Mouse Colon Cancer Research Articles

Baoyuan Jiedu Decoction Alleviates Cancer-Induced Myotube Atrophy by Regulating Mitochondrial Dynamics Through p38 MAPK/PGC-1α Signaling Pathway

Cancer cachexia is a multifactorial syndrome characterized by continuous body wasting and loss of skeletal muscle. Impaired mitochondria function is closely associated with muscle atrophy in cancer cachexia. Our previous study confirmed the effectiveness of Baoyuan Jiedu decoction (BJD) in inhibiting cancer-induced muscle atrophy in an in vivo model. However, little is known about its mechanisms in regulating mitochondria dysfunction. In this study, we evaluated the therapeutic effect and action mechanisms of BJD against atrophy both in the Lewis-conditioned medium induced C2C12 myotube atrophy model and in a BALB/c mice xenograft model using mouse colon cancer C26 cells. The mitochondrial content was tested by 10-Non-ylacridine orange staining. Expressions of related proteins and mRNAs were detected by western blotting (WB) and qPCR, respectively. As a result, 18 major components were identified in BJD by ultra-high performance liquid chromatography-quadrupole (UHPLC-Q) Exactive analysis. As shown in the in vitro results, BJD treatment prevented prominent myotube atrophy and increased the myotube diameter of C2C12 cells. Besides, BJD treatment increased mitochondrial content and ATPase activity. Furthermore, the protein and mRNA expressions that were related to mitochondrial functions and generation such as cytochrome-c oxidase IV, Cytochrome C, nuclear respiratory factor 1, and mitochondrial transcription factor A were significantly increased in BJD treatment compared to the control group. The in vivo results showed that BJD treatment prevented body weight loss and improved the gastrocnemius index in cachexia mice. Moreover, the expressions of Atrogin-1 and muscle RING-finger protein-1 were decreased by BJD treatment. Mechanically, BJD increased the expression of peroxisome proliferator-activated receptor-gamma coactivator 1, and consistently, inhibited the expression of p38 MAPK and its phosphorylation both in vivo and in vitro. Taken together, this study identified that BJD effectively relieved cancer-induced myotube atrophy and provided a potential mechanism for BJD in regulating mitochondrial dynamics through p38 MAPK/PGC-1α signaling pathway.

Intratumorally CpG immunotherapy with carbon nanotubes inhibits local tumor growth and liver metastasis by suppressing the epithelial-mesenchymal transition of colon cancer cells.

Colon cancer liver metastasis accounts for the major cause of death of colon cancer patients. Previous study reported a carbon nanotubes (CNT)-conjugated CpG complex (CNT-CpG), which displayed a significant antitumor effect in gliomas. However, whether CNT-CpG could limit colon tumor growth and suppress the colon cancer liver metastasis has not been evaluated. In this study, we report CNT enhances CpG uptake in mouse colon cancer cells. Results demonstrated only CpG with CNT conjugation showed significant activation of NF-κB signal. Moreover, intratumorally delivery of CNT-CpG successfully suppressed local xenograft tumor growth and liver metastasis. CNT-CpG treatments cured 75% of mice and inhibited local tumor growth, significantly prolonged survival outcomes and limited liver metastatic tumor nodules from colon cancer cells. Using human colon cancer cell line, HCT116, we observed significantly inhibitory effects of CNT-CpG on cell growth, invasion and migration. Importantly, CNT-CpG treatment blocked the epithelial to mesenchymal transition (EMT). We compared the mRNA levels of EMT markers of colon cancer cells without or with CNT-CpG treatment from in-vitro and in-vivo models. Consistent results demonstrated expression of epithelial marker, E-cadherin was upregulated by CNT-CpG. In contrast, three mesenchymal markers, snail, fibronectin and vimentin were significantly suppressed by CNT-CpG treatment compared with control or free CpG. In summary, our data suggest CNT-CpG is an effective therapeutic approach against local colon tumor and their liver metastasis. This study presents the CNT-CpG complex as a promising therapeutic target for developing novel therapies against both local colon tumors and liver metastatic tumors.

Krill Oil Perturbs Proliferation and Migration of Mouse Colon Cancer Cells in vitro by Impeding Extracellular Signal-Regulated Protein Kinase Signaling Pathway.

