Colorectal Xenografts Research Articles

Self-assembled peptide-paclitaxel nanoparticles for enhancing therapeutic efficacy in colorectal cancer.

Chemotherapy is one of the main treatments for colorectal cancer, but systemic toxicity severely limits its clinical use. Packaging hydrophobic chemotherapeutic drugs in targeted nanoparticles greatly improve their efficacy and reduce side effects. We previously identified a novel colorectal cancer specific binding peptide P-LPK (LPKTVSSDMSLN) from phage display peptide library. Here we designed a self-assembled paclitaxel (PTX)-loaded nanoparticle (LPK-PTX NPs). LPK-PTX NPs displayed a superior intracellular internalization and improved tumor cytotoxicity in vitro. Cy5.5-labeled LPK-PTX NPs showed much higher tumor accumulation in colorectal cancer-bearing mice. Furthermore, LPK-PTX NPs exhibit enhanced antitumor activity and decreased systemic toxicity in colorectal cancer patient-derived xenografts (PDX) model. The excellent in vitro and in vivo antitumor efficacy proves the improved targeting drug delivery, suggesting that peptide P-LPK has potential to provide a novel approach for enhanced drug delivery with negligible systemic toxicity.

Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

Acid Sphingomyelinase-Ceramide Induced Vascular Injury Determines Colorectal Cancer Stem Cell Fate.

It is unknown whether cancer stem cells respond differentially to treatment compared with progeny, potentially providing therapeutic vulnerabilities. Our program pioneered use of ultra-high single dose radiotherapy, which cures diverse metastatic diseases at a higher rate (90-95%) than conventional fractionation (~65%). Single dose radiotherapy engages a distinct biology involving microvascular acid sphingomyelinase/ceramide signaling, which, via NADPH oxidase-2-dependent perfusion defects, initiates an adaptive tumor SUMO Stress Response that globally-inactivates homologous recombination repair of double stand breaks, conferring cure. Accumulating data show diverse stem cells display heightened-dependence on homologous recombination repair to repair resolve double stand breaks. Here we use colorectal cancer patient-derived xenografts containing logarithmically-increased Lgr5+ stem cells to explore whether optimizing engagement of this acid sphingomyelinase dependent biology enhances stem cell dependent tumor cure. We show radioresistant colorectal cancer patient-derived xenograft CLR27-2 contains radioresistant microvasculature and stem cells, whereas radiosensitive colorectal cancer patient-derived xenograft CLR1-1 contains radiosensitive microvasculature and stem cells. Pharmacologic or gene therapy enhancement of single dose radiotherapy-induced acid sphingomyelinase/ceramide-mediated microvascular dysfunction dramatically sensitizes CLR27-2 homologous recombination repair inactivation, converting Lgr5+ cells from the most resistant to most sensitive patient-derived xenograft population, yielding tumor cure. We posit homologous recombination repair represents a vulnerability determining colorectal cancer stem cell fate, approachable therapeutically using single dose radiotherapy.

A novel anti-PD-L1/IL-15 immunocytokine overcomes resistance to PD-L1 blockade and elicits potent antitumor immunity

Despite the demonstrated immense potential of immune checkpoint inhibitors in various types of cancers, only a minority of patients respond to these therapies. Immunocytokines designed to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME) and block the immune checkpoint simultaneously may provide a strategic advantage over the combination of two single agents. To increase the response rate to checkpoint blockade, in this study, we developed a novel immunocytokine (LH01) composed of the antibody against programmed death-ligand 1 (PD-L1) fused to interleukin (IL)-15 receptor alpha-sushi domain/IL-15 complex. We demonstrate that LH01 efficiently binds mouse or human PD-L1 and maintains IL-15 stimulatory activity. In syngeneic mouse models, LH01 showed improved antitumor efficacy and safety versus anti-PD-L1 plus LH02 (Fc-sushi-IL15) combination and overcame resistance to anti-PD-L1 treatment. Mechanistically, the dual anti-immunosuppressive function of LH01 activated both the innate and adaptive immune responses and induced a favorable and immunostimulatory TME. Furthermore, combination therapy with LH01 and bevacizumab exerts synergistic antitumor effects in an HT29 colorectal xenograft model. Collectively, our results provide supporting evidence that fusion of anti-PD-L1 and IL-15 might be a potent strategy to treat patients with cold tumors or resistance to checkpoint blockade.

