statistical analysis was performed by Student t-test for independent samples with equal variances. The level of significance was set at p < 0.05 as *, and p < 0.01 as **.To evaluate the in vitro cytotoxicity of irrigation fluids, we used intra operative fluids such as water, normal saline and glycine to challenge HTB9, RT4, T24 and UMUC3. Cells were treated in suspension manner to mimic the floating of residual cancer cells inside urinary bladder. Water incubation significantly reduced total number of cells counts in the four CaB cell lines Fig. A. NormalAfter combining with hyperthermia, we observed that water decreased total number of cells counts in the four CaB cell lines Fig. A. HypertTo investigate the cytotoxicity response of hyperthermia drug treatment, we incubated CaB cell suspensions in 10 µg/ml, 100 µg/ml or 1000 µg/ml MMC at 37 °C or 43 °C for one hour. We observed that MMC had a dose-dependent effect; 1000 µg/ml MMC at 43 °C had the most toxic effect against all four CaB cell lines Fig. . Cell ki9. Taoka et al. showed that sterile water induced cytocidal effects on bladder cancer cell lines as well10. Hereby, we provided a more comprehensive study on the effects of intra-operative and post-operative environment on bladder cancer recurrence, as we believed early recurrence of bladder cancer occur as soon as during resection of tumor, when residual floating cancer cells start to reimplant onto the bladder wall. In our study, we investigated the effects of common irrigating fluids and mitomycin C, with or without hyperthermia, on bladder cancer cell lines. Four CaB cell lines in three subtypes, RT4 , HTB9/5637  and T24 and UMUC3 (non-type) were used to explore their unique cellular reaction in different culture reagents. In order to mimic residual cancer cells floating intravesically, we performed incubation of suspended cells in different irrigation fluids and we suggested this can maximally mimicked the 3D environment inside urinary bladder.There are studies in recent years investigating the possibilities to reduce bladder cancer recurrence during and after surgical resection. Among all, van der Heijden et al. proposed a synergism of MMC and hyperthermia could significantly decrease proliferation rates of bladder cancer cell lines. evaluated the performance of osmotic cytolysis by distilled water and mitomycin to reduce CaB recurrence. Results show that both distilled water and mitomycin led to significant cell death after 10 min to all cell lines. Nonetheless, viability of all cell lines treated with distilled water decreased to 3–5%, yet only 3 out of the 4 cell lines with survival dropped to below 5%11. Together with our study findings, water is shown to have strong cytotoxic effects and is promising to prevent bladder cancer cells reimplantation during surgery and recurrence. Flow cytometric results also revealed the mechanisms of cell deaths induced by sterile water which necrosis was mainly observed. To explain the phenomenon physically, water is strongly hypotonic to tumour cells, which lack cell wall protection. While surrounding by hypotonic solution, water moves into the cells from outside to balance the concentration on both side of the cells until equilibrium and equal concentration are reached. Therefore, prolong exposure of water may exert cytocidal effects of hypotonic shock to bladder cancer cell lines12. In extreme situation, water keeps moving into the cells, destroying normal function, bursting the cells and eventually causing cell death.We found that water possessed the strongest toxicity to all 4 bladder cancer cell lines, in terms of proliferation, adhesion, invasion and clonal formation. Fechner et al13, suggesting that cells including but not limited to cancer cells were sensitive to glycine irrigation. These studies are reflection of our in vitro flow cytometric results of glycine on all cell lines. Besides, 1.5% glycine is a weakly ionized solution, contains 15 g/L glycine, having osmolarity of 200 mOsmol/L is slightly hypotonic to normal in vivo physiologic osmolarity condition. The weaker hypotonic property of glycine than sterile water may explain its limited cytotoxic effect to CaB cancer cell lines.On the other hand, our study showed that normal saline possessed a weaker cytotoxic effect, therefore, could hardly prevent the reimplantation of cancer cells. Isotonic normal saline is 0.9% (9 g/L) sodium chloride, having osmolarity around 300 mOsm/L, is closely approximate to the osmolarity of sodium chloride in blood and human cells (about 290 mOsm/L). Normal saline is indeed a favorable environment for cancer cells to survive and reimplant to the bladder, which in turn may lead to tumour recurrence. For 1.5% glycine, our results showed that it had a weaker cytotoxic effect than distilled water. We observed that bladder cancer cell lines maintained proliferation in terms of tumorigenic properties upon glycine irrigation. This demonstrated that 1.5% glycine may favoured cancer cells reimplantation in bladder in vivo after resection. Nonetheless, results from flow cytometry demonstrated that glycine could induce necrosis in all CaB cell lines at both normothermic and hyperthermic conditions. Several studies indicated the usage of glycine as an irrigation fluid was toxic, a xenografts study demonstrated that intravenous injection of glycine caused more tissue damages and higher mortality14. A comparison between sterile water and MMC as irrigating agents in vivo after resection was done by Bijalwan et al. Results showed that continuous bladder irrigation with sterile water after resection had comparable results to immediate intravesical MMC in terms of preventing tumour recurrence in NMIBC. Moreover, authors observed sterile water as irrigating agent could reduce adverse effects significantly when compared to MMC instillation. Besides, the recurrence-free rates were both low for sterile water-treated and MMC-treated groups though the study had a small sample size15.To sum up, continuous bladder irrigation with normal saline may in fact potentiate the risk of tumour reimplantation and it should be avoided as far as possible. While water is hypotonic, using it as irrigating fluid during transurethral resection surgery is not ideal due to the risk of transurethral resection syndrome. However, continuous intravesical water irrigation immediately after the surgery can be a cheap but effective method in minimising the risk of tumour reimplantation16. Besides, direct cytotoxicity associated with heat exposure is related to exposure time. It was shown that one hour exposure at 43 °C induced a large extent of irreversible cytotoxicity, 30% more cell death17, therefore, it was expected that the cytotoxicity to CaB cell lines induced by water and glycine were at least similar or even higher under hyperthermic condition. Intriguingly, our results showed that saline under hyperthermic condition did not affect cytotoxicity as in water and glycine. In fact, upon hyperthermic saline resulted in an attenuated inhibition to 2 out of 4 CaB cell lines, T24 and UMUC3, in terms of invasion and colony formation. T24 and UMUC3 both belong to non-type CaB cell lines (Warrick 2016) under molecular characterization, and the mechanism behind this diverse response of hyperthermic normal