CT26 Colon Carcinoma Cells Research Articles

Radiation-induced change of PD-1/PD-L1 immune checkpoint in mouse colon cancer models

Abstract Background Radiotherapy (RT), a major part of anti-cancer treatment, directly kills tumor cells, and subsequent anti-tumor immune responses are up-regulated. However, immunologic impacts of RT on PD-1/PD-L1 immune checkpoint activity has not been much investigated. This study evaluated RT-induced alterations of the PD-1/PD-L1 checkpoint molecules based on murine colon carcinoma models. Methods CT26 colon carcinoma cell line was subcutaneously inoculated on the right hind leg of BALB/c mice. Based on tumor growth curves after irradiation of 15 Gy x 1 fx or 5 Gy x 3 fx, mouse tumors were surgically resected on 4 different time points: “Pre-RT”, non-irradiated status just prior to initiation of RT; “Early”, the early phase of RT response; “Nadir”, representing minimal tumor volume; and “Regrowth”, with regrown tumors after RT. PD-L1 expression on tumor cells, PD-1 expression on tumor-infiltrating CD4+ and CD8+ T cells, and proportions of tumor-infiltrating CD4+ and CD8+ T cell populations were estimated using flow cytometry analysis. Results PD-L1 expression on mouse tumor cells surged within a few days after completion of RT, followed by abrupt decreases on the “Nadir” and “Regrowth” phases (P Conclusions This study verified radiation-induced immunologic shift and dynamic change of the PD-1/PD-L1 checkpoint activity and tumor-infiltrating lymphocytes. The change was maximal at the early phase of RT response, which highlights the need of concurrent combinatory strategy of PD-L1 blockade and RT. Legal entity responsible for the study The author. Funding National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (NRF-2019R1C1C1007994). Disclosure The author has declared no conflicts of interest.

Engineering of a novel subnanomolar affinity fibronectin III domain binder targeting human programmed death-ligand 1.

The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5mg/L and a protein mass of 12kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38±0.26nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

1221P - Anti-PD-L1/IL-15 fusion protein generates robust adaptive immune gene signatures in tumours leading to tumour inhibition and memory responses

BackgroundWe have generated a therapeutic anti-PD-L1/IL-15 fusion protein (KD033) by combining a proprietary, fully human, high affinity anti-PD-L1 antibody with human IL-15. We have also generated a surrogate KD033 by combining an anti-mouse PD-L1 surrogate antibody with human IL-15. Both KD033 and KD033 surrogate significantly increased CD8+ T, NK and NKT cells in peripheral blood in monkeys and mice respectively. KD033 surrogate treatment resulted in tumor clearance and generated memory responses in syngeneic mouse models of colon carcinoma. We examined the extent of memory responses in these KD033 surrogate-treated tumor-free mice and evaluated the molecular mechanisms of KD033 surrogate efficacy. MethodsMouse CT26 colon carcinoma cells were grown subcutaneously in Balb/c mice prior to intravenous treatments with fusion proteins. Tumor-free mice were re-challenged with tumors from the same genetic background. For immune gene expression analysis, tumors were isolated 7 days after treatments and analyzed using the mouse PanCancer IO 360 Gene Expression Panel from Nanostring. ResultsKD033 surrogate treatment was associated with increased cytotoxic lymphocyte infiltrations in tumors and the microenvironment. One hundred percent (100%) of KD033 surrogate-treated tumor-free animals rejected the same tumor without any additional treatments. Mice re-challenged with unrelated tumors from the same background were rejected or demonstrated reduction of tumor growth without further treatments. KD033 surrogate-treated tumors showed significant increases in expression of genes involved in cytokine, adaptive and inflammatory pathways and upregulation of immune gene signatures involving not only cytotoxic cells but also dendritic and B cells. ConclusionsKD033 surrogate inhibited tumor growth inhibition and demonstrated evidence of epitope spreading. Broad activation of innate and adaptive immune responses in tumors was observed. These observations strongly support KD033 clinical development. A phase I clinical trial of KD033 is planned in 2H 2019. Legal entity responsible for the studyAnimal studies were conducted for Kadmon by Crown Bioscience Inc. with approved SOP and IACUC protocol. Nanostring platform analysis was done for Kadmon by Canopy Biosciences. FundingKadmon Corporation, LLC. DisclosureS. Martomo: Full / Part-time employment: Kadmon Corporation. X. Feng: Full / Part-time employment: Kadmon Corporation. D. Lu: Full / Part-time employment: Kadmon Corporation. Z. Polonskaya: Full / Part-time employment: Kadmon Corporation. X. Luna: Full / Part-time employment: Kadmon Corporation. M.V. Poyurovsky: Full / Part-time employment: Kadmon Corporation. K. McCracken: Full / Part-time employment: Kadmon Corporation. F. Miyara: Full / Part-time employment: Kadmon Corporation. L. Li: Full / Part-time employment: Kadmon Corporation. S. Aggarwal: Full / Part-time employment: Kadmon Corporation. J. Patel: Full / Part-time employment: Kadmon Corporation.

