Mouse Model Of Colon Cancer Research Articles

MUC1-induced immunosuppression in colon cancer can be reversed by blocking the PD1/PDL1 signaling pathway.

Mucin1 (MUC1) upregulation in colon cancer has been linked to poor patient outcomes and advanced stage at diagnosis. This is partially due to MUC1-mediated inhibition of T-cell proliferation affecting efficient lysis by cytotoxic lymphocytes, which contributes to escape from immune surveillance. In the present study, human colorectal cancer tissues were collected, and MUC1-positive and MUC1-negative colon cancer mouse models were prepared; subsequently, the number and function of immune cells in tumor tissues were measured using flow cytometry. The present study revealed that MUC1, as a tumor-associated antigen, can recruit more tumor-infiltrating lymphocytes into the tumor microenvironment compared with MUC1-negative colon cancer, but that these cells could not serve antitumor roles. Conversely, the present study demonstrated that MUC1-positive colon cancer attracted more regulatory T cells (Treg cells), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) to the tumor site than MUC1-negative colon cancer. Furthermore, the data suggested that programmed death protein 1 (PD1)-programmed death ligand 1 (PDL1) expression is greater in MUC1-positive colon cancer. Blocking the PD1-PDL1 signaling pathway reduced the percentage of Treg cells, MDSCs and TAMs in the tumor microenvironment, enhanced T-cell cytotoxicity and inhibited tumor growth, prolonging the survival time of MUC1-positive tumor-bearing mice. Therefore, the present study elucidated the role of MUC1 in tumor immune escape and provides a foundation for the application of PDL1 inhibitors to MUC1-positive colon cancer.

Discovery of oxyepiberberine as a novel tubulin polymerization inhibitor and an anti-colon cancer agent against LS-1034 cells.

Coptis chinensis Franch. has been extensively used in traditional Chinese medicine. The chemical structure of oxyepiberberine, as an alkaloid isolated from Coptis chinensis Franch., has been previously studied. However, anti-cancer effects and underlying mechanisms of oxyepiberberine need to be explored. This study aimed to investigate the anti-cancer effects and underlying mechanisms of oxyepiberberine on LS-1034 human colon cancer cells. The anti-proliferative effects of six derivatives of oxyepiberberine on colon cancer cells were assessed. Among six derivatives, oxyepiberberine showed the greatest anti-proliferative effect on LS-1034 cells with an IC50 value of 1.36μM. Oxyepiberberine also induced apoptosis and inhibited migration of LS-1034 cells in a concentration-dependent manner. Importantly, oxyepiberberine was identified as a potent tubulin polymerization inhibitor. The tubulin polymerization inhibitory effects of oxyepiberberine in a concentration-dependent manner with an IC50 value of 1.26μM were observed. A xenograft mouse model of colon cancer showed that oxyepiberberine could suppress tumor growth without an obvious toxicity. Conclusion Oxyepiberberine was found as a novel tubulin polymerization inhibitor, and it could be a promising agent to treat colon cancer.

Perfluorocarbon@Porphyrin Nanoparticles for Tumor Hypoxia Relief to Enhance Photodynamic Therapy against Liver Metastasis of Colon Cancer

Photodynamic therapy (PDT) shows great promise for the treatment of colon cancer. However, practically, it is a great challenge to use a nanocarrier for the codelivery of both the photosensitizer and oxygen to improve PDT against PDT-induced hypoxia, which is closely related to tumor metastasis. Hence, an effective strategy was proposed to develop an oxygen self-supplemented PDT nanocarrier based on the ultrasonic dispersion of perfluorooctyl bromide (PFOB) liquid into the preformed porphyrin grafted lipid (PGL) nanoparticles (NPs) with high porphyrin loading content of 38.5%, followed by entrapping oxygen. Interestingly, the orderly arranging mode of porphyrins and alkyl chains in PGL NPs not only guarantees a high efficacy of singlet oxygen generation but also reduces fluorescence loss of porphyrins to enable PGL NPs to be highly fluorescent. More importantly, PFOB liquid was stabilized inside PGL NPs with an ultrahigh loading content of 98.15% due to the strong hydrophobic interaction between PGL and PFOB molecules, facilitating efficient oxygen delivery. Both in vitro and in vivo results demonstrated that the obtained O2@PFOB@PGL NPs could act as a prominent oxygen reservoir and effectively replenish oxygen into the hypoxic tumors with no need for external stimulation, conducive to augmented singlet oxygen generation, hypoxia relief, and subsequent downregulation of COX-2 expression. As a result, the use of O2@PFOB@PGL NPs for hypoxia relief dramatically inhibits tumor growth and liver metastasis in an HT-29 colon cancer mouse model. In addition, the O2@PFOB@PGL NPs could serve as a bimodal contrast agent to enhance fluorescence and CT imaging, visualizing nanoparticle accumulation to guide the subsequent laser irradiation for precise PDT.

