Male Nude Mice Research Articles

Pharmacokinetics and Anti-Tumor Efficacy of PEGylated Liposomes Co-Loaded with Cisplatin and Mifepristone.

Although cisplatin is an effective chemotherapy drug used against many types of cancer, it has poor bioavailability, produces severe adverse effects, and frequently leads to tumor resistance. Consequently, more effective formulations are needed. The co-administration of cisplatin with mifepristone, which counters an efflux pump drug-resistance mechanism in tumor cells, has shown important synergism, but without resolving the problem of poor bioavailability. Specificity to tumor tissue and bioavailability have been improved by co-encapsulating cisplatin and mifepristone in a liposomal formulation (L-Cis/MF), which needs further research to complete pre-clinical requirements. The aim of this current contribution was to conduct a pharmacokinetic study of cisplatin and mifepristone in male Wistar rats after administration of L-Cis/MF and the conventional (unencapsulated) formulation. Additionally, the capacity of L-Cis/MF to reduce tumor growth in male nude mice was evaluated following the implantation of xenografts of non-small-cell lung cancer. The better pharmacokinetics (higher plasma concentration) of cisplatin and mifepristone when injected in the liposomal versus the conventional formulation correlated with greater efficacy in controlling tumor growth. Future research on L-Cis/MF will characterize its molecular mechanisms and apply it to other types of cancer affected by the synergism of cisplatin and mifepristone.

Molecular MR Imaging of Prostate Cancer by Specified Iron Oxide Nanoparticles With PSMA-11 Peptides: A Preclinical Study.

Prostate-specific membrane antigen (PSMA) can provide a prostate cancer (PCa) detection approach in positron emission tomography (PET) using Food and Drug Administration (FDA)-approved PSMA-11 peptide. There are some studies evaluated magnetic-nanoprobes for PSMA detection by MRI, using non-FDA-approved ligands including antibodies or peptides, which are not as specific as PSMA-11. To assess targeted iron oxide nanoparticles (IONPs) by PSMA-11 peptides as a potential specific nano-molecular probes to investigate a PSMA+ PCa-xenograft model by MRI. Prospective. Twenty male C57BL6 nude mice induced subcutaneously PSMA+ LNCaP cell line tumor. 1.5 T, T2-W Fast Spin echo and T2*-W Gradient echo. Coated IONPs with Carboxymethylated-dextran (DNPs) and with bovine serum albumin (BNPs), as well as, targeted DNPs with PSMA-11-HYNIC peptide (TDNPs) and targeted BNPs with PSMA-11-HBED peptide (TBNPs) were injected intravenously with dose 2.8 mg Fe/kg. Coronal T2-W and the T2*-W images were obtained before and 4 hours and 6 hours post-injection. Signal intensity (SI) and relative signal enhancement (RSE) were computed in two- and three-dimensional analyses. Histological analysis of tumors was evaluated, and the Fe distribution within the body based on atomic absorption spectroscopy was calculated. One-way ANOVA followed by Tukey's multiple comparison test, Paired-samples T-test, P < 0.05 was considered significant. A reduction in T2-W SI was achieved as 22 ± 7%, 59 ± 3%, 65 ± 5%, and 78 ± 3% respectively for BNPs, TBNPs, DNPs, and TDNPs 6 hours post-injection. The most difference between targeted and non-targeted groups was observed at 6 hours for PSMA-11-HBED, and at 4 hours for PSMA-11-HYNIC. RSE indicated 88.6 ± 3.1% and 80.7 ± 3.2% enhanced contrast between tumor and muscle region for TBNPs and TDNPs on T2*-W images. Both TBNPs and TDNPs are promising novel nano-molecular probes for PSMA+ PCa tumor detection. The injection dose of non-targeted IONPs can be reduced by using targeted nanoprobes three times for BNPs and two times for DNPs. 1 TECHNICAL EFFICACY: Stage 1.

Naringin Inhibits Colorectal Carcinogenesis by Inhibiting Viability of Colorectal Cancer Cells.

