Human Colorectal Cancer Xenograft Research Articles

Structure-Inherent Tumor-Targeted IR-783 for Near-Infrared Fluorescence-Guided Photothermal Therapy.

IR-783, a commercially available near-infrared (NIR) heptamethine cyanine dye, has been used for selective tumor imaging in breast, prostate, cervical, and brain cancers in vitro and in vivo. Although the molecular mechanism behind the structure-inherent tumor targeting of IR-783 has not been well-demonstrated, IR-783 has unique properties such as a good water solubility and low cytotoxicity compared with other commercial heptamethine cyanine dyes. The goal of this study is to evaluate the phototherapeutic efficacy of IR-783 as a tumor-targeted photothermal agent in human colorectal cancer xenografts. The results demonstrate that IR-783 shows both the subcellular localization in HT-29 cancer cells and preferential accumulation in HT-29 xenografted tumors 24 h after its intravenous administration. Furthermore, the IR-783 dye reveals the superior capability to convert NIR light into heat energy under 808 nm NIR laser irradiation in vitro and in vivo, thereby inducing cancer cell death. Taken together, these findings suggest that water-soluble anionic IR-783 can be used as a bifunctional phototherapeutic agent for the targeted imaging and photothermal therapy (PTT) of colorectal cancer. Therefore, this work provides a simple and effective approach to develop biocompatible, hydrophilic, and tumor-targetable PTT agents for targeted cancer phototherapy.

Exploration of novel isoxazole-fused quinone derivatives as anti-colorectal cancer agents through inhibiting STAT3 and elevating ROS level

Colorectal cancer (CRC) is trending to be a major health problem throughout the world. Therapeutics with dual modes of action have shown latent capacity to create ideal anti-tumor activity. Signal transducer and activator of transcription 3 (STAT3) has been proved to be a potential target for the development of anti-colon cancer drug. In addition, modulation of tumor redox homeostasis through deploying exogenous reactive oxygen species (ROS)-enhancing agents has been widely applied as anti-tumor strategy. Thus, simultaneously targeting STAT3 and modulation ROS balance would offer a fresh avenue to combat CRC. In this work, we designed and synthesized a novel series of isoxazole-fused quinones, which were evaluated for their preliminary anti-proliferative activity against HCT116 cells. Among these quinones, compound 41 exerted excellent in vitro anti-tumor effect against HCT116 cell line with an IC50 value of 10.18 ± 0.4 nM. Compound 41 was proved to bind to STAT3 by using Bio-Layer Interferometry (BLI) assay, and can significantly inhibit phosphorylation of STAT3. It also elevated ROS of HCT116 cells by acting as a substrate of NQO1. Mitochondrial dysfunction, apoptosis, and cell cycle arrest, which was caused by compound 41, might be partially due to the inhibition of STAT3 phosphorylation and ROS production induced by 41. Moreover, it exhibited ideal anti-tumor activity in human colorectal cancer xenograft model and good safety profiles in vivo. Overall, this study provided a novel quinone derivative 41 with excellent anti-tumor activity by inhibiting STAT3 and elevating ROS level, and gave insights into designing novel anti-tumor therapeutics by simultaneously modulation of STAT3 and ROS.

The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

UBXN11 Predicts as a Poor Index for Colorectal Cancer and Contributes to the Tumorigenesis by Activating NF-κB Signaling.

The complex mechanisms of colorectal cancer (CRC) pathogenesis and progression remain poorly understood. This study endeavors to unravel the role of UBXN11within the context of CRC. UBXN11 expression level in CRC, stomach adenocarcinoma and esophageal carcinoma, and the overall survival in corresponding cancers were analyzed using UALCAN database. Human CRC cell lines and xenograft mouse model with UBXN11 overexpression were established to investigate the pathological role of UBXN11 in CRC progression. Luciferase assay, qPCR, and Western blot were performed to dissect the interaction between UBXN11 and NF-κB signaling. Heightened UBXN11 expression was observed in various digestive tract tumors, which was positively correlated with the reduced overall survival rates in CRC patients. Overexpression of UBXN11 significantly enhanced CRC cell proliferation in vitro and promoted tumor growth in vivo. Mechanistically, UBXN11 promoted CRC tumorigenesis through increasing the activation of NF-κB signaling pathway. This study underscores the pivotal role of UBXN11 in CRC progression and paves the way for novel therapeutic strategies for CRC treatment.