Redox-Sensitive Nanocomplex for Targeted Delivery of Melittin.

Although peptide therapeutics have been explored for decades, the successful delivery of potent peptides in vitro and in vivo remains challenging due to the poor stability, low cell permeability, and off-target effects. We developed a redox sensitive polymer-based nanocomplex which can efficiently and stably deliver the peptide drug melittin for cancer therapy. The nanocomplex selectively targets cancer cells through lactobionic acid mediated endocytosis and releases melittin intracellularly upon the trigger of elevated redox potential. In vivo study proved that the targeted nanocomplex shows excellent potency in inhibiting tumor growth in a xenograft colon cancer mouse model. Thus, the polymer/melittin nanocomplexes will provide a new approach for melittin based cancer therapy.

Immunogenic chemotherapy in two mouse colon cancer models.

Aside from the induction of cell death, some anticancer chemotherapeutic drugs can modulate antitumor immune responses. In this study, we examined the anticancer effects of 5‐fluorouracil (5‐FU) and oxaliplatin (L‐OHP), which are standard chemotherapeutic drugs for colon cancer, combined with cyclophosphamide (CP) in two mouse colon cancer models (CT26 and MC38 colon adenocarcinoma models). In the CT26 model, two injections of 5‐FU/L‐OHP and CP significantly suppressed the growth of subcutaneously established CT26 tumors compared with either 5‐FU/L‐OHP or CP, without a significant loss of body weight. The anticancer effect was weakened in nude mice. Cured mice acquired protective immunity against CT26, and CT26‐specific cytotoxic T cells (CTLs) were induced from their spleen cells. Analysis of tumor‐infiltrating immune cells revealed that 5‐FU/L‐OHP treatment with or without CP increased the proportion of CD8+ T cells at tumor sites. The 5‐FU/L‐OHP treatment decreased the proportion of granulocytic myeloid‐derived suppressor cells (MDSCs) and increased monocytic MDSCs in tumor sites, whereas the addition of CP treatment reversed these changes. In the MC38 model, although significant anticancer effects of the triple combination therapy were seen, additional treatment with anti‐PD‐1 antibody increased the number of cured mice. These mice exhibited protective immunity against MC38, and MC38‐specific CTLs were generated from their spleen cells. Together, these results indicate that the antitumor effects of the combination of 5‐FU/L‐OHP and CP mainly depend on host T cells; moreover, the therapeutic efficacy can be effectively boosted by immune checkpoint blockade.

A Natural Flavone Tricin from Grains Can Alleviate Tumor Growth and Lung Metastasis in Colorectal Tumor Mice.

Tricin, a flavone isolated from rice bran, has been shown to be chemopreventive in a colorectal cancer (CRC) mouse model. This study aimed to illustrate the inhibitory activities of tricin in colon cancer cells and in a metastatic CRC mouse model. BALB/c mice injected with mouse Colon26-Luc cells into the rectum wall were treated with tricin (37.5 mg/kg) daily for 18 days. Orthotopic colon tumor growth and metastasis to lungs were assessed by in vivo bioluminescence imaging. Results showed that tricin suppressed Colon-Luc cells motility and downregulated phosphorylated Akt, Erk1/2 and NF-κB expressions of human colon cancer HT-29 cells. While tricin treatment suppressed tumor growth and lung metastasis as well as altered the populations of myeloid-derived suppressor cells and regulatory T cells in spleens. In summary, the tumor microenvironment modulatory and anti-metastatic effects of tricin in colon cancer mouse model were shown for the first time, suggesting the potential development of tricin-containing food supplements for CRC patients.

