Cancer Xenograft Mouse Research Articles

Qingdai Decoction suppresses prostate cancer growth in lethal-stage prostate cancer models.

Contemporary therapy for advanced castration-resistant prostate cancer (CRPC) employs reagents such as enzalutamide and abiraterone acetate targeting the androgen receptor (AR) transcription axis only provide a temporary response and rapidly develop resistance. Additionally, neuroendocrine prostate cancer (NEPC) is an AR pathway-independent and lethal-stage prostate cancer with no standard therapy. Qingdai Decoction (QDT), a traditional Chinese medicine formula, has various pharmacological activities and was widely used for the treatment of different diseases including prostatitis which may contribute to prostate cancer development. This study aims to explore the anti-tumor role and potential mechanism of QDT on prostate cancer. CRPC prostate cancer cell models and xenograft mice models were established for research. The effect of TCMs on cancer growth and metastasis were determined by CCK-8, wound-healing assays and the PC3-xenografted mice model. The toxicity of QDT in the major organs was investigated by H&E staining. The compound-target network was analyzed with network pharmacology. The correlation of QDT targets with prostate cancer patient's prognosis was analyzed with multiple prostate cancer patient cohorts. The expression of related proteins and mRNA were detected by western blot and real-time PCR. The gene knockdown was achieved with CRISPR-Cas13 technology. By integrating functional screening, network pharmacology analysis, CRISPR-Cas13 directed RNA targeting, and molecular biology validation in different prostate cancer models and clinical prostate cancer cohorts, we found that Qingdai Decoction (QDT), a Traditional Chinese Medicine, can repress cancer growth in advanced prostate cancer models in vitro and in vivo in an AR independent manner by targeting NOS3, TGFB1, and NCOA2. This study not only identified QDT as a novel drug for lethal-stage prostate cancer treatment but also provided an extensive Integrative research paradigm for investigating the roles and mechanisms of TCMs for the treatment of other diseases.

Noninvasive Strategies for the Treatment of Tiny Liver Cancer: Integrating Photothermal Therapy and Multimodality Imaging EpCAM-Guided Nanoparticles

Surgical resection and ablation therapy have been shown to achieve the purpose of a radical cure for liver cancer with a size of less than 3 cm; however, tiny liver cancer lesions of diameters smaller than 2 cm remain challenging to diagnose and cure due to the failure of the generation of new blood vessels within tumors. Emerging evidence has revealed that optical molecular imaging combined with nanoprobes can detect tiny cancer from the perspective of molecular and cellular levels and kill cancer cells by the photothermal effect of nanoparticles in real time, thereby achieving radical goals. In the present study, we designed and synthesized multicomponent and multifunctional ICG-CuS-Gd@BSA-EpCAM nanoparticles (NPs) with a potent antineoplastic effect on tiny liver cancer. Using subcutaneous and orthotopic liver cancer xenograft mouse models, we found that the components of the NPs, including ICG and CuS-Gd@BSA, showed synergistic photothermal effects on the eradication of tiny liver cancer. We also found that the ICG-CuS-Gd@BSA-EpCAM NPs exhibited triple-modal functions of fluorescence imaging, magnetic resonance imaging, and photoacoustic imaging, with targeted detection and photothermal treatment of tiny liver cancer under near-infrared light irradiation. Together, our study demonstrates that the ICG-CuS-Gd@BSA-EpCAM NPs in combination with optical imaging technique might be a potential approach for detecting and noninvasively and radically curing tiny liver cancer by the photothermal effect.

Preclinical Evaluation of ON203, A Novel Bioengineered mAb Targeting Oxidized Macrophage Migration Inhibitory Factor as an Anticancer Therapeutic.

High levels of macrophage migration inhibitory factor (MIF) in patients with cancer are associated with poor prognosis. Its redox-dependent conformational isoform, termed oxidized MIF (oxMIF), is a promising tumor target due to its selective occurrence in tumor lesions and at inflammatory sites. A first-generation anti-oxMIF mAb, imalumab, was investigated in clinical trials in patients with advanced solid tumors, where it was well tolerated and showed signs of efficacy. However, imalumab has a short half-life in humans, increased aggregation propensity, and an unfavorable pharmacokinetic profile. Here, we aimed to optimize imalumab by improving its physicochemical characteristics and boosting its effector functions. Point mutations introduced into the variable regions reduced hydrophobicity and the antibodies' aggregation potential, and increased plasma half-life and tumor accumulation in vivo, while retaining affinity and specificity to oxMIF. The introduction of mutations into the Fc region known to increase antibody-dependent cellular cytotoxicity resulted in enhanced effector functions of the novel antibodies in vitro, whereas reduced cytokine release from human peripheral blood mononuclear cells in the absence of target antigen by the engineered anti-oxMIF mAb ON203 versus imalumab reveals a favorable in vitro safety profile. In vivo, ON203 mAb demonstrated superior efficacy over imalumab in both prophylactic and established prostate cancer (PC3) mouse xenograft models. In summary, our data highlight the potential of the second-generation anti-oxMIF mAb ON203 as a promising immunotherapy for patients with solid tumors, warranting clinical evaluation.