saline to T24 and UMUC3 is uncertain and need further examination.To further investigate the cytotoxic effects induced by irrigating fluids under hyperthermia, we repeated the proliferation assay, cell adhesion assay, invasion assay and colonies formation assay on the four CaB cell lines at 43 °C. Similar to our results in the normothermic experiments, the strongest cytotoxicity was observed in water under hyperthermic condition. Water as a strong hypotonic fluid, its toxicity effect was maintained and augmented under high temperature on cell growth and other cancer properties as well as necrotic effects were also observed in four all cell lines tested. Our results also showed that hyperthermia could increase the in vitro toxicity of glycine on all cell lines. Treating cells in 43 °C glycine for one hour could greatly suppress cell adhesion, invasion and colonies formation in HTB9, RT4, T24 and UMUC3 and amplified the effects of glycine-induced necrosis. Hyperthermia alone can induce protein unfolding, ROS generation and DNA replication and repair alteration, eventually causes cell death9. From our results, we demonstrated that hyperthermia at 43 °C in synergy with MMC resulted in a stronger cytotoxic effect to all 4 CaB cancer cell lines than MMC treatment in 37 °C. On the other hand, we observed MMC concentration of 1000 µg/mL exhibited the strongest inhibition in proliferation and cancer properties of the four CaB cell lines. Nonetheless, the mode of cell death induced by one hour incubation of MMC at different concentrations were mainly occurred as necrosis but not apoptosis. Several previous studies reported the in vitro and in vivo effects of MMC at different concentrations20. Yoshida et al. observed DNA fragmentation, which is hallmark of apoptosis, was not surfaced upon addition of MMC to their CaB-derived spheroids, suggesting apoptosis did not occur, therefore, the authors concluded that high dose MMC (1 mg/ml) did not induce apoptosis, but necrosis and autophagy20. One explanation to our observations is that tumour cells may uptake higher amount of MMC and are less resistant to the drugs under hyperthermia treatment. Besides, MMC may have increase cytotoxic activities under higher temperatures, as heat can destroy cell membrane fluidity, weaken or denature cellular proteins, losing protein function will eventually causing decrease in proliferation and increase in apoptosis of cancer cells. Moreover, another signature of our results was the cell death induced by irrigation fluids or MMC at normothermic and hyperthermic conditions was mainly necrosis. Qian et al. evaluated heat stress-induced necrosis rates at rat cardiomyocyte. Results indicated a transient exposure at 43 °C (< 3 h) mainly induced necrotic cell death and apoptosis surfaced only after 4 h exposure21, therefore, we suggested other types of cell death such as necrosis and autophagy were induced upon heat stress and treating cells with sterile water, 1.5% glycine and high dose MMC. Our results supported the use of hyperthermic chemotherapy in the clinical setting to optimise the oncological outcomes of NMINC following TURBT.Intravesical maintenance chemotherapy is a standard treatment for patients with NMIBC. Although it shows a lower rate of adverse events, its treatment efficacy has been proven to be inferior to intravesical BCG therapy, especially for high-risk NMIBC patients. In recent years, people have developed device-assisted technology which can enhance chemotherapy efficiency. MMC is a naturally occurring bio-reductive alkylating agent that attacks and destroys cancerous cells, and the standard clinical concentration is 1000 µg/ml in final volume 20 ml to be instilled into bladder for an hour incubation. However, we found that 1000 µg/ml MMC under normothermic condition could not significantly reduce cell adhesion ability of non-typing cells, T24 and UMUC3. Cell adhesion plays a pivotal role in cancer progression and metastasis. Hyperthermia has been shown to boost the efficacy of cytotoxic agent treatments. Malignant cancerous cells are also more heat sensitive than normal cells or tissues11. Secondly, the mechanisms underlying the synergistic effects of hyperthermia and MMC in different cancer cell lines require further investigations. The synergistic effects may vary depending on the concentrations of MMC and the type of cell lines being tested. In our results, UMUC-3 exhibited the greatest drug resistance towards MMC, even under hyperthermia treatment, in terms of immediate toxicity effects. Thirdly, although the CaB cell lines utilized represented different stages and grades of bladder tumour, they are still limited in mimicking the in vivo conditions. For example, cell lines cannot illustrate the interactions between bladder tumour and surrounding cells. Besides, morphology and physiological parameters may alter upon prolong culture of cancer cells on plastic surface. Therefore, more advance models should be considered before applying our results in clinical practices. For example, 3D organoid cultures serve as a better to mimic cell–cell interactions and in vivo human environment. Further studies can be applied on immortalized cell line derived-organoid or even patient primary tumour-derived organoid models to validate our results.Our study investigated the intra-operative and post-operative intravesical environment in preventing CaB cell lines reimplantation. The cytotoxic effects of water as an irrigation agent to CaB cancer cell lines, and the synergistic effect of hyperthermia and MMC in inhibiting proliferation of cancer cells in vitro were explored. The results are promising in translating into clinical studies and applications. Nonetheless, there are also some limitations in our study. First of all, our results on the in vitro setting might not be replicated in the in vivo setting. Further investigations using animal models could be considered before translating into clinical practiceUrinary bladder cancer is a common cancer in worldwide. Although TURBT is currently the standard to resect NMIBC tumour, it accompanies with high recurrence rate due to residual cancer cell reimplantation. Hereby, we investigated intra-operative and post-operative environments in reducing bladder cancer recurrence. In our study, we investigated the cytotoxic effects of different irrigating fluids to CaB cell lines, in terms of tumorigenic properties. Our results demonstrated that water is the most cytotoxic agents to CaB cell lines when compared to glycine and saline, which could only barely kill cancer cells or even maintained their tumorigenic properties. We further investigated the synergic effects of hyperthermia and irrigating agents as well as MMC. Our results demonstrated that water still possessed the highest cytotoxic effects to CaB cell lines under hyperthermic condition. Besides, synergism among hyperthermia and MMC also exhibited high efficacy in killing cancer cells. Our results are promising in studying how different conditions, during resection and after surgery, may contributed to bladder cancer recurrence. Nonetheless, further experiments should be carried out, for instance, xenograft models, before translating our results into clinical settings.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Legends.