Abstract 3966: The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer

Abstract Purpose: Obesity is associated with an increased risk of colon cancer. Our current study examines whether moderate weight loss or treatment with the non-steroidal anti-inflammatory drug sulindac suppresses the tumor-promoting effects of obesity in a mouse model of colon cancer. Methods: Male FVB mice were treated with azoxymethane (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) diet. After 15 weeks, there was an interim sacrifice of 5 mice/group. The DIO mice were then further randomized to remain on DIO or switch to control diet to induce weight loss and become formerly obese mice (FOB), and within each diet group (control, DIO, and FOB), half the mice were randomized to start sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were euthanized. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P<0.05), while FOB mice had intermediate levels of adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had 10-fold greater tumor multiplicity relative to control mice (P=0.07). At study endpoint, DIO mice had greater tumor multiplicity compared to both control (P<0.05) and FOB (P<0.01) mice, which did not significantly differ from each other. Sulindac reduced tumor multiplicity in both control (P<0.05) and DIO (P<0.0001) mice, but not FOB mice. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus control (P<0.05) and DIO+sulindac (P<0.01) mice, but not in comparison to FOB mice. Gene expression in paired colon tumor and mesenteric adipose tissue samples was assessed by microarray. Ingenuity pathway analysis (IPA) of the significant (P<0.05) differentially expressed tumor genes indicated that both weight loss and sulindac reduced signaling related to matrix metalloproteinases (MMPs) and inflammation. In the visceral adipose tissue, IPA demonstrated that weight loss reduced signaling related to B cell receptors and glucose/fatty acid metabolism while sulindac decreased MMP, prostaglandin, and PPAR signaling. Additional analyses to explore the potential role of cross-talk between these two tissues are being performed, and key findings will be validated in the tissues at the protein level and via mechanistic cell culture experiments. Conclusions: Both moderate weight loss and sulindac treatment completely reverse the effects of chronic obesity on colon tumorigenesis, the latter independent of any change in adiposity. Our findings suggest that an investigation regarding the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted, particularly for those unable to achieve moderate weight loss. Citation Format: Laura W. Bowers, Arunima Punjala, Alison Q. Jeffries, Stephanie A. Montgomery, Andrew J. Dannenberg, Stephen D. Hursting. The procancer effects of obesity can be reversed by moderate weight loss or an anti-inflammatory drug in a mouse model of colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3966.

Abstract 1475: Blockade of IL17-GCSF-Bv8 inflammation axis ameliorate resistant to anti-angiogenic therapy for colon cancer

Abstract Anti-angiogenic therapy in combination with chemotherapy is one of the standard strategies for metastatic/unresectable colon cancers. Therefore, resistance to anti-angiogenic therapy causes a critical problem for the treatment of colon cancer patients. While some resistant mechanisms of anti-angiogenic therapy were proposed, it is not yet completely unveiled. The aim of this study was to investigate the resistant mechanism against anti-angiogenic therapy using spontaneous colon cancer mouse models. We established a mouse model of anti-VEGF resistant colon cancer. Genetically-engineered Apc/Smad4 knockout mice developed several intestinal tumors that are sensitive to anti-VEGF neutralizing antibody. When Apc/Smad4 mice were treated with dextran sulfate sodium (DSS) (DSS-Apc/Smad4 mice), they developed colon tumors that were resistant to anti-VEGF antibody. Sera from DSS-Apc/Smad4 mice showed significant upregulation of granulocyte-colony stimulating factor (GCSF) compared to water-drinking Apc/Smad4 mice. Colon tumors from DSS-Apc/Smad4 mice also showed GCSF upregulation. It also showed upregulation of IL17, one of inflammatory cytokines upstream of GCSF, and Bv8, one of angiogenic factors downstream of GCSF, suggesting that IL17-GCSF-Bv8 inflammation axis contributes to obtaining anti-VEGF resistant. Immunostaining of the tumors from DSS-Apc/Smad4 mice revealed significant accumulation of neutrophils in the tumors. Neutrophils from tumor-bearing mice showed upregulation of Bv8 expression when stimulated with GCSF. Immunostaining confirmed that neutrophils in the tumors were positive for Bv8. When DSS-Apc/Smad4 mice were treated with either anti-IL17, anti-GCSF, or anti-BV8 neutralizing antibody in combination with anti-VEGF antibody, colonic tumor formation was significantly suppressed. We also performed ELISA analysis using human serum samples from subcohort of AVF2107 trial in which metastatic colon cancer patients were treated with chemotherapy (IFL with or without bevacizumab). Higher Bv8 serum levels by ELISA were significantly correlated with poor overall survival in human colon cancer patients. Collectively, inflammatory neutrophils in the tumor microenvironment of colon cancer provide anti-VEGF resistant through IL17-GCSF-Bv8 axis, and blockade of this pathway ameliorate resistance to anti-angiogenic therapy of colon cancer mouse models. Citation Format: Yoshiro Itatani, Takamasa Yamamoto, Napoleone Ferrara. Blockade of IL17-GCSF-Bv8 inflammation axis ameliorate resistant to anti-angiogenic therapy for colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1475.

