Preclinical modeling in glioblastoma patient-derived xenograft (GBM PDX) xenografts to guide clinical development of lisavanbulin—a novel tumor checkpoint controller targeting microtubules

BackgroundGlioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts.MethodsMice bearing intracranial tumors received lisavanbulin +/−RT +/−TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis.ResultsLisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01).ConclusionsLisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients.     

Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In this study, the relationship between MGMT protein expression and tumor response to TMZ was evaluated in four GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors displayed elevated basal MGMT protein expression, but only two of these were resistant to TMZ therapy (tumors GBM43 and GBM44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low-dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT nonmethylated tumors, and in these tumors, protracted dosing regimens may not be effective.     

Thermographic assessment of tumor growth in mouse xenografts

In human breast tumors, a 1-2 degrees C increase in skin surface temperature is usually observed at the periphery; it has been proposed that this change is due to the hypervascularity and increased blood flow resulting from tumor-associated angiogenesis. Here we tested the hypothesis that thermal imaging might represent a useful adjunctive technique in monitoring the growth dynamics of human tumor xenografts. Xenografts were established in immunocomprised nude mice using MDA-MB-231 or MCF7 breast cancer cells. We exploited the inherent noncontact and noninvasive advantages of infrared thermography to detect skin surface temperature changes. Continuous thermographic investigation was performed to detect and monitor tumor growth in vivo and high resolution digital images were analyzed to measure the tumor temperature dynamics. In contrast to the skin temperature increases associated with human breast cancer, a consistent temperature decrease was found in the xenograft mice. In one case, a smaller secondary tumor, otherwise undetectable, was clearly evident by thermal imaging. The tumors were cooler than the surrounding tissue with a maximum temperature reduction of 1.5 degrees C for MDA-MB-231 tumor and 3 degrees C for MCF7 tumors observed on day 14. In addition, the temperature of the xenograft tumors decreased progressively as they grew throughout the observation period. It was demonstrated that thermographic imaging could detect temperature changes as small as 0.1 degrees C on the skin surface at an early stage of tumor development. The findings of the study indicate that thermographic imaging might have considerable potential in monitoring human tumor xenografts and their response to anticancer drugs.     

Patient-derived bladder cancer xenografts in the preclinical development of novel targeted therapies

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients'' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.     

七氟烷通过抑制PI3K磷酸化抑制结肠癌SW480 细胞的增殖和侵袭以及裸鼠移植瘤的生长

目的：探讨七氟烷（sevoflurane）通过调控PI3K磷酸化对结肠癌SW480 细胞增殖和侵袭以及裸鼠移植瘤生长的影响及其机制。方法：采用七氟烷处理结肠癌SW480 细胞,将细胞随机分为对照组、0.5%七氟烷组、1.0%七氟烷组和2.0%七氟烷组进行后续实验。用克隆形成实验检测SW480 细胞的增殖能力,RT-PCR 检测细胞中MDM2、survivin mRNA表达水平,Transwell小室法检测细胞的侵袭能力,Western blotting 检测细胞中MDM2、survivin、VEGF、PI3K、p-PI3K、AKT、p-AKT 蛋白的表达水平。并加入PI3K激活剂740 Y-P 进行验证。建立裸鼠SW480 细胞移植瘤模型,称取肿瘤质量,免疫组化检测移植瘤组织中MDM2 和VEGF阳性表达率。结果：与对照组和低剂量组比较,1.0%和2.0%七氟烷组SW480 细胞的克隆形成率显著降低（均P<0.01）;细胞中MDM2 mRNA和蛋白水平显著升高（均P<0.01）,survivin mRNA和蛋白水平显著降低（均P<0.01）;SW480 细胞侵袭数显著降低（P<0.01）,VEGF、p-PI3K/PI3K、p-AKT/AKT 蛋白水平显著降低（均P<0.01）。740Y-P 可逆转七氟烷对SW480 细胞增殖、侵袭及PI3K/AKT信号通路相关蛋白表达的影响。2.0%七氟烷组小鼠移植瘤质量显著降低（P<0.01）,瘤组织中MDM2 阳性表达率显著升高（P<0.01）、VEGF阳性表达率显著降低（P<0.01）。结论：七氟烷抑制结肠癌SW480 细胞的增殖和侵袭以及裸鼠移植瘤的生长,其作用机制可能是通过抑制PI3K磷酸化实现的。     