Hepatocellular carcinoma (HCC) is the sixth most common and the fourth most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented potentially novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short–half-life proteins. High-throughput expression screening identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report — for the first time to our knowledge — the effectiveness of omacetaxine in HCC. In addition, we elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC. Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide, with approximately 765,000 new cases each year , 2. The The development of effective systemic therapies against HCC has been hindered, at least in part, by a paucity of representative HCC models. Recent work by us and others demonstrated that 3-dimensional (3D) human patient-derived organoid (PDO) models accurately capture original HCC tissue architecture while maintaining the genomic, epigenetic, and molecular heterogeneity features of original tumor source material, even after long-term expansion –17. In aOmacetaxine mepusuccinate (formerly known as homoharringtonine) is approved by the FDA for relapsed/refractory or accelerated-phase chronic myelogenous leukemia (CML) that is resistant to 2 or more tyrosine kinase inhibitors (TKIs) . Unlike We previously reported the establishment of 10 HCC PDO lines . AverageWe developed 2 distinct HCC PDX models to establish in vivo drug effectiveness documented in human HCC PDOs in vitro. H&E staining results in 1 primary HCC tumor specimen, its matched PDO, and its matched PDX . H&E stahttps://doi.org/10.1172/jci.insight.138197DS1). p.P33R has a score of 0.247 and is seen in HCC PDO26, -30, -34, -36, -40, -65, -67, and -77. In addition, p.P33R was also seen in PDX26, which is to be expected since HCC PDX26 was established by implantation of HCC PDO26 in NOD–SCID–IL-2Rγ chain–deficient (NSG) mice. This finding argues that PDX models established from PDO cells are representative for the cells of origin. All samples except for PDX50 had at least 1 APOB mutation scoring greater than zero, and PDX26, as well as PDO30, –34-4, and –36-3 had 1 ranked 0.1 or higher. Similarly, all samples except for PDX50 had at least 1 RP1L1 mutation scoring above zero. At least 1 IL6ST mutation ranked 0.1 or higher in several samples: PDX26, and PDO40, -59, –65-2, –67-1, –67-2, and –80-4. Two samples had more than 1 MET mutation ranked 0.1 or higher; 3 more samples had 1 MET mutation scoring above zero. All samples except for PDX50 and PDO67-1, –67-2, and –71-1 exhibit at least 1 CCDN1 mutation, though none of these mutations are scored as functional by CHASMplus. There was less evidence for other common drivers. For example, 26% of HCCs have CTTNB1 mutations . CHASMplus is a computational method to predict driver missense mutations, which is uniquely powered to identify rare driver mutations within the long-tail . Based on this score, we hypothesize that ARID1A could be a driver gene in this patient. Exonic mutations in APOB, RP1L1, and p53 are notably absent, as are several other exonic mutations found in the other samples. These data underline the great diversity of genetic alterations seen in HCC samples who appear otherwise fairly similar in regards to standard clinical pathology staining.PDX26, which was established by implanting PDO26-3 cells into NSG mice, appears more similar to the majority of PDOs. PDX26 displayed mutations in IL6ST, as well as APOB, ARID1A, AXIN1, EEF1A1, RP1L1, p53, and TSC1.50). As shown in 50 curves for each of the 40 HCC PDOs, as well as for the 2 human and 1 mouse normal liver organoid lines. While there was variability, which was expected due to functional (drug response) heterogeneity among patients, the IC50 curves demonstrated that all 40 HCC PDOs were sensitive to omacetaxine at nanomolar concentrations. The calculated average IC50 across all 40 HCC PDO lines was 31.4 nM, which is less than the maximal concentration (Cmax) of omacetaxine in FDA-qualifying human studies of 46 nM (25.1 ng/mL). The IC50 values for all the 40 HCC PDO lines are shown in 50 of 170, 240, and 534 nm  in CML patients, which explains why there were no observed liver toxicities in these CML patients. It is furthermore reassuring that the average IC50 in HCC organoid lines (31.4 nm) is 5.5- to 17-fold smaller than the IC50 of omacetaxine in normal liver organoid lines. These data indicate that omacetaxine has a wide therapeutic index that makes it an attractive option for patients with HCC.d 534 nm . These cFACS demonstrated that omacetaxine inhibited incorporation of BrdU into newly synthesized DNA during S-phase. In fact, omacetaxine inhibited proliferation in all 6 HCC PDO lines tested . To furtPrevious studies, mostly in acute myelogenous leukemia (AML)/CML disease models, showed that omacetaxine inhibits protein synthesis by blocking formation of the first peptide bond during polypeptide synthesis , 30. In cMYC, β-catenin, cyclin D1, XIAP, and MET are short–half-life proteins that have previously been reported to be downregulated by omacetaxine in CML , 31, 32.To further explore the mechanism of action for omacetaxine and its influence on gene expression, 5 HCC PDO lines were treated with 100 nM omacetaxine or control, respectively. mRNA was extracted, and real-time PCR arrays were performed with a panel of 203 genes. The heatmap of gene expression for these 203 genes is shown in PDX models of human cancer are well documented to recapitulate primary tumor biology, as well as response to treatment , 38. To Furthermore, we also noted that the control 26-3 HCC PDX mice developed several liver and lung metastatic sites . ImportaTo verify the mechanism of omacetaxine against HCC in vivo, omacetaxine-treated HCC PDX tumors, as well as matched controls (tumors treated with the negative control), were harvested, and frozen section slides were made. We found that proliferation was less, as measured by Ki-67 protein expression . ApoptosWe have demonstrated that omacetaxine induces (a) global downregulation of protein synthesis  and (b) Few HCC patients are diagnosed at early enough stages to be eligible for curative-intent treatments, such as surgical resection, ablation, or liver transplantation . PatientSuccessful establishment of HCC PDOs has been reported by us and others , 15. In DNA sequencing shows strong evidence that APOB, RP1L1, and p53 mutations are driver mutations across the majority of PDO and PDX lines established and analyzed here. While PDX26 was more similar to the majority of PDO lines, PDX50 exhibited a unique mutational signature: ARID1A1 is a likely driver gene, and mutations in p53, APOB, and RP1L1 are notably absent. The mutational profiles identified across these samples argue that these PDO and PDX lines are representative for mutational profiles and driver mutations generally found in HCC patients. Furthermore, the fact that PDX50 is mutationally divergent from the majority of the HCC PDO lines analyzed in this study, and yet responds well to omacetaxine, highlights the strength of a nontargeted therapeutic. Interestingly, the same conclusion was reached in CML patients, and the FDA approval is specifically for CML patients who had been treated with targeted therapies. Some of these patients who had been treated with TKIs and developed resistance, intolerance, or insufficient response or developed progression still demonstrated a good response to omacetaxine. The fairly uniform good response to omacetaxine across all 40 HCC PDO and 2 PDX lines, coupled with a variety of mutational profiles in these PDO/PDX lines, argue that — similar to CML — omacetaxine could be positioned to HCC patients who are either intolerant to or progress while on targeted/immunotherapies.Omacetaxine was originally found in Chinese herbal extracts from the bark of the Chinese plum yew, Cephalotaxus . This baOmacetaxine is a protein translation inhibitor . This drOf great translational importance, omacetaxine induces toxicity in HCC cells at much lower doses that in normal human liver cells. In our study, the dose required to induce toxicity in human normal liver cells is 5.5- to 17-fold higher than the dose required to induce toxicity across all 40 HCC PDO lines. Future studies are required to establish a dosing regimen for HCC, as well as the positioning of omacetaxine in the therapeutic arsenal for HCC.Fresh human HCC tissue was obtained from procedures (surgery or needle biopsy) performed at the Johns Hopkins Hospital (JHH).HCC PDOs and normal human liver organoids, as well as mouse normal liver organoids, were established as described . DetailsIn this study, we established 2 different HCC PDX models. Five- to 6-week-old female NSG (The Jackson Laboratory) were utilized. Details are included in Supplemental Data.Fifteen of the 40 PDO and 2 PDX lines established in our lab had DNA available for exome sequencing. The DNA was extracted using QiAamp DNA min kit DNA (Qiagen); DNA sequencing was performed by Novogene. Normalization and data quality control were performed with Trim Galore!  and FastFor variants within known HCC driver genes, we performed an additional level of analysis to distinguish between driver mutations and passenger mutations without functional impact. We used CHASMplus  to prediCounts of exonic variants within driver genes –62 were 50 calculation curves are included in Supplemental Data.A panel of 129 FDA-approved anticancer drugs obtained from the NCI was utilized for drug screening on HCC PDOs, as described . Details3. The size of the PDX50 tumors was 6.06 ± 1.71 mm3. Mice were treated with either vehicle or omacetaxine at a dose of 0.5 mg/kg via i.p. injection daily until day 30 (for HCC PDX26) or day 45 (for PDX50). S.c. tumor volumes were measured with a Vernier caliper twice a week. Animals were then sacrificed, and tumor weights were recorded. The presence of lung and liver metastases was verified by histology for both PDX models. For survival curves experiment, another 8 HCC PDX26 mice were randomized into 2 groups (n = 4 mice per group). Mice were treated from day 7 after the tumor was implanted until the mouse died. Further details of materials and methods are in Supplemental Data.Six HCC PDX26 mice and 6 PDX50 mice were each randomized to 2 groups. The treatment started at day 7 after implantation. At that point, the size of the PDX26 tumors was 8.54 ± 2.181 mmP value was determined with a 2-tailed Student’s t test or 1-way ANOVA. A Kaplan–Meyer method and log-rank test was used for xenograft survival curve analysis. A 2-sided P < 0.05 or P < 0.001 was considered statistically significant.All graphs were generated using Microsoft Excel, R software, and GraphPad Prism 7.0 (GraphPad). Data were presented as mean ± SD or mean ± SEM as specified in figure legends. The biological replicates are shown in each figure. Statistical significance with the Patients were consented under an approved IRB from Johns Hopkins University School of Medicine. The diagnosis of HCC was confirmed by a liver pathologist at JHH. All mouse procedures were performed with approval from the Animal Care and Use Committee (ACUC) at Johns Hopkins University.LL, JSB, and FMS designed experiments. LL, GH, HH, and PD performed experiments. LL, GH, MGL, KO, JSB, and FMS analyzed data. MJW, JH, BP, RB, WRB, RNW, AMC, CLW, CG, SK, NSA, and MY provided patient-derived specimens and clinical correlations. LL, MGL, JBS, and FMS wrote the manuscript, MJW, BP, CG, SJM, LME, JSB, and FMS revised the manuscript. All authors approved the manuscript.