Dietary fructose-induced gut dysbiosis promotes mouse hippocampal neuroinflammation: a benefit of short-chain fatty acids

BackgroundWestern-style diets arouse neuroinflammation and impair emotional and cognitive behavior in humans and animals. Our previous study showed that a high-fructose diet caused the hippocampal neuroinflammatory response and neuronal loss in animals, but the underlying mechanisms remained elusive. Here, alterations in the gut microbiota and intestinal epithelial barrier were investigated as the causes of hippocampal neuroinflammation induced by high-fructose diet.ResultsA high-fructose diet caused the hippocampal neuroinflammatory response, reactive gliosis, and neuronal loss in C57BL/6N mice. Depletion of the gut microbiota using broad-spectrum antibiotics suppressed the hippocampal neuroinflammatory response in fructose-fed mice, but these animals still exhibited neuronal loss. Gut microbiota compositional alteration, short-chain fatty acids (SCFAs) reduction, intestinal epithelial barrier impairment, NOD-like receptor family pyrin domain-containing 6 (NLRP6) inflammasome dysfunction, high levels of serum endotoxin, and FITC-dextran were observed in fructose-fed mice. Of note, SCFAs, as well as pioglitazone (a selective peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist), shaped the gut microbiota and ameliorated intestinal epithelial barrier impairment and NLRP6 inflammasome dysfunction in fructose-fed mice. Moreover, SCFAs-mediated NLRP6 inflammasome activation was inhibited by histamine (a bacterial metabolite) in ex vivo colonic explants and suppressed in murine CT26 colon carcinoma cells transfected with NLRP6 siRNA. However, pioglitazone and GW9662 (a PPAR-γ antagonist) exerted no impact on SCFAs-mediated NLRP6 inflammasome activation in ex vivo colonic explants, suggesting that SCFAs may stimulate NLRP6 inflammasome independently of PPAR-γ activation. SCFAs and pioglitazone prevented fructose-induced hippocampal neuroinflammatory response and neuronal loss in mice. Additionally, SCFAs activated colonic NLRP6 inflammasome and increased DCX+ newborn neurons in the hippocampal DG of control mice.ConclusionsOur findings reveal that gut dysbiosis is a critical factor for a high-fructose diet-induced hippocampal neuroinflammation in C57BL/6N mice possibly mediated by impairing intestinal epithelial barrier. Mechanistically, the defective colonic NLRP6 inflammasome is responsible for intestinal epithelial barrier impairment. SCFAs can stimulate NLRP6 inflammasome and ameliorate the impairment of intestinal epithelial barrier, resulting in the protection against a high-fructose diet-induced hippocampal neuroinflammation and neuronal loss. This study addresses a gap in the understanding of neuronal injury associated with Western-style diets. A new intervention strategy for reducing the risk of neurodegenerative diseases through SCFAs supplementation or dietary fiber consumption is emphasized.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