Development of an antibody-ligand fusion protein scFvCD16A-sc4-1BBL in Komagataella phaffii with stimulatory activity for Natural Killer cells

BackgroundNatural killer (NK) cell-based immunotherapies have demonstrated substantial potential for the treatment of hematologic malignancies. However, its application is limited due to the difficulty in the production of a large number of NK cells in vitro and the insufficient therapeutic efficacy against solid tumors in vivo. Engineered antibodies or fusion proteins targeting activating receptors and costimulatory molecules of NK cells have been developed to encounter these problems. They are mostly produced in mammalian cells with high cost and long processing times. Yeast systems, such as Komagataella phaffii, present a convenient manipulation of microbial systems with the key advantages of improved folding machinery and low cost.ResultsIn this study, we designed an antibody fusion protein scFvCD16A-sc4-1BBL, composed of the single chain variant fragment (scFv) of anti-CD16A antibody and the three extracellular domains (ECDs) of human 4-1BBL in a single-chain format (sc) with the GS linker, aiming to boost NK cell proliferation and activation. This protein complex was produced in the K. phaffii X33 system and purified by affinity chromatography and size exclusion chromatography. The scFvCD16A-sc4-1BBL complex showed comparable binding abilities to its two targets human CD16A and 4-1BB as its two parental moieties (scFvCD16A and monomer ECD (mn)4-1BBL). scFvCD16A-sc4-1BBL specifically stimulated the expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells in vitro. Furthermore, in the ovarian cancer xenograft mouse model, adoptive NK cell infusion combined with intraperitoneal (i.p) injection of scFvCD16A-sc4-1BBL further reduced the tumor burden and prolonged the survival time of mice.ConclusionOur studies demonstrate the feasibility of the expression of the antibody fusion protein scFvCD16A-sc4-1BBL in K. phaffii with favourable properties. scFvCD16A-sc4-1BBL stimulates PBMC-derived NK cell expansion in vitro and improves the antitumor activity of adoptively transferred NK cells in a murine model of ovarian cancer and may serve as a synergistic drug for NK immunotherapy in future research and applications.

Abstract 5091: Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer

Abstract Since the appearance of oxaliplatin, FOLFOX therapy is positioned as the standard of care for colorectal cancer (CRC). And after the advent of capecitabine, an oral prodrug of 5-FU, both FOLFOX and CAPOX remain the first-line treatment for local and advanced CRC. Even though oral 5-FU prodrug has approved more than two decades ago, orally available oxaliplatin has not been developed yet. Oral chemotherapy has garnered attention in the era of cancer immunotherapy because oral form of the therapy makes metronomic therapy feasible. In this regard, we invented orally absorbable oxaliplatin exploiting complexation with bile acid moiety. The invention encouraged to expand the dosing of combination treatment of 5-FU and oxaliplatin, the most established chemotherapy combination of CRC, into metronomic scheduling. Oral CAPOX therapy is developed to administrate 5-FU and oxaliplatin combination (FOX) metronomically for chemo-immunotherapy. Metronomic dosage successfully induced immunogenic cancer cell death in colorectal cancer xenograft mouse model not exerting toxicity to other organs abolishing immune suppressive effect of chemotherapy. Oral CAPOX boosted tumor immunity translating immune microenvironment. Immune cell population and activation was analyzed using flow cytometry. Suppressive immune cell population decreased after Oral CAPOX treatment. Especially, immune-suppressive macrophage population diminished. Macrophages became immunogenic and presented antigen more frequently. Improved tumor microenvironment combined with anti-PD1 treatment activated and proliferated T cells locally and systemically. Oral CAPOX and anti-PD1 combination therapy activated cytotoxic T cell in tumor tissue and tumor draining lymph node. This therapy also activated NK cells systemically. Oral CAPOX and anti-PD1 combination treatment regressed established tumor completely in more than 90% of the mouse showing its capacity for clinical usage. Liver is the region which makes CRC least immunogenic among metastatic site and the organ CRC is metastasized most abundantly. Immuno-suppresive macrophage in liver is known for the factor discriminating liver metastasized CRC from other organs metastasized one suppressing T cells. Because Oral CAPOX therapy converts macrophage more immunogenic, Oral CAPOX therapy could reverse immunosuppressive tumor microenvironment of liver metastasized colorectal cancer. Oral CAPOX and anti-PD1 combination is treated liver metastasized CRC model and showed standing out antitumor efficacy, and T cell activation. Citation Format: Seong Jin Park, Seho Kweon, Moyo Knowledge Mudhibadhi, Ha Rin Kim, Youngro Byun. Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer. [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 5091.