To explore the therapeutic effect of naringin on colorectal cancer (CRC) and the related mechanism. Cell counting kit-8 (CCK-8) assay and annexin V-FITC/PI assay were used to detect the effect of naringin (50-400 µg/mL) on cell proliferation and apoptosis of CRC cells, respectively. The scratch wound assay and transwell migration assay were used to assess the effect of naringin on CRC cell migration. Four-week-old male nude mice were injected with HCT116 cells subcutaneously to establish the tumor xenograft model. Naringin was injected intraperitoneally at 50 mg/(kg·d), with solvent and 5-fluorouracil treatment as control. The width and length of the tumors were measured and recorded every 6 days, and tumor tissues were photographed and weighed on the last day of the 24-d observation period. Immunohistochemical staining for caspase-3, proliferating cell nuclear antigen and TUNEL assay were used to evaluate the effect of naringin on cell proliferation and apoptosis in tumor tissues. The body weight, food and water intake of mice were recorded, and the major organs in different treatment groups were weighed on the last day and stained with hematoxylin and eosin for histological analysis. Meanwhile, the routine blood indicators were recorded. CCK-8 and annexin V-FITC/PI results confirmed that naringin (100, 200, and 400 µg/mL) could inhibit proliferation and promote apoptosis. The scratch wound assay and transwell migration assay results confirmed the inhibitory activity of naringin against CRC cells migration. In vivo results demonstrated the inhibitory effect of naringin on tumor growth with good bio-compatibility. Naringin inhibited colorectal carcinogenesis by inhibiting viability of CRC cells.

#4158 URINE DERIVED STEM CELL AMELIORATES RENAL FIBROSIS VIA KLOTHO ACTIVATION

Abstract Background and Aims After renal IRI, regeneration and recovery of the renal tubular cell occurs. However, if the renal repair process is maladaptive, it progresses to renal fibrosis. The role of stem cells in kidney regeneration or fibrosis has not been fully elucidated. we evaluated the urine drived stem cells (UDSC) for renal inflammation and fibrosis after renal ischemia reperfusion (IR). Method 10 week old balb/c nude male mice were used. sham, sham with UDSC, IR, IR with UDSC. UDSC were infused 3 times via tail vain at 6, 7, 8th day after renal IR. Urine NGAL/creatinine (Cr) were checked. The kidneys tissue were harvested at day 14 day. In vitro, TGF-β treated HK2 cell were co-cultured with UDSC. Klotho siRNA silencing was performed in UDSC. Results Urinary NGAL/Cr were significantly increased in IR mice after 14 day IR, compared to sham mice. Urinary NGAL/Cr significantly decreased in UDSC treated IR mice, compared to IR mice. In H&amp;E stain, renal tubulo-interstitial injury were significantly decreased in UDSC treated IR mice, compared to IR mice. In masson trichrom stain, renal fibrosis area were were significantly decreased in UDSC treated IR mice, compared to IR mice. The renal expression of TGF-β, α-SMA, and collagne IV were significantly decreased in UDSC treated IR mice, compared to IR mice. The renal expression of Klotho were increased in UDSC treated IR mice, compared to IR mice. in vitro, UDSCs were stem cells that expressed Klotho protein more strongly than other mesenchymal stem cells (MSCs). UDSCs also suppressed fibrosis by inhibiting transforming growth factor (TGF)-β in HK-2 human renal proximal tubule cells in an in vitro model. Klotho siRNA silencing reduced the TGF-β-inhibiting ability of UDSCs. Conclusion UDSC attenuate renal fibrosis after renal IR. Klotho-secretion of UDSC play a role in these anti- fibrotic effects.

Human Adipose-Derived Endothelial Progenitor Cells Accelerate Epithelialization of Radiation Ulcers in Nude Mice.

Cotransplantation of adipose-derived stem cells (ASCs) and endothelial progenitor cells has shown superior angiogenic effects compared with ASCs alone in recent animal studies. However, endothelial progenitor cells could only be collected from blood vessels or bone marrow. Thus, the authors have established a method for purifying adipose-derived endothelial progenitor cells (AEPCs). The authors hypothesized that AEPCs would enhance the therapeutic effect of ASCs on radiation ulcers. Seven-week-old male nude mice (BALB/cAJcl-nu/nu) were irradiated on the dorsal skin (total 40 Gy); 12 weeks later, 6-mm-diameter wounds were created. The mice were then treated with subcutaneous injection of human ASCs [1 × 10 5 ( n = 4)], human AEPCs [2 × 10 5 or 5 × 10 5 ( n = 5)], combinations of those [ASCs 1 × 10 5 plus AEPCs 2 × 10 5 ( n = 4) or 5 × 10 5 ( n = 5)], or only vehicle ( n = 7). The nonirradiated group was also prepared as a control ( n = 6). The days required for macroscopic epithelialization was compared, and immunostaining for human-derived cells and vascular endothelial cells was performed at day 28. AEPC-ASC combination-treated groups healed faster than the ASC-treated group (14 ± 0 days versus 17 ± 2 days; P < 0.01). Engraftment of the injected cells could not be confirmed. Only the nonirradiated mice had significantly higher vascular density (0.988 ± 0.183 × 10 -5 /µm -2 versus 0.474 ± 0.092 × 10 -5 /µm 2 ; P = 0.02). The results suggested therapeutic potentials of AEPCs and an enhanced effect of combination with ASCs. This study is a xenogenic transplantation model, and further validation in an autologous transplantation model is needed. Human AEPCs and their combination with ASCs accelerated epithelialization of radiation ulcers in nude mice. The authors suggest that administration of humoral factors secreted from AEPCs (eg, treatment with culture-conditioned media) could be used for the same purpose.