Abstract B066: Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics

Abstract The immune system, which is profoundly affected by the microbiota composition and activity, plays an anti-tumorigenic role by surveying for and killing cancer cells. Indeed, recent studies found the microbiota to be essential for the success of cancer immunotherapy treatments in mice, while the bacterial effectors remain unknown. We developed an innovative high throughput screening system to isolate microbiota-derived effectors that modulate the ability of immune cells to kill cancer cells. We utilize the mouse CT26 colon carcinoma cell line as targeT-cells and the GSW11 specific CCD2Z T-cell hybridoma cell line as the effector cells. Bacterial DNA extracted from fecal contents of mice bearing colorectal tumors served for generation of metagenomics libraries. Supernatnants of the metagenomic clones were added to the CT26-CCD2Z co-culture and CCD2Z activation was measured using a LacZ reporter, regulated by NFAT. We identified about 40 clones that activate CCD2Z cells. We performed secondary screens and tested the activation of CD8+ T-cells sorted from mouse spleens. We sequenced the metagenomic DNA of several clones and analyzed the coding sequences. Currently, we are sub-cloning the clones to find the microbial genes responsible for CD8+ T-cell activation and design strategies for isolation of active molecules from these bacterial supernatnats. Finally, we hope that our future results could be taken to the clinic and serve for diagnosis, prognosis and therapeutics. Citation Format: Lior Lobel, Wendy S. Garrett. Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B066.

H2O2-responsive nano-prodrug for podophyllotoxin delivery.

The tumor microenvironment is different from that of normal tissue; therefore, the development of a prodrug that retains its efficacy in the tumor microenvironment can be useful in enhancing the anticancer properties of podophyllotoxin. An innovative podophyllotoxin prodrug (POD-PEG) was designed by linking podophyllotoxin to poly(ethylene glycol)(n) monomethacrylate with a H2O2-responsive oxalate ester bond. POD-PEG can self-assemble into stable nanoparticles (POD-PEG NPs). In vitro experiments demonstrated that the POD-PEG NPs can be activated by hydrogen peroxide resulting in podophyllotoxin release and are highly toxic against colon carcinoma CT26 cells. In vivo biodistribution studies demonstrate that PEGylated POD-PEG NPs are capable of prolonging blood circulation. Intravenous injection of POD-PEG NPs into CT26 tumor-bearing Balb/c mice resulted in a significantly enhanced therapeutic efficacy against tumors, with no significant systemic toxicity. Therefore, this H2O2-responsive prodrug delivery system exhibits good biosafety and provides a novel strategy for the development of drug delivery systems.

Knock-down the clock gene can lead to colon carcinoma CT26 cell proliferation arrest through p53-dependent pathway and c-myc gene

ABSTRACTColorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality of the world. Recently, the studies about over-expression circadian locomotor output cycles kaput gene will promote CRC progression and inhibit tumor cell apoptosis in vitro through the AKT-pathway (an antiapoptotic pathway have been reported). However, it remains to be studied the molecular mechanism of proliferation in CRC. In our present study, we attempt to study the clock gene which inhibits proliferation of CRC CT26 cells through p53-dependent pathway when the clock gene is suppressed in the CRC cell line CT26. Lentiviral vector binds to RNA target sequence, knocking down clock genes in CT26 cells with short hairpin RNA. For detecting their proliferation rates, we used CCK8 assay, plate clone formation assay, Western blot and mouse tumorigenicity assay. The results revealed that knocking down the clock gene has a negative effect on CT26 cell proliferation, depicted that low expression of clock gene reduced cylinD1 and c-myc expression, improved P21 and P53 expression in vitro and inhibited the growth of tumor in vivo. In conclusion, while silencing the clock gene can depress CT26 cell growth through p53-dependent pathway and c-myc.

Thermosensitive liposomes for triggered release of cytotoxic proteins

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells.Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Abstract 3811: Targeted Interleukin 2 and synergy with immune check-point inhibitors