熊果酸对荷人骨肉瘤裸小鼠移植瘤生长的影响

目的:研究熊果酸(ursolic acid,UA)对荷人骨肉瘤裸小鼠移植瘤生长的抑制作用及其机制探讨.方法:采用裸小鼠背部皮下注射接种人骨肉瘤MG-63细胞的方法建立荷人骨肉瘤裸小鼠模型并给予不同剂量熊果酸[5、10、20mg/(kg·d)]和顺铂[2mg/(kg·d)]进行干预治疗,疗程5天.称取瘤重并计算抑瘤率,HE染色观察肿瘤组织形态结构改变.TUNEL染色检测细胞凋亡状况并计算细胞凋亡指数(apoptosis index,AI).免疫组织化学染色(immunohistochemistry,IHC)观察肿瘤组织中caspase-3、Bax、bcl-2蛋白表达并进行半定量分析.结果:较模型组,熊果酸干预组荷人骨肉瘤裸小鼠移植瘤呈现细胞皱缩、片状坏死等病理性改变,凋亡细胞数量明显增多、AI显著升高,瘤体重量显著减轻、抑瘤率显著升高,caspase-3和Bax蛋白表达显著上调,bcl-2蛋白表达显著下调,Bax/bcl-2比值显著升高,熊果酸上述作用均具有一定的剂量依赖性.结论:熊果酸可能通过促进肿瘤细胞凋亡而对荷人骨肉瘤裸小鼠移植瘤生长起到一定的抑制作用,其作用机制可能与熊果酸能够调节凋亡相关蛋白(caspase-3、Bax、bcl-2)并提高Bax/bcl-2比值有关.     

IL-18对Lewis肺癌小鼠移植瘤生长的抑制作用

目的：观察IL-18对Lewis肺癌小鼠移植瘤生长的抑制作用。方法：建立Lewis肺癌小鼠皮下移植瘤模型16只，按随机数字法分为IL-18治疗组、荷瘤模型组，每组8只，分别给予IL-18、生理盐水于接种第7日起腹腔注射。观察IL-18对移植瘤体积变化及瘤重的影响。结果：IL-18对小鼠健康状况无明显影响，但对小鼠移植瘤生长有明显抑制作用，肿瘤抑制率为75％。结论：IL-18可抑制Lewis肺癌小鼠移植瘤的生长，其机制值得深入研究。     

The allosteric AKT inhibitor,MK2206, decreases tumor growth and invasion in patient derived xenografts of endometrial cancer

The purpose of this study was to test the effect of MK2206, an allosteric inhibitor of AKT, on the growth and invasion of patient-derived xenografts (PDX) of endometrial cancer. Three PDX lines, USC1 (uterine serous), EEC2 (endometrioid grade 2) and EEC4 (endometrioid grade 3) of endometrial cancer were grafted under the renal capsule of NSG mice. After 2 weeks of tumor growth the mice were treated with vehicle or 120mg/kg MK2206 twice a week for 3 weeks. Growth of all 3 PDX lines of different type and grade was significantly inhibited in response to MK2206 compared with vehicle control. Histological analysis revealed invasion and spread of EEC2 and EEC4 tumors were significantly decreased with MK2206 treatment. Immunohistochemical analysis showed a decrease in Ki67 in EEC2 upon MK2206 treatment, while USC1 and EEC4 tumors did not show differences in Ki67 levels. PR levels were evident in EEC2 which dramatically increased upon MK2206 treatment. In vitro analysis of EEC4 and AN3CA cells showed a dose-dependent decrease in p(Ser473)-AKT and p(Thr308)-AKT with MK2206. Invasion of EEC4 and AN3CA cells also significantly decreased after 36h and 72h of MK2206 treatment. PDX tumors provide an appropriate model for the testing of compounds that incorporates the heterogeneous nature of endometrial cancer. Further studies to determine efficacy of MK2206 alone or in combination with other compounds can also identify predictors of response to these pathway inhibitors.     

Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth,invasion, and epithelial-to-mesenchymal transition

Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas.     

