CT26 Colorectal Cancer Cells Research Articles

Improved Antitumor Efficacy of Combined Vaccine Based on the Induced HUVECs and DC-CT26 Against Colorectal Carcinoma.

Angiogenesis is essential for the development, growth, and metastasis of solid tumors. Vaccination with viable human umbilical vein endothelial cells (HUVECs) has been used for antitumor angiogenesis. However, the limited immune response induced by HUVECs hinders their clinical application. In the present study, we found that HUVECs induced by a tumor microenvironment using the supernatant of murine CT26 colorectal cancer cells exerted a better antiangiogenic effect than HUVECs themselves. The inhibitory effect on tumor growth in the induced HUVEC group was significantly better than that of the HUVEC group, and the induced HUVEC group showed a strong inhibition in CD31-positive microvessel density in the tumor tissues. Moreover, the level of anti-induced HUVEC membrane protein antibody in mouse serum was profoundly higher in the induced HUVEC group than in the HUVEC group. Based on this, the antitumor effect of a vaccine with a combination of induced HUVECs and dendritic cell-loading CT26 antigen (DC-CT26) was evaluated. Notably, the microvessel density of tumor specimens was significantly lower in the combined vaccine group than in the control groups. Furthermore, the spleen index, the killing effect of cytotoxic T lymphocytes (CTLs), and the concentration of interferon-γ in the serum were enhanced in the combined vaccine group. Based on these results, the combined vaccine targeting both tumor angiogenesis and tumor cells may be an attractive and effective cancer immunotherapy strategy.

Egg white coated alginate nanoparticles with electron sprayer for potential anticancer application

To development biomimetic strategy for enhanced cancer therapy, the paclitaxel loaded egg white/sodium alginate nanoparticles were prepared by electronic spray method. Their appearance and particle size were observed by transmission electron microscope and particle size analyzer. The drug release behavior of nanoparticles was determined by high performance liquid chromatography (HPLC). The toxicity, morphology and interaction of nanoparticles to cells were studied by means of MTT, live/dead staining and laser scanning confocal microscopy. The results show that the nanoparticles have good particle size dispersion, regular morphology, good drug release performance, good biocompatibility and in vitro effective inhibition of CT26 colorectal cancer cells. These results demonstrated that the good biocompatibility of egg white coated sodium alginate nanoparticles and PTX loaded these nanoparticles would show potential application for cancer therapy.

Inhibition of colorectal cancer liver metastasis in BALB/c mice following intratumoral injection of oncolytic herpes simplex virus type 2 for the induction of specific antitumor immunity

Liver metastasis represents the most prominent metastasis of colorectal cancer (CRC) and is the leading cause of CRC mortality, making the early prevention of this event very important. While current CRC therapies include surgery, radiotherapy and chemotherapy, no effective treatment option for CRC liver metastasis (CRLM) exists. Furthermore, the effects of currently available metastatic CRC drugs are frequently limited by their toxicity and side effects. Oncolytic herpes simplex virus type 2 (oHSV2) selectively infects tumor cells and also induces an antitumor immune response. The present study investigated the cytopathic effects of oHSV2 on CT-26 cells in vitro and tested its inhibitory effect on CRLM. In vitro experimental data demonstrated that oHSV2 effectively inhibited the growth of CT-26 cells. In vivo study data demonstrated that treatment with oHSV2 alone slowed the growth of subcutaneous xenograft tumors without inducing weight loss and also inhibited CRLM by increasing the numbers of cluster of differentiation (CD)4+ T, CD8+ T and natural killer cells. In summary, oHSV2 shows potential as a safe and effective therapeutic agent for inhibiting the metastasis of CT-26 CRC cells to the liver.

Stromal Cell PD-L1 Inhibits CD8+ T-cell Antitumor Immune Responses and Promotes Colon Cancer.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFα-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8+ granzyme B-secreting T cells in vitro, and the inhibition of CD8+ effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome from human colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8+ antitumor immune responses and potentiation of colorectal cancer progression. Cancer Immunol Res; 6(11); 1426-41. ©2018 AACR.