Lactococcus lactis HkyuLL 10 suppresses colorectal tumourigenesis and restores gut microbiota through its generated alpha-mannosidase

ObjectiveProbiotic Lactococcus lactis is known to confer health benefits to humans. Here, we aimed to investigate the role of L. lactis in colorectal cancer (CRC).DesignL. lactis abundance was evaluated in...

Abstract 56: Preclinical in vivo characterization underpinning LGR5-targeting CAR-T cells as a cancer immunotherapy

Abstract Incidences of early-onset colorectal cancer (CRC) have been accelerating worldwide. To begin to address the lack of therapeutic options for CRC, we have developed a CAR-T cell therapy specific for leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5). LGR5 is known to potentiate Wnt/Β-catenin signaling and mark cells-of-origin in intestinal cancers. Extensive screening has demonstrated elevated expression of LGR5 in CRC, which is correlated with reduced patient survival. In CRC, LGR5 has been intimately linked with metastasis, cell survival and perturbation of chemotherapeutic interventions. We have developed a 9-day preclinical manufacturing method to generate LGR5-targeting CAR-T cells with a minimally differentiated T cell phenotype, which was concomitant with significant cytotoxic and activation marker expression. In human CRC xenograft mouse models, LGR5-targeting CAR-T cells eradicated tumors at doses as low as 800,000 CAR+ cells, and were capable of significantly inhibiting advanced tumor growth following delayed administration. Robust CAR-T cell-mediated immunological protection was observed in rechallenge experiments, in which secondary tumor growth was undetectable in mice which had tumors rejected following previous CAR-T cell administration. LGR5 protein and mRNA expression was also detected in a diverse range of cancer families, including ovarian, brain, liver and stomach, expanding upon the clinical indications with which LGR5-targeting CAR-T cells may be harnessed. These results contribute to a preclinical body of work that culminated in a Phase I/IIa clinical trial in metastatic colorectal cancer patients, which was approved for initiation by the FDA and commenced in 2023 (NCT05759728). Citation Format: Jade Foeng, Timona Tyllis, Caitlin Abbott, Dylan McPeake, Emma Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Claudine Bonder, Iain Comerford, Tim Sadlon, Simon Barry, Shaun McColl. Preclinical in vivo characterization underpinning LGR5-targeting CAR-T cells as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 56.

Assessment of chemotherapy resistance changes in human colorectal cancer xenografts in rats based on MRI histogram features.

We investigated the value of magnetic resonance imaging (MRI) histogram features, a non-invasive method, in assessing the changes in chemoresistance of colorectal cancer xenografts in rats. A total of 50 tumor-bearing mice with colorectal cancer were randomly divided into two groups: control group and 5-fluorouracil (5-FU) group. The MRI histogram characteristics and the expression levels of p53 protein and MRP1 were obtained at 24 h, 48 h, 72 h, 120 h, and 168 h after treatment. Sixty highly repeatable MRI histogram features were obtained. There were 16 MRI histogram parameters and MRP1 resistance protein differences between groups. At 24 h after treatment, the MRI histogram texture parameters of T2-weighted imaging (T2WI) images (10%, 90%, median, energy, and RootMeanSquared) and D images (10% and Range) were positively correlated with MRP1 (r = 0.925, p = 0.005). At 48 h after treatment, histogram texture parameters of apparent diffusion coefficient (ADC) images (Energy) were positively correlated with the presence of MRP1 resistance protein (r = 0.900, p = 0.037). There was no statistically significant difference between MRI histogram features and p53 protein expression level. MRI histogram texture parameters based on T2WI, D, and ADC maps can help to predict the change of 5-FU resistance in colorectal cancer in the early stage and provide important reference significance for clinical treatment.