Abrogation of PIK3CA or PIK3R1 reduces proliferation, migration, and invasion in glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is a highly invasive and deadly brain tumor. Tumor cell invasion makes complete surgical resection impossible and reduces the efficacy of other therapies. Genome-wide analyses of mutations, copy-number changes, and expression patterns have provided new insights into genetic abnormalities common in GBM. We analyzed published data and identified the invasion and motility pathways most frequently altered in GBM. These were most notably the focal adhesion and integrin signaling, and extracellular matrix interactions pathways. We mapped alterations in each of these pathways and found that they included the catalytic PIK3CA and regulatory PIK3R1 subunit genes of the class IA PI3K. Knockdown of either of these genes separately in GBM cell lines by lentiviral-mediated shRNA expression resulted in decreased proliferation, migration, and invasion in all lines tested. FAK activity was reduced by knockdown of either PIK3CA or PIK3R1, and MMP2 levels were reduced by knockdown of PIK3R1. We conclude that PIK3R1, like PIK3CA, is a potential therapeutic target in GBM and that it also influences tumor cell growth and motility.     

Inhibitory effects of CAI in glioblastoma growth and invasion

While distant metastases are rare in patients with primary brain malignancies, local growth and invasion are common and life-threatening.Regional infiltration is responsible for the failure of local therapies,resulting in tumor recurrence, progression, and death. The process of invasion requires cellular adhesion, local proteolysis and migration. CAI, carboxyamide-triazole, is an anticanceragent developed as an inhibitor of selected signal transduction pathways.Studies on the effects of CAI on human glioblastoma growth and invasiveness are presented. CAI inhibited proliferation of 6 of 8 cell lines tested in a dose-dependent fashion in vitro (IC₅₀ range 1.5—44 M), with no effect on the U373 line. Inhibition of adhesion to tissue culture plastic was observed for the H4, T98G, and U373 lines pretreated with CAI; H4 and T98G were inhibited in adhesion to collagentype IV. Incubation with CAI decreased production of the 72 kDa and 92 kDa type IV collagenases in all cell lines, ranging from 16 to 93% inhibition. These observations show that the effects of CAI on cell line behavior can vary between lines that are similar in origin. Despite variability in the inhibitory effects for proliferation and adhesion, CAI is consistently able to inhibit the invasive phenotype of all glioma cell lines in vitro using the Matrigel barrier assay (IC₅₀range 13—28 M). These observations suggest that CAI may have benefit in the treatment of gliomas and high grade astrocytomas.     

Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo

Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50 %). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70 % killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.     

Flaxseed attenuates the tumor growth stimulating effect of soy protein in ovariectomized athymic mice with MCF-7 human breast cancer xenografts

In several epidemiological studies, a phytoestrogen-rich diet containing lignans and isoflavones is associated with reduced breast cancer risk, but experimental findings are controversial. In postmenopausal mammary cancer xenograft model, flaxseed (FS), a rich source of plant lignans, reduced breast cancer growth, while soy protein (SP), a rich source of isoflavones, enhanced it. The intake of phytoestrogens is increasing particularly among postmenopausal women, emphasizing the importance of elucidating their interactive effects on breast cancer. Our study determined the effect of FS and SP diets, alone and in combination, on the established human breast cancer MCF-7 tumor growth in ovariectomized athymic nude mice. Tumor bearing mice were divided into 4 groups and fed for 25 weeks either the basal diet (BD), or BD supplemented with 10% FS, 20% SP or 10% FS and 20% SP. After estrogen deprivation, FS regressed the tumor size similar to that of control. SP initially regressed the tumors but starting at week 13, the tumors regressed significantly less than in control and 43% of the tumors were regrowing until the end of the experiment and were significantly larger in size than in control. The combination of SP with FS reduced the tumor growth similar to that of control, as suggested also by the reduced tumor cell proliferation index. In conclusion, dietary FS did not stimulate the growth of estrogen responsive MCF-7 cancers in ovariectomized mice, while long-term consumption of SP did. Furthermore, FS reduced the tumor growth stimulating effect of SP to the same level as control, suggesting tumor growth attenuating effect of FS.     

Clonal heterogeneity influences drug responsiveness in renal cancer assessed by ex vivo drug testing of multiple patient-derived cancer cells

Renal cell cancer (RCC) has become a prototype example of the extensive intratumor heterogeneity and clonal evolution of human cancers. However, there is little direct evidence on how the genetic heterogeneity impacts on drug response profiles of the cancer cells. Our goal was to determine how genomic clonal evolution impacts drug responses. Finding from our study could help to define the challenge that clonal evolution poses on cancer therapy. We established multiple patient-derived cells (PDCs) from different tumor regions of four RCC patients, verified their clonal relationship to each other and to the uncultured tumor tissue by genome sequencing. Furthermore, comprehensive drug-sensitivity testing with 460 oncological drugs was performed on all PDC clones. The PDCs retained many cancer-specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes. The drug testing highlighted vulnerability in the PDCs toward approved RCC drugs, such as the mTOR-inhibitor temsirolimus, but also novel sensitivities were uncovered. The individual PDC clones from different tumor regions in a patient showed distinct drug–response profiles, suggesting that genomic heterogeneity contributes to the variability in drug responses. Studies of multiple PDCs from a patient with cancer are informative for elucidating cancer heterogeneity and for the determination on how the genomic evolution is manifested in cancer drug responsiveness. This approach could facilitate tailoring of drugs and drug combinations to individual patients.     