Abstract 4083: In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal cancer

Abstract While CAR-T cells have demonstrated significant efficacy in the treatment of haematological malignancies, there are many challenges to overcome in developing CAR-T cell therapies against solid tumours. In particular, the scarcity of recognised effective target antigens in solid tumours remains a significant hurdle. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a cancer stem cell marker, which mediates important roles in tumor initiation and metastasis. Upregulated LGR5 expression in malignant cell contexts highlights this surface receptor as a promising target for CAR-T cell therapy. In this study, we evaluated the efficacy of LGR5-targeting CAR-T cells against a range of human cancers. We demonstrate that LGR5 is expressed, both in vitro and in vivo, by numerous human cancers, including colorectal, ovarian, hepatic, brain, and pancreatic cancer. LGR5-targeting CAR-T cells effectively kill LGR5-expressing cancer cells in in vitro cytotoxicity assays and display significant anti-tumor activity in vivo when administered at 1, 2 or 3 weeks post-tumor inoculation in a subcutaneous human colorectal cancer xenograft mouse model. Furthermore, LGR5-targeting CAR-T cells that induce primary tumor rejection confer complete protection against a secondary tumor challenge 5 weeks post-T cell transfer. In vivo spatiotemporal distribution and repeated dosing studies demonstrate that LGR5-targeting CAR-T cells accumulate at the tumor site until tumor elimination, do not persist in off-target organs by d28 post-tumor inoculation and are safe and well-tolerated under the conditions tested. Together, these results provide the foundation for furthering the development of LGR5-targeting CAR-T cells for use in the clinic against colorectal cancers and other solid tumours. Citation Format: Dylan McPeake, Timona Tyllis, Caitlin Abbott, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Elaheh Rohani-Rad, Silvana Napoli, Timothy Sadlon, Simon Barry, Shaun McColl. In vivo efficacy of LGR5-targeting CAR-T cell therapies developed for the treatment of colorectal cancer. [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 4083.

Abstract 6126: Antitumor effect of Iso-mukaadial acetate in MCF-7 human cancer xenograft models

Abstract Antitumor drugs used today have shown significant efficacy and are derived from natural products such as plants. Iso-mukaadial acetate has previously been shown to possess anticancer properties by inducing apoptosis. The purpose of this study was to investigate the therapeutic effect of Iso-mukaadial acetate in the breast cancer xenograft mice model. Female athymic mice were used and were inoculated with breast cancer cells subcutaneously after culturing. Group one served as a negative control group (no treatments) and group two positive control group (cisplatin) which was administered intravenously. Iso-mukaadial acetate treatment was administered orally to group three(100 mg/kg) and group four (500 mg/kg). Blood was collected (70 µL) from the tail vein on day zero, day seven and day 14. Tumor regression was measured, and a pharmacokinetic study was conducted. Estimation of Serum Parameters for Hepatic and Renal Indices were examined using an Automated Chemistry Analyzer. Histopathological analysis was conducted to evaluate morphological changes before and after treatment under a light microscope. Tumor shrinkage was observed after treatment. In conclusion, Iso-mukaadial acetate inhibited tumor progression in breast cancer athymic mice without major side effects. Citation Format: Portia Raphela-Choma, Lesetja R. Motadi, Mpho S. Choene. Antitumor effect of Iso-mukaadial acetate in MCF-7 human cancer xenograft models [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 6126.

Abstract 1862: Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models

Abstract Delta-like 1 homolog (DLK-1), a non-canonical notch ligand, is a membrane protein with 6 tandem EGF-like motifs in extracellular region. While the expression of DLK-1 is restricted in some neuroendocrine tissues such as adrenal gland and hypophysis in normal adult tissues, its over-expression in cancers have been reported in a variety of cancer types, such as hepatocellular carcinoma (HCC), small cell lung carcinoma, ovarian carcinoma, adrenal cortical carcinoma, pancreatic carcinoma, gastrointestinal stromal tumor, etc. Interestingly, it has been suggested that DLK-1 is a marker of cancer stem cells in HCC. CBA-1205 is a humanized antibody (IgG1/κ) targeting DLK-1, which is glycoengineered by GlymaxX® to potentiate antibody-dependent cellular cytotoxicity (ADCC) activity. CBA-1205 showed no toxicology issues in cynomolgus monkey GLP studies and the Phase 1 clinical trial in patients with solid tumors is under way (JapicCTI-205384). Previously, we demonstrated potent anti-tumor efficacy of CBA-1205 as a monotherapy in multiple human DLK-1 positive cancer xenograft mouse models including HCC (AACR 2019 annual meeting). Lenvatinib is an oral multi-kinase inhibitor which has been used for 1st line therapy of unresectable advanced HCC globally. The purpose of present study is to evaluate the combinatorial efficacy of CBA-1205 with lenvatinib in two HCC xenograft models (Hep3B and HepG2). Hep3B model was sensitive to lenvatinib monotherapy; oral daily dosing of lenvatinib at 3 mg/kg treatment showed 50% tumor growth inhibition (TGI) and at 10 mg/kg treatment showed more than 80% TGI. In contrast HepG2 model was less sensitive to Lenvatinib; TGI at 3 mg/kg treatment group was 20 % and that for 10 mg/kg treatment group was 50 %. When CBA-1205 (1 mg/kg, twice a week, total 4 times) was intraperitoneally administered in combination with 3 mg/kg of lenvatinib (daily, total 10 times) in Hep3B xenograft mice, synergistic and long-lasting anti-tumor efficacy was observed compared either with CBA-1205 treatment alone or lenvatinib treatment alone. Tumor growth of all mice (N=8) treated with CBA-1205 and lenvatinib showed almost complete inhibition even 2 weeks after the last dosing. More importantly, 4 out of 8 mice in CBA-1205 and lenvatinib treatment group showed tumor regression. Similar results were obtained with CBA-1205 at 1 mg/kg dosage in combination with lenvatinib at 10 mg/kg dosage in HepG2 xenograft model. In conclusion, we demonstrated synergistic and long-lasting anti-tumor efficacy of CBA-1205 in combination with lenvatinib in two different HCC pre-clinical models suggesting that the combination could be a new treatment option for unresectable advanced HCC. Citation Format: Koji Nakamura, Izumi Sakaguchi, Kota Takahashi. Synergistic and long-lasing anti-tumor efficacy of CBA-1205, a novel glycoengineered humanized antibody targeting DLK-1, in combination with lenvatinib in human HCC xenograft models [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 1862.