Investigational cyclin-dependent kinase 4/6 inhibitor GLR2007 demonstrates activity against isocitrate dehydrogenase wild-type glioblastoma and other solid tumors in mice xenograft models.

Cyclin-dependent kinases, CDK4 and CDK6, are essential in regulating the cell cycle, which is disrupted in cancers like isocitrate dehydrogenase wild-type glioblastoma (GBM). Currently marketed CDK4/6 inhibitors, including abemaciclib, have shown preclinical efficacy in solid tumors, but factors such as poor blood–brain barrier (BBB) penetration limit their efficacy in GBM. GLR2007 is an investigational CDK4/6 inhibitor with the potential for improved BBB penetration. In vitro assays were used to assess the potency and inhibition of CDK4/6 enzymatic activity of GLR2007. Using in vivo assays, the distribution of radiolabeled GLR2007 in rats was determined through quantitative whole-body autoradiography. The antitumor efficacy of GLR2007 was evaluated in human GBM and breast cancer orthotopic mice xenograft models, and human lung, colorectal, and liver cancer in a subcutaneous xenograft model. In tumor cell line proliferation assays, GLR2007 inhibited proliferation at lower concentration values than abemaciclib in 19 of 20 GBM, five of seven breast, 20 of 21 lung, and 24 of 24 liver cancer cell lines. Total levels of radiolabeled GLR2007 in the brains of rats exceeded those in plasma by 2.3–4.5-fold from 2–6 hours after dosing. A xenograft model showed that, compared with vehicle control, 50 mg/kg GLR2007 induced 95.9% tumor growth inhibition (TGI) (P<0.001) in GBM orthotopic xenografts, 81.4% TGI (P=0.037) in breast cancer orthotopic xenografts, and 91.5% TGI (P<0.001) in colorectal cancer subcutaneous xenografts. These studies show possible BBB penetration of GLR2007 and demonstrate its potential as a CDK4/6 inhibitor for the treatment of solid tumors, including GBM.

Abstract 6043: Chromatin remodeling in patient derived colorectal cancer models

Abstract Patient-derived models of cancer (PDMCs) are becoming the gold standard preclinical models for therapeutic development and precision oncology. However, how tumor cells evolve in PDMC and the implication on therapeutic response remains largely unclear. Herein, we leveraged ATAC-seq to compare the chromatin accessibility landscapes of matched sets of colorectal cancer (CRC) patient-derived organoids (PDOs), patient-derived xenografts (PDXs), PDO-derived PDX (PDOXs), and original patient tumors (PTs), supplemented by mRNA-seq for the transcriptomics information. In the study, nine surgically resected CRC tumor specimens were recruited at Duke. For a PT-PDMCs set, part of the original patient tumor was prepared for sequencing, and the remainders from the same specimens were subjected to derive PDXs and PDOs. Throughout the study, eight PDOs were generated, and three subcutaneous PDXs grew. Besides, we also developed six patient-derived xenografts through organoids. In total, we successfully established 6 matched PT-PDO-xenograft sets, including 3 sets that have both conventional PDXs and PDOXs. The global chromatin accessibility derived from PDMCs and PTs ATAC-seq libraries presented broad congruence (79.6% peaks remained unchanged), suggesting that PDMCs mostly retained their epigenetic identity of patient tumors. Nevertheless, paired differential analysis identified alterations in chromatin accessibility between PTs and PDMCs. We discovered two principal remodeling axes. The first one separates PT from all PDMCs, and the second separate in vitro and in vivo models. Interestingly, PDOXs are much more similar to PDXs than to PDOs, indicating that the PDMCs environment is the driving force for chromatin remodeling. Single cell multiome sequencing (10X Genomics) also agreed that the PDOXs are more similar to PDXs compared to PDOs. Based on our findings, PDOXs may be a good substitution for conventional PDXs. Between in vitro and in vivo models, bivariate genomic footprinting analysis was used to further identify transcription factors (TF) that drive chromatin remodeling differentially. Our data suggested KLF14 and EGR2 have enriched motif-binding footprints in PDOXs from all patient cases. Their silencing enhanced CRC growth, which partially explains why PDXs tend to grow slower than PDOs. Downstream genes of EGR2 and KLF14 have also been analyzed. One of the targets, EPHA4, has been reported it would influence the drug sensitivities. Collectively, patient CRC cells undergo both common and distinct chromatin remodeling in PDOs and PDXs/PDOXs, driven by their respective microenvironments. The datasets allow researchers to look into chromatin accessibility, gene expression, transcriptional factor binding, and pathways that are altered in PDMC compared to original tumors, which will shed light on their predictability for therapeutic profiling. Citation Format: Kun Xiang, Ergang Wang, Qiang Huang, David Hsu, Xiling Shen. Chromatin remodeling in patient derived colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6043.