Vasculogenic Mesodermal Cells Derived From Induced Pluripotent Stem Cells Improve Limb Perfusion and Decrease Necrosis in Murine Models of Hindlimb Ischemia

Critical limb-threatening ischemia (CLTI) is a national health care priority as there has been a 50% increase in amputations from 2010 to 2018. Unlike other cell products that simply secrete proangiogenic growth factors, induced pluripotent stem cell (iPSC)-derived mesodermal cells have the capacity to differentiate into endothelial cells and smooth muscle cells and form functioning blood vessels in vivo. Here we discuss the effects of intramuscular injection of human iPSC mesodermal cells (VSC100) on limb perfusion and limb necrosis. iPSCs were produced using methods including Yamanaka factors (Broxmeyer et al [21393480]). The iPSC cells were differentiated using sequential Activin A, BMP4, vascular endothelial growth factor, and fibroblast growth factor 2 to promote mesodermal and endothelial cell differentiation. Murine CLTI models were created via excision of the common femoral artery in male BALB-C Nude mice, aged 6 to 8 weeks. After creation of the CLTI model mice were randomized to 1 × 106 VSC100 cells labeled with td-tomato (red fluorescence) or vehicle control injected into the gastrocnemius and gracilis muscles (n = 10/group) at 7 to 14 days postinduction of ischemia. Limb perfusion was measured with laser speckle contrast imaging up to day 64, qualitative analysis of VSC100 differentiation into capillaries was assessed with immunohistochemical staining for isolectin B4 and human CD31 and confocal microscopy to detect td-tomato. Degree of tissue loss of the index limb was quantified with established necrosis scores. VSC100-treated BALB-C nude mice showed a significant increase in limb perfusion as compared to vehicle controls with average percent blood flow/contralateral limb ratios of 45% in treated groups compared to 25% in control (P < .001) at day 64 (Fig 1). Limb necrosis scores showed similar results, in that necrosis was statistically significantly reduced in iPSC-treated vs vehicle control treated groups (Fig 2). Immunohistochemical analysis showed qualitative significant deposition of cells of interest in our CLTI models. iPSC-derived VSC100 is a novel approach to treating CTLI as the cells are capable of forming functioning blood vessels in ischemia skeletal muscle, improve tissue perfusion, and mitigate tissue necrosis. These early results and data support further translational work to support development of an investigational new drug for future clinical trial studies.Fig 2Clinical scoring ratio of chronic limb-threatening ischemia (CLTI) in control vs treated groups. Mouse population same as in Fig 1 (n = 10/group).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Effect of overexpression of Rac1 on radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice, and the underlying mechanism

Purpose: To study the effect of overexpression of Rac1 on the radiosensitivity of transplanted nasopharyngeal carcinoma (NPC) in nude mice, and the mechanism involved. &#x0D; Methods: Forty Lucky SPF-grade male thymus-free nude mice were used. Mice were divided into 4 groups: overexpression control, overexpression Rac1, Rac1 inhibition, and inhibition control groups, each with 10 mice. The prepared cell lines were treated with 6 MV x-ray. Before and after radiation, the growth of tumors in each group was monitored. Histomorphological images of nude mice tumors were obtained using hematoxylin and eosin (H&amp;E) stains. Protein expressions of p67, P47, and Rac1 were evaluated bu Western blotting. &#x0D; Results: Transplanted tumor growth slowed down after 20 days. Growth rate was significantly higher in Rac1 and Rac1 overexpression groups than in overexpression and inhibition control groups (p &lt; 0.05). Overexpression of Rac1 resulted in more cell necrosis, incomplete cellular structure, severe nuclear fragmentation, nuclear pyknosis, and cytotoxic red staining in endoscopic tumor tissues (p &lt; 0.05). There were significantly lower expression levels of p67, P47, and Rac1 in Rac1 invasion group than in invasion control and overexpression control groups, while the expression levels of p67 and P47 were significantly higher in overexpression Rac1 group and IR overexpression Rac1 group than in inhibition control (p &lt; 0.05). However, concentrations of p67 and P47 were significantly higher in overexpression Rac1 and IR overexpression Rac1 groups than in inhibition control mice. &#x0D; Conclusion: Rac1 increases the radiosensitivity of NPC xenografts in nude mice via a mechanism related to the regulation of expressions of proteins associated with Rac1/NADPH signaling pathway. Thus, Rac1 is potentially a new target for radio-sensitization of NPC.