Enhanced Therapeutic Activity of Non-Internalizing Small-Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX in Combination with Targeted Interleukin-2.

Purpose: Antibody-drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues.Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload.Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX.Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Clin Cancer Res; 24(15); 3656-67. ©2018 AACR.

Effects of β-caryophyllene on arginine ADP-ribosyltransferase 1-mediated regulation of glycolysis in colorectal cancer under high-glucose conditions.

Type2 diabetes mellitus (T2DM) is associated with an increased risk of the development of colorectal cancer(CRC). A previous study revealed that the levels of arginine-specific mono-ADP-ribosyltransferase1 (ART1) in CRC tissues from patients with T2DM were higher than in non-diabetic patients. Hyperglycemia, which is a risk factor of cancer, is a common feature of T2DM; however, the effects of ART1 on glycolysis and energy metabolism in CRC cells under high-glucose conditions remains to be elucidated. β-caryophyllene (BCP) has been reported to exert anticancer and hypoglycemic effects. In the present study, CT26 cells were cultured under a high-glucose conditions and the expression levels of relevant factors were detected by western blotting. Cell Counting Kit-8 assay, flow cytometry, Hoechst33258 staining, ATP assay and lactic acid assay were used to detect the proliferation, apoptosis and energy metabolism of CT26 cells. To observe the effects of ART1 and BCP on tumor growth invivo, CT26 cell tumors were successfully transplanted into BALB/c mice with T2DM. The results demonstrated that overexpression of ART1 may increase glycolysis and energy metabolism in CT26 CRC cells under high glucose conditions by regulating the protein kinaseB/mammalian target of rapamycin/c‑Myc signaling pathway and the expression of glycolytic enzymes. BCP inhibited the effects induced by ART1, which may be due to a BCP-induced reduction in the expression levels of ART1 via nuclear factor-κB. Therefore, ART1 may be considered a therapeutic target for the treatment of diabetic patients with CRC.

Non-thermal plasma induces immunogenic cell death in vivo in murine CT26 colorectal tumors

ABSTRACTImmunogenic cell death is characterized by the emission of danger signals that facilitate activation of an adaptive immune response against dead-cell antigens. In the case of cancer therapy, tumor cells undergoing immunogenic death promote cancer-specific immunity. Identification, characterization, and optimization of stimuli that induce immunogenic cancer cell death has tremendous potential to improve the outcomes of cancer therapy. In this study, we show that non-thermal, atmospheric pressure plasma can be operated to induce immunogenic cell death in an animal model of colorectal cancer. In vitro, plasma treatment of CT26 colorectal cancer cells induced the release of classic danger signals. Treated cells were used to create a whole-cell vaccine which elicited protective immunity in the CT26 tumor mouse model. Moreover, plasma treatment of subcutaneous tumors elicited emission of danger signals and recruitment of antigen presenting cells into tumors. An increase in T cell responses targeting the colorectal cancer-specific antigen guanylyl cyclase C (GUCY2C) were also observed. This study provides the first evidence that non-thermal plasma is a bone fide inducer of immunogenic cell death and highlights its potential for clinical translation for cancer immunotherapy.

Abstract 758: Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins

Abstract In an attempt to improve the therapeutic index of cancer chemotherapy, monoclonal antibodies and small organic ligands have been proposed as delivery vehicles, allowing the construction of antibody-drug conjugates (ADCs) and of small molecule-drug conjugates (SMDCs). Four ADC products have gained marketing authorization for cancer therapy, while SMDCs are still being investigated in clinical trials. SMDCs could in principle offer certain advantages compared to ADCs, such as a more rapid and uniform diffusion into the tumor mass, lower cost-of-goods and lack of immunogenicity, but a direct head-to-head comparison has not yet been reported. Here we describe a novel small molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product features a peptide linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. We compared the therapeutic efficacy of the novel SMDC and of an anti-CAIX ADC, which were injected at equimolar doses in nude mice bearing SKRC-52 renal cell carcinoma xenografts. Both products showed a potent anti-cancer activity, although no complete tumor eradication was observed. Instead, when the SMDC was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering interleukin-2 to the tumor neo-vasculature), all treated mice in the combination group could be rendered tumor-free, in a process which favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously-grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. These findings may be of clinical significance, since carbonic anhydrase IX is over-expressed in the majority of clear-cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of interleukin-2 helps potentiate the action of targeted cytotoxics leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Citation Format: Samuele Cazzamalli, Alberto Dal Corso, Fontaine Widmayer, Dario Neri. Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 758.