Abstract 5574: In vivo characterization of a novel CAR-T cell therapy directed towards LGR5 for the treatment of colorectal cancer

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer and incidence rates are rapidly increasing among young adults. Despite current treatments, the 5-year survival rate is approximately 65%, highlighting an urgent need for new therapies. Leucine-rich repeat-containing G-protein coupled receptor (LGR5) is a promising antigen target for chimeric antigen receptor (CAR)-T cell therapy, which may represent a novel effective treatment for CRC. LGR5 binds R-spondins and functions to enhance Wnt and β-catenin signalling involved in maintenance and proliferation of adult stem cells. Expression of LGR5 has been previously reported on cancer stem cells in CRC, and has been demonstrated to play a crucial role in CRC tumor initiation and metastasis. Flow cytometric analysis of LGR5 cell surface expression on in vitro and ex vivo human CRC tumour cells revealed a subpopulation of LGR5+ cells. Six LGR5-targetting CD3+ CAR-T cell variants were developed, which differed in CAR variable heavy (VH) and variable light (VL) chain orientation and hinge length. As preliminary experimentation identified that four of the six CAR-T cell variants displayed robust anti-tumour cytotoxicity in vitro, these CAR-T cell variants were tested for in vivo anti-tumour efficacy using a subcutaneous human colorectal tumor xenograft mouse model. Pre-injection phenotyping after freeze-thawing of the CAR-T cells revealed greater than 94% transduction efficiency, low expression of co-inhibitory receptors CTLA-4 and PD-1 and a high frequency of central memory T cells (CD62L+ CD45RO+). Additionally, a high frequency of the CD8+ compartment of these CAR-T cells expressed perforin, CD107a, Granzyme B and TNFα. For each variant, intravenous injection of 20x106 LGR5-targeting CAR-T cells induced significant tumor regression and gave 100-day survival post-tumor injection. Ex vivo analyses of tumors and spleens via flow cytometry at endpoint indicated CAR-T cell persistence to 100-days post-tumor injection. Of the CAR-T cells isolated from tumours ex vivo, high level expression of co-inhibitory receptors PD-1, CTLA-4, and TIM-3 was observed. The splenic T cell population was enriched for central memory T cells and expressed high levels of PD-1. Complete tumor remission was achieved in 100% of mice receiving the VH-VL orientation, medium hinge CAR construct (designated CNA3103), prompting further investigation of this variant. Intravenous injection of 5x106 CNA3103 CAR-T cells was sufficient to induce tumour remission and 100-day survival. Similarly, complete tumour rejection was achieved with a dose of 2.5x106 CNA3103 CAR-T cells in a 30-day in vivo experiment. These results demonstrate that LGR5-targeting CAR-T cells exhibit potent in vivo anti-tumor efficacy and position LGR5 as a promising CAR target antigen, prompting investigation of LGR5-targeting CAR-T cells as a therapeutic for CRC in future clinical trials. Citation Format: Dylan J. McPeake, Timona S. Tyllis, Jade Foeng, Veronika Bandara, Caitlin A. Abbott, Batjargal Gundsambuu, Elaheh Rohani-Rad, Silvana Napoli, Timothy Sadlon, Simon C. Barry, Shaun R. McColl. In vivo characterization of a novel CAR-T cell therapy directed towards LGR5 for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5574.