Abstract 5335: CA102N suppresses the growth of mouse colon cancer by inhibition of PI3K pathway and immune modulation

Abstract It is well documented that COX-2 inhibitors have the potential in preventing or treating colorectal cancer. However, the cardiovascular toxicity of COX-2 inhibitors hampered further development of these inhibitors in cancer management. CA102N is a covalently bound conjugate of the biological polymer sodium hyaluronate (NaHA) and nimesulide (Nim) aiming to deliver Nim directly to the tumor tissues to limit the systemic toxicity. Here, we report the antitumor effect of CA102N in colon cancer and the possible mechanism associated with its antitumor activity. The antitumor efficacy of CA102N was tested in the CT-26 syngeneic Balb/c mouse colon cancer model. The effect of CA102N on inflammation was examined by serum cytokines, intratumor and urinary COX-2 metabolites. Immune modulation was determined by profiling TILs with flow cytometry. Cell signaling proteins were measured by western blot analysis. CA102N (50 mg/kg and 200 mg/kg) i.v. injected mice carrying CT26 mouse colon tumor showed significantly reduced average tumor weight (726.1 ± 335.2 mg and 633.4 ± 453.5 mg, respectively) compared to that of the vehicle control treated mice (1392.0 ± 523.8, p < 0.05). The reduction of tumor growth by CA102N in this particular tumor model was comparable to the mice having received TAS102 (100 mg/kg) p.o. daily for two weeks. Intratumor COX-2 metabolite analysis showed that CA102N (200 mg/kg) reduced levels of both PGE2 and PGI2 by 31% and 58% (p< 0.05), respectively compared to that of control group. The levels of urinary COX-2 metabolites in CA102N treated CT26 tumor bearing mice were similar to that of control mice. Results of serum cytokine analysis suggest that levels of MCP-1 (109.9 ± 24.3 pg/ml) and IL6 (18.0 ± 3.8 pg/ml) in CA102N (50 mg/kg) treated mice were significantly lower than that of control group (175.4 ± 21.0 pg/ml) and (123.3 ± 55.8 pg/ml), respectively (p < 0.05). Furthermore, TILs profiling showed that FOXP3+ T-regulatory immune cell population was reduced by almost 63% in CA102N (50 mg/kg) treated mice whereas the number of CD8+ T cells in CA102N (50 mg/kg) treated tumors was about 2-fold higher than that of the control group. In contrast, CA102N (200 mg/kg) treatment neither altered the serum cytokines and nor increased CD8+ T-cell populations in the tumor. Additionally, the protein expression of pAkt and pS6 in CA102N (200 mg/kg) treated tumor tissues were significantly down-regulated by more than 50% compared to that of control group, while CA102N (50 mg/kg) treatment only slightly altered the abundance of these proteins, suggesting antitumor activity of CA102N in different doses can potentially be mediated by differential mechanisms. Together, these findings suggest that the lower dose of CA102N exerts antitumor activity in mouse colon tumor model by immune modulation whereas the antitumor activity of the higher dose of CA102N might be mediated through inhibition of PI3kinase pathway on tumor tissues. Citation Format: Peiying Yang, Tara Conway, Patrea Rhea, Bo Wei, Eskouhie Tchaparian. CA102N suppresses the growth of mouse colon cancer by inhibition of PI3K pathway and immune modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5335.