The radiation- and chemo-sensitizing capacity of diclofenac can be predicted by a decreased lactate metabolism and stress response

BackgroundAn enhanced aerobic glycolysis (“Warburg effect”) associated with an increase in lactic acid in the tumor microenvironment contributes to tumor aggressiveness and resistance to radiation and chemotherapy. We investigated the radiation- and chemo-sensitizing effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac in different cancer cell types.MethodsThe effects of a non-lethal concentration of diclofenac was investigated on c-MYC and Lactate Dehydrogenase (LDH) protein expression/activity and the Heat shock Protein (HSP)/stress response in human colorectal (LS174T, LoVo), lung (A549), breast (MDA-MB-231) and pancreatic (COLO357) carcinoma cells. Radiation- and chemo-sensitization of diclofenac was determined using clonogenic cell survival assays and a murine xenograft tumor model.ResultsA non-lethal concentration of diclofenac decreases c-MYC protein expression and LDH activity, reduces cytosolic Heat Shock Factor 1 (HSF1), Hsp70 and Hsp27 levels and membrane Hsp70 positivity in LS174T and LoVo colorectal cancer cells, but not in A549 lung carcinoma cells, MDA-MB-231 breast cancer cells and COLO357 pancreatic adenocarcinoma cells. The impaired lactate metabolism and stress response in diclofenac-sensitive colorectal cancer cells was associated with a significantly increased sensitivity to radiation and 5Fluorouracil in vitro, and in a human colorectal cancer xenograft mouse model diclofenac causes radiosensitization.ConclusionThese findings suggest that a decrease in the LDH activity and/or stress response upon diclofenac treatment predicts its radiation/chemo-sensitizing capacity.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

M4IDP stimulates ROS elevation through inhibition of mevalonate pathway and pentose phosphate pathway to inhibit colon cancer cells

Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.

Knockdown of BUB1B Inhibits the Proliferation, Migration, and Invasion of Colorectal Cancer by Regulating the JNK/c-Jun Signaling Pathway.

Background: Colorectal cancer (CRC) ranks as the third most common cancer, accounting for a significant number of cancer-related deaths worldwide every year. Yet, the molecular mechanisms responsible for the progression of this malignancy are not fully understood. Numerous studies indicate that BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) plays a role in the progression of various malignant tumors. However, the specific biological functions and the detailed mechanisms of how BUB1B influences CRC are still not completely known. This study aimed to explore the expression and role of BUB1B in CRC. Materials and Methods: To achieve this, the expression levels of BUB1B in human CRC tissues and cell lines were examined using real-time polymerase chain reaction and Western blotting. The role and associated mechanisms of BUB1B in CRC cell progression were assessed both in vitro and in vivo using RNA interference. Results: The findings of this study revealed an elevated expression of BUB1B in both CRC tissues and cell lines. The silencing of BUB1B in CRC cell lines notably inhibited cell proliferation, migration, and invasion, leading to cell cycle arrest and apoptosis. In addition, the knockdown of BUB1B inhibited the JNK/c-Jun signaling pathway, increased the expression of proapoptotic proteins, and decreased the expression of antiapoptotic proteins. The effects of BUB1B knockdown on CRC cell progression were reversed by the JNK activator PAF(C-16). Conclusions: In summary, the suppression of BUB1B hindered malignant tumor progression and heightened apoptosis and cell cycle arrest in CRC cells via the JNK/c-Jun pathway. Importantly, the removal of BUB1B expression curtailed tumor growth in human CRC xenografts in nude mice, suggesting its potential as a promising therapeutic target for CRC patients. ClinicalTrials.gov ID: No.2019 K-C086.

Pro-inflammatory Cytokines Promote the Occurrence and Development of Colitis-associated Colorectal Cancer by Inhibiting miR-615-5p.

Patients with ulcerative colitis (UC) may be prone to colitis-associated colorectal cancer (CAC), but there is still a poor understanding of the underlying mechanism so far. This study intended to clarify the role of pro-inflammatory cytokines and miR-615-5p in this process. This experiment first detected miR-615-5p expressions in paraffin-embedded sections of colonic tissues from patients with UC and CAC. Then, we investigated the mechanism through which pro-inflammatory cytokines affected miR-615-5p. Furthermore, in vivo and in vitro tests were performed to identify how miR-615-5p affected colorectal cancer (CRC). Dual-luciferase reporter assay was then employed to identify the targeting relationship between miR-615-5p and stanniocalcin-1 (STC1). The miR-615-5p was lowly expressed in both cancerous and noncancerous colonic tissues of patients with CAC. Pro-inflammatory cytokines downregulated miR-615-5p expression. Overexpression of miR-615-5p reduced the proliferation and migration of CRC cells and had a certain therapeutic effect on in human CRC xenograft mice. Stanniocalcin-1 was identified to be a target gene of miR-615-5p and was involved in the effect of miR-615-5p on CRC. During the progression from UC to CAC, pro-inflammatory cytokines downregulate miR-615-5p, which may induce the upregulation of STC1, and promote the occurrence and development of tumors. These findings offer new insights into the mechanism of CAC and may indicate novel tumor markers or therapeutic targets.