Abstract 2566: Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies

Abstract Background: RXC004, a potent and selective inhibitor of the Wnt pathway regulator Porcupine, is being investigated in phase 2 studies in patients with advanced cancers (NCT04907851 and NCT04907539). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Frizzled (Fzd) on the cell surface increasing Wnt-ligand dependent signaling. These alterations, which have been shown to be sensitive to Porcupine inhibitors, are implicated in colorectal, gastric and pancreatic cancer. Pre-clinical in vivo data on the effects of RXC004 in combination with clinically relevant anti-cancer therapies have not previously been described in genetically-defined models of cancer. Methods: RXC004 was evaluated as a monotherapy, and in combination with clinically relevant chemotherapy regimens, in an RSPO-fusion colorectal xenograft model (SNU-1411). RXC004, dosed at 5mg/kg QD (5 days on/2 days off), was evaluated in combination with either the clinically relevant colorectal triplet chemotherapy regimen (5-fluorouracil 5-FU (25mg/kg), irinotecan (50mg/kg) and oxaliplatin (5mg/kg)) or doublet chemotherapy regimen (5-FU (25mg/kg), irinotecan (50mg/kg)). Each chemotherapy regimen was dosed once a week for 3 to 4 weeks either alone or in combination with RXC004, which was dosed from day 8 onwards at 5mg/kg QD (5 days on/2 days off). Efficacy was measured by tumor volume and survival endpoint. In addition, RXC004 was studied in vitro, as monotherapy and in combination with a PARP inhibitor in genetically selected cell lines with both RSPO-fusion and RNF43 mutation. Combination and monotherapy effects on proliferation and effects on downstream signaling were investigated. Results: RXC004 in combination with the triplet chemotherapy regimen led to significantly increased survival compared to either treatment alone (p&amp;lt;0.005) and significant tumor volume reduction measured at Day 22 (p&amp;lt;0.001). RXC004 in combination with the doublet chemotherapy also increased survival compared to either treatment alone (p&amp;lt;0.05), and the combination led to significantly decreased tumor volume measured at Day 25 (p&amp;lt;0.05). RXC004 treatment in genetically defined cell lines with RSPO fusions or RNF43 mutations led to a significant reduction in BRCA1 and BRCA2 gene expression. In in vitro proliferation studies a synergistic effect of RXC004 and a PARP inhibitor was demonstrated in genetically defined cell lines. Conclusion: These data demonstrate that RXC004 in combination with clinically relevant standard of care chemotherapy regimens can lead to potential benefit over chemotherapy alone. The RXC004 induced downregulation of DNA repair pathway genes observed in vitro could contribute to the beneficial effect seen in combination with a PARP inhibitor in vitro as well as the combination effect observed in vivo with standard of care chemotherapy agents. Citation Format: James Kelly, Simon Woodcock, Inder Bhamra, Cliff Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2566.