Abstract Systemic high-dose Interleukin 2 (IL2) is able to elicit complete responses in patients suffering from metastatic melanoma. However, the majority of patients cannot receive the whole treatment dose due to severe toxicities. Other approved drugs for the treatment of metastatic melanoma include chemotherapeutics (e.g. Dacarbazine), a protein kinase B-raf inhibitor (Vemurafenib) and an immune check-point inhibitor (Ipilimumab, an anti CTLA-4 antibody). These drugs provide a clear benefit to patients, but cancer cures are still rare. Targeting IL2 to the site of disease represents a promising strategy to circumvent the toxicity and maximize the efficacy of this immunomodulatory payload. Various formats of targeted IL2 fusion proteins are currently in clinical development. Furthermore, recent successful approaches in the field of immunotherapy focus on the inhibition of check-point pathways, aiming to release the immunosuppressive brakes of the immune system. To explore the possibility of improving therapeutic efficacy of the single agents, a targeted version of IL2 (namely F8-IL2, where the F8 antibody recognizes the alternatively-spliced extra-domain A of fibronectin) was investigated in combination with an anti PD-1, anti PD-L1, or anti CTLA-4 antibody in an immunocompetent mouse model of CT26 colon carcinoma. In a first study, the combination of F8-IL2 and CTLA-4 blockade led to significant tumor growth retardation (curing 3/ 5 mice). Increasing the dose of F8-IL2 from 30 to 45 µg led to a 100 % cure rate with no signs of toxicity (body weight loss <5 %). Moreover, all cured mice acquired protective long-term immunity, which prevented tumor growth upon subsequent rechallenge with CT26 colon carcinoma cells. Furthermore, high dose F8-IL2 combined with the anti PD-1 antibody showed better therapeutic results than the respective monotherapies. In contrast, combination of F8-IL2 with the anti PD-L1 antibody did not improve the therapeutic outcome. Taken together, these data provide a strong rationale for the clinical use of targeted IL2 in combination with Ipilimumab. Citation Format: Cornelia Deborah Hutmacher-Berndt, Dario Neri. Targeted Interleukin 2 and synergy with immune check-point inhibitors [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 3811.

PO-428 Radiotherapy, immunocytokines and immune checkpoint inhibitors: finding the optimal combination

IntroductionAlthough immunotherapy is currently changing cancer treatment practice, primary resistance is still seen partly due to insufficient immunogenic priming. Radiotherapy (RT) induces the release of tumour antigens and thereby increases tumour immunogenicity. Therefore, combination of these therapies has high potential. Here, we investigated the therapeutic outcome of combining a single dose RT with either immunocytokines (L19-IL2) or immune checkpoints inhibitors (ICIs) (aCTLA-4, aPD-L1 and aPD-1), aiming to further activate and/or prolong the RT-elicited immune response.Material and methodsBalb/c or C57BL/6 mice were injected in the right flank with either C51 and CT26 colon carcinoma or Lewis lung carcinoma (LLC) cells. Upon an average volume of 200mm3, animals were randomised in different treatment groups: RT+vehicle/L19-IL2+IgG or RT+vehicle/L19-IL2+aPD L1/aPD1/aCTLA-4. Tumours were irradiated with 5Gy (C51 and CT26) or 10Gy (LLC), as the latter is known to be poorly immunogenic. Vehicle/L19-IL2 (1 mg/kg), aCTLA-4 and IgG (both 10 mg/kg) were given i.v. on day 1, 3 and 5 after RT; aPD-1, aPD-L1 and IgG (all 10 mg/kg) were given i.p. 1, 3, 5, 7 and 9 days after RT. Blood was collected before and after treatment for immune-profiling. Tumour response was quantified as time to reach 4 times starting tumour volume (T4xSV).Results and discussionsIn the CT26 model, RT +L19-IL2, RT +aCTLA-4 and RT +aPD L1 resulted in higher T4xSV compared to RT only (p<0.001, p<0.01 and p<0.05). In the C51 model solely RT +L19-IL2 was better (p<0.001) compared with RT only. RT +L19-IL2 efficacy was significantly (p<0.001) better compared to RT +ICIs in the C51, but not in the CT26 and LLC models. Adding ICIs to 5Gy+L19-IL2 did not result in improved outcome in either of the two models. Conversely, in the LLC model, the triple combination of RT +L19-IL2+aPD L1 yielded a better outcome compared to RT +L19-IL2 (p<0.05) and RT +aPD L1 (p<0.001). Similar results were observed for aCTLA-4 and aPD-1. Overall, we found an ICI-driven increase and decrease of circulating CD4 +and CD8+T cells respectively and an increase of PD-L1 associated with L19-IL2 therapy in all three models.ConclusionOur results show a larger therapeutic effect when RT is combined with L19-IL2 as compared to ICIs in the majority of the models, yet trimodal therapy was also beneficial in one model, revealing a tumor-model dependency of treatment. Immunological profiling of tumours is undergoing to explore the mechanisms underlying differential responses.