Jujuboside B suppresses angiogenesis and tumor growth via blocking VEGFR2 signaling pathway

Jujuboside B (JuB), one of the main active triterpenoid saponins from the traditional Chinese medicine Ziziphus jujuba, possesses a wide range of pharmacological activities. However, it is unknown whether JuB can inhibit tumor angiogenesis, a crucial step in solid tumor growth. In this study, we found that JuB significantly inhibited the proliferation, migration, and tube formation of human umbilical vein endothelial cells in a dose-dependent manner. JuB also suppressed angiogenesis in chick embryo chorioallantoic membranes and Matrigel plugs. Moreover, through angiogenesis inhibition, JuB delayed the growth of human HCT-15 colorectal cancer xenograft in mice. Western blot assay demonstrated that JuB inhibited the phosphorylation of VEGFR2 and its key downstream protein kinases, such as Akt, FAK, Src, and PLCγ1. In conclusion, the antiangiogenic potency and molecular mechanism of JuB are revealed for the first time, indicating that this triterpene saponin may be further explored as a potential drug candidate or lead compound for antiangiogenic cancer therapy.

Phase I first-in-human trial of ABT-301, an oral pan-HDAC inhibitor, in patients with advanced solid tumors.

e15137 Background: ABT-301 (previously known as MPT0E028) is a novel, oral pan-histone deacetylases (HDAC) inhibitor with potent inhibitory activity for HDAC 1, 2, 3 (class I), HDAC 6, 10 (class IIb), modest activity for HDAC 8 (class I) and HDAC 11 (class IV), but no activity for HDAC 4, 5, 7, 9 (class IIa). Preclinical studies showed that ABT-301 had a stronger apoptotic activity and inhibited HDAC activity more potently than vorinostat. ABT-301 exerts antitumor activity as a monotherapy in human colorectal cancer and B-cell lymphoma xenografts and synergistic antitumor activity with immune checkpoint inhibitor in CT26 syngeneic tumor model. Methods: This was a 3+3 Phase 1 dose escalation study to determine the safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and pharmacokinetic (PK) /pharmacodynamic (PD) profiles for the ABT-301 in patients with locally advanced or metastatic solid cancers. 5 cohorts of 3 or 6 patients received ABT-301 once daily at gradually escalating doses starting from 50 mg. Results: 23 subjects (6 sarcomas and 17 carcinomas) were treated at 5 dose levels (3 at 50, 100, and 250 mg respectively, 6 at 150 mg, and 8 at 200 mg). Cycle 1 DLTs occurred in 5 subjects: 1 at 50 mg (G3 mucositis); 2 at 200 mg (G3 abdominal pain and G4 thrombocytopenia); 2 at 250 mg (G3 vomiting and G4 thrombocytopenia). The MTD was 150 mg. The most common treatment-related adverse events were thrombocytopenia (44%), anemia (18%), nausea (61%), vomiting (44%), anorexia (22%), hyperglycemia (22%), fatigue (18%), and mucositis (18%), which were generally G1 to G2 in severity. No cardiac abnormalities were observed. 1 had a partial response (PR), and 8 achieved SD, resulting in a disease control rate (DCR) of 39%. The PR (a patient with eccrine ductal carcinoma) lasted 24 weeks whereas 5/8 SD patients achieved a sustained condition of 16 to 53 weeks (hepatocellular carcinoma, 53 w; thymic cancer, 49 w; salivary gland carcinoma, 27 w; endometrial stromal carcinoma, 25 w; renal cell carcinoma, 16 w). DCR for 9 patients at MTD was 78% (7/9; 1 PR and 6 SD). Plasma PK showed a dose-dependent increase in ABT-301 exposure. Cmax is 5.5 µM and AUC0-inf is 11.5 µM*h at 150 mg. Increased acetylated histone 3 was observed across all cohorts. Conclusions: ABT-301 was well-tolerated at a daily dose of up to 150 mg and demonstrated objective responses and long-term SD across multiple tumor types at MTD. The safety profile of ABT-301 was superior to known HDAC inhibitors with only predictable HDAC inhibitor-related toxicities observed but no cardiac toxicity, neutropenia, nor lymphopenia elicited. Non-clinical and clinical results suggested the potential clinical development of ABT-301 in combination with myelosuppressive agents and immunotherapies. Phase 2 studies combining PD-1/PD-L1 blockade are being designed. Clinical trial information: NCT02350868 .