Viscum album) alone and in combination with mebendazole in an in vitro model of canine high-grade astrocytoma using the cell line SDT-3G. SDT-3G cells were exposed to a range of concentrations of mistletoe extract alone to obtain an IC50. In separate experiments, cells were exposed to mebendazole at a previously determined IC50 (0.03 µM) alone or in conjunction with varying concentrations of mistletoe extract to determine the additive effects. The IC50 for mistletoe alone was 5.644 ± 0.09 SD μg/mL. The addition of mistletoe at 5 μg/mL to mebendazole at 0.03 µM led to increased cell death compared to what would be expected for each drug separately. The cytotoxicity of mistletoe in vitro and its additive effect with mebendazole support future expanded in vitro and in vivo studies in dogs and supply early evidence that this may be a useful adjunct therapeutic agent for use in glioma-bearing dogs. To the authors’ knowledge, this is the first published report of Viscum album extract in canine glioma. Malignant gliomas are associated with extremely poor clinical outcomes in both humans and dogs, and novel therapies are needed. Glioma-bearing canine patients may serve as promising preclinical models for human therapies, including complementary medicine. The objective of this study was to evaluate the effects of mistletoe extract ( Malignant gliomas represent a significant proportion of both human and canine primary brain tumors and have a similarly poor prognosis despite treatment in both species ,3,4,5,6.MGMT and, therefore, do not respond optimally to TMZ treatment [MGMT promoter methylation status of canine malignant gliomas, but preliminary epigenetic investigations into a small subset of tumors suggests no evidence of significant MGMT promoter methylation in those tumors . Additionally, little is known about the efficacy of TMZ in canine glioma patients [The traditional standard of care for treating glioblastoma, the most common and malignant form of glioma in humans, includes surgery followed by radiation and chemotherapy . The mosreatment ,19,20. Vpatients ,22, althpatients ,23. Therpatients  and is e2-M phase of the cell cycle and ultimately triggering apoptosis [A promising alternative or adjunct treatment undergoing investigation in treating malignant gliomas is mebendazole (MBZ), a benzimidazole (BZ) anthelmintic that has been used successfully for many years to treat intracranial and intestinal helminth infections. The anthelmintic effects of BZ are primarily mediated by binding to tubulin and preventing its polymerization , and thipoptosis . In its poptosis . Since ipoptosis ,28. MBZ poptosis . Oral abpoptosis . Our labpoptosis . Viscum album) extract for use in oncologic complementary medicine has grown in the last decades, especially in its indigenous European countries, due to its ability to repeatedly demonstrate significant cytotoxicity in many different human tumor cell lines, including leukemia, lymphoma, breast cancer, glioma, and others in vitro [Interest in European mistletoe ; survival in canine mammary carcinoma patients treated with surgery and mistletoe vs. surgery alone was not statistically significantly improved but showed a trend toward improvement. In the Italian language-only literature there is a study evaluating the use of mistletoe in dogs with transmissible venereal tumor that showed a reduction in vincristine dose in dogs being treated with Viscum album [hey grow ,52. For patients , there wum album . In orde2. Prior to experiments, subconfluent cells were trypsinized and counted manually using the trypan blue method. Then, 3500 cells/well were seeded into 96-well plates and allowed to attach for approximately 18 h prior to the addition of test drugs.The canine high-grade astrocytoma cell line SDT-3G (a generous gift from Dr. Pete Dickinson) was originally classified as a grade IV astrocytoma/glioblastoma and was derived from a spontaneous primary tumor in a 12-year-old male English Bulldog. This cell line has copy number alterations in EGFR (gain), AKT (gain), PIK3CA (gain), CDKN2A/B (homozygous loss), and NF1 (homozygous loss) and lacks TP53 mutations . SDT-3G Viscum album extract  was provided as a gift from the manufacturer. The standardized Strength H is a 5% solution of the entire plant extract, containing 50 mg/mL of Viscum album in an aqueous solution of water, NaCl, and NaHCO3. This equates to 13.7 μg/mL of the lectin active ingredient [gredient . When diIn the experiments evaluating additive effects of mistletoe extract and MBZ, one “high” concentration of mistletoe (“HMT”), 5 μg/mL, and one “low” concentration (“LMT”), 2.5 ng/mL, were separately supplemented to the MBZ at IC50 concentration of 0.03 μM, for 72 h, followed by analysis of cell viability using the MTT assay. ® M200 plate reader, Tecan Group Limited, Männendorf, Switzerland) at 570 nm with a reference of 630 nm subtracted for background effect [The MTT -2,5-diphenyltetrazolium bromide) cell viability assay provides an estimate of the number of metabolically active cells; viable cells have the ability to convert MTT to a formazan product that produces a colorimetric reaction detected by absorbance around 570 nm. Following exposure of cells to drugs for 72 h, MTT reagent was added to experimental and control wells and allowed to incubate for 4 h at 37 °C. Then, cells were lysed and solubilized by the addition of DMSO. Absorbance from the colorimetric reaction was read in an automated plate reader , with log-transformation of drug concentrations followed by generation of IC50 values using a four-parameter, variable-slope, curve fit, nonlinear regression analysis with automatic outlier exclusion. Comparison of effects across groups was measured by ANOVA with statistical significance set at Viscum album on this cell line is dose-dependent, has a low standard deviation among technical replicates in each experiment, and is highly repeatable across experiments performed on different days at different cell passages. Exposure of the SDT-3G canine high-grade astrocytoma cell line to mistletoe extract alone for 72 h generated an average IC50 of 5.644 ± 0.09 SD μg/mL . The genViscum album cytotoxicity experiments. The “low” dose was based on an in vitro study showing significant apoptosis after administration of ML-1 purified mistletoe lectin with a concentration of 2.5 ng/mL to human leukemic cells [Exposure of the canine SDT-3G canine malignant glioma cell line to MBZ at its IC50 (0.03 μM) for 72 h alone and in conjunction with either a high  mistletoe concentration or low , while the addition of MBZ with HMT generated a significant further decrease in cell viability as compared to MBZ-only treated cells  or mistletoe-only treated cells at the same dose  (p = 0.98). Cells exposed to MBZ at its IC50 showed the expected approximately 50% decrease in cell viability as compared to untreated cells  . TreatmeViscum album alone indicated that mistletoe extract does have a dose-dependent cytotoxic effect on the canine SDT-3G malignant glioma cell line in vitro. In pharmacokinetic studies, the Cmax refers to the peak serum concentration that a drug achieves after administration of a single dose, while the Tmax (or area under the curve) is the amount of time the drug is at the Cmax in serum. Although there is little primary pharmacokinetic data for mistletoe, in one study in which healthy