Evolutionary predictability of genetic versus nongenetic resistance to anticancer drugs in melanoma

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Overexpression of SCLIP promotes growth and motility in glioblastoma cells

SCLIP, a microtubule-destabilizing phosphoprotein, is known to be involved in the development of the central nervous system (CNS). It has been well established that there are notable parallels between normal development and tumorigenesis, especially in glioma. However, no studies have examined the significance of SCLIP in gliomagenesis. To address this, we investigated the expression of SCLIP and its roles in the development of gliomas. Notably, we found that SCLIP was highly expressed in various grades of glioma samples, as compared with normal brain tissues. Overexpression of SCLIP dramatically stimulated tumor cell migration and invasion as well as proliferation and downregulation of SCLIP showed opposite effects, establishing an important oncogenic role for this gene. Furthermore, we revealed that STAT3 was required to maintain SCLIP stability, suggesting that overexpression of STAT3 may be a critical step to facilitate microtubule dynamics and subsequently promotes migration and invasion of glioma cells. Taken together, our findings demonstrate that SCLIP plays an important role in glioma pathology, and may represent a novel therapeutic strategy against human glioma.     

Artificial Intelligence Role in Subclassifying Cytology of Thyroid Follicular Neoplasm

Objective: Fine needle aspiration cytology has higher sensitivity and predictive value for diagnosis of thyroid nodules than any other single diagnostic methods.  In the Bethesda system for reporting thyroid, the category IV, encompasses both adenoma and carcinoma, but it is not possible to differentiate both lesions in the cytology practice and can be only differentiated after resection. In this work, we aim at exploring the ability of a convolutional neural network (CNN) model to sub-classifying cytological images of Bethesda category IV diagnosis into follicular adenoma and follicular carcinoma. Methods: We used a cohort of cytology cases n= 43 with extracted images n= 886 to train CNN model aiming to sub-classify follicular neoplasm (Bethesda category IV) into either follicular adenoma or follicular carcinoma. Result: In our study, the model subclassification of follicular neoplasm into follicular adenoma (n = 28/43, images n = 527/886) from follicular carcinoma (n = 15/43, images n= 359/886), has achieved an accuracy of 78%, with a sensitivity of 88.4%, and a specificity of 64% and an area under the curve (AUC) score of 0.87 for each of follicular adenoma and follicular carcinoma. Conclusion: Our CNN model has achieved high sensitivity in recognizing follicular adenoma amongest cytology smears of follciualr neoplasms, thus it can be used as an ancillary technique in the subcalssification of Bethesda Iv category cytology smears.     

Decursin inhibits cell growth and autophagic flux in gastric cancer via suppression of cathepsin C

Autophagy plays an important role in the survival of cancer cells under stressful conditions, such as nutrient or oxygen deficiency. Therefore, autophagy inhibition is being considered as a novel therapeutic strategy for cancer. Decursin is a natural compound derived from Angelica gigas; it has been used in the treatment of various diseases, including cancer. However, the mechanism by which decursin regulates autophagy in gastric cancer and other carcinomas remains unclear. Here, we demonstrated that decursin reduced the growth and induced cell cycle arrest in gastric cancer cells in vitro. Decursin blocked autophagic flux by reducing the expression of lysosomal protein cathepsin C (CTSC) and attenuating its activity, thereby causing autophagic dysregulation (i.e., accumulation of LC3 and SQSTM1). Decursin also inhibited cell proliferation and cell cycle progression by inhibiting CTSC and E2F3, both of which were linked to gastric cancer aggressiveness. The antitumor effects of decursin were confirmed in vivo. We established spheroid and patient-derived organoid models and found that decursin decreased the growth of spheroids and patient-derived gastric organoids, as well as modulated the expression of CTSC and autophagy-related proteins. Hence, our findings uncovered a previously unknown mechanism by which decursin regulates cell growth and autophagy and suggests that decursin may act as a potential therapeutic agent that simultaneously inhibits cell growth and autophagy.