Abstract 3946: Radiolabeled αvβ3 analog 177Lu-EB-RGD is an effective therapeutic agent in thyroid cancer xenograft mouse model

Abstract Background: Integrins are cell adhesion receptors consisting of 18α and 8β subunits. A subset of integrins (αvβ3) recognizes Arg-Gly-Asp (RGD) peptide motifs which are involved in the neovascularization and progression of various cancers. The aim of the study was to investigate if αvβ3 can serve as a molecular target for the treatment of thyroid cancer (TC) with a novel radiolabeled RGD analog 177Lu-EB-RGD. Methods: Integrin αvβ3 mRNA and/or protein expression was evaluated in 496 TC included in The Cancer Genome Atlas, tissue microarray including 70 TC and 10 normal thyroid samples, and 14 TC cell lines. BRAF-like or RAS-like expression profile was determined through standard BRS scores ranging from -1 to 0 for BRAF-like and 0 to 1 for RAS-like TC. The association between BRS and the αvβ3 expression was tested by the Spearman correlation (r). Nude mice xenografts developing αvβ3 expressing TC after subcutaneous injection of 5*10^6 TC cells were subjected to monotherapy with 0.5 mCi 177Lu-EB-RGD (177Lu) or in combination with Lenvatinib (177Lu+L). The therapeutic efficacy of 177Lu-EB-RGD was compared with standard-of-care Lenvatinib alone (L) and placebo (P). The continuous data were presented as medians with [25-75% interquartile ranges] and compared using Kruskal-Wallis test. The mixed-effects models were used for longitudinal data analysis with adjusted p≤ 0.05 considered statistically significant. Results: We found a moderate negative correlation between BRS and αv (r=-0.5, p<0.001), and β3 (r=-0.27, p<0.001), revealing that BRAF-like tumors have a higher mRNA expression of αvβ3 integrins. Consistently, the highest αvβ3 mRNA and/or protein expression was found in the BRAF-like TC cell lines OCUT2 (BRS=-0.56), TPC1 (BRS=-0.4), K1 (BRS=-0.29), and Hurthle TC cell line XTC1 (BRS=-0.46). The immunostaining revealed a higher αvβ3 expression in papillary TC compared with follicular TC (p=0.002), and in normal thyroid (p<0.001). Poorly differentiated TC had a similar αvβ3 expression to papillary TC (p=0.14). In the thyroid cancer xenograft mouse model, all treatment modalities were more effective than the placebo in decreasing tumor size as early as 5 days after therapy initiation. A significant difference in growth curve and tumor volume was maintained at the study endpoint (L 0.584 cm3 [0.196-0.984] vs P 0.911 cm3 [0.183-1.68], p=0.001; 177Lu 0.259 cm3 [0.103-0.376] vs P, p<0.001; and 177Lu+L 0.274 cm3 [0.108-0.406] vs P, p<0.001). The combination therapy (177Lu+L) resulted in decreased tumor volume as compared with monotherapy with Lenvatinib (p=0.05) but had a similar effect as compared with 177Lu-EB-RGD monotherapy (p=0.99). Conclusions: The radiolabeled αvβ3 analog 177Lu-EB-RGD has potent growth inhibitory effects in TC characterized by a high integrins expression. The αvβ3 integrin could potentially serve as a molecular target for therapy with radiolabeled RGD analogs in TC. Citation Format: Sonam Kumari, Zhantong Wang, Shilpa Thakur, Laura Abaandou, Oksana Gavrilova, Huiyan Lu, Lixin Lang, Dale Kiesewetter, Vasyl Vasko, Joanna Klubo-Gwiezdzinska. Radiolabeled αvβ3 analog 177Lu-EB-RGD is an effective therapeutic agent in thyroid cancer xenograft mouse model. [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 3946.