Abstract 547: Vasculogenic Mesodermal Cells Derived From Induced Pluripotent Stem Cells Improve Limb Perfusion And Decrease Necrosis in Murine Models of Hindlimb Ischemia

Objectives: Critical Limb Threatening Ischemia (CLTI) is a health care priority with a 50% increase in amputations from 2010 to 2018. Unlike other cell products that simply secrete pro-angiogenic growth factors Induced pluripotent stem cells (iPSC) derived mesodermal cells can differentiate into endothelial and smooth muscle cells and form functioning blood vessels in vivo . We discuss the effects of intramuscular injection of human iPSC mesodermal cells (VSC100) on limb perfusion and limb necrosis. Methods: IPSCs were produced using methods including Yamanaka factors (Broxmeyer H. et al [21393480}). iPSC cells were differentiated using sequential Activin A, BMP4, VEGF, and FGF2 to promote mesodermal and endothelial cell (EC) differentiation. Murine CLTI models were created via excision of the common femoral artery in male BALB-C Nude mice, aged 6-8 weeks. After creation of the CLTI model mice were randomized to 1x10 6 VSC100 cells labeled with td-tomato (red fluorescence) or vehicle control injected into the gastrocnemius and gracilis muscles (N=10/group) at 7-14 days postinduction of ischemia. Limb perfusion was measured with Laser Speckle Contrast Imaging (LSCI) up to Day 64, qualitative analysis of VSC100 differentiation into capillaries was assessed with immunohistochemical staining for Isolectin B4 and human CD31 and confocal microscopy to detect td-tomato. Degree of tissue loss of the index limb was quantified with established necrosis scores. Results: VSC100 treated mice showed a significant increase in limb perfusion compared to controls, average percent blood flow/contralateral limb ratios of 45% in treated groups compared to 25% in control (p &lt; 0.001) at day 64 (Figure 1). Limb necrosis scores showed necrosis was statistically significantly reduced in iPSC treated versus vehicle control treated groups (Figure 2). Immunohistochemical analysis showed qualitative significant deposition of cells of interest in our CLTI models. Conclusions: iPSC derived VSC100 is a novel approach to treating CTLI as the cells can form functioning blood vessels in ischemic skeletal muscle, improve tissue perfusion, and mitigate tissue necrosis. These early results are being used for FDA applications for clinical trials utilizing iPSCs in CLTI patients.

Abstract 5986: The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study

Abstract INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV&amp;lt;20 mm3) in 60% population in comparison to either of the cohorts post 30 days of treatment (control: 2000 mm3, P- 1200 mm3; N- 1500 mm3, p&amp;lt;0.05). Compared to control sequential P+N treatment in vivo i) reduced anti-apoptotic BcLxL mRNA expression to 2.5 fold ii) Cleaved PARP increased 10 folds and iii) BcLxL levels decreased 2 fold. CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5986.

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI&amp;gt;50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 465.

E3 Ubiquitin Ligase MARCH8 Promotes Pancreatic Cancer Growth and Metastasis by Activating STAT3 via Degradation of PTPN4.

The role E3 ubiquitin ligase membrane-associated RING-CH 8 (MARCH8) has not been studied in pancreatic cancer. Pancreatic cancer cell lines and the normal pancreatic cells were tested in vitro studies and male athymic nude mice were tested in vivo studies. Measuring cell viability by Cell Counting Kit-8 assay (CCK8), 5-ethynyl-2'- deoxyuridine (Edu) staining, and colony formation assay. Wound healing assay was implemented for cell migration and Transwell assay was performed for cell invasion to evaluate the histological status by hematoxylin and eosin staining and to detect the protein ubiquitination by ubiquitination assay. The protein expression was determined by immunohistochemistry staining and western blotting, and mRNA expression was measured by quantitative reverse transcription polymerase chain reaction. The expression of MARCH8 was increased whereas PTPN4 was decreased in pancreatic cancer cells. Overexpression of MARCH8 promoted the growth, migration, and invasion of cells, and knockdown of PTPN4 had the similar effects both in vitro and in vivo. MARCH8 promoted PTPN4 protein degradation through ubiquitination. Moreover, PTPN4 suppressed the transcription activities of STAT3 by impairing the level of pSTAT3 (705), while inhibition of PTPN4 activated phosphorylation of STAT3. MARCH8 promoted pancreatic cancer growth and invasion through mediating the degradation of PTPN4 and activated the phosphorylation of STAT3.

Pseurotin A Validation as a Metastatic Castration-Resistant Prostate Cancer Recurrence-Suppressing Lead via PCSK9-LDLR Axis Modulation.