Abstract 6793: In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human colorectal cancer

Abstract Background: The development of novel biotherapies for metastatic colorectal cancer (CRC), a fatal disease when advanced, is an urgent medical need. Oncolytic viruses (OVs) are novel cancer biotherapies, and the oncolytic measles virus (MV) has demonstrated safety and antitumor activity in early clinical studies. Triptolide (TRP), a diterpenoid epoxide extracted from the thunder god vine, and Minnelide (MN), its water-soluble compound, are novel agents associated with potent antitumor, anti-proliferative, pro-apoptotic, antiangiogenic, and ER stress inducing effects. The objectives of this study are to characterize the in vitro and in vivo effects and molecular mechanisms of measles virotherapy in combination with TRP and MN in CRC. Methods: The in vitro effects of TRP, MV-GFP (Edmonston strain of Measles virus expressing eGFP), MV-CD46-muPA (dual targeted MV against the murine uPA receptor and human CD46), or virus-triptolide combinations on tumor cell cytotoxicity were assessed on HT-29, HCT116 and SW620 CRC cells. Molecular and mechanistic characterization of TRP’s effects alone and combination with MV in HT29 cells was analyzed by the Reverse Phase Protein Array (RPPA) and validated by western blot analysis. In vivo effects (tumor progression and survival) of MN alone and in combination with MV vectors using different treatment schemes were assessed in HT-29 tumor bearing NSG mice, and tumor correlative studies were performed. Results: MV and TRP had single agent antitumor activity against human CRC cell lines. MV-TRP combinations were associated with increased in vitro cytotoxicity compared to single agent treatments. Functional proteomics analysis showed that TRP-MV combinations were associated with inhibition of survival and proliferation pathways, such as AKT, ERK, inhibition of HSP70 as well as induction of autophagy, at a higher level than each agent alone. In vivo, both a 7-days MN treatment and a 21-days MN treatment significantly improved long term tumor control and significantly improved survival, compared to each agent alone. Tumor studies showed that MN increased MV tumor deposition, as well as enhancement in apoptosis and inhibition of proliferation. Molecular characterization of oncogenic pathways modulated by MV-Minnelide combinations (Nanostring) are underway and will be presented at the meeting. Conclusions: Our results demonstrated that, in human CRC xenografts, Minnelide safely and significantly enhances measles virus oncolysis in vivo, leading to improved antitumor effects and prolonged survival. These effects were associated with enhanced antiproliferative and pro-apoptotic effects with the MV-Minnelide combination, suggesting in vivo synergy. Studies characterizing the molecular mechanisms of in vivo synergy and assessing this promising combination in immunocompetent CRC models are underway. Citation Format: Valery A. Chavez, Floritza Bustamante, Carolina Merchan-Mendes, Ashok Saluja, Jaime Merchan. In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human 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 6793.

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 2439: RPL27 augments growth and stemness properties in colorectal cancer through PLK1 signaling

Abstract Background: In 2020, colorectal cancer (CRC) had the second highest mortality rate worldwide and was the third most common cancer type among various carcinomas, accounting for 10.0% and 9.4% of all carcinomas, respectively. Although the expression of ribosomal protein L27 (RPL27) is upregulated during CRC development, oncogenic roles of RPL27 have not yet been elucidated. We aimed in this study to investigate the effect of RPL27 targeting on CRC growth and whether RPL27 gained an extra-ribosomal function during CRC development. Methods: Human CRC cell lines, HCT116 and HT29, were transfected with RPL27-specific siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and Western blotting. To obtain insights into the molecular changes in response to RPL27 knockdown, global changes in gene expression were examined using RNA sequencing. Results: We observed that inhibition of RPL27 expression suppresses CRC cell growth and long-term colony formation through an induction of apoptotic cell death. Targeting RPL27 also significantly inhibited the growth of human CRC xenografts in nude mice. Noticeably, polo-like kinase 1 (PLK1), which is known to play an important role in mitotic cell cycle progression and stemness, was commonly down-regulated in both HCT116 and HT29 cells with RPL27 depletion. Western blot analysis confirmed that RPL27 silencing reduces the levels of PLK1 protein and G2/M-associated regulators, such as p-CDC25C, CDK1, and cyclin B1. Reflecting these molecular responses, knockdown of RPL27 reduced the migration and invasion abilities of CRC cells. In terms of CSC phenotypic changes, RPL27 knockdown suppressed sphere forming capacity of isolated CD133+ CSC population accompanying with the reduction in CD133 and PLK1 levels. Conclusion: Taken together, these findings indicate that RPL27 contributes to promotion of CRC growth and stemness through PLK1 signaling and that targeting RPL27 could be a next-line therapeutic strategy for both primary CRC treatment and prevention of metastasis and/or recurrence. Citation Format: Keon Uk Park, Ilseon Hwang, Hun Mo Ryoo, Yun-Han Lee, So-Young Park. RPL27 augments growth and stemness properties in colorectal cancer through PLK1 signaling [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 2439.