human volunteers were given a single subcutaneous administration of the product containing 20 μg of natural lectins, there was considerable interindividual variability, with a plasma Cmax range of 188 to 2970 pg/mL and a Tmax range of 0.3 to 336 h [The initial study of the cytotoxicity of to 336 h . There ito 336 h , rodent to 336 h , and humto 336 h ,59. There are no published pharmacokinetic data on mistletoe in dogs and almost no published safety data; however, one toxicology study on recombinant ML-1 showed no genotoxic or mutagenic effects at doses up to 1000 ng/kg intravenously, and repeated subcutaneous or intravenous dosing of rats and dogs up to 1000 ng/kg did not produce any specific target-organ toxicity. In dogs administered 10,000 ng/kg intravenously daily for 1 week, there were bleeding complications; local reactions at the sites of subcutaneous injections occurred at doses greater than 50 ng/mL . This work represents the first published data regarding the efficacy of mistletoe in canine malignant glioma. While these initial results seem promising, the study has limitations and many questions remain regarding the safety and efficacy of mistletoe in tumor-bearing dogs and people. The IC50 data from the present study, in conjunction with other published experimental data, suggest that alternatives to systemic delivery of whole-extract products  may be necessary to achieve therapeutically efficacious levels for direct cytotoxicity. However, patients may still benefit from the immunomodulatory or additive effects even at sub-IC50 levels, but that will require appropriate experimental investigation in clinical trials in glioma-bearing dogs. Additionally, in vitro investigation in multiple canine glioma cell lines and in vitro and in vivo evaluation of mistletoe’s additive effects with multiple other chemotherapeutic combinations used in canine glioma such as temozolomide and lomustine would provide valuable information. With regard to the possibility of local administration, it is interesting to note that in one study, syngeneic mouse models that carried mammary carcinoma subcutaneously in both hind legs experienced more mistletoe-induced cytotoxicity in the tumor nearest the flank injection site than in the contralateral tumor or metastatic tumors, suggesting some direct regional effects in addition to the systemic ones . Treatme


Marugan, Silvia Muro, Zhiyuan Li, Roscoe Brady, Wei ZhengThe authors reported an error in the Results section of this article. In the first paragraph of the first heading, “Generation of iPSC Lines From NPA Patient Fibroblasts and NSC Differentiation”, the first sentence showed the names of the two cell lines switched. The sentence should have read as follows:GM16195 and HT139B and HT139E from GM13205 .Two NPA patient fibroblast lines were reprogrammed to iPSCs, and two iPSC colonies were established for each patient line as HT138C, HT138F from The two bolded cell line names were incorrect and are now corrected.The authors regret the error and apologize for any inconvenience this may have caused.


Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50 = 21 μM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.Verdinexor (KPT‐335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose‐dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC These data will serve as the foundation for potential future clinical studies investigating verdinexor in dogs with OS.22.1Verdinexor was graciously donated by Anivive Lifesciences . Stock solutions of verdinexor were prepared in 100% DMSO, stored at −20°C, and diluted in the indicated culture medium for cell line treatments.2.2l‐glutamate, 1% penicillin/streptomycin, 1% 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, 1% non‐essential amino acids, and 1% sodium pyruvate (Gibco). Cells were cultured in a humidified incubator containing 5% CO2 at 37°C. Cells were routinely tested for mycoplasma and if positive, treated with Plasmocin  and re‐tested to ensure complete eradication.The canine cell lines OSCA‐8, OSCA‐16, OSCA‐40 were provided by Dr Jaime Modiano . The Abrams cell line was provided by Dr Doug Thamm . The D17 cell line (Cat.# ATCC CCL‐183) was purchased from the American Type Culture Collection . Normal canine osteoblasts (Cat.#Cn406‐05) were obtained from Cell Applications, Inc. . Cell lines were cultured in Gibco (Thermo Fisher Scientific) RPMI 1640 , DMEM medium , or CnOb growth medium . Media was supplemented with 10% foetal bovine serum, 1% Canine OS tumours were collected from patients treated at The Ohio State University College of Veterinary Medicine Veterinary Medical Center (OSU‐CVM VMC) in accordance with established hospital protocols and approved by the Institutional Animal care and Use Committee (IACUC #2010A0015). Fresh tumours were flash frozen in liquid nitrogen and confirmed to be OS by board certified veterinary anatomic pathologists at OSU‐CVM VMC.2.3Six cell lines were used in this study, and all cell lines were previously established and validated. Two cell lines were purchased directly from commercial culture collections, as outlined above. Cells were not subjected to species verification testing; however, morphology under light microscopy and growth kinetics remained consistent throughout the experiments.2.4RNA was extracted from canine OS cell lines and tumours using TRIzol reagent (Invitrogen) and purified using the RNeasy Mini Kit  according to manufacturer's instructions. cDNA was made from 2 μg of total RNA using Superscript III (Invitrogen). Published canine XPO1 primers and 18S primers were used with Fast SYBR Green Master Mix (Applied Biosciences) and qRT‐PCR was performed using the Applied Biosystems StepOne Plus Detection System .2.5OS cells were collected, washed with 1X Dulbecco's phosphate‐buffered saline (Gibco) and re‐suspended in complete lysis buffer consisting of 20 mM Tris‐HCL pH 8.0, 137 nM NaCl, 10% glycerol, 1% IPEGAL CA‐630, 10 mM ethylenediaminetetraacetic acid, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride for 1 hour at 4°C for protein extraction. Frozen tumours were pulverized and re‐suspended in complete lysis buffer for 1 hour at 4°C. Extracted protein was quantified using the Bradford assay and 75 μg protein was separated by SDS‐PAGE and transferred to PVDF membranes. Membranes were blocked in TBST containing 5% non‐fat dry milk for 1 hour at room temperature and incubated overnight with anti‐XPO1  at 4°C. Membranes were incubated in horseradish peroxidase linked anti‐mouse secondary antibody , washed, and exposed to substrate . Blots were stripped , washed, and re‐probed for β‐actin  overnight at 4°C. Band intensities were calculated with ImageJ software  and relative intensity of XPO1 was determined by dividing by β‐actin.2.63 cells were seeded in 96‐well plates and incubated in media overnight. Cells were treated with 0.1% DMSO (control) or varying concentrations of verdinexor (0.001‐10 μM) for 96 hours. Media was removed after 96 hours and plates were frozen overnight at −80°C. Fluorescence was measured with the UV Spectromax M2 plate reader  with 485 nm excitation/530 nm emission detection. Cell proliferation was calculated as a percentage of the control wells. All treatments were evaluated in triplicate in a least three independent experiments. The concentration of verdinexor that inhibited cell viability by 50% (IC50) was determined using the GraphPad Prism v8.3.0 .The effect of verdinexor on canine OS cell viability was assessed with the CyQUANT Cell Proliferation Assay Kit . 