Metastatic castration-resistant prostate cancer (mCRPC) cells can de novo biosynthesize their own cholesterol and overexpress proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 proved to contribute to mCRPC cell motility since PCSK9 knockdown (KD) in mCRPC CWR-R1ca cells led to notable reductions in cell migration and colony formation. Human tissue microarray results proved a higher immunohistoscore in patients ≥ 65 years old, and PCSK9 proved to be expressed higher at an early Gleason score of ≤7. The fermentation product pseurotin A (PS) suppressed PCSK9 expression, protein-protein interactions with LDLR, and breast and prostate cancer recurrences. PS suppressed migration and colony formation of the CWR-R1ca cells. The progression and metastasis of the CWR-R1ca-Luc cells subcutaneously (sc) xenografted into male nude mice fed a high-fat diet (HFD, 11% fat content) showed nearly 2-fold tumor volume, metastasis, serum cholesterol, low-density lipoprotein cholesterol (LDL-C), prostate-specific antigen (PSA), and PCSK9 levels versus mice fed a regular chow diet. Daily oral PS 10 mg/kg treatments prevented the locoregional and distant tumor recurrence of CWR-R1ca-Luc engrafted into nude mice after primary tumor surgical excision. PS-treated mice showed a significant reduction in serum cholesterol, LDL-C, PCSK9, and PSA levels. These results comprehensively validate PS as an mCRPC recurrence-suppressive lead by modulating the PCSK9-LDLR axis.

Preparation and bone repair capability of a new plastic bone filler material

To prepare a new plastic bone filler material with adhesive carrier and matrix particles derived from human bone, and evaluate its safety and osteoinductive ability through animal tests. The human long bones donated voluntarily were prepared into decalcified bone matrix (DBM) by crushing, cleaning, and demineralization, and then the DBM was prepared into bone matrix gelatin (BMG) by warm bath method, and the BMG and DBM were mixed to prepare the experimental group's plastic bone filler material; DBM was used as control group. Fifteen healthy male thymus-free nude mice aged 6-9 weeks were used to prepare intermuscular space between gluteus medius and gluteus maximus muscles, and all of them were implanted with experimental group materials. The animals were sacrificed at 1, 4, and 6 weeks after operation, and the ectopic osteogenic effect was evaluated by HE staining. Eight 9-month-old Japanese large-ear rabbits were selected to prepare 6-mm-diameter defects at the condyles of both hind legs, and the left and right sides were filled with the materials of the experimental group and the control group respectively. The animals were sacrificed at 12 and 26 weeks after operation, and the effect of bone defect repair were evaluated by Micro-CT and HE staining. In ectopic osteogenesis experiment, HE staining showed that a large number of chondrocytes could be observed at 1 week after operation, and obvious newly formed cartilage tissue could be observed at 4 and 6 weeks after operation. For the rabbit condyle bone filling experiment, HE staining showed that at 12 weeks after operation, part of the materials were absorbed, and new cartilage could be observed in both experimental and control groups; at 26 weeks after operation, the most of the materials were absorbed, and large amount of new bone could be observed in the 2 groups, while new bone unit structure could be observed in the experimental group. Micro-CT observation showed that the bone formation rate and area of the experimental group were better than those of the control group. The measurement of bone morphometric parameters showed that the parameters at 26 weeks after operation in both groups were significantly higher than those at 12 weeks after operation ( P<0.05). At 12 weeks after operation, the bone mineral density and bone volume fraction in the experimental group were significantly higher than those in the control group ( P<0.05), and there was no significant difference between the two groups in trabecular thickness ( P>0.05). At 26 weeks after operation, the bone mineral density of the experimental group was significantly higher than that of the control group ( P<0.05). There was no significant difference in bone volume fraction and trabecular thickness between the two groups ( P>0.05). The new plastic bone filler material is an excellent bone filler material with good biosafety and osteoinductive activity.

Murine toxicology and pharmacokinetics of lead next generation galeterone analog, VNPP433-3β

VNPP433-3β (compound 2, (3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Compound 2 is currently in development as potential new therapeutic for prostate and pancreatic cancers. The preliminary toxicity study reveals that the compound 2 was better tolerated by the normal male CD-1 mice than the male Nude mice. The maximum tolerated dose (MTD) in the Nude mice was estimated to be between 25 < 50 mg/kg. After oral dosing of compound 2 to male and female rats, the plasma concentration versus time curves were very consistent between animals and the AUClast increased with dose. Many plasmas concentration versus time curves profiles were nearly flat over 24 hr., suggesting extended absorption from the GI tract. Consequently, reliable values for half-life and AUCinf were not determined. Calculated oral bioavailability (using oral AUClast and excluding the outlier IV animal) ranged from 32 to 47 %. This should be considered a minimum value since the contribution to true AUC beyond 24 hr. is clearly not zero. Clearly, these toxicology and pharmacokinetics parameters pave the way for understanding the anticancer pharmacological actions and provide a meaningful basis for further preclinical development and eventual clinical development.