Abstract 1043: Patient-derived and cell line xenograft growth in the B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumor cell line xeno- (CDX) and allografts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data were also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models; however, the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, athymic nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in athymic nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in athymic nude mice. In study A, no tumor growth was seen in athymic nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models. Citation Format: Jamie L. Naden. Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [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 1043.

Abstract 3512: Impact of IDO inhibitor on tryptophan and kynurenine pathway reflected in the tumor microenvironment and highlighted using quantitative mass spectrometry imaging

Abstract Introduction: Indoleamine-2,3-dioxygenase (IDO1) is an enzyme which converts tryptophan (Trp) into kynurenine (Kyn) in many cell types. IDO1 has a critical role in immunosuppressive mechanisms that permit tumor cells to escape the immune system when Trp and Kyn level decrease and increase in the microenvironment, respectively. Therefore, IDO1 has been one of the first targets that have really been purposed for immuno-oncology (I-O) applications and immunotherapies development. A potent and selective IDO1 inhibitor such as Epacadostat (EPA) can significantly enhance the antitumor activity by restoring the body's natural ability to recognize and to fight cancer. In this study, using quantitative mass spectrometry imaging (QMSI, we went further than only quantifying the metabolites and the drug. Both target exposure and engagement studies were handled in CT26 mouse tumor model, plasma and whole blood. Methods: Undifferentiated colon carcinoma CT26 cell line was implanted into mice. Tumors, plasma and blood were sampled from two groups: one treated group (3 mice, 100mg/Kg of EPA 2 hours before sampling), and one control group of 2 mice. All samples were stored at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) matrix mixed to internal standards was sprayed onto tumor tissue sections of 10 µm thickness using the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) at 120 µm spatial resolution in full scan negative mode. Acquired data were treated with MultimagingTM software (ImaBiotech). Histological staining was applied afterwards. Quantitative results of Trp, Kyn and EPA in whole tumors, plasma and blood were then confirmed by LC-MS/MS analysis. Results: Using both QMSI and LC-MS/MS technologies, quantification was realized for the two endogenous metabolites and the drug. Thus, using a potent and selective IDO1 inhibitor, EPA, CT26 model validation was possible. As expected, a decrease of Kyn/Trp ratio was noticed in treated CT26 tumors (-24%), plasma (-57%) and blood (-45%) compared to control samples. By inhibiting IDO1 and decreasing Kyn in tumor cells, EPA increases and restores the proliferation, activation and regulation of various immune cells. This promoted infiltration of active immune cells into the tumor microenvironment is an important feature of many immunotherapies. It is marked by glycolysis signatures and adenosine-inosine metabolism. Citation Format: Lauranne Poncelet, Rima Ait-Belkacem, Bruno Gomes, Stefan Linehan, Gregory Hamm, Jonathan Stauber. Impact of IDO inhibitor on tryptophan and kynurenine pathway reflected in the tumor microenvironment and highlighted using quantitative mass spectrometry imaging [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 3512.

Abstract 5485: A promising pathway in immuno-oncology: CD73-adenosine axis highlighted by quantitative mass spectrometry imaging