1 × 10Drug combination studies were completed with verdinexor and doxorubicin in the D17, OSCA‐40, and Abrams cell lines using the fixed‐ratio method. Combination index (CI) was calculated with the CompuSyn software  to determine synergistic effects using the median‐effect method of Chou and Talalay.2.73 OS cells were seed in 96‐well plates and incubated with media containing either 0.1% DMSO (control) or 1 or 10 μM concentrations of verdinexor for 48 hours. Caspase‐3/7 substrate solution was added to all wells and incubated in 5% CO2 at 37°C for 18 hours. Fluorescence was measured on the UV Spectromax M2 plate reader (Molecular Devices) using 354 nm excitation/442 nm emission detection. All treatments were evaluated in triplicate in a least three independent experiments.Caspase‐3/7 pathway activation following verdinexor treatment was measured by the SensoLyte Homogeneous AMC Caspase‐3/7 Assay Kit . 1 × 102.83 canine OS cells were plated in 96‐well collagen covered glass bottom plates and incubated overnight. For γH2A.X experiments, cells were incubated in media containing 0.1% DMSO, 1 μM doxorubicin for 2 hours, 1 μM verdinexor for 48 hours, or 1 μM doxorubicin for 2 hours followed by recovery in 1 μM verdinexor or 0.1% DMSO for 48 hours. Media was aspirated, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton‐X. Cells were blocked in blocking buffer (1× PBS/2% BSA/0.1% Triton‐X) for 60 minutes and incubated with anti‐γH2A.X  at a dilution of 1:500 overnight at 4°C. Secondary donkey anti‐mouse antibody  was applied for 1 hour. Cells were stained with DAPI for nuclear visualization and γH2A.X fluorescence was detected by immunofluorescence microscopy using a Nikon Ti2E microscope fitted with a Prime 95B camera. Data analysis was performed using custom MATLAB software .4.0 × 102.9P < .05 were considered statistically significant.Data analysis was performed using the software program GraphPad Prism v8.3.0 . Normality of the data was assessed by the Shapiro‐Wilk and/or the D'Agostino & Pearson tests. All experiments were performed three times in at least three independent experiments unless noted elsewhere. A one‐way ANOVA with Dunnett's multiple comparison test was used to evaluate differences in apoptosis and XPO1 gene expression. Values of 33.1XPO1 transcript expression was quantified via qRT‐PCR in 21 primary canine OS tumours and XPO1 protein expression was assessed by Western blotting in eight primary canine OS tumours to determine if XPO1 represents a relative target for therapeutic intervention. XPO1 is overexpressed in the majority primary OS tumours at the RNA and protein level when compared with normal canine osteoblasts Figure . XPO1 ex3.250 values for the canine OS cell lines were in low nanomolar concentrations, ranging from 30 to 74 nM. Interestingly, the IC50 concentration of verdinexor for normal canine osteoblasts was 21 μM, indicating that normal osteoblasts are significantly less sensitive to verdinexor compared with canine OS cell lines ; however, sensitivity to verdinexor did not directly correlate with basal XPO1 expression levels. Prior studies evaluating the XPO1 inhibition in a panel of sensitive and resistant cell lines found that intrinsic cellular resistance was not attributed to XPO1 occupancy by selinexor, suggesting that cancer cell responsiveness to XPO1 inhibition depends on the modulation of signalling pathways downstream of XPO1.50 value of 21 μM. Importantly, the canine OS cell line IC50 concentrations were physiologically relevant as previous verdinexor pharmacokinetic profiles established a Cmax of 253 ng/mL (or 572 nM) in dogs receiving oral verdinexor.XPO1 transcript and protein were overexpressed in the majority of canine OS tumours and cell lines as compared with normal canine osteoblast cells. Concordant with our data, XPO1 is overexpressed in human OS compared with normal tissues.50 concentrations of verdinexor. These findings suggest that decreased OS cell proliferation with verdinexor treatment is primarily attributed to cytostatic and/or cytotoxic effects rather than apoptosis. Additional experiments are necessary to determine the mechanism by which verdinexor inhibits proliferation in OS cell lines.Verdinexor decreased OS cell proliferation which correlated with modest, but statistically significant dose‐dependent increases in caspase‐3/7 enzymatic activity in canine OS cell lines. No differences in caspase‐3/7 enzymatic activity were detected in normal osteoblasts at both concentrations of verdinexor tested. Although these data demonstrate that canine OS cells were significantly more sensitive to the effects of verdinexor in an apoptotic manner, the minimal to mild increases in caspase‐3/7 activity in OS cells treated with verdinexor (1‐10 μM) were discordant given the low nanomolar ICTo determine the effects of verdinexor on XPO1 inhibition in canine OS cells, we evaluated XPO1 transcript and protein expression 24 hours following treatment with verdinexor. We observed decreased XPO1 expression in all four cell lines at 0.1 and 1 μM verdinexor when compared with vehicle treated cells with the exception of the Abrams cell line. At 0.1 μM, Abrams cells expressed more XPO1 than vehicle treated cells; however, at 1.0 μM, XPO1 expression was reduced. Prior in vitro studies have documented that cellular exposure to SINE compounds leads to a time‐dependent accumulation of nuclear proteins; however, the nuclear protein abundance progressively decreases as a consequence of enhanced ubiquitination and proteasome‐dependent XPO1 degradation.To determine if a combinational approach would enhance the chemotherapeutic efficacy of doxorubicin, a topoisomerase II inhibitor routinely used in the treatment of canine and human OS, OS cell lines were treated with verdinexor and doxorubicin and potential synergism was assessed. Studies performed in human multiple myeloma and acute myeloid leukaemia cell lines demonstrate therapeutic synergistic effects using a combination of selinexor and topoisomerase II inhibitors.To evaluate the mechanism by which doxorubicin and verdinexor work synergistically in canine OS cells, cells were pre‐treated with doxorubicin to induce DNA damage and recovered in media with or without verdinexor for 24 hours. To visualize DNA damage, we used the γH2A.X marker, a histone that is phosphorylated in response to double‐stranded DNA breaks.5In summary, XPO1 is a relevant target for therapeutic intervention in canine OS as XPO1 is overexpressed in canine OS tumours and cell lines. Inhibition of XPO1 using the novel orally bioavailable XPO1 inhibitor, verdinexor results in inhibition of cellular proliferation in canine OS cell lines at biologically achievable concentrations. Verdinexor upregulated XPO1 mRNA while downregulating XPO1 protein, indicative of on‐target drug effects. Combinatorial treatment using doxorubicin and verdinexor demonstrated synergistic activity in canine OS cells, possibly through hindrance of the DNA damage repair process. Together, these findings provide important pre‐clinical data supporting future clinical investigations of verdinexor in combination with doxorubicin dogs with OS.The authors declare no potential conflict of interest.