Abstract 1483: A novel anti-Tie2 antibody stabilizes vessel and increases tumor infiltrated lymphocytes

Abstract Objective: The delivery of drugs and immune cells into the tumor microenvironment is limited due to abnormal tumor vessel which is characterized as low oxygen concentration, low perfusion rate and leakage. It leads to diminish the efficacy of drugs and radiation therapy. Traditional anti-angiogenesis strategies attempt to reduce the tumor vascular supply, but their success is restricted by insufficient efficacy or development of resistance. To solve the problem, we try to using active vessel stabilizer, which can normalize the abnormal condition of tumor vessel. Background: Tie2 acts as cell-surface receptor for Ang1/Ang2 and regulates angiogenesis, endothelial cell survival, proliferation, migration, as well as maintenance of vascular quiescence. Especially, Ang1-Tie2 signaling pathway has been known to promote blood vessel maturation and stabilization. Methods: We have developed a series of novel human IgG monoclonal antibody that binds to human-TIE2 with a high affinity (Kd ~0.1 nM). The antibody binds to human TIE2 extracellular domain and activates its associated function in a ligand independent way. The mode of action was studied in vitro with human umbilical vein endothelial cells (HUVEC) and animal models. The vessel stabilization was investigated with laser induced choroidal neovascularization (CNV). The effect on tumor infiltrated lymphocyte was also studied in mouse colon cancer model. Results: Anti-Tie2 antibody increased human endothelial cell migration like Ang1 and inhibited VEGF-induced vascular leakage, contributing to vessel normalization. It binds to Tie2 in a ligand independent way and thus it can stabilize vessel as an agonist. Both p-VEGFR and p-VE-Cadherin levels are also significantly decreased on VEGF-induced VEGFR signaling pathway by anti-Tie2 antibody, suggesting that it can maintain cell to cell junction. The vessel stabilization effect was demonstrated in a mouse model of laser induced CNV. Moreover, in syngeneic CT26 mouse models, anti-Tie2 antibody showed anti-tumor effect in which it significantly reduced the tumor growth and synergistically when it combined with anti PD-1 antibody. The tumor infiltrating lymphocytes were increased in CT26 tumor, especially CD8+ T cells and MDSC when anti-Tie2 was administered. Conclusion: We developed active vessels stabilizer targeting Tie2, having an effect of vessel stabilization in a ligand independent way. It inhibited VEGF-induced vascular leakage through decrease of p-VEGFR and VE-cadherin. This active vessel stabilizer could help deliver drug and lymphocyte into the TME. The therapeutic efficacy of immune checkpoint inhibitors can be increased when it combined with this active vessel stabilizer. Thus, this active vessel stabilizer can change cold tumor into hot tumor. Citation Format: Eun-Ah Lee, Beomyong Park, Nu Ri Kang, Youngae Lee, Do-yun Kim, Sang Soon Byun, Jin Yong An, Cheon Ho Park, Weon Sup Lee, Joo Hyoung Lee. A novel anti-Tie2 antibody stabilizes vessel and increases tumor infiltrated lymphocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1483.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4380.