Abstract B036: Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding

Abstract Osteosarcoma (OS) is the most common primary bone cancer worldwide where it mainly affects adolescents. Metastatic OS has a 20% survival rate and treatment options remain poor, thus understanding mechanisms that contribute to OS metastasis is critical for improved patient outcomes. Platelets, commonly recognized for their role in hemostasis, have been shown to promote metastasis in several cancer models. However, most previous studies investigating platelet contributions to metastasis have focused on carcinomas while their role in osteosarcoma remains poorly understood. Previous studies by our lab have shown that platelet interactions with breast cancer cells resulted in increased migration in vitro and increased lung metastases in vivo. Thus, we hypothesized that OS cell migration will be enhanced in the presence of platelets, and that platelet depletion in vivo will inhibit OS lung seeding. Cell suspensions of SaOs2 and MG63.3 cells in serum free media were fluorescently stained and seeded, in triplicates, in the top chamber of an 8um pore Fluoroblock migration assay wells. In the bottom chamber, media and either Tyrode’s buffer or 1.5x106 isolated human platelets were added. The presence of platelets resulted in a 3.5- and 4.6-fold increase of migrated MG63.3 and SaOs2 cells respectively. For in vivo analysis, cohorts of eight-week-old, male Nod Scid-Gamma (NSG), Nod Scid, and Nude mice were randomly assigned to either the control or platelet depletion group. Mice were depleted of platelets by administration of Anti-Platelet glycoprotein Ib alpha (GPIbα) antibody and, 5x105 GFP transduced MG63.3 cells were injected via tail vein. 24 hrs later, the mice were euthanized, and lungs were collected and microscopically imaged. Platelet depletion in NSG mice resulted in a 48% reduction in MG63.3 lung seeding compared to the isotype control recipient mice. This result was more dramatic in the less immunocompromised Nude and Nod Scid mice with a 61% and 87% reduction of MG63.3 lung seeding in the platelet depleted mice respectively. Finally, RNAseq and was performed on seven OS cell lines co-cultured with isolated mouse platelets, and alterations in gene expression were compared to control cells. Gene Enrichment Set Analysis (GSEA) of the data indicated that platelet co-incubation significantly upregulated pathways related to inflammatory responses, Tumor Necrosis Factor (TNF)-a signaling, and Interleukin (IL)-6 signaling. These results suggest that platelet secreted factors facilitate OS cell migration in vitro while an immune dependent mechanism is involved in the role of platelets in the in vivo lung seeding of OS cells. Citation Format: Olivia K. Travis, Ruishan Wang, Myriam Labelle. Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B036.

Calorie restriction remodels gut microbiota and suppresses tumorigenesis of colorectal cancer in mice.

Colorectal cancer (CRC) is one of the most common cancers worldwide and the consumption of a high-calorie diet is one of its risk factors. Calorie restriction (CR) slows tumor growth in a variety of cancers, including colorectal cancer; however, the mechanism behind this remains unknown. In the present study, CR effectively reduced the tumor volume and weight in a xenograft BALB/c male nude mouse model. In addition, tumor immunohistochemistry revealed that the CR group had significantly higher expression of Bax (P<0.001) and significantly lower levels of Bcl2 (P<0.0001) and Ki67 (P<0.001) compared with control group. Furthermore, data from 16S ribosomal (r)RNA sequencing implied that CR was able to reprogram the microbiota structure, characterized by increased Lactobacillus constituent ratio (P<0.05), with amelioration of microbial dysbiosis caused by CRC. Further receiver operating characteristic curves demonstrated that the bacteria Bacteroides [area under the curve (AUC)=0.800], Lactobacillus (AUC=0.760) and Roseburia (AUC=0.720) served key roles in suppression of CRC in the mouse model. The functional prediction of intestinal flora indicated 'cyanoamino acid metabolism' (P<0.01), 'replication initiation protein REP (rolling circle plasmid replication)' (P<0.01), 'tRNA G10 N-methylase Trm11' (P<0.01) and 'uncharacterized protein with cyclophilin fold, contains DUF369 domain' (P<0.05) were downregulated in CR group. These findings implied that CR suppressed CRC in mice and altered the gut microbiota.