The prevalence of colorectal cancer (CRC) continues to increase. Treatment of CRC remains a significant clinical challenge, and effective therapies for advanced CRC are desperately needed. Increasing attention and ongoing research efforts have focused on krill oil that may provide health benefits to the human body. Here we report that krill oil exerts in vitro anticancer activity through a direct inhibition on proliferation, colony formation, migration, and invasion of mouse colon cancer cells. Krill oil inhibited the proliferation and colony formation of CT-26 colon cancer cells by causing G0/G1 cell cycle arrest and apoptosis. Cell cycle arrest was attributable to reduction of cyclin D1 levels in krill oil-treated cells. Further studies revealed that krill oil induced mitochondrial-dependent apoptosis of CT-26 cells, including loss of mitochondrial membrane potential, increased cytosolic calcium levels, activation of caspase-3, and downregulation of anti-apoptotic proteins MCL-1 and BCL-XL. Krill oil suppressed migration of CT-26 cells by disrupting the microfilaments and microtubules. Extracellular signal-regulated protein kinase (ERK) plays crucial roles in regulating proliferation and migration of cancer cells. We found that krill oil attenuated the activation of ERK signaling pathway to exert the effects on cell cycle, apoptosis, and migration of colon cancer cells. We speculate that polyunsaturated fatty acids of krill oil may dampen ERK activation by decreasing the phospholipid saturation of cell membrane. Although findings from in vitro studies may not necessarily translate in vivo, our study provides insights into the possibility that krill oil or its components could have therapeutic potential in 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.

Effects of oral bovine lactoferrin on a mouse model of inflammation associated colon cancer

Patients with ulcerative colitis or colonic Crohn's disease have a significantly increased risk of developing colorectal cancer. Bovine lactoferrin (bLF) reportedly inhibited the development of colon cancer in rats and mice, and in a placebo controlled trial, ingestion of bLF inhibited the growth of intestinal polyps. In addition, in a case study, a patient with Crohn's disease was reported to have remained in remission for over 7 years while ingesting 1g of bLF daily. Thus, bLF has an inhibitory effect on colon carcinogenesis, and it may also promote remission of Crohn's disease. The purpose of this study was to investigate the effects of bLF in a mouse model of colorectal cancer related to irritable bowel disease (IBD). The mice were divided into 4 groups: (i) no treatment; (ii) treated with bLF only; (iii) treated with azoxymethane plus dextran sulfate sodium (AOM + DSS); and (iv) treated with AOM + DSS + bLF. AOM was used to initiate intestinal cancer, and DSS was used to induce IBD-like inflammation in the intestine of the C57BL/6 mice. At the end of the study, the mice treated with AOM + DSS + bLF had a better fecal score, fewer lesions in the colon, and less weight loss than the mice treated with AOM + DSS without bLF. However, there were no statistically significant differences between the two groups with respect to tumor burden.

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.

Modification of In Vitro and In Vivo Antioxidant Activity by Consumption of Cooked Chickpea in a Colon Cancer Model.

Chickpea has been classified as a nutraceutical food due to its phytochemical compounds, showing antioxidant, anti-inflammatory, and anticancer activity. To investigate this, we evaluated the effect of cooking on the nutritional and non-nutritional composition and the in vitro and in vivo antioxidant activity of chickpea seed. The latter was determined by the variation in the concentration of nitric oxide (NO), oxidized carbonyl groups (CO), malondialdehyde (MDA), and the expression of 4-hydroxy-2-nonenal (4-HNE) in the colon of male BALB/c mice fed with a standard diet with 10 and 20% cooked chickpea (CC). We induced colon cancer in mice by administering azoxymethane/dextran sulfate sodium (AOM/DSS); for the evaluation, these were sacrificed 1, 7, and 14 weeks after the induction. Results show that cooking does not significantly modify (p < 0.05) nutritional compounds; however, it decreases the concentration of non-nutritional ones and, consequently, in vitro antioxidant activity. The in vivo evaluation showed that animals administered with AOM/DSS presented higher concentrations of NO, CO, MDA, and 4-HNE than those in animals without AOM/DSS administration. However, in the three evaluated times, these markers were significantly reduced (p < 0.05) with CC consumption. The best effect on the oxidation markers was with the 20% CC diet, demonstrating the antioxidant potential of CC.

Potent half-sandwich Ru(Ⅱ) N^N (aryl-BIAN) complexes: Lysosome-mediated apoptosis, invitro and invivo anticancer activities.

Herein a new series of organometallic half-sandwich Ru(Ⅱ) complexes bearing aryl-BIAN chelating ligands with various electron-withdrawing and electron-donating substituents have been developed as theranostic agents. All the complexes display much higher anti-proliferative potency than the clinical chemotherapeutic drug cisplatin towards seven cancer cell lines. The anti-proliferative efficacy of these complexes is correlated to their electron-withdrawing ability. Interestingly, complex Ru1 also potently suppresses cancer cell migration invitro and effectively inhibit tumor growth invivo in a CT26 colon cancer mouse xenograft model. Mechanisms of action studies display that Ru1 can favorably accumulate in lysosome and exerts anti-cancer potency by inducing a series of events related to lysosomal dysfunction in CT26cells. Interestingly, inhibition of lysosomal enzymes leads to suppression of cytotoxicity and apoptosis induced by Ru1. Our results elucidate that complex Ru1 can elicit cytotoxicity through lysosome-mediated apoptosis invitro and suppress tumor growth invivo.