Abstract Introduction The CD73-Adenosine (CD73-ADO) axis constitutes one of the most promising pathways in immuno-oncology. CD73 catalyzes the conversion of adenosine monophosphate (AMP) to ADO. CD73 is believed to play a role in mediating the inhibitory function of regulatory B and T cells. It is widely expressed and up-regulated in many cancerous tissues. The presence of extracellular ADO within tumor microenvironment has been described as an immunosuppressive halo surrounding the tumor, permeating the tumor microenvironment and preventing antitumor immunity. In this study, the endogenous level of AMP and ADO were analyzed by quantitative mass spectrometry imaging (QMSI) and LC-MS/MS in CT26 tumor mouse model. Clones with different CD73 expression levels were selected (low, medium &amp; high), which mimed different CD73-inhibitors efficacies. Then, ADO metabolic pathway was targeted in order to highlight the tumor microenvironment response linked to a mimic immunotherapy effect. Methods Undifferentiated colon carcinoma CT26 cell line was implanted into C57BL/6 mice. Then, tumors were harvested and frozen at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) and 2,5-dihydroxybenzoic acid (2,5-DHB) matrices mixed to internal standards were sprayed onto tumor sections of 10 µm thickness with the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) and analyses were set at 120µm spatial resolution in full scan positive and negative modes. Acquired data were treated with MultimagingTM software (ImaBiotech). All quantitative results were confirmed by LC-MS/MS analysis. Histological and immunohistochemistry staining anti-CD73 were applied on adjacent section. Results A validation of CT26 mouse model occurred since an increase of ADO/AMP ratio of 77% was relative to the high CD73 expression. In other words, CT26 mouse models with high CD73 expression showed higher ADO/AMP ratio (2.1) than CT26 medium (1.7) and low (1.6) clones. Then, as an immunosuppressive halo, the impact of ADO expression on tumor microenvironment was also characterized using MSI analysis. A large number of metabolites that are implicated in this pathway were highlighted (ADP, IMP, GMP, Guanosine, Inosine, Hypoxanthine, Xanthine, Uric acid⋯) and differences were also observed. Conclusion The combination of all these data extended our understanding on the relative contribution of ADO signaling in suppressing antitumor immunity. Because the development of immunotherapies such as CD73 inhibitor requires a deep understanding of the interplay between the immune system and cancer cells in the tumor microenvironment, these immune/tumor metabolites and nutrients can now be followed by QMSI as biomarkers of response to treatment towards enhancing immunotherapies efficacy. Citation Format: Lauranne Poncelet, Rima Ait-Belkacem, Bruno Gomes, Stefan Linehan, Jonathan Stauber. A promising pathway in immuno-oncology: CD73-adenosine axis highlighted by quantitative mass spectrometry imaging [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 5485.

Combination Treatment of Stereotactic Body Radiation Therapy and Immature Dendritic Cell Vaccination for Augmentation of Local and Systemic Effects.

PurposeThe purpose of this study was to investigate the efficacy of stereotactic body radiation therapy (SBRT) as a tumor-associated antigen (TAA) presentation method for dendritic cell (DC) sensitization and evaluate its effect in combination with immunotherapy using an intratumoral injection of immature DCs (iDCs).Methodsand MaterialsCT-26 colon carcinoma cell was used as a cancer cell line. Annexin V staining and phagocytosis assays were performed to determine the appropriate radiation dose and incubation time to generate TAAs. BALB/c mice were used for in vivo experiments. Cancer cells were injected into the right legs and left flanks to generate primary and metastatic tumors, respectively. The mice were subjected to radiation therapy (RT) alone, intradermal injection of electroporated DCs alone, or RT in combination with iDC intratumoral injection (RT/iDC). Tumor growth measurement and survival rate analysis were performed. Enzyme-linked immunospot and cytotoxicity assays were performed to observe the effect of different treatments on the immune system.ResultsAnnexin V staining and phagocytosis assays showed that 15 Gy radiation dose and 48 hours of incubation was appropriate for subsequent experiments. Maximum DC sensitization and T-cell stimulation was observed with RT as compared to other TAA preparation methods. In vivo assays revealed statistically significant delay in the growth of both primary and metastatic tumors in the RT/iDC group. The overall survival rate was the highest in the RT/iDC group.ConclusionThe combination of SBRT and iDC vaccination may enhance treatment effects. Clinical trials and further studies are warranted in the future.

PO-215 Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Introduction Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumour cell line xeno- (CDX) and allo-grafts in the R2G2 immunodeficient mouse model. Material and methods The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumour growth data was also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Results and discussions Colorectal PDX tissue was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in into 4 sections of R2G2 mice each, and 100% of mice developed either one or two tumours. The human esophageal adenocarcinoma OE33 cells were implanted into both flanks of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumour growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in R2G2 mice and take rate was 90%. Growth of two allogeneic tumour lines was also examined. The mouse colon carcinoma CT26 cells were implanted in R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. Conclusion These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumour models research, with high take rates and quick growth of allogeneic models.

Translating phototherapeutic indices from in vitro to in vivo photodynamic therapy with bacteriochlorins.