Abstract 477: Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers

Abstract Background: KRAS is the most frequently mutated oncogene with high prevalence in non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and pancreatic cancers (PAC). FDA currently approved sotorasib provides breakthrough therapy for cancer patients with KRASG12C mutation, however there is still high unmet medical need for new agents that target a broader KRAS mutated tumors. A new opportunity emerges to develop a pan KRAS inhibitor by suppressing the upstream guanine nucleotide exchange factor (GEF) protein son of sevenless 1 (SOS1). SOS1 is a key activator of KRAS and facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state. Binding to its catalytic domain, small molecule SOS1 inhibitor prevents KRAS activation and suppresses cancer cell proliferation. Material and Methods: Regor’s unique Computer Accelerated Rational Design (CARD) technology platform was applied to identify potent and selective SOS1 inhibitors. Biochemical assays and cellular assays were utilized to drive the structure-activity relationship (SAR). In vitro and in vivo target engagement were confirmed. In vivo efficacy study data were generated using lung and pancreatic cancer xenograft mouse models with KRAS mutation. Results: In vitro, RGT-018 blocked the interaction of KRAS::SOS1 with high selectivity, and inhibited proliferation of a broad spectrum of mutant KRAS-driven cancer cells as a single agent. Robust anti-proliferation activity was observed when RGT-018 was combined with MEK, KRASG12C, EGFR or CDK2/4/6 inhibitors in vitro. Oral administration of RGT-018 inhibited tumor growth and suppressed KRAS signaling pathway activation in tumor xenografts. Furthermore, combination with MEK or KRASG12C inhibitors led to profound tumor regression. Conclusions: The pharmacological properties of RGT-018 represent an attractive drug candidate with oral bioavailability for combination with targeted agents to treat a broader patient population driven by mutant KRAS. Citation Format: Fei Xiao, Kailiang Wang, Xinjuan Wang, Huijuan Li, Zhilong Hu, Wei Huang, Xiaoming Ren, Teng Feng, Lili Yao, Jing Lin, Chunlai Li, Liufeng Mei, Zhuanzhuan Zhang, Yangyang Liu, Xi Chen, Xiaotian Zhu, Wenge Zhong, Zhi Xie. Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers [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 477.

Abstract 585: Pretargeted radioimmunotherapy with a novel anti-oxMIF/HSG bispecific antibody and a Lu177-loaded HSG radioligand results in significant tumor regression in murine models of cancer

Abstract Background: Radioimmunotherapy (RIT) of patients with solid tumors poses a challenge due to the slow blood clearance and delayed tumor uptake of directly radiolabeled antibodies that can cause high radiation exposure to normal tissues. Pretargeting techniques utilizing bispecific antibodies (bsmAbs) overcome these limitations by administering the tumor-targeting antibody and the radioactive payload sequentially. First, the bsmAb is injected and allowed to accumulate in the tumor. Once >95% bsmAbs have cleared from circulation, a small radioligand with high affinity to the bispecific antibody is administered. Due to its pharmacokinetic profile, the radioligand quickly binds to the tumor-trapped bsmAbs while excess radioligand is rapidly excreted by the kidneys. Thereby, high tumor/non-tumor ratios are achieved. Oxidized Macrophage Migration Inhibitory Factor (oxMIF) is the disease-related structural isoform of the pleiotropic cytokine MIF and is specifically present in tumor tissue. Here we show preclinical efficacy of an anti-oxMIF x anti-HSG (histamine-succinyl-glycyl) bsmAb designed for pretargeted RIT (PRIT). Methods: Infrared dye-labeled cON-05, an anti-oxMIF x anti-HSG bsmAb, was administered to Balb/c mice bearing subcutaneous CT26 cancer syngrafts to assess tumor uptake and retention by infrared imaging and its pharmacokinetics by ELISA. A PRIT regimen using cON-05 in conjunction with a Lu177-loaded di-HSG peptide was examined in Balb/c mice bearing subcutaneous (s.c.) CT26 colon cancer syngrafts and in Balb/c nude mice bearing s.c. CFPAC-1 pancreatic cancer xenografts. Results: Infrared dye-labeled cON-05 accumulated in the tumor and was retained for >7 days. cON-05 was found to clear from circulation following a biexponential decay with only 1-2% of the injected dose remaining in plasma after 3-5 days post-injection. Significant tumor regression and survival benefit (100% cON-05 vs. 0% vehicle) were achieved in mice bearing CT26 colorectal tumors, if cON-05 (5 mg/kg) was administered 3 days prior to applying Lu177-di-HSG. In the same model, when changing the pretargeting interval to 5 days, significant tumor growth inhibition (TGI; 29% and 44% on day 9) and survival benefit (15 and >21 days cON-05 vs. 9 days vehicle) could be demonstrated for 2.5 and 5 mg/kg cON-05. In a CFPAC-1 pancreatic cancer mouse xenograft model, 38% TGI on day 28 was achieved upon injection of 5 mg/kg cON-05 followed by Lu177-di-HSG 5 days later. Conclusion: cON-05 is a novel anti-oxMIF x anti-HSG bsmAb designed for PRIT. cON-05 shows good tumor penetration and retention and tumor growth inhibition in conjunction with Lu177-di-HSG peptide in colorectal and pancreatic cancer mouse models. PRIT with cON-05 might offer new options for patients with difficult-to-treat tumors (e.g., pancreatic, gastric, head & neck) Citation Format: Alexander Schinagl, Irina Mirkina, Alejandro Puchol Tarazona. Pretargeted radioimmunotherapy with a novel anti-oxMIF/HSG bispecific antibody and a Lu177-loaded HSG radioligand results in significant tumor regression in murine models of cancer [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 585.