Study of the suppression of a tumor growth expressing a carcinoembryonic antigen with a new high-tech drug carplasmin (CAR-T therapy) in Balb/c nude mice

Introduction. Adoptive immunotherapy based on chimeric antigen receptors (CAR) is considered as a promising direction in the treatment of solid malignant tumors. To produce genetically modified human T-lymphocytes, lenti/retroviral transduction is currently most often used. However, safety concerns associated with the viral vector production and possible unwanted genome modification limit the clinical utility of CAR-T cells. Therefore, non-viral transfection methods, in particular electroporation, using of DNA or RNA vectors, are being actively studied as a method for producing CAR-T lymphocytes.Aim. To evaluate in vivo antitumor activity of the new high-tech drug carplasmin, intended for CAR-T therapy of tumors expressing carcinoembryonic antigen (CEA). Materials and methods. Carplasmin was obtained by electroporation of activated human lymphocytes with plasmid DNA carrying the third generation CAR gene specific to CEA. The study was performed on a human colorectal cancer xenograft model obtained by intraperitoneal injection of CEA-positive HCT116 cell line to athymic Balb/c nude mice. Carplasmin treatment was carried out once a week, starting from the third day after HCT116 cell inoculation. Mice in the two control groups were treated with either electroporated lymphocytes without plasmid addition (pulse-lymphocytes) or RPMI-1640 culture medium (group without treatment).Results. In vivo, carplasmin demonstrated a pronounced antitumor effect. Seven weekly injections of the drug to inoculated mice led to a prominent effect of antitumor therapy: 80 % of the animals in the experimental group survived (with 40 % of the mice had a complete remission without signs of a detectable tumor), compared to 100 % death in the control group (without treatment).Conclusion. The results of preclinical efficacy studies demonstrate that carplasmin is a promising drug for the treatment of CEA-positive intraperitoneal tumors.

MicroRNA 483-3p overexpression unleashes invasive growth of metastatic colorectal cancer via NDRG1 downregulation and ensuing activation of the ERBB3/AKT axis.

In colorectal cancer, the mechanisms underlying tumor aggressiveness require further elucidation. Taking advantage of a large panel of human metastatic colorectal cancer xenografts and matched stem-like cell cultures (m-colospheres), here we show that the overexpression of microRNA 483-3p (miRNA-483-3p; also known as MIR-483-3p), encoded by a frequently amplified gene locus, confers an aggressive phenotype. In m-colospheres, endogenous or ectopic miRNA-483-3p overexpression increased proliferative response, invasiveness, stem cell frequency, and resistance to differentiation. Transcriptomic analyses and functional validation found that miRNA-483-3p directly targets NDRG1, known as a metastasis suppressor involved in EGFR family downregulation. Mechanistically, miRNA-483-3p overexpression induced the signaling pathway triggered by ERBB3, including AKT and GSK3β, and led to the activation of transcription factors regulating epithelial-mesenchymal transition (EMT). Consistently, treatment with selective anti-ERBB3 antibodies counteracted the invasive growth of miRNA-483-3p-overexpressing m-colospheres. In human colorectal tumors, miRNA-483-3p expression inversely correlated with NDRG1 and directly correlated with EMT transcription factor expression and poor prognosis. These results unveil a previously unrecognized link between miRNA-483-3p, NDRG1, and ERBB3-AKT signaling that can directly support colorectal cancer invasion and is amenable to therapeutic targeting.