Abstract 3367: Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies

Abstract Cancer immunotherapy with immune checkpoint inhibitors which enhances T-cell regulatory pathways has provided unprecedented benefit to cancer patients. Programmed death 1 ligand (PD-L1) being expressed by cancer cells binds to programmed death-1 (PD-1) to avoid anti-tumor activity of immune cells. Antibodies to disrupt the interaction have been developed for cancer therapeutics. YBL-006 is a new anti-PD-1 human IgG4 monoclonal antibody. Here, we described in vitro & in vivo studies to evaluate pharmacological effect of YBL-006 along with non-clinical studies of pharmacokinetics, toxicokinetics & tissue cross reactivity (TCR). YBL-006 was bound specifically to human PD-1 among receptors of B7 family by ELISA. When affinity to recombinant PD-1 of various species was measured using surface plasmon response system, KD of YBL-006 was 0.372 nM to human PD-1 & 0.070 nM to cynomolgus monkey PD-1 which is higher affinity than nivolumab (1.37 nM & 2.50 nM) & pembrolizumab (1.44 nM & 0.817 nM). It did bind to mouse PD-1 with less affinity than to human PD1, while nivolumab & pembrolizumab did not bind to mouse PD-1. YBL-006 was able to inhibit interaction between PD-1 & both PD-L1 & PD-L2 which was confirmed in competitive binding assay. Inhibition of PD-1 by YBL-006 increased the level of IFN-γ in a mixed lymphocyte reaction model. In a syngeneic mouse model of MC38 colon cancer, weight of tumors in mice treated with YBL-006 was less than that in mice with human IgG control by 25%. In a humanized mouse model (miXeno mouse) of HCC827 non-small cell lung cancer, volume of tumors in YBL-006-treated mice was smaller (35 % less comparing to control mice) than that in nivolumab-treated mice (24 % less). Pharmacokinetic analysis in monkey showed that exposures to YBL-006 increased dose-dependently & in a dose-proportional manner. Maximum mean serum concentrations (Cmax) of YBL-006 were reached (Tmax) between 0.5- & 1.0-hour post onset of infusion & YBL-006 mean serum concentrations slowly declined at a mean estimated t1/2 value of 70.8~110 hours. Clearance & mean volume of distribution suggest that YBL-006 was mainly distributed throughout the blood. In 1-month IV infusion toxicity study in monkey, there was no toxicity when they were infused up to 100 mg/kg/dose of YBL-006, but anti-drug antibody was observed (7 out of 22 monkeys). There was no potential toxicity in TCR study. YBL-006 is an anti-PD-1 antibody with high affinity, promising anti-tumor activity in animal models, & favorable safety profile. First-in-human phase I trial to investigate the safety & efficacy of YBL-006 in advanced solid cancer will be held in 2020. Citation Format: Jaebong Yoon, Hyunmi Lee, Eunyoung Jeon, Jinsil Lee, Eunyoung Shim, Hyunju Lee, Eunjin Lee, Younggyu Cho, Bumchan Park, Hanseung Lee, Youngwoo Park. Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3367.

Abstract 973: Telomere stress potentiates host STING-dependent anti-tumor immunity

Abstract Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells but not in normal cells. Here we show that treatment with a telomerase-mediated telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression in innate and adaptive immune-dependent manners in syngeneic and humanized mouse colon cancer models. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/IFN-I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8 T-cell activation. Moreover, 6-thio-dG overcomes resistances to checkpoint blockade in advanced mouse cancer models. Together, our results unveil how 6-thio-dG induced telomere stress increases innate sensing and adaptive anti-tumor immunity and provide a strong rationale for sequentially combining telomerase directed telomere-targeted therapy with immunotherapy. Citation Format: Anli Zhang, Ilgen Mender, Zhenhua Ren, Chuanhui Han, Yafang Deng, Silvia Siteni, Huiyu Li, Jerry W. Shay, Yang-Xin Fu. Telomere stress potentiates host STING-dependent anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 973.

Ketogenic Diet Elicits Antitumor Properties through Inducing Oxidative Stress, Inhibiting MMP-9 Expression, and Rebalancing M1/M2 Tumor-Associated Macrophage Phenotype in a Mouse Model of Colon Cancer.

Many advanced cancers are characterized by metabolic disorders. A dietary therapeutic strategy was proposed to inhibit tumor growth through administration of low-carbohydrate, average-protein, and high-fat diet, which is also known as ketogenic diet (KD). In vivo antitumor efficacy of KD on transplanted CT26+ tumor cells in BALB/c mice was investigated. The results showed that the KD group had significantly higher blood β-hydroxybutyrate and lower blood glucose levels when compared with the normal diet group. Meanwhile, KD increased intratumor oxidative stress, and TUNEL staining showed KD-induced apoptosis against tumor cells. Interestingly, the distribution of CD16/32+ and iNOS+ M1 tumor-associated macrophages (TAMs) increased in the KD-treated group, with concomitantly less arginase-1+ M2 TAMs. Moreover, KD treatment downregulated the protein expression of matrix metalloproteinase-9 in CT26+ tumor-bearing mice. Western blot analysis demonstrated that the expression levels of HDAC3/PKM2/NF-κB 65/p-Stat3 proteins were reduced in the KD-treated group. Taken together, our results indicated that KD can prevent the progression of colon tumor via inducing intratumor oxidative stress, inhibiting the expression of the MMP-9, and enhancing M2 to M1 TAM polarization. A novel potential mechanism was identified that KD can prevent the progression of colon cancer by regulating the expression of HDAC3/PKM2/NF-κB65/p-Stat3 axis.