Abstract 72: Radiation induces LAP, latency-associated peptide of TGF-beta, on the surface of lymphoid cells in the tumor microenvironment

Abstract LAP, latency-associated peptide of TGFβ, is a protein that is co-synthesized with TGFβ and serves to effectively cage the cytokine, holding it in a latent state in the extracellular matrix and on the surface of immunosuppressive cell types. In the tumor microenvironment, activation of the LAP-TGFβ complex by integrins or proteases releases mature TGFβ, producing an immunosuppressive environment and fostering EMT. Anti-LAP antibodies have been shown to be effective at slowing tumor growth in mouse models (Gabriely et al., 2017) and offer a promising new approach to treating cancer. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and through reducing the number of LAP+ inhibitory cells. Radiation is used as a first line therapy for many forms of cancer and is known to be a potent inducer of TGFβ production. This study was designed to determine the effects of radiation on the expression of LAP in the tumor microenvironment. 30 Balb/c mice were inoculated sc in the right flank with 106 CT26 colorectal cancer cells. When the mean tumor volume was approximately 300 mm3, half of the mice received a single dose of 20 Gy of targeted ionizing radiation. Groups of 3 mice from each cohort were sacrificed 1, 2, 3, 7 and 14 days after radiation and spleen and TILs collected for analysis by flow cytometry. As expected, radiation reduced tumor growth, with mean tumor volumes of 250 mm3 at day 14 vs 3600 mm3 in the non-irradiated control group. This was accompanied by a marked increase in CD45+ cells in the tumor which started on day 2 post-radiation and peaked on day 7, when 84% of live cells recovered from tumors were CD45+ (vs 25% in the non-irradiated controls). Radiation induced a significant (p<0.001) increase in CD8+ T cell numbers in the tumor, and prevented the increase in M2 macrophage numbers that occurred in the non-irradiated tumors. Radiation increased LAP expression on multiple cell types. Most notably, expression of LAP on DC and M2 macrophages increased steadily in response to radiation, with 33% of DC and 64% of M2 macrophages expressing LAP, significantly more than in control animals (10%, p=0.007 and 25%, p=0.01, respectively). In addition, 30% of M-MDSC from irradiated tumors expressed LAP, compared to 6% of non-irradiated tumors (p=0.03); expression on M-MDSC peaked at day 7. These data are the first demonstration that radiation increases cell-associated latent TGFβ in the tumor microenvironment. Radiation significantly altered both the distribution of immune cells in the tumor microenvironment and the expression of LAP on the surface of those cells. The increased LAP/TGFβ expression is anticipated to offset the effects of increased CD8+ effector cells. These observations suggest testing the use of an anti-LAP antibody in combination with radiation therapy to enhance effective immune responses against the tumor. Citation Format: Stavros Kopsiaftis, Xiufeng Song, Patricia E. Rao, Barbara S. Fox. Radiation induces LAP, latency-associated peptide of TGF-beta, on the surface of lymphoid cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 72.

Human serum albumin binding and synergistic effects of gefitinib in combination with regorafenib on colorectal cancer cell lines

This study aimed to evaluate the combination effect of gefitinib (GEF) and regorafenib (REG) against HCT116, CT26 and SW948 colorectal cancer cell lines. Results showed synergistic effects on HCT116 and CT26 cells, while the additive effect was observed on SW948 cells. Combination of GEF and REG induced sub-G1 peak as the apoptotic population on HCT116 cells, through flow cytometry histogram. Downregulation of AKT1 and TGFB2 and upregulation of CASP3 were observed in the combination of GEF and REG in HCT116 cells, using quantitative real-time PCR analysis. HSA binding properties exhibit that the first drug increased binding affinity between the second drug and HSA; as a result, HSA could transport both drugs. Thus, we hope this study creates a promising strategy to treat colorectal cancer.