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 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 4124: The combination of PARP inhibitor talazoparib with low-dose temozolomide results in increased cell lethality in BRCA1/2 wild-type melanoma, small cell lung cancer, ovarian, and colon cancer cell lines and mouse xenografts via the formation of DNA double-strand breaks during S-phase

Abstract Background: Previous studies of PARP inhibitors have shown efficacy in tumors with defects in homologous recombination repair, particularly in persons with ovarian and breast cancers with germline deleterious BRCA1/2 mutations (gBRCA1/2+). There is much interest in expanding the use of PARP inhibitors beyond these populations, with one strategy being the creation of DNA damage with cytotoxic chemotherapy with concomitant inhibition of its repair with a PARP inhibitor. Murai, et al, have also shown that PARP is trapped on the DNA at the site of damage in the presence of a PARP inhibitor. In April 2016, my colleague Dr Zev Wainberg presented Phase I safety and efficacy data at AACR (abstract CT011) with PARP inhibitor talazoparib in combination with alkylating agent temozolomide in cancer patients without gBRCA1/2+, demonstrating this to be a promising combination. Methods: We evaluated the molecular mechanisms underlying cancer cell lethality with talazoparib plus temozolomide in melanoma, small cell lung, ovarian, and colorectal cancer cell lines and mouse xenografts. We determined growth-adjusted IC50s for talazoparib in multiple histologies to choose cell lines of low and high sensitivity to the PARP inhibitor as monotherapy. We studied percent growth inhibition of talazoparib, temozolomide, and combination therapy at increasing doses. We investigated cell cycle dynamics at 24 hours and 48 hours; PARP inhibitors have been shown to activate the G2/M cell cycle checkpoint, which could confound growth arrest and DNA repair studies. We performed western blot analysis to identify phosphorylated histone H2AX as a marker of DNA double-strand breaks. We used FACS to determine apoptotic fractions and to correlate phospho-H2AX formation with cell cycle phase. We used confocal microscopy to correlate phospho-H2AX with nuclear PARP. We used mouse xenografts to demonstrate sensitivity of tumors to temozolomide, talazoparib, and combination therapy. Results: The combination of temozolomide with talazoparib results in the formation of DNA double-strand breaks during S-phase, which results in increased cell lethality in BRCA1/2 wild-type cell lines and mouse xenografts of melanoma, colorectal, ovarian, and small cell lung histologies even when those cell lines are not sensitive to either talazoparib or temozolomide alone. We were unable to demonstrate colocalization of PARP and phospho-H2AX due to PARP's high expression in the nucleus during S-phase. We were also not able to demonstrate PARP-trapping by western blot at doses utilized in human patients, but this is likely due to lack of assay sensitivity. Conclusions: Taking into consideration both the molecular data demonstrating efficacy of talazoparib plus temozolomide therapy in BRCA1/2 wild-type cell lines and mouse xenografts and the Phase I trial data showing safety and efficacy with this combination, PARP inhibitors likely have utility beyond treatment of gBRCA1/2+ patients. Use of the alkylating agent might be particularly important, as it is recognized during S-phase and trapping of PARP on the DNA could result in collapse of a stalled replication fork into a DNA double-strand break. Citation Format: Kelly Elizabeth McCann, Erika von Euw, Neil O'Brien, Dennis Slamon. The combination of PARP inhibitor talazoparib with low-dose temozolomide results in increased cell lethality in BRCA1/2 wild-type melanoma, small cell lung cancer, ovarian, and colon cancer cell lines and mouse xenografts via the formation of DNA double-strand breaks during S-phase [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 4124.