In vivo near-infrared fluorescence imaging of gastric cancer in an MKN-45 gastric cancer xenograft mouse model using intraoperative ureteral identification agent ASP5354.

Accurate intraoperative identification of gastric cancer lesions and determination of the extent of resection are important for curability and function preservation. This study aimed to investigate the potential of the near-infrared fluorescence (NIRF) imaging agent ASP5354 for in vivo fluorescence imaging of gastric cancer. The capability of ASP5354 was evaluated using an MKN-45 human gastric cancer xenograft mouse model. A single dose of ASP5354 was intravenously administered to the mice at a concentration of 120nmol (0.37mg)/kg body weight. In vivo NIRF images of the mouse backs were obtained using an NIRF camera system. Moreover, the cancer tissues were dissected, and the NIRF intensity in the tissue sections was measured using the NIRF camera system. ASP5354 uptake in MKN-45 cells was assessed in vitro using the NIRF microscope. The NIRF signal of ASP5354 was selectively detected in gastric cancer tissues immediately after the intravenous administration of ASP5354. The cancer tissues emitted stronger NIRF signals than adjacent normal tissues. The difference in the NIRF intensity between the normal and cancer tissues was clearly observed at the boundary between them in the macrolevel NIRF images. Cancer tissues can be distinguished from normal tissues based on the measurement of the NIRF of ASP5354, using an NIRF camera system. ASP5354 is a promising agent for NIRF imaging of gastric cancer tissues.

Reprogramming anchorage dependency by adherent-to-suspension transition promotes metastatic dissemination

BackgroundAlthough metastasis is the foremost cause of cancer-related death, a specialized mechanism that reprograms anchorage dependency of solid tumor cells into circulating tumor cells (CTCs) during metastatic dissemination remains a critical area of challenge.MethodsWe analyzed blood cell-specific transcripts and selected key Adherent-to-Suspension Transition (AST) factors that are competent to reprogram anchorage dependency of adherent cells into suspension cells in an inducible and reversible manner. The mechanisms of AST were evaluated by a series of in vitro and in vivo assays. Paired samples of primary tumors, CTCs, and metastatic tumors were collected from breast cancer and melanoma mouse xenograft models and patients with de novo metastasis. Analyses of single-cell RNA sequencing (scRNA-seq) and tissue staining were performed to validate the role of AST factors in CTCs. Loss-of-function experiments were performed by shRNA knockdown, gene editing, and pharmacological inhibition to block metastasis and prolong survival.ResultsWe discovered a biological phenomenon referred to as AST that reprograms adherent cells into suspension cells via defined hematopoietic transcriptional regulators, which are hijacked by solid tumor cells to disseminate into CTCs. Induction of AST in adherent cells 1) suppress global integrin/ECM gene expression via Hippo-YAP/TEAD inhibition to evoke spontaneous cell–matrix dissociation and 2) upregulate globin genes that prevent oxidative stress to acquire anoikis resistance, in the absence of lineage differentiation. During dissemination, we uncover the critical roles of AST factors in CTCs derived from patients with de novo metastasis and mouse models. Pharmacological blockade of AST factors via thalidomide derivatives in breast cancer and melanoma cells abrogated CTC formation and suppressed lung metastases without affecting the primary tumor growth.ConclusionWe demonstrate that suspension cells can directly arise from adherent cells by the addition of defined hematopoietic factors that confer metastatic traits. Furthermore, our findings expand the prevailing cancer treatment paradigm toward direct intervention within the metastatic spread of cancer.

Baicalein suppresses HER2-mediated malignant transformation of HER2-overexpressing ovarian cancer cells by downregulating HER2 gene expression.