Abstract LB192: Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene

Abstract DNA methyltransferase (DNMT) 1 is an enzyme responsible for maintaining DNA methylation pattern during DNA replication. However, its role on the methylation of tumor suppressor genes and DNA damage in relation to antitumor activity of DNMT inhibitors remains controversial. Here, we investigated the effects of DNMT inhibitors in human colorectal and breast cancer cell lines and animal models with intact (DNMT1+/+) and disrupted DNMT1 (DNMT1-/-) gene. Treatment with 5 µM of 4’-thio-5-aza-2’-deoxycytidine (aza-TdC), decitabine and azacytidine all inhibited DNMT1 expression and cell growth (94.0%/97.1%, 39.9%/58.6% and 69.2/91.3), and induced apoptosis (66.9%/62.5%, 30.9%/23.8% and 66.2/43.1%) in DNMT1+/+ HCT116/MCF7 cells; in contrast, the DNMT inhibitors had less/little activities in DNMT1-/- HCT116/MCF7 cells (P&amp;lt;0.001 each). γH2AX and G2/M cell-cycle arrest were induced by the inhibitors and greater degree of induction was produced by aza-TdC than decitabine in DNMT1+/+ cells (P&amp;lt;0.001), with activation of checkpoint kinase 1 and upregulation of p21. Both demethylation and increased acetylation of CDKN2A and CDKN2B gene promoters together with re-expression of p16INK4A and p15INK4B proteins were detected in DNMT1-/- versusDNMT1+/+ (demethylation only) cells upon exposure to decitabine for 72h. Compared with saline treatment, tumor growth was significantly inhibited by aza-TdC (90%; n=18 mice) and decitabine (62%; n=18) in DNMT1+/+ colorectal xenograft tumors, alongside decreasing expression of DNMT1 (51% and 44%) and an increase in p21 (85% and 48%). Similarly, tumor growth delays (86%; n=15 and 57%; n=14) were associated with the reduction of DNMT1 and augmentation of p21 in bladder PDX tumors. The findings suggest that DNMT inhibitors induce DNA damage and higher anti-tumor activity of aza-TdC is proportional to a greater magnitude of induction of DNA damage, p21 and cell cycle arrest without reactivating the expression of p16INK4A and p15INK4B. The gene re-expression coupled to demethylation and acetylation corresponds to the less anti-tumor activity by decitabine in DNMT1-/- cells. Thus, it is the DNA damage effects, rather than re-expression of the tumor suppressors, that are associated with antitumor activity of the DNMT inhibitors in cancer cells and/or xenograft and PDX tumors. Citation Format: Angelo B. Laranjeira, Melinda Hollingshead, Dat Nguyen, Robert Kinders, Michael Difilippantonio, James H. Doroshow, Sherry X. Yang. Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB192.

NCI 9938: Phase I clinical trial of ATR inhibitor berzosertib (M6620, VX-970) in combination with irinotecan in patients with advanced solid tumors.

3012 Background: Ataxia telangiectasia and Rad3 related (ATR) is activated in response to replication stress from topoisomerase 1 inhibitors. Selective ATR inhibition with berzosertib potentiates the efficacy of irinotecan in colorectal mouse xenograft models. We hypothesized that berzosertib in combination with irinotecan is well tolerated, modulates the DNA damage repair response to irinotecan, and the combination is associated with clinical activity. Methods: This phase I study utilized a modified Storer’s up and down dose escalation design. Dose Levels (DLs) combined berzosertib 60 to 270 mg/m2 with irinotecan 180 mg/m2, every 2 weeks in a 4-week cycle. The primary endpoint was identification of the maximum tolerated dose (MTD) and recommended phase II dose (RP2D). Activity, pharmacokinetics (PK), and pharmacodynamics (PD) were secondary endpoints. The identification of molecular subpopulations sensitized to the combination was exploratory. Results: Between July 2016 and July 2021, 51 patients (pts) enrolled, of whom 50 received treatment. Pts most commonly had colorectal cancer (CRC, 39%), pancreatic cancer (24%), small cell lung cancer (SCLC, 6%) and non-small cell lung cancer (6%). The median number of prior lines of therapy was 4 (range, 2 to 11). In Stage I, 1 of 3 evaluable pts experienced dose-limiting toxicity (DLT) of grade 3 lung infection at DL3 (berzosertib 180 mg/m2 - irinotecan 180 mg/m2), and Stage II was initiated enrolling cohorts of 5 pts. In Stage II, 4 of the first 11 pts treated at DL4 (berzosertib 270 mg/m2 - irinotecan 180 mg/m2) were unable to complete the DLT evaluation period due to clinically significant toxicity not meeting DLT criteria: grade 2 diarrhea (1 pt), grade 3 diarrhea (1 pt), and grade 3 neutrophil decrease (2 pts). The protocol was amended to limit dose escalation beyond DL4. At DL4, 1 of 21 evaluable pts experienced DLT (grade 4 febrile neutropenia). Most common treatment-related grade ≥ 3 toxicities were neutrophil decrease (34%), lymphocyte decrease (30%), WBC decrease (28%), anemia (20%), diarrhea (16%), fatigue (8%) and hypokalemia (8%). 2 partial responses were observed, occurring in pts with pancreatic cancer and ATM alterations: 32% decrease in an ATM E11828/ATM K1109* tumor lasting 15.3 months and 68% decrease in an ATM R3008H/germline ATM R1882* tumor ongoing at 11 months. An additional pt with ATM S214fs*40 mutant colorectal cancer (CRC) experienced a 26% decrease lasting 7.5 months. Conclusions: Berzosertib 270 mg/m2 - irinotecan 180 mg/m2 was declared the RP2D. The combination is associated with manageable side effects and promising disease activity in ATM mutant solid tumors. PK and PD studies are in process. Tumor biopsy studies are planned in a 15 pt dose expansion cohort at DL4, enrolling pts with CRC, pancreatic cancer, SCLC and DNA damage repair deficient tumors. Clinical trial information: NCT02595931.