The aim of this Special Issue was to discuss novel findings regarding the different mechanisms involved in the progression of neuroblastoma (NB), which represents the most common pediatric extra-cranial solid tumor. Indeed, high-risk NB patients usually display metastatic tumors, with a high frequency of relapse and a survival rate less than 50% . Thus, nwt) undergo cell death through apoptosis, whereas NB cell lines with mutated p53 (p53mut) follow a different mechanism. In addition, they demonstrated cell cycle arrest in G0/G1 for p53wt NB cell lines, whereas mutated p53 determined an arrest of the cell cycle in the G2/M phase, with an increased DNA content. Accordingly, CRL proteins are increased in NB cell lines after treatment with pevonedistat, but WEE1, a protein involved in cell cycle progression, was increased in p53mut NB cell lines and decreased in those with p53wt. However, preclinical studies performed with orthotopic xenografts of NB demonstrated that pevonedistat is effective in reducing tumor volume, independently of the p53 status of the NB cell lines injected [In this view, Foster and coworkers analyzed the effects of pevonedistat, an inhibitor of NEDD8, on NB cell lines in vitro, and in preclinical models of NB . NEDD8 iTwo interesting papers have addressed the role of long non-coding RNAs (lncRNAs), a heterogeneous group of RNA molecules that contribute to various cellular processes in normal and disease states, and are dysregulated in the majority of human cancers, including NB ,4. lncRNBaldini and coworkers summarized, in their review data, the lncRNAs that have been described in NB . The expNMYC-amplified NB cell lines than in those with a single copy of NMYC. Furthermore, they found MIAT expression in formalin-fixed paraffin-embedded tissues, with higher MIAT expression in tumor samples from high-risk patients than in those from intermediate-risk patients, and low to absent expression in samples from benign tumors (ganglioneuroma and ganglioneuroblastoma). Since MIAT expression is connected to NMYC status, the authors investigated the effects of MIAT silencing, and they found that MIAT-specific siRNA induced apoptosis in NMYC-amplified NB cell lines, and inhibited cell growth in NB cell lines with a single copy of NMYC. In addition, MIAT silencing down-regulated NYMIC expression in NMYC-amplified NB cell lines, and c-myc expression in NB cell lines with a single copy of NMYC. Accordingly, silencing of MIAT down-regulated the transcription of both NMYC and c-myc downstream genes, such as ornithine decarboxylase, in both NMYC-amplified and non-amplified NB cell lines [NMYC down-regulation, induced cell cycle arrest of NB cells in the G0/G1 and G2/M phases, with a reduction in cells in the S phase. Accordingly, the proliferation index and migration of NB cells are inhibited by MIAT silencing. Finally, they observed a reduction in glycolytic activity and respiratory capacity in NB cells subjected to MIAT silencing [In this view, the study performed by Feriancikova et al. was focused on myocardial infarction-associated transcript (MIAT), which contributes to the development of physiological and pathological processes, including human cancers . In tumoll lines . Furtherilencing . All theNMYC in NB, which are both up-regulated and associated with poor prognosis. They also described the signaling pathways activated by different ALK isoforms, and they summarized data regarding alterations of these pathways during neural crest formation, which may be involved in malignant transformation, and they hypothesized a role of ALK in the migration and proliferation of neural crest cells [The review by Wulf and coworkers summarized data regarding the role of anaplastic lymphoma kinase (ALK), which may be amplified and/or mutated in NB . Pleiotrst cells . On the st cells .Olivera and coworkers addressed the interesting field of pharmacogenetics (PGx), in the context of NB . PGx, wh+ and Nmyc− NB cells). Furthermore, NB cells may transfer EVs to immune cell populations , or may acquire EVs from them, and the effects of this bi-directional cross-talk may lead to increased or decreased tumor aggressiveness [We contributed to this Special Issue by summarizing and discussing the role of tumor-derived extracellular vesicles (EVs) in NB . EVs aresiveness . In conclusion, the main topics of this Special Issue, which are summarized in


Our results show anion-dependentvesicle release, vesicle opening, and vesicular content, providingunderstanding of the pharmacological and pathological processes inducedby inorganic ions.Hofmeistereffectshave often been ignored in living organisms,although they affect the activity and functions of biological molecules.Herein, amperometry has been applied to show that the vesicular content,dynamics of exocytosis and vesicles opening, depend on the anionicspecies treatment. Compared to 100 μM Cl It has been studied in biologicalsystems , but Hofmeister effects have often been ignored in living organisms,although they are generally based on various aqueous salts includingnumerous anions and cations, which are listed in the Hofmeister series.For anions, ClO4–, SCN–, and NO3– can act as inhibitors ofiodide transport which might affect the function of the thyroid,10 ClO4– also can affect the release ofinsulin,11 and Br– hasbeen applied as a drug to treat epilepsy.13 Hofmeister series anions have been reported to affect in vitro aggregationof several amyloidogenic proteins, which are involved in the onsetof neurodegenerative disease.18 Recently, we observed that counteranions in the stimulation solutionalter the dynamics of exocytosis that can be explained by immediateHofmeister effects on lipid bilayers of the cell membrane.19 Of particular interest, however, is the investigationof the Hofmeister effect in living biological models .The Hofmeister series is theearliest specific ion effect, known for more than a century.–: Cl–, Br–, NO3–, ClO4–, SCN–) for 3 h before amperometric methods. Weused intracellular vesicle impact electrochemical cytometry (IVIEC)to detect the opening of vesicles on the electrode in the intracellularenvironment, and simultaneously applied single cell amperometry (SCA)to monitor the exocytotic process at cells triggered with 30-s 30mM KCl stimulation solution (see Supporting Information S1). Combined with these two methods, we quantified the catecholaminesstored in vesicles and compared them to exocytotic release, calculatingthe fraction of catecholamine released in each event. In addition,the high temporal resolution of amperometry allows the dynamics ofvesicle release and vesicle opening to be measured.In this paper, chromaffin cells were treatedwith 100 μMKX  in each vesicle can be quantified from eachcurrent transient by Faraday’s law. A log-normalized frequencyhistogram of the molecular count in each vesicle is shown in Nmolecules per vesicle obtained across the Hofmeisterseries . Vesicularcatecholamine content is dominated by two competing pathways: thecatecholamine transport into the vesicles by the vesicular monoaminetransporter and extracellular inactivation by monoamine oxidases andcatechol-O-methyltransferase.21 We assume that the