Properties of melanoma mitochondria to form tumor nodules in the body of experimental animals

The aim hereof is to develop a method for revealing the properties of mitochondria isolated from melanoma of the skin to form melanoma tumor nodules in the body of mice in parenteral transplantation of melanoma mitochondria. Materials and methods. We used experimental animals according to the following design: intraperitoneal transplantation with B16/F10 melanoma mitochondria in the Balb/c Nude male mice, n = 8. All animals underwent a one-time transplantation of B16/F10 melanoma mitochondria based on 5.2 mg of protein per 1 animal in 0.4 ml of saline solution. The Balb/c Nude male mice were used as the references, which were transplanted with 0.4 ml of saline solution once into the muscle and intraperitoneally. Morphological examination of sections of the internal organs and the B16/F10 melanoma foci was carried out after specimen paraffin embedding and hematoxylin-eosin staining with microscopy of specimens with Axiovert (Carl 44 Zeiss, Germany) and Axiovision 4 image visualization software (Carl Zeiss, Germany). Results. Upon intraperitoneal transplantation of mitochondria isolated from melanoma to the Balb/c Nude mice, in the autopsies in 100% of the animals after 2 weeks of the experiment the total damage to the internal organs by the tumor nodules was revealed. The morphological examination of the internal organs of the Nude mice 2 weeks after the intraperitoneal transplantation of B16 melanoma mitochondria demonstrated a tumor lesion of the visceral tissue and the abdominal organs. Conclusion. Thus, the use of parenteral transplantation of mitochondria isolated from B16/F10 melanoma in the C57BL/6 and Balb/c Nude male mice caused the growth and development of melanoma foci in the body. Our study revealed previously unknown properties of mitochondria isolated from B16/F10 skin melanoma, consisting in their ability to induce the development of melanoma tumor nodules in the body of animals upon the tumor cell parenteral transplantation.

MYBPC1 is a key regulator for laryngeal carcinoma formation.

Laryngeal carcinoma represents one of the most common types of tumor of the respiratory tract. The aim of the present study was to evaluate the functions of myosin-binding protein C1 (MYBPC1) in the progression of laryngeal carcinoma and to unravel the potential underlying molecular mechanism(s). Significantly differentially expressed mRNAs and miRNAs were analyzed, and potential genes were verified using clinically recruited patients with laryngeal carcinoma. The human laryngeal carcinoma cell lines TU686, TU212 and AMC-HN-8, as well as the control nasopharyngeal epithelial cell line NP69, were selected for the functional analysis of MYBPC1. The interaction between MYBPC1 and miR-451a was also explored in depth. The functions of MYBPC1 in the laryngeal carcinoma cell lines were examined using colony formation assay, cell proliferation and invasion assays, and via measuring the extent of apoptosis. The intracellular function of MYBPC1 was subsequently confirmed by constructing an in vivo xenograft model through the subcutaneous injection of laryngeal carcinoma cells into 4-week-old male nude mice. Compared with normal tissue, MYBPC1 was found to be the most significantly downregulated gene, whereas activating transcription factor-2 (ATF-2) was the most significantly upregulated one. At the same time, miR-451a was found to be the most significantly downregulated miRNA in laryngeal squamous cell carcinoma tissues. According to the WHO classification system, we found that the level of MYBPC1 was significantly decreased in grade IV tissues compared with grade II and grade III tissues, a finding that was consistent with the observed activity of miR-451a. MiR-451a was found to cause a marked enhancement of the activity of MYBPC1 in TU212 cells, which in turn was attenuated by ATF overexpression, suggesting that miR-451a could indirectly modulate the function of MYBPC1 through the ATF2-dependent signaling axis. MYBPC1 suppressed the invasion of cells induced by ATF2 in laryngeal carcinoma cells. Moreover, subcutaneous injection of MYBPC1 to construct an in vivo xenograft mouse model enabled rescue of the mice from laryngeal carcinoma formation. Taken together, the results of the present study have shown that MYBPC1 fulfills a pivotal role in laryngeal carcinoma formation, and these findings may provide both a new avenue for research planning and a potential therapeutic target for laryngeal carcinoma.

CDK 4/6 Inhibitors are Potent Radiosensitizers in Retinoblastoma Protein Positive Meningiomas