The upregulation of the HER2 oncogene is associated with a variety of human cancers and is associated with poor prognosis. Baicalein is reported to have anti-tumor activity, but the molecular mechanism of this effect in HER2-positive cancer cells has not been studied. In this study, our data showed that baicalein can inhibit the proliferation and transformation potential of ovarian cancer cells overexpressing HER2. Baicalein treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level. Baicalein acted on ovarian cancer cells overexpressing HER2 to downregulate the PI3K/Akt signaling pathway downstream of HER2 and inhibit the expression or activity of downstream targets, such as VEGF and cyclin D1 and MMP2. Oral administration of baicalein supplemented with a pharmaceutical excipient significantly inhibited the growth of HER2-overexpressing ovarian SKOV-3 cancer xenografts in mice. These results suggest that downregulation of HER2 gene expression by baicalein at the transcriptional level contributes to inhibit the in vitro and in vivo proliferation and HER2-mediated malignant transformation of HER2-overexpressing ovarian cancer cells.

Preclinical Activity of 4-Demethyl-4-cholesteryloxycarbonylpenclomedine in Melanoma.

Temozolomide plays a role in treating melanoma refractory to immunomodulatory and mitogen-activated protein kinase-targeted approaches, but its efficacy is limited. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate. Its mechanism of action is considered to be via alkylation/adduct formation with N7-guanine. It demonstrated activity in intracranial implanted human glioma and breast cancer xenograft mouse models. The activity of DM-CHOC-PEN in melanoma models was assessed. B-16 melanoma cells were exposed to DM-CHOC-PEN at different concentrations to assess proliferation and survival. B-16 cells were implanted subcutaneously into the flank of adult female C57BL mice which were then were treated with 200 mg/kg DM-CHOC-PEN intraperitoneally daily for 5 days in the setting of palpable subcutaneous tumor. Survival was compared to mice treated with temozolomide or saline. Five mice were treated per group. In vitro, the respective half-maximal inhibitory concentrations of DM-CHOC-PEN and temozolomide were 0.5 and ≥3.0 μg/ml. Floating, heavily melanotic cells formed and these cells were separated, analyzed, and contained 10-90 ng DM-CHOC-PEN per 105 cells. The improvement in survival of mice treated with DM-CHOC-PEN or temozolomide relative to saline controls was 142% and 78%, respectively. Longer survival was seen with DM-CHOC-PEN in a C57BL murine model relative to temozolomide and saline-treated controls, supporting the development of clinical trials assessing the efficacy of DM-CHOC-PEN as treatment for metastatic melanoma.

NAT10 drives cisplatin chemoresistance by enhancing ac4C-associated DNA repair in bladder cancer.

Epitranscriptomic RNA modifications constitute a critical gene regulatory component that can affect cancer progression. Among these, the RNA N4-acetylcytidine (ac4C) modification, which is mediated by the ac4C writer N-acetyltransferase 10 (NAT10), regulates the stabilization of mRNA. Here, we identified that the ac4C modification is induced upon cisplatin treatment and correlates with chemoresistance in bladder cancer (BCa). Both in vitro and in vivo, NAT10 promoted cisplatin chemoresistance in BCa cells by enhancing DNA damage repair. Mechanistically, NAT10 bound and stabilized AHNAK mRNA by protecting it from exonucleases, and AHNAK-mediated DNA damage repair was required for NAT10-induced cisplatin resistance. Clinically, NAT10 overexpression was associated with chemoresistance, recurrence, and worse clinical outcome in BCa patients. Cisplatin-induced NFκB signaling activation was required for the upregulation of NAT10 expression, and NFκB p65 directly bound to the NAT10 promoter to activate transcription. Moreover, pharmacological inhibition of NAT10 with Remodelin sensitized BCa organoids and mouse xenografts to cisplatin. Overall, the present study uncovered a mechanism of NAT10-mediated mRNA stabilization in BCa, laying the foundation for NAT10 as a therapeutic target to overcome cisplatin resistance in BCa.

An integrated approach of network pharmacology, molecular docking, and experimental verification uncovers kaempferol as the effective modulator of HSD17B1 for treatment of endometrial cancer

BackgroundEndometrial cancer (EC) is one of the most common gynecological malignancies globally, and the development of innovative, effective drugs against EC remains a key issue. Phytoestrogen kaempferol exhibits anti-cancer effects, but the action mechanisms are still unclear.MethodMTT assays, colony-forming assays, flow cytometry, scratch healing, and transwell assays were used to evaluate the proliferation, apoptosis, cell cycle, migration, and invasion of both ER-subtype EC cells. Xenograft experiments were used to assess the effects of kaempferol inhibition on tumor growth. Next-generation RNA sequencing was used to compare the gene expression levels in vehicle-treated versus kaempferol-treated Ishikawa and HEC-1-A cells. A network pharmacology and molecular docking technique were applied to identify the anti-cancer mechanism of kaempferol, including the building of target-pathway network. GO analysis and KEGG pathway enrichment analysis were used to identify cancer-related targets. Finally, the study validated the mRNA and protein expression using real-time quantitative PCR, western blotting, and immunohistochemical analysis.ResultsKaempferol was found to suppress the proliferation, promote apoptosis, and limit the tumor-forming, scratch healing, invasion, and migration capacities of EC cells. Kaempferol inhibited tumor growth and promotes apoptosis in a human endometrial cancer xenograft mouse model. No significant toxicity of kaempferol was found in human monocytes and normal cell lines at non-cytotoxic concentrations. No adverse effects or significant changes in body weight or organ coefficients were observed in 3–7 weeks’ kaempferol-treated animals. The RNA sequencing, network pharmacology, and molecular docking approaches identified the overall survival-related differentially expressed gene HSD17B1. Interestingly, kaempferol upregulated HSD17B1 expression and sensitivity in ER-negative EC cells. Kaempferol differentially regulated PPARG expression in EC cells of different ER subtypes, independent of its effect on ESR1. HSD17B1 and HSD17B1-associated genes, such as ESR1, ESRRA, PPARG, AKT1, and AKR1C1\\2\\3, were involved in several estrogen metabolism pathways, such as steroid binding, 17-beta-hydroxysteroid dehydrogenase (NADP+) activity, steroid hormone biosynthesis, and regulation of hormone levels. The molecular basis of the effects of kaempferol treatment was evaluated.ConclusionsKaempferol is a novel therapeutic candidate for EC via HSD17B1-related estrogen metabolism pathways. These results provide new insights into the efficiency of the medical translation of phytoestrogens.

