Mouse Colon Adenocarcinoma Research Articles

Biosynthesis of cobalt oxide nanoparticles and their cytotoxicity against cancer cells mouse colon adenocarcinoma cell line

The application of plant extracts for the biosynthesis of cobalt oxide nanoparticles is eco-friendly, more economical, less toxic, and more biocompatible. In this study, we used Rose hip extract to synthesize cobalt oxide nanoparticles (Co3O4 NPs). The biosynthesized cobalt oxide nanoparticles were characterized by FT-IR spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM). The PXRD and FTIR analyses confirmed the successful synthesis and the characteristic bands of Co3O4 NPs, respectively. The FESEM image showed that the Co3O4 NPs were approximately spherical morphology with a mean size of 90.77 ± 32.35 nm. The EDX analysis showed the elemental composition contains carbon in the formulation of Co3O4 NPs. The results showed that the plant extracts can be used to synthesiz cobalt oxide nanoparticles with good quality at nanoscale sizes. The as-prepared Co3O4 NPs exhibited excellent cell toxicity properties against mouse colon adenocarcinoma cells, indicating that biosynthesized cobalt oxide nanoparticles have potential applications in cancer and nanomedicine.

Optimizing the Design and Geometry of T Cell-Engaging Bispecific Antibodies Targeting CEA in Colorectal Cancer.

Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.

Abstract 2835: Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing

Abstract Cancer cachexia is a wasting syndrome characterized primarily by loss of skeletal muscle and adipose tissue mass and contributes greatly to overall cancer-related mortality (for recent review see Argiles 2023). An estimated 25-75% of cancer patients develop cachexia which leads to impacts on therapeutic tolerability, well-being, and eventually overall survival (Bossi 2021). Some preclinical mouse and rat cancer models manifest cachexia as they progress. In this study we established a syngeneic (immune competent) and a xenograft (immune incompetent) tumor model of cachexia with the intention of comparing the models with respect to typical study endpoints and to provide a platform for testing novel therapeutic agents aimed at inhibiting or reversing the effects of cachexia. The Colon26 syngeneic mouse colon adenocarcinoma model was reported as an experimental cancer cachexia model by Tanaka, et. al. in 1990. The model was developed in male BALB/c × DBA/2 F1 (first generation cross) mice. We typically implant Colon26 in female BALB/c mice for preclinical drug testing of anticancer agents and noticed a strong tendency for mice bearing Colon26 tumors to develop signs of cachexia on study, but not always consistently. A review of the literature showed there are clear gender differences with male cancer patients having higher prevalence of cachexia and greater muscle loss compared with female patients (Zhong 2020). Based on this we implanted Colon26 tumors in male BALB/c mice to establish the cachexia model. The HT1080 human fibrosarcoma model HT1080 was characterized as a cachexic model by Bernardo, et. al. in female severe combined immunodeficiency disease (SCID) mice. We developed the HT1080 model in male SCID mice based on the literature and previous experience. Both models were characterized by multiple endpoints including overall body weight loss, skeletal muscle weight loss, reduction of adipose tissue, and cardiac muscle loss, as well as tracking a broad panel of relevant cytokine levels at study endpoint. Both models generate robust wasting of skeletal muscle and adipose tissue providing an opportunity to assess novel anti-cachexia therapies. Citation Format: Kyle Draheim, Diana Gietl, Karyn Shinn, William Durham, Fei Zhao, Caitlin Thompson, Alexis Baldwin, Glenn Harris, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2835.

Novel neoantigen peptides and tumor regression in the MC38 colorectal cancer model.