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 &amp;lt; 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&amp;lt; 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 &amp;lt; 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 6371: Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo

Abstract Nitric Oxide (NO) acts as a signaling molecule in the body in different pathologic conditions, including cancer. Beyond Air is developing an innovative gaseous Nitric Oxide (gNO) based tumor ablation method. The highly diffusible nature of NO may ease its spreading inside the tumor, while its short half-life may limit its effect to the tumor site and not affect the surrounding area. According to our preliminary data, exposure of gNO at 10,000-50,000ppm for a very short time (in some cases one minute or less) efficiently eradicates breast, colon, lung and prostate cancers and shows a potent mouse tumor's destruction in-vivo. Methods: Mouse colon and breast cancer cell lines were exposed to gNO at 150-50,000 ppm for 10 seconds - 180 seconds. Cell viability was examined 24hrs later by XTT assay. Mouse colon tumors were treated by gNO at 10,000ppm - 50,000ppm intratumorally by hypodermic needles or local exposure of the tumor using a plastic box or tube. Results: To test if gNO at 150-50,000ppm can decrease cancer cell viability, cells were exposed (I) directly to NO in a 96 well plate or (II) in an acrylic glass box. Cell viability was tested 24hrs later by XTT. According to our findings, less than 3% of 4T1 cells remain viable when exposed to 10,000ppm NO for 3 minutes or 50,000ppm NO for 1 minute in a 96-well plate. ~1% of CT26 cells remain viable after exposure to 50,000ppm NO for 10 seconds in a 96 well plate; while less than 10% remain viable after 1 minute of exposure of 50,000ppm NO in a box. To test if gNO at 10,000-50,000ppm can destroy mouse colon cancer tumors in-vivo, tumors were exposed to gNO locally by hypodermic needles or a plastic cover. Air treated tumor bearing mice served as the control group. A complete tumor destruction was observed when the skin was removed from the tumor and the entire tumor was exposed to 10,000ppm gNO for 10 minutes. Conclusion: In the current work, gNO at high concentration showed a substantial cytotoxic effect on cancer cells in-vitro and in-vivo. The proposed project represents development of a novel treatment for cancer. Our innovative gNO based treatment may serve to treat solid tumors locally or be used as an adjunctive therapy for surgery and can be synergized with common anticancer therapies, such as chemotherapy and radiotherapy, to further enhance the effect. Citation Format: Hila Confino, Steve Lisi, Rinat Kalaora, Matan Goldshtein, Amir Avniel. Gaseous nitric oxide at high concentration is a powerful anti-tumor agent both in-vitro and in-vivo [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 6371.

Abstract 4755: Chemoprevention and pharmacokinetics study using Gasdermin D knock out induced colon cancer model mouse treated with FAS inhibitors

Abstract (Introduction) It is well known that cancer cell harness different metabolic pathway from normal cells more efficiently. Recent studies indicate disorder of lipid metabolism has happen even in the benign cell, and that cause to be malignancy. Our research group focus attention on irregular accumulation of fatty acid in cancer cells. This time, we analysis several studies using Gasdermin D knock out induced colon cancer model mouse treated with FAS inhibitors. 1) metabolism analysis for mouse colon cancer. 2) Colon cancer treatment and chemoprevention by FAS inhibitor 3)Pharmacokinetics status of FAS inhibitor (colon cancer model mouse) Gasdermin D (GSDM D), pylotosis key factor, knockout mouse treat with AOM and DSS. 3month after all mouse has several big tumors (patent in Japan 2016-69775) (Result) 1) There are Many famous long chain fatty acids in cancer tissue. And also Very Long Chain Fatty Acid (:VLCFA) can detected by this analysis. 2) C75 and Kampo (include Berberine) can decrease tumor number and size. And they has chemoprevention effects, too. 3) Almost 40% FAS activity can reduce by everyday FAS inhibitor treatment. (Conclusion) AS metabolic disorders seem to induce colon cancer carcinogenesis, this target therapy can work for that prevention and treatment. Citation Format: Hajime Orita, Hyeoncheol Lee, Toshiyuki Kobayashi, Tetsu Fukunaga. Chemoprevention and pharmacokinetics study using Gasdermin D knock out induced colon cancer model mouse treated with FAS inhibitors [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 4755.

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 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.

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.

Apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression

Increasing studies confirm that anti-angiogenesis can increase the effectiveness of immunotherapy. In this study, we found that an angiogenesis inhibitor apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Apatinib treatment upregulated PD-L1 expression in various colon cancer cells both at the mRNA and protein levels. Further, apatinib-treated cancer cells hampered activation and IFN-γ secretion of T cells in the co-culture system, which was reversed by the anti-PD-1 antibody. Based on this, the combination of apatinib with anti-PD-1 on colon cancer growth in mice was examined. The combination treatment showed more significant inhibition on the growth of transplanted tumors in mice than single-drug treatment. Overall, our study here showed the enhancement of anti-PD-1 antitumor efficacy in a syngeneic mouse model (CT-26 cells in Balb/c) by the angiogenesis inhibitor apatinib via upregulating PD-L1 expression as well as angiogenesis inhibition, which may provide a rationale for the combination of apatinib and anti-PD-1 antibody for colorectal cancer treatment in the clinic.

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.