<h3>Purpose/Objective(s)</h3> Meningiomas are the most common primary brain tumor. Preclinical efficacy of CDK 4/6 inhibitors, routinely used in breast cancer, has been demonstrated against retinoblastoma protein (RB) positive meningiomas. We investigated three CDK 4/6 inhibitors to identify the most potent radiosensitizer as a candidate drug for the next NRG trial. <h3>Materials/Methods</h3> Three experiments were performed: <b>#1</b>). PK experiment to determine the clinically relevant dose (for human trials) of CDK 4/6 inhibitors in six-week-old male athymic nude mice. The ‘<i>Back Translation Method</i>' was used (Spilker et al., Cancer Res 2018). The drug doses given orally for 4 days were: Ribociclib (RBCL) 50 and 150 mg/kg daily, Palbociclib (PBCL) 30mg/kg daily, Abemaciclib (ABCL) 25 and 75 mg/kg daily. Blood samples (25 mice x 4) were collected at 1, 4, 7 and 24 hours and PK analysis was conducted by Pfizer; <b>#2</b>). To compare the effectiveness of the 3 CDK 4/6 inhibitors administered at clinically relevant doses (<b>from</b> <i>#1</i>) for 14 days +/- whole brain radiotherapy (WBRT) (20 Gy/10fr) against orthotopic meningioma CH157 xenografts; <b>#3</b>). To confirm the efficacy of the most potent inhibitor (<b>from #2</b>), +/- WBRT (10 Gy/5fr) against a second orthotopic meningioma (IOMM-Lee) xenografts. Study End Points (Experiments 2 and 3): Bioluminescence Imaging (BLI) for tumor growth and overall survival. RNA-seq was done to detect differential tumor gene expression <b>(#3</b>). <h3>Results</h3> <b>Experiment 1:</b> The clinically relevant mouse drug doses were 15 mg/kg BID (PBCL), 150 mg/kg QD (RBCL), and 15 mg/kg BID (ABCL). <b>Experiment 2:</b> Median overall survival in days was 12, 14, 15, 17, 17, 16, 19, and 26, for Control, PBCL, RBCL, ABCL, RT, RT + ABCL, RT + PBCL and RT + RBCL, respectively. Hazard ratios for death, using RT + RBCL as reference were: 68.68, 7.08, 3.19, and 2.52 for Controls (P = 1.03e-08), RT+ ABCL (P = 5.42e-04), RT+ PBCL (P = 0.02) and RT (P = 0.068), respectively. The log BLI was consistently lower in RT + RBCL. <b>Experiment 3:</b> Median overall survival was 19, 29, 27, and 59 days for Control, RT, RBCL, and RT + RBCL, respectively. Hazard ratios for death using RT + RBCL as reference were: 70.9, 5.21, 4.85 for Controls (P = 1.90e-10), RT (P = 0.001), and RBCL (P = 0.002), respectively. Log BLI was consistently lower in RT + RBCL. RNA-seq showed significant down-regulation of interferon-beta and -gamma response pathways in all treatment arms compared to controls. Treatment-specific changes include: up-regulation of cell-cell adhesion, gliogenesis (RBCL), upregulation of MAPK signaling pathway and neuronal death (RT) and upregulation of mTOR signaling pathway, glycolysis and gluconeogenesis, and negative regulation of cell proliferation (RT+RBCL). <h3>Conclusion</h3> Among the CDK 4/6 inhibitors administered at human clinically relevant doses, <i>Ribociclib</i> was the most potent radiosensitizer against meningioma xenografts. The combination of Ribociclib and RT will be considered for the next NRG protocol for high-risk biomarker positive (RB protein) primary and recurrent meningiomas.

Comparison of Cytotoxic and Ototoxic Effects of Lipoplatin and Cisplatin in Neuroblastoma In Vivo Tumor Model.

Background:This study aimed to compare the cytotoxic, cytostatic, and ototoxic effects of lipoplatin compared to cisplatin application in the subcutaneous xenograft nude mouse neuroblastoma tumor model.Methods:In this study, C1300 neuroblastoma cells were administered subcutaneously to 21 male nude mice. When the tumor reached 150 mm3 diameter, mice were randomized into 3 groups. Saline, cisplatin, and lipoplatin were given intraperitoneally. The auditory function tests were performed before administration and 72 hours after administration. Mice were sacrificed and the tumor and cochlea were removed after 72 hours. Histopathologic evaluation of necrosis and apoptosis was determined by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Cyclooxygenase 2, superoxide dismutase 2, and inducible nitric oxide synthase levels were determined by immunohistochemistry in tissue samples.Results:Apoptosis and necrosis rates were higher in lipoplatin group than in cisplatin group (P = .035 and P = .010, respectively) in tumor tissue. In the spiral ganglion, apoptosis and necrosis were lower in the lipoplatin group than in cisplatin group (P = .002 and P = .002, respectively). Cyclooxygenase 2 pattern in the cochlea was positive in both control and lipoplatin group and negative in cisplatin group (P = .001). Superoxide dismutase 2 and inducible nitric oxide synthase 2 protein expressions showed no difference between groups. The auditory functions were similar to baseline values and had a better threshold value in lipoplatin group than cisplatin group.Conclusion:For the treatment of neuroblastoma, the use of lipoplatin seems to be beneficial in reducing side effects of cisplatin. We recommend that the mechanism of these properties of lipoplatin should be evaluated in further studies.