28 Background: Neoantigen vaccine is an emerging modality to treat cancer by unleashing cytotoxic T cells against tumor-specific mutant antigens. Neoantigens are de-novo changes in tumor cells and are hence more immunogenic and less susceptible to off-target toxicities. In building our own pipeline for neoantigen discovery, we used the mouse colon adenocarcinoma cell line MC38 as a validation model. Methods: We sequenced MC38 cells by whole exome and RNA sequencing to shortlist variants. Candidates were confirmed by sanger sequencing, then analyzed for binding affinity to MHC class I H2-Kb and H2-Db. We selected top 10 epitopes for facile modification to maximize immunogenicity by taking into account peptide characteristics, such as hydrophobicity. In-vivo immunogenicity was tested by subcutaneous injections of pooled peptides in C57BL/6J mice. Amongst the 10 peptides tested, we picked five that elicited greatest IFNγ secretion in re-stimulated splenocytes. Predicted binding affinity did not correlate with in-vivo immunogenicity. We inoculated MC38 cells into the left flank of C57BL/6J mice and started treatment four days later. Mice were injected twice weekly with PBS (group 1), CpG only (group 2), anti-PDL1 only (group 3), Peptide pool + CpG (group 4) and Peptide pool + CpG + anti-PDL1 (group 5) (n=8 per group). CpG ODN 1826 is a TLR9 agonist and acts as an adjuvant, while anti-PDL1 is an immune checkpoint inhibitor. We monitored tumor size, animal weights and food intake every 2-3 days. Results: In control group 1, tumor size exponentially increased by 36 folds on the 18th day of treatment. Groups 2, 3, 4 and 5 saw 16, 13, 8 and 5-fold increases in tumor size respectively, 21 days post-treatment. No tumors were found in 3/7 mice in group 5 upon sacrifice, suggesting tumor regression. Tumor control in groups 4 and 5 corroborated with significant IFNγ secretion from neoantigen re-stimulated splenocytes. We observed 38-45 fold expansion in the number of CD8+ KLRG1+ short-lived effector cells and significant increase in PD-1+ TIM3+ exhausted T cells in group 4 and 5 relative to control. Reps1 and Adpgk neoantigen peptides are widely reported and used in MC38 syngenic model. However, they were not included because both were undetected by sequencing. Cell line authentication by STR profiling confirmed that our cells matched that of MC38 from Kerafast. Conclusions: Using an in-house pipeline, we derived a set of novel neoantigen peptides that robustly controlled tumor growth in the MC38 tumor challenge model. In-silico binding predictions did not correlate with in-vivo patterns of IFNγ secretion. In-vivo tests for IFNγ induction after peptide vaccination is a better measure for potential in tumor control. Upregulation of T-cell exhaustion markers after peptide vaccination explains the improved response with anti-PDL1. Reps1 and Adpgk mutations were absent in our cells, which calls caution to researchers conducting similar research.

Abstract 2932: Datopotamab deruxtecan (Dato-DXd) enhances antitumor response to PD-1/PD-L1 inhibitors in TROP2-expressing tumors in mice

Abstract Background: Trophoblast cell surface antigen 2 (TROP2) is highly expressed in various epithelial tumors, including non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC), and correlates with poor prognosis. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I (Topo I) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd has shown encouraging antitumor activity and a manageable safety profile in patients with NSCLC and TNBC as part of the first-in-human, Phase 1 study (NCT03401385). To enhance clinical responses, clinical trials evaluating combination therapy of Dato-DXd with PD-1/PD-L1 inhibitors in NSCLC and TNBC are currently ongoing (e.g. NCT04526691 and NCT03742102). Here we report preclinical evidence for supporting increased antitumor activity of Dato-DXd in combination with PD-1/PD-L1 inhibitors using a syngeneic mouse tumor model and its impact on tumor immunity. Methods: MC38 mouse colon adenocarcinoma cells stably transfected with human TROP2 (hTROP2_MC38) were inoculated subcutaneously in immunocompetent C57BL6 mice or immunodeficient nude mice and the antitumor activity of Dato-DXd with or without mouse PD-1/PD-L1 inhibitors was evaluated. The tumor infiltrating immune cells in hTROP2_MC38 tumors were analyzed by flow cytometry at 3 days and 10 days after dosing. In addition, the impact of CD8+ T cell depletion on the antitumor activity of Dato-DXd against hTROP2_MC38 tumors in C57BL6 mice was evaluated. Results: Dato-DXd showed stronger antitumor activity against hTROP2_MC38 tumors in immunocompetent C57BL6 mice than in immunodeficient nude mice, which suggests that immune cells play an important role in the mechanism of action (MoA) of Dato-DXd. The combination of Dato-DXd and mouse PD-1/PD-L1 inhibitors enhanced antitumor activity compared to monotherapy against hTROP2_MC38 tumors in C57BL6 mice. Activation of the dendritic cells in hTROP2_MC38 tumors in C57BL6 mice was observed after Dato-DXd monotherapy or combination therapy with mouse PD-1/PD-L1 inhibitors, which led to the increase of tumor infiltrating CD8+ T cells. Involvement of CD8+ T cells in the MoA of Dato-DXd was also supported by the result that CD8+ T cell depletion in C57BL6 mice decreased the antitumor activity of Dato-DXd against hTROP2_MC38 tumors. In addition, activation of tumor infiltrating NK cells and macrophages was observed after Dato-DXd monotherapy or combination therapy, suggesting the potential effect on innate immunity. Conclusion: These results suggest that Dato-DXd may stimulate tumor immunity and sensitize tumors to PD-1/PD-L1 inhibitors. The combination of Dato-DXd and PD-1/PD-L1 blockers could be a valuable therapy for patients with TROP2-positive tumors. Citation Format: Daisuke Okajima, Satoru Yasuda, Takami Suzuki, Michiko Kitamura, Junko Yamaguchi, Takanori Maejima, Tsuyoshi Karibe, Tadashi Toki, Penny Phillips, Toshinori Agatsuma. Datopotamab deruxtecan (Dato-DXd) enhances antitumor response to PD-1/PD-L1 inhibitors in TROP2-expressing tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2932.