Targeted Delivery of CXCL9 and OX40L by Mesenchymal Stem Cells Elicits Potent Antitumor Immunity

Major obstacles in immunotherapies include toxicities associated with systemic administration of therapeutic agents, as well as low tumor lymphocyte infiltration that hampers the efficacies. In this study, we report a mesenchymal stem cell (MSC)-based immunotherapeutic strategy in which MSCs specifically deliver T/natural killer (NK) cell-targeting chemokine CXCL9 and immunostimulatory factor OX40 ligand (OX40L)/tumor necrosis factor superfamily member 4 (TNFSF4) to tumor sites in syngeneic subcutaneous and azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced spontaneous colon cancer mouse models. This approach generated potent local antitumor immunity by increasing the ratios of tumor-infiltrating CD8+ T and NK cells and production of antitumor cytokines and cytolytic proteins in the tumor microenvironment. Moreover, it improved the efficacy of programmed death-1 (PD-1) blockade in a syngeneic mouse model and significantly suppressed the growth of major histocompatibility complex class I (MHC class I)-deficient tumors. Our MSC-based immunotherapeutic strategy simultaneously recruits and activates immune effector cells at the tumor site, thus overcoming the problems with toxicities of systemic therapeutic agents and low lymphocyte infiltration of solid tumors.

Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)–GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2−/− colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

Effect of actively targeted copolymer coating on solid tumors eradication by gold nanorods-induced hyperthermia.

Efforts in the field of anticancer therapy are increasingly focusing on the development of localized and selective treatments. Photothermal therapy (PTT) can lead to a spatially confined death of cancer cells, exploiting an increasing in temperature generated after UV-NIR irradiation of peculiar materials. Herein, a new actively targeted gold-based drug delivery system, named PHEA-LA-Fol-AuNRs/Iri, was explored for hyperthermia and chemotherapy colon cancer treatment. Gold nanorods were stabilized using a folate-derivative of α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA-LA-PEG-FA) as coating agent and then loaded with the antineoplastic drug irinotecan (Iri). The efficacy of empty and irinotecan-bearing systems was investigated in vitro on human colon cancer (HCT116) cell line, as well as in vivo, employing a xenograft mouse model of colon cancer. After laser treatment, both nanostructures tested induced a considerable deceleration in tumor growth overtime, achieving the total eradication of the cancer when the nanosystems produced were intratumorally administered. Biodistribution data showed that the polymer coated nanorods were able to preferentially accumulate in the tumor site. Considering the excellent stability in aqueous media, the capacity to reach the tumor site and, finally, the in vivo efficacy, PHEA-LA-Fol-AuNRs/Iri might be recommended as an effective tool in the chemotherapy and PTT of colon cancer.

Abstract A46: Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer

Abstract Purpose: We previously showed diet-induced obesity (DIO) increases (and calorie restriction decreases) systemic markers of inflammation and azoxymethane (AOM)-induced colon carcinogenesis in FVB/N mice. Our current study examines whether moderate weight loss via low-fat diet (LFD) and/or treatment with the nonsteroidal anti-inflammatory drug sulindac reverses the inflammatory and tumor-promoting effects of DIO in the AOM-induced mouse model of colon cancer. Methods: Male FVB/N mice were treated with AOM (10 mg/kg i.p.) for 5 weeks, then randomized to a control (10% kcal from fat) or DIO (60% kcal from fat) diet. After 15 weeks on diet, 5 mice/group were killed and their tissues collected for interim timepoint analysis. The remaining DIO mice were further randomized to either remain on DIO or switch to control diet to induce weight loss (formerly obese [FOb] mice), and within each diet group (control, DIO, and FOb), half the mice were randomized to receive sulindac treatment (140 ppm in the diet). Eight weeks later, all mice were killed and their serum, tumors, and other tissues collected for analysis. Results: The DIO mice, compared to controls, had significantly greater body weight and body fat (P<0.05), while FOb mice had intermediate levels of weight and adiposity. Sulindac did not affect adiposity in any diet group. At the interim timepoint, DIO mice had ~10-fold greater tumor multiplicity relative to control mice (p<0.01) and Fob mice (p<0.01). The control and FOb groups did not significantly differ from each other. At study termination sulindac significantly reduced tumor multiplicity in both control (P<0.05) and DIO (P<0.0001) mice, but not FOb mice. In the mice that received sulindac, tumor multiplicity did not significantly differ between diet groups. Tumor multiplicity in the DIO+sulindac, FOb, and FOb+sulindac groups was also significantly lower than the interim timepoint DIO mice (P<0.05), suggesting the interventions induced tumor regression. Serum levels of several inflammation-related proteins, including interleukin 6, chemokine (C-X-C motif) ligand 1, monocyte chemoattractant protein 1, and vascular endothelial growth factor, were elevated in DIO mice versus controls (P<0.05), but did not differ between FOb and control mice. Sulindac treatment significantly reduced serum levels of these proteins in DIO mice (P<0.01). Gene expression in paired colon tumor and mesenteric adipose tissue samples is currently being assessed by microarray analysis to investigate crosstalk between these tissues and inform the design of future studies on the mechanism(s) mediating sulindac’s effects. Conclusions: Both moderate weight loss and sulindac treatment reverse the effects of chronic obesity on AOM-induced colon tumorigenesis. The anticancer effects of sulindac were independent of any change in adiposity. Given the challenges of losing weight, our findings suggest that further investigation of the effects of NSAID treatment on colon cancer risk and/or progression in obese patients is warranted. Citation Format: Stephen D. Hursting, Laura W. Bowers, Arunima Punjala, Andrew J. Dannenberg. Dietary weight loss and sulindac treatment each reverse obesity-associated inflammation and tumorigenesis in a mouse model of carcinogen-induced colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A46.