increased vesicular content might be inducedby the inhibition of enzymes via the chaotropic effect. Chaotropicanions have been shown to affect catecholamine transport across thevesicular membrane, membrane potential, and intravesicular pH,24 which will affect the vesicular amine concentration.TypicalIVIEC amperometric traces for vesicle opening are shownin – orSCN– and in Figure S2 for Br–, NO3–, orClO4–. Upon stimulation, the vesiclemembrane fuses with the cell membrane and releases part of the vesiclecontent, which is recorded as a trace of current transients representingexocytotic release events. The number of release events (Nevents) was calculated for each condition . Compared to Cl–, the Nevents decreases significantlywhen the cells were treated with SCN– . Similarly,a log-normalized frequency histogram of Nmolecules after the exposure of each anion is shown in Nmolecules released from chromaffin cells after the exposureto the different anions is also compared and shown in p valuesin Table S3). There is no significant differencebetween the release molecule count after the exposure of kosmotropicanions (Cl–) and chaotropic anions . Interestingly, a decreasein the number of molecules released is observed after the exposureof Br–, whereas an increase in molecules releasedis observed after exposure to NO3–. Asthe molecular count for catecholamine release and the vesicle contentare both obtained here, the release fraction can be calculated . Comparing the fraction releasedafter the exposure of kosmotropic anions ,we found a smaller release fraction for chaotropic anions . Interestingly,Br– decreases the release and release fraction (36%),which might reverse the seizure activity and help to understand themechanism of epilepsy treatment.25Typical SCA amperometrictraces of exocytosis from chromaffin cellsare shown in 27 We analyzed the dynamicparameters  of current spikesin SCA amperometric traces, including Imax, the peak amplitude, t1/2, the halfpeak width, trise, the rise time, and tfall, the fall time, and compared them in p values are listed in Tables S4–S7). Exocytosis at treated chromaffin cells with different anions displaysa larger Imax as the anion is changedfrom kosmotropic to chaotropic anions ,30 actin,32 and dynamin33) and enzymes )34 regulate exocytosis at the cell membrane. Anions can interact withproteins via ion-pairing, hydrogen bonding, π-anion, and chaotropiceffects.37 The chaotropic anionsprefer to interact with the hydrophobic regions of proteins directlymodifying the activity and increasing solubility of proteins. However,kosmotropic anions are thought to interact with proteins via mediatingwater molecules. In this case, chaotropes (SCN–)help to unfold proteins and induce a salting-in effect. By comparison,kosmotropes (Cl–) lead to the stabilization of thefolded state and cause a salting-out effect. Therefore, it seems plausiblethat chaotropic anions can increase the formation of the SNARE complexin the cell membrane by decreasing the protein–protein interactionbetween SNAREs and other noncognate SNAREs,39 resulting in a shorter duration. Moreover, the activity of PKC canbe inhibited by chaotropic anions via increasing its solubility becauseit is water-soluble in the inactive state.41 As a result, the activation of actin fragmentation causes a tighteractin network which results in a less stable fusion and shorter exocytoticevents before the pore closes again.43 The looseningassembly around microtubules of dynamin induced by adsorption of chaotropicanions might lead to a shorter duration of exocytosis events.44 Interestingly, the effect of chaotropic anionscontrasts the effects of Zn2+ (kosmotropes).45 This is, again, consistent with the Hofmeistereffect.An opposite effect inexocytotic dynamics has been observed inchromaffin cells, which is induced by the chaotropic effects on thelipids bilayer.Ifoot and tfoot , representing the current amplitudeand lifetime, respectively. The released molecules during the prespikefoot can be calculated via the area of the foot part. Values for Ifoot, tfoot, Nmolecules(foot), and Nfoot/Nevents decrease significantlyafter the exposure of chaotropic anions  as shown in Figure S5 . The value for Ifoot decreases (ClO4–) or remains constant (SCN–) after exposure of chaotropic anions, even though the vesicle contentincreases simultaneously, suggesting a smaller initial pore,46 which could be induced by a tighter actin networkthan after exposure to Cl–. Moreover, the decreased tfoot, Nfoot/Nevents, and Nmolecules in the foot indicate that the chaotropic anions induce a less stablefusion pore, which is consistent with the results from the main peak.It could be induced by a tighter actin network and looser dynaminafter interacting with chaotropic anions as mentioned in the proposedmechanism.To support the proposed mechanism, we analyzed the parametersofprespike feet from SCA experiments to provide insights into the initialfusion pore after treatment with different anions, including p values are listed in Tables S12–S15). There is no significant difference observedbetween the values of Imax obtained fordifferent anions treated cells . This is opposite to the effect of chaotropicanions on the dynamics of exocytosis, but is consistent with the previousobservation.19 We assume that this is dueto the adsorption of chaotropic anions on the dense-core and vesicularlipid bilayer, and chaotropic anions affect the association of adrenalinewith intravesicular matrix species  astheir colligative properties are strongly dependent on pH,47 which leads to a slower vesicle opening eventon the electrode. These results suggest the chaotropic anions affectdifferent targets by varying their treatment mode, concentration,and time, which could be helpful to understand the chemical interactionsand use to regulate release fraction.Peak parameters for vesicle opening obtained by IVIECare summarizedand analyzed in ed cells 4A. Howev4– and SCN–). However, catecholamine release during exocytosis remains nearlythe same, resulting in a smaller release fraction which has been implicatedas an important factor as part of regulated exocytosis in plasticityand cognition.51 Further, chaotropic anions shorten the duration time of the membranefusion pore during exocytosis. We assume the chaotropic anions canaffect the physicochemical properties of proteins via chaotropic effects,including an increase in the formation of SNAREs, decrease in theactivity of PKC, tightening of the structure of the actin network,and loosening of the structure of dynamin, all resulting in a shorterexocytotic process. Our results suggest the process of vesicle release,and the amount vesicular content in living cells can be regulatedby Hofmeister effects, which might be helpful to understand the manypharmacological and pathological processes induced by ions.In conclusion, amperometricmeasurements show that micromolar concentrationsof chaotropic anions not only trigger changes in the dynamics of exocytosisbut also, importantly, vesicle content in secreting cells, a noveldiscovery. The vesicle catecholamine content in chromaffin cells issignificantly increased after 3-h exposure to 100 μM chaotropicanions (e.g., ClO