To compare hydrophilic and lipophilic bacteriochlorin photosensitizers in the photodynamic therapy of cancer, and relate their properties and in vitro phototoxicities to the efficacy of in vivo PDT treatments. Photochemical characterization of a hydrophilic bacteriochlorin (F2 BOH) photosensitizer, and its use in PDT was compared with the performance of a closely related but water-insoluble bacteriochlorin (F2 BMet or redaporfin). Biodistribution, pharmacokinetics, skin photosensitivity, PDT efficacy and immune responses of two bacteriochlorins were compared. PDT in vitro employed CT26 colon carcinoma cells. BALB/c mice bearing CT26 cells were treated according to a protocol where the illumination of the subcutaneous tumor is performed 15 minute after intravenous administration of the photosensitizer, while it is in the vascular compartment (vascular-PDT). F2 BOH has photochemical properties comparable to redaporfin and both are promising photosensitizers for PDT. Although, F2 BOH is 10 times less phototoxic in vitro than redaporfin, the phototoxicity of F2 BOH in vascular-PDT is comparable to that of redaporfin. This is consistent with the fact that the vasculature is the main target of vascular-PDT. F2 BOH-PDT led to long-term cures and stimulation of the immune system. F2 BOH is soluble in aqueous media, photostable, has a convenient elimination half-life of 44 hours and leads to very low skin photosensitivity one week after administration. F2 BOH and redaporfin are both very phototoxic in vascular-PDT, but this could not be anticipated from their widely different phototherapeutic indices in vitro. PDT with F2 BOH enabled long-term cures of BALB/c mice with subcutaneously implanted CT26 tumors, and the cured mice rejected tumor re-inoculation one year after the treatment. Lasers Surg. Med. 50:451-459, 2018. © 2018 Wiley Periodicals, Inc.

Patient‐derived and cell line xenograft growth in the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient‐derived (PDX) and tumor cell line xeno‐ (CDX) and allo‐grafts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data was also collected from two allografts of murine colorectal cancer (CT26) and B‐cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumor growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B‐cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel hydro- and lipo-philic selenium zinc(II) phthalocyanines: Synthesis, photophysical properties and photodynamic effects on CT26 colon carcinoma cells

The synthesis and photochemical properties of two novel selenium zinc(II) phthalocyanines, a lipid-soluble 2,9(10),16(17),23(24)-tetrakis [(2-dimethylamino)ethylselanyl]phthalocyaninato zinc(II) (4) and its water-soluble quaternized derivative 2,9(10),16(17),23(24)-tetrakis [(2- trimethylammonium)ethylselanyl]phthalocyaninato zinc(II) tetraiodide (5), were investigated. Maximum absorption values were 689 nm and 684 nm for 4 and 5 in DMF, respectively. In addition, phthalocyanines were revealed to be very efficient singlet oxygen generators with high values of ΦΔ 0.74 and 0.84 for 4 and 5 in DMF, and they were photostable over the irradiation times studied. The photodynamic effect were evaluated on CT26 colon carcinoma cells. After light exposure, 4 and 5 were found to be cytotoxic, and IC50 values were 0.5 ± 0.1 μM and 2.3 ± 0.6 μM, respectively. The production of a greater amount of reactive oxygen species after phthalocyanines irradiation would be responsible for its potent phototoxic action on CT26 colon carcinoma cells.

Acylated and unacylated ghrelin inhibit apoptosis in myoblasts cocultured with colon carcinoma cells.

Cancer cachexia is a life‑threatening syndrome associated with myofiber damage. Tumor factors impair muscle regeneration by promoting myoblast apoptosis. Ghrelin is a multifunctional hormone with an anti‑apoptotic effect, but its mechanism of action is not fully understood. In the present study, we investigated whether the coculturing of C2C12 myoblasts with CT26 colon carcinoma cells may induce myoblast apoptosis, and whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) may exert anti‑apoptotic effects. We found that the coculture induced myoblast apoptosis and increased tumor necrosis factor (TNF)‑α concentrations in the culture medium. Moreover, the coculture increased c‑Jun N‑terminal kinase (JNK) activity, suppressed Akt activity, increased the mitochondrial Bax/Bcl‑2 ratio, impaired mitochondrial membrane potential (Δψm), increased the cytosolic cytochromec levels, and activated the caspase‑3/poly (ADP‑ribose) polymerase (PARP) cascade in myoblasts. We also found that either AG or UnAG inhibited these changes. The present study describes a novel invitro model that can be employed to investigate cancer‑induced myoblast apoptosis, and our findings suggest a possible use for AG and UnAG in treating cancer cachexia.