MiR-15a regulation of endothelial radiation sensitivity in colorectal cancer.

709 Background: Radiation dose escalation causes significant changes within the tumor microenvironment (TME) to enhance tumor cell death including altered microRNA (miR) levels. Among endothelial miRs, we identified miR-15a exhibits dose dependent differential regulation. miR-15a targets a key determinant of endothelial cell (EC) radiosensitivity, acid sphingomyelinase (SMPD1), an enzyme that drives rapid EC apoptosis via enhanced ceramide production. In colorectal cancer (CRC) (n = 182 patients), high miR-15a is associated with worse 5-year progression free and overall survival. miR-15a also affects immune function by promoting a pro-inflammatory TME milieu. We hypothesized miR-15a inhibition will increase tumor cell death through preservation of EC SMPD1, enhancing endothelial apoptosis and inflammatory cytokine upregulation. Methods: Using TaqMan Human miR panels, miRs were profiled in human umbilical vein ECs (HUVECs) after single 2 vs 20 Gy treatment. miR-target prediction programs identified miRs targeting SMPD1. In vitro gain and loss of function studies were performed with miR transfections in HUVECs and CT26 CRC cells. CXCL10 expression was measured by qRT-PCR. Caspase 1 activation was measured by a luminescence based assay. A CT26 syngeneic CRC flank murine model was used for in vivo miR-15a inhibitor assessment administered via tail vein injection unencapsulated or encapsulated in vascular-targeted 7C1 nanoparticles. Results: Among miRs targeting SMPD1, miR-15a exhibited the greatest differential change in HUVECs 6h post-IR between low and high dose radiation. Lower dose was associated with higher miR-15a and vice versa. Further, miR-15a levels inversely correlated with SMPD1. Exogenous miR-15a significantly decreased SMPD1 mRNA and protein. miR-15a inhibition decreased proliferation in both HUVECs and CT26 cells and increased apoptosis when combined with radiation. miR-15a inhibition increased endothelial CXCL10 expression and caspase-1 activation. Both systemic and vascular-targeted miR-15a inhibitor significantly diminished tumor growth in vivo. Conclusions: Our data suggests inhibition of vascular miR-15a is sufficient to decrease tumor growth likely due to rescue of endothelial SMPD1.

Non-Thermal Plasma Induced Immunogenic Cell Death In Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal diagnosis, with a 5-year survival rate of approximately 7%. PDAC often presents late, when the only curative option, surgery, in no longer viable. Even after curative surgery in patients with resectable disease, early recurrence and disease progression often occur. Non-thermal plasma therapy represents a novel, non-invasive, immunotherapeutic approach for management of PDAC. We have previously reported plasma induced immunogenic cell death (ICD) of CT-26 colorectal cancer cells in vitro. Moreover, we have shown, that a whole-cell vaccine produced by in vitro treatment of cancer cells with plasma has resulted in protective immunity in a mouse model of colorectal cancer. Here we tested the ability of plasma to induce ICD in a radiation and multi-drug resistant human epithelial pancreatic adenocarcinoma cell line, Panc-02, with a nanosecond pulsed dielectric barrier discharge (nspDBD). We report (Figure 1) decreased cell viability in a dose-dependent fashion in vitro. Additionally, ICD, as evidenced by increased expression of Calreticulin (CRT), a damage associated molecular pattern (DAMP) protein, is shown in Table 1. Mitoxantrone (MTX) is a candidate positive control for ICD in the Panc-02 cell line with a 45-fold increase in CRT expression associated with an approximate 20-25% decrease in cell viability. Cisplatin resulted in a 2-fold increase in CRT expression and an approximate 20-25% decrease in cell viability. The cell viability results were dose-independent for both MTX and Cisplatin. Vaccination results of C57B/6 mice with plasma treated Panc-02 cells will be presented. Download : Download high-res image (212KB) Download : Download full-size image Figure 1: Plasma Induced Panc-02 cell viability via PI assay.