Inhibition of cancer stem cells with TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase.

e15105 Background: Cancer stem cells (CSCs) are believed to be responsible for both therapy resistance and metastasis. Until now, these resistant CSCs have not been well-characterized, and potential treatments targeting these cells have yet to be identified. Epigenetic modifiers, such as KDM4 histone demethylase, have been shown to play an important role in CSC renewal and maintenance. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that has promising pre-clinical and pharmacologic properties as a possible cancer therapeutic. Methods: Effects of TACH101 on CSC population were evaluated in the colorectal cancer SU60 xenograft model. Mice engrafted with SU60 cells were treated with TACH101 or vehicle. The day after the last dose, tumors from each group were dissociated into single cells and used to perform (1) flow cytometry analysis of the tumorigenic cell population using markers CD44 and EpCAM, (2) single cell gene expression analysis using quantitative reverse transcription polymerase chain reaction (RT-qPCR), and (3) functional tumorigenicity assays (limiting dilution assays). Results: TACH101 administration reduced SU60 tumor growth by 62% compared to vehicle-treated mice (p &lt; 0.0005). Flow cytometry analysis of SU60 tumor cells showed the proportion of cells with cancer stem cell features (CD44HighEpCAM+) was 2.5-fold lower in TACH101-treated mice as compared to vehicle-treated animals. Single cell gene expression analysis followed by hierarchical clustering based on expression of immature and mature cell markers showed that TACH101-treated tumors had a significant reduction in the proportion of progenitor-like cells (p = 0.01) and an increase in number of both intermediate-like and differentiated-like cells although the increase was not significant (p = 0.07). In limiting dilution assay experiments, result showed that tumors were bigger/grew faster in animals receiving tumor cells derived from vehicle-treated animals compared to TACH101-treated animals. Furthermore, TACH101 was shown to reduce the number of tumor-initiating cancer stem cells by 4.4-fold. Further evaluation of TACH101 across additional xenograft models including esophageal, gastric, breast, and lymphoma showed tumor growth inhibition of up to 100%. TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human trials. Conclusions: TACH101 inhibited proliferation of and tumor initiation by CSCs. As therapy resistance and metastatic dissemination are grave consequences of CSC activity, TACH101 provides a novel approach to eradicating this population and shows broad applicability as a potential therapeutic agent. Preparations to advance TACH101 into clinical trials are underway.

Evaluation of antitumor effects of VEGFR-2 inhibitor F16in a colorectal xenograft model.