Near-infrared photoimmunotherapy is effective treatment for colorectal cancer in orthotopic nude-mouse models.

Photoimmunotherapy (PIT) employs the use of a near-infrared (NIR) laser to activate an antibody conjugated to a NIR-activatable dye to induce cancer cell death. PIT has shown to be effective in a number of studies, however, there are no data on its use in colorectal cancer in an orthotopic model. Humanized anti-CEA antibody (M5A) was conjugated to NIR-activatable IRDye700DX (M5A-700). PIT was validated in vitro with a colon cancer cell-line, using a laser intensity of either 4 J/cm2, 8 J/cm2, or 16 J/cm2. Orthotopic colon cancer mouse models were established by surgical implantation of LS174T tumor fragments onto the cecum. M5A-700 was administered and PIT was performed 24 hours later using a 690 nm laser. Repeat PIT was performed after 7 days in one group. Control mice received laser treatment only. In vitro PIT demonstrated tumor cell death in a laser intensity dose-dependent fashion. In orthotopic models, control mice demonstrated persistent tumor growth. Mice that underwent PIT one time had tumor growth arrested for one week, after which re-growth occurred. The group that received repeated PIT exposure had persistent inhibition of tumor growth. PIT arrests tumor growth in colon cancer orthotopic nude-mouse models. Repeated PIT arrests colon cancer growth for a longer period of time. PIT may be a useful therapy in the future as an adjunct to surgical resection or as primary therapy to suppress tumor progression.

Dynamic alterations in serum IgG N-glycan profiles in the development of colitis-associated colon Cancer in mouse model

BackgroundAlternative glycosylation of serum IgG has been shown to be closely associated with colorectal cancer (CRC). Currently, a dynamic study which can not only minimize the influence of genetic background, environment and other interfering factors during cancer development, but also focus on investigating carcinogenic characteristics of IgG glycan is lacking. MethodsSerum IgG N-glycans were characterized at four stages of CRC development by ultra-performance liquid chromatography in a typical colitis-related CRC mouse model induced by azoxymethane-dextran sodium sulfate. Furthermore, the expression of related glycosyltransferases in splenic B lymphocytes at the corresponding time was also assessed. ResultsThe relative abundance of seven IgG glycans, which can be classified as monoantennary, core fucose, sialic acid, galactose and bisecting, was changed during tumor growth. The abundance of some glycans was altered during the first stage of cancer induction. Correspondingly, the expression of glycosyltransferases in splenic B lymphocytes and different tissues in cancer groups was also decreased compared to that in controls. ConclusionsThis study represents the comprehensive analysis of IgG glycosylation in the dynamic process of colitis-associated CRC. To our knowledge, this is the first report that the expression of glycosyltransferases in mouse splenic B lymphocytes is consistent or inconsistent with the alterations of IgG N-glycans, and the variation tendency is tissue nonspecific. General SignificanceProviding a novel approach to identify the IgG glycans related to the development of CRC and laying a foundation for research on structure and function of glycans using mouse.