A Novel Homodimer Peptide–Drug Conjugate Improves the Efficacy of HER2-Positive Breast Cancer Therapy

Tumor-targeting peptide-drug conjugates (PDCs) have become a focus of research in recent years. However, due to the instability of peptides and their short in vivo effective half-life, they have limited clinical application. Herein, we propose a new DOX PDC based on a homodimer HER-2-targeting peptide and acid-sensitive hydrazone bond, which could enhance the anti-tumor effect of DOX and reduce systemic toxicities. The PDC could accurately deliver DOX into HER2-positive SKBR-3 cells, with it showing 2.9 times higher cellular uptake than free DOX and enhanced cytotoxicity with respect to IC50 of 140 nM (vs. 410 nM for free DOX). In vitro assays showed that the PDC had high cellular internalization efficiency and cytotoxicity. In vivo anti-tumor experiments indicated that the PDC could significantly inhibit the growth of HER2-positive breast cancer xenografts in mice and reduce the side effects of DOX. In summary, we constructed a novel PDC molecule targeting HER2-positive tumors, which may overcome some deficiencies of DOX in breast cancer therapy.

6-Phosphogluconate dehydrogenase inhibition arrests growth and induces apoptosis in gastric cancer via AMPK activation and oxidative stress.

Poor outcomes in advanced gastric cancer necessitate alternative therapeutic strategies. 6-Phosphogluconate dehydrogenase (6-PGDH), an enzyme that catalyzes the decarboxylation step in the oxidative pentose phosphate pathway, has been identified as a promising therapeutic target in many cancers. In this study, we systematically investigated the expression and function of 6-PGDH in gastric cancer. We found that 6-PGDH expression and activity were aberrantly elevated in gastric cancer tissues compared to their adjacent normal tissues. 6-PGDH knockdown using two independent shRNAs resulted in minimal 6-PGDH levels and activity, decreased growth, and enhanced gastric cancer cell sensitivity to 5-flurorouracil. However, 6-PGDH knockdown did not affect the cancer cells. Mechanistic studies showed that 6-PGDH inhibition disrupted lipid biosynthesis and redox homeostasis in gastric cancer, inhibited growth, and induced apoptosis. Notably, the in vitro findings were validated using an in vivo gastric cancer xenograft mouse model. This study established that 6-PGDH is broadly elevated in gastric cancer patients and that 6-PGDH inhibition can sensitize gastric cancer cells in response to chemotherapy.

A Novel Tri-Functional Liposome Re-Educates "Cold Tumor" and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade.

Immunotherapy has been regarded as a breakthrough in cancer treatment and achieved great success. However, the poor response rate is still a formidable challenge of current immunotherapies, especially in solid tumors without sufficient infiltration of immune cells, also known as "cold tumor." SAR405is a highly specific VPS34inhibitor and has been suggested as a potential approach converting "cold tumor" into "hot tumor" by inhibiting autophagy. In this study, a tri-functional doxorubicin (DOX) plus SAR405liposome system is established and further modified with a novel anti-PD-L1peptide JY4for targeted delivery (DOX-SAR-JY4LIPO ). The data here demonstrate that in a lung cancer xenograft mouse model, by facilitating the tumoral enrichment of both SAR405and DOX, DOX-SAR-JY4LIPO effectively increases the infiltration of cytotoxic lymphocytes in the tumor by synergizing DOX-induced immunogenic cell death (ICD) and SAR405-mediated upregulation of chemokines including CCL5and CXCL10. As results, DOX-SAR-JY4LIPO significantly inhibits tumor growth, metastasis, and resurrection by re-educating immunosuppressive tumor microenvironment. In conclusion, this study not only proves the concept of inhibiting autophagy for better immune infiltration in the tumor but also presents a novel tri-functional liposomal system that overcomes the deficiencies of current therapies and holds great promise in cancer immunotherapy.