Colorectal cancer(CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5μM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 valueof 9.52 ± 1.49µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showeda reduction in CD31 levels also in F16 treated groups. These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.

Luteolin Synergistically Enhances Antitumor Activity of Oxaliplatin in Colorectal Carcinoma via AMPK Inhibition.

Luteolin is a naturally-occurring polyphenolic compound that is known to have antioxidative and antitumor activities in vitro. This study aimed to examine the in vivo anticancer efficacy of luteolin in conjunction with oxaliplatin treatment using a colorectal carcinoma xenograft mouse model. HCT116 human colorectal carcinoma cells were subcutaneously implanted into BALB/c nude mice, followed by the intraperitoneal administration of luteolin at a dose of 50 mg/kg body weight (BW)/day with or without oxaliplatin at a dose of 10 mg/kg BW/day three times per week for a total of 3 weeks. The combined luteolin and oxaliplatin treatment resulted in the synergistic suppression of the growth of HCT116 xenograft tumors when compared to treatment with luteolin or oxaliplatin alone. In addition, the combined treatment significantly increased the expression of cleaved PARP and p53 in the xenograft tumors compared with the vehicle control, but only marginally affected the level of heme oxygenase-1 (HO-1). These results indicated that luteolin treatment retarded oxaliplatin-induced tumor growth by facilitating apoptotic cell death and inhibiting HO-1-mediated cytoprotection. Therefore, these findings suggest the synergistic potential of dietary luteolin in conjunction with conventional chemotherapy for the treatment of colorectal cancer.

Characterization of Anti-Angiogenic Chemo-Sensitization via Longitudinal Ultrasound Localization Microscopy in Colorectal Carcinoma Tumor Xenografts.

Objective:Super-resolution ultrasound localization microscopy (ULM) has unprecedented vascular resolution at clinically relevant imaging penetration depths. This technology can potentially screen for the transient microvascular changes that are thought to be critical to the synergistic effect(s) of combined chemotherapy-antiangiogenic agent regimens for cancer.Methods:In this paper, we apply this technology to a high-throughput colorectal carcinoma xenograft model treated with either the antiangiogenic agent sorafenib, FOLFOX-6 chemotherapy, a combination of the two treatments, or vehicle control.Results:Longitudinal ULM demonstrated morphological changes in the antiangiogenic treated cohorts, and evidence of vascular disruption caused by chemotherapy. Gold-standard histological measurements revealed reduced levels of hypoxia in the sorafenib treated cohort for both of the human cell lines tested (HCT-116 and HT-29). Therapy resistance was associated with an increase in tumor vascular fractal dimension as measured by a box-counting technique on ULM images.Conclusion:These results imply that the morphological changes evident on ULM signify a functional change in the tumor microvasculature, which may be indicative of chemo-sensitivity.Significance:ULM provides additional utility for tumor therapy response evaluation by offering a myriad of morphological and functional quantitative indices for gauging treatment effect(s).

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

Cabozantinib Enhances Anti-PD1 Activity and Elicits a Neutrophil-Based Immune Response in Hepatocellular Carcinoma.