Effects of EphB4 receptor expression on colorectal cancer cells, tumor growth, vascularization and composition

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide. Increased expression of the molecular target, EphB4 receptor, has been observed in several cancer types. However, studies on the role of EphB4 receptor in CRC have yielded contradictory results. The aim of this study was to investigate the influence of EphB4 expression levels on CRC cell behavior and its contribution to tumor growth and vascularization.Methods: SW480, LIM2405 and CT26 CRC cell lines were transfected with EphB4 expression vector. High EphB4 expressing cells were compared to low EphB4 expressing empty vector controls. Proliferation and migration assays as well as EphrinB2-Fc cell stimulations were conducted in vitro and subcutaneous xenografts of CRC were analyzed in vivo.Results: High EphB4 expression enhanced migratory ability of these CRC cell lines in vitro and contributed to a significant increase in tumor growth and vascularization in vivo. Tumours induced with high EphB4 expressing SW480 and LIM2405 cells yielded homogenous masses densely packed with cancer cells. EphrinB2-Fc cell stimulations induced cell clustering of high EphB4 expressing SW480 and LIM2405 in vitro.Conclusion: These results suggest that with enhanced vascularization and an increase in migratory abilities, the high EphB4 expressing cells may be able to metastasize more readily.

Dependence of Immunogenic Modulation of Tumor Cells by Proton Radiation on the Linear Energy Transfer

Radiation therapy has been shown to induce immune-mediated tumor regression by multiple mechanisms. One of the mechanisms is to trigger an immunogenic phenotype characterized by upregulation of cell surface immune co-stimulators, antigen presenting machinery and checkpoint molecules that primes cells for subsequent elimination by cytotoxic T cells. The LET of the proton beam varies with the depth of dose deposition. Prior studies comparing proton radiation to photon radiation have failed to identify a stronger immunogenic phenotype with protons. However, the linear energy transfer (LET) was comparable for both modalities. We asked whether immunogenic phenotype induction would be different with higher LET, one that is typically noted towards the end of the Bragg peak of protons. Murine CT26 colorectal cancer cells were irradiated with 8Gy proton beams at 1.09keV/um (low), 2.58keV/um (medium) and 7.7keV/um (high) LET. 48hours after irradiation the cells were stained for surface expression of OX40L, ICOSL, CD40, CD70, 41BBL (immune co-stimulators), MHC-I, Calreticulin (antigen presenting machinery), ICAM-1 (cell adhesion molecule), PDL-1, CTLA-4 (immune checkpoint molecules) using a multiplexed flow staining panel. The treated cells were also plated on 8 well glass chamber slides and immunofluorescence (IF) staining for 41BBL, OX40L, CD40, MHC-1 and calreticulin was evaluated. Freshly extracted mouse splenocytes were used as biological positive control for both flow cytometry and immunofluorescence. We observed increase in percentage expression of OX40L, CD40, ICAM-1 and MHC-I, and drop in PDL-1 expression in the cells irradiated in the high LET region compared to low LET region of the proton beam as shown in the following table.Abstract 3444Treatment groupsMarker % expressionControl8Gy Low LET8Gy Medium LET8Gy High LETOX40L0.70.2360.6187.79CD4073.1383.292.5596.73MHC-I91.1195.1195.998.62ICAM-13.125.660.9627.42PDL-110.3256.4650.2138.36 Open table in a new tab This data is the average of independent experiments performed under same conditions. The other immune markers did not show statistically significant difference between the low and high LET proton radiation although the expression increased compared to the untreated controls. There was increased expression of 41BBL, OX40L, CD40, Calreticulin and MHC-I in the high LET group compared to low LET on immunofluorescence staining, which was assessed qualitatively. High LET proton radiation can be used to stimulate better immunogenic phenotype in tumor cells compared to low LET proton radiation. This preliminary data will form the foundation for future in vivo testing on animal models, to see if high LET proton therapy improves the local control and elicits a stronger systemic immune response compared to low LET radiation.

Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72 hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established.

Identification and Characterization of MEDI4736, an Antagonistic Anti–PD-L1 Monoclonal Antibody

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

Vascular Endothelial Growth Factor C Disrupts the Endothelial Lymphatic Barrier to Promote Colorectal Cancer Invasion

Colorectal cancer (CRC) is highly metastatic. Metastases spread directly into local tissue or invade distant organs via blood and lymphatic vessels, but the role of lymphangiogenesis in CRC progression has not been determined. Lymphangiogenesis is induced via vascular endothelial growth factor C (VEGFC) activation of its receptor, VEGFR3; high levels of VEGFC have been measured in colorectal tumors undergoing lymphangiogenesis and correlated with metastasis. We investigated VEGFC signaling and lymphatic barriers in human tumor tissues and mice with orthotopic colorectal tumors. We performed immunohistochemical, immunoblot, and real-time polymerase chain reaction analyses of colorectal tumor specimens collected from patients; healthy intestinal tissues collected during operations of patients without CRC were used as controls. CT26 CRC cells were injected into the distal posterior rectum of BALB/c-nude mice. Mice were given injections of an antibody against VEGFR3 or an adenovirus encoding human VEGFC before orthotopic tumors and metastases formed. Lymph node, lung, and liver tissues were collected and evaluated by flow cytometry. We measured expression of vascular endothelial cadherin (CDH5) on lymphatic vessels in mice and in human intestinal lymphatic endothelial cells. Levels of podoplanin (a marker of lymphatic vessels), VEGFC, and VEGFR3 were increased in colorectal tumor tissues, compared with controls. Mice that expressed VEGFC from the adenoviral vector had increased lymphatic vessel density and more metastases in lymph nodes, lungs, and livers, compared with control mice. Anti-VEGFR3 antibody reduced numbers of lymphatic vessels in colons and prevented metastasis. Expression of VEGFC compromised the lymphatic endothelial barrier in mice and endothelial cells, reducing expression of CDH5, increasing permeability, and increasing trans-endothelial migration by CRC cells. Opposite effects were observed in mice and cells when VEGFR3 was blocked. VEGFC signaling via VEGFR3 promotes lymphangiogenesis and metastasis by orthotopic colorectal tumors in mice and reduces lymphatic endothelial barrier integrity. Levels of VEGFC and markers of lymphatic vessels are increased in CRC tissues from patients, compared with healthy intestine. Strategies to block VEGFR3 might be developed to prevent CRC metastasis in patients.

Isolation of colorectal cancer stem-like cells.

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.

Intraperitoneal administration of a tumor-associated antigen SART3, CD40L, and GM-CSF gene-loaded polyplex micelle elicits a vaccine effect in mouse tumor models.

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.

HOCl Oxidation-modified CT26 Cell Vaccine Inhibits Colon Tumor Growth in a Mouse Model

Despite progress in elucidating mechanisms associated with colorectal cancer and improvement of treatment methods, it remains a frequent cause of death worldwide. New and more effective therapies are therefore urgently needed. Recent studies have shown that immunogenicity of whole ovarian tumor cells and subsequent T cell response were potentiated by oxidation modification with hypochlorous acid (HOCl) in vitro and ex vivo. These results prompted us to investigate the protective antitumor response with an HOCl treated CT26 colorectal cancer cell vaccine in an in vivo mouse model. Administration of HOCl modified vaccine triggered robust antitumor immunity to autologous tumor cells in mice and prolonged survival period significantly. In addition, increased necrosis and apoptosis were found in tumor tissue from the oxidation group. Interestingly, ELISPOT assays showed that specific T cell responses were not elicited in response to the immunizing cellular antigen, in contrast to raising sera antibody titer and antibody binding activity shown by ELISA assay and flow cytometry. Further evaluation of the mechanisms underlying HOCl modified vaccine mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results combined with previous studies suggest that HOCl oxidation modified whole cell vaccine has wide applicability as a cancer vaccine because it can target both T cell- and B cell-specific responses. It may thus represent a promising approach for the immunotherapy of colorectal cancer.