Immune checkpoint inhibitors combined with antiangiogenic agents produce benefits in the treatment of advanced hepatocellular carcinoma (HCC). We investigated the efficacy and immunomodulatory activity of cabozantinib alone and combined with anti-PD1 in experimental models of HCC, and explored the potential target population that might benefit from this combination. C57BL/6J mice bearing subcutaneous Hepa1-6 or Hep53.4 tumors received cabozantinib, anti-PD1, their combination, or placebo. Tumor and blood samples were analyzed by flow cytometry, IHC, transcriptome, and cytokine profiling. Cabozantinib-related effects were validated in a colorectal cancer patient-derived xenograft model. Transcriptomic data from three human HCC cohorts (cohort 1: n = 167, cohort 2: n = 57, The Cancer Genome Atlas: n = 319) were used to cluster patients according to neutrophil features, and assess their impact on survival. The combination of cabozantinib and anti-PD1 showed increased antitumor efficacy compared with monotherapy and placebo (P < 0.05). Cabozantinib alone significantly increased neutrophil infiltration and reduced intratumor CD8+PD1+ T-cell proportions, while the combination with anti-PD1 further stimulated both effects and significantly decreased regulatory T cell (Treg) infiltration (all P < 0.05). In blood, cabozantinib and especially combination increased the proportions of overall T cells (P < 0.01) and memory/effector T cells (P < 0.05), while lowering the neutrophil-to-lymphocyte ratio (P < 0.001 for combination). Unsupervised clustering of human HCCs revealed that high tumor enrichment in neutrophil features observed with the treatment combination was linked to less aggressive tumors with more differentiated and less proliferative phenotypes. Cabozantinib in combination with anti-PD1 enhanced antitumor immunity by bringing together innate neutrophil-driven and adaptive immune responses, a mechanism of action which favors this approach for HCC treatment.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5–7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Inhibition of growth of colorectal cancer patient-derived subcutaneous xenografts using combined Wnt signaling pathway inhibitor and 5‑fluorouracil

Purpose of the study. Was to analyze antitumor efficacy of the XAV 939 Wnt signaling pathway inhibitor and its combination with 5 fluorouracil in subcutaneous xenografts derived from patients with colorectal cancer.Materials and methods. Antitumor efficacy of the agents and their combination was studied in xenografts derived from patients with colorectal cancer and subcutaneously implanted in immunodeficient Balb/c Nude mice. All animals with tumors were divided into 4 groups (n = 5): group 1 received 5 fluorouracil 25 mg/kg, group 2 – XAV 939 25 mg/kg, group 3–5 fluorouracil and XAV 939 combination at the same dosages, group 4 was control. Criteria for the efficacy of the tested agents and their combination included tumor growth rate and tumor growth inhibition rate (TGI %).Results. The mean volumes of xenografts and tumor growth rate in the group receiving a combination of 5 fluorouracil and XAV 939 were 335.2 ± 40.7 mm3 , being lower than the averages of xenografts in controls – 609.3 ± 69.5 mm3 (p &lt; 0.05). The mean volumes of xenografts in the group receiving 5 fluorouracil monotherapy were 601.9 ± 45.5 mm3 , in the group with the XAV 939 monotherapy – 527.9 ± 258.6 mm3 . The highest TGI (44.99 %) was registered in the group receiving a combination of 5 fluorouracil and XAV 939.Conclusion. The study revealed the ability of combined XAV 939 Wnt signaling pathway inhibitor and 5 fluorouracil to inhibit the growth of subcutaneous xenografts derived from patients with colorectal cancer.

Emodin-8-O-β-D-glucopyranoside, a natural hydroxyanthraquinone glycoside from plant, suppresses cancer cell proliferation via p21-CDKs-Rb axis

Emodin-8-O-β-D-glucopyranoside (EG), a natural hydroxyanthraquinone glycoside from some traditional medicinal plants, has been demonstrated to have potential antitumor effects in our previous studies. Herein, we confirm that EG remains stable in the cell culture medium. It suppresses cell viability and proliferation and induces G1 cell cycle arrest in human colorectal cancer and neuroblastoma cells in vitro. EG inhibits tumor growth in human colorectal cancer cell HCT 116-bearing xenograft mice with low toxicity in the liver and kidney. The transcriptome analysis shows that the p53 signaling pathway is the most enriched cellular pathway and EG affects the proliferation of HCT 116 cells through modulating cell cycle related genes, such as CDKN1A and Cyclin-dependent kinases (CDKs). We demonstrate that the protein expression level of p21 was up-regulated, and CDK1/CDK2 were reduced significantly in both HCT 116 and SH-SY5Y cells after EG treatment. The switch from hypo- to hyper-phosphorylated Retinoblastoma (Rb), which is believed as a result of activated CDKs, was inhibited when cells were treated with EG. These findings indicate that EG suppresses cancer cell proliferation via p21-CDKs-Rb axis.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5′-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.