Abstract 1641: Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice

Abstract Interleukin 21 (IL21) binding to its receptor IL21R, results in downstream activation of multiple signaling molecules, including JAK1, JAK3, STAT1, and STAT3. Intriguingly, IL21 has been implicated in both positively and negatively regulating immune responses in a cell-type specific manner, suggesting drug candidates that target IL21/IL21R signaling has therapeutic potential. Specifically, recent reports suggest that antibodies targeting PD-1 fused to IL-21 has a synergistic anti-tumor effect. To generate a suitable animal model for in vivo assessment of a human anti-PD-1-IL21 fusion protein, Biocytogen developed humanized PD-1 and IL21R mice (B-hIL21R mice and B-hPD-1/hIL21R mice) by replacing the murine Il21r and PD-1 gene with the human IL21R and PD-1 counterparts via homologous recombination. Our data shows that human IL21R was detectable in sorted cells from homozygous B-hIL21R and B-hPD-1/hIL21R mice, while human PD-1 was detectable in B-hPD-1/hIL21R mice compared to wild type mice. Furthermore, STAT3 phosphorylation in splenocytes was successfully induced with mouse IL21 or human IL21 in homozygous B-hIL21R mice. In addition, percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hIL21R mice and B-hPD-1/hIL21R mice were similar to those in wild type mice, demonstrating that introduction of human IL21R and PD-1 genes in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. To evaluate in vivo efficacy of anti-mPD-1-hIL21 and anti-hPD-1-hIL21 fusion proteins, mouse colon adenocarcinoma MC38 cells were subcutaneously implanted into homozygous B-hIL21R mice (female, 6-7-week-old, n=6) or B-hPD-1/hIL21R mice (female, 6-7-week-old, n=6). Mice were grouped and treated when tumor volume reached approximately 100 mm3. Results showed that anti-PD-1-hIL21 fusion proteins were efficient at inhibiting tumor growth in B-hIL21R and B-hPD-1/hIL21R mice. Altogether, B-hIL21R and B-hPD-1/hIL21R mice are promising humanized mouse models for preclinical in vivo studies to assess novel drug candidates. Keywords: IL21/IL21R, humanized mice, fusion protein Citation Format: Ruili Lyu, Zhixia Li, Leila Kokabee, Jiawei Yao, Yanhui Nie, Chaoshe Guo. Evaluating in vivo efficacy of bi-functional fusion protein anti-PD-1-IL21 in humanized B-hIL21R mice and B-hPD-1/hIL21R mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1641.

Abstract 1642: Evaluating in vivo efficacy of anti-VSIG4 antibodies in humanized B-hVSIG4 mice

Abstract V-set and immunoglobulin domain containing 4 (VSIG4) protein is an immune checkpoint molecule that is related to the B7 family of immune regulatory proteins. B7 family members have been implicated in both positive and negative regulation of T cell activation, particularly VSIG4 is a known negative regulator of T cell proliferation and IL-2 production. Pathological consequences of VSIG4 expression are associated with inflammatory disorders, lung cancer development, high-grade glioma, and multiple myeloma, suggesting VSIG4 is a potential target for drug development. Therefore, Biocytogen developed humanized VSIG4 mice (B-hVSIG4 mice) by replacing the murine Vsig4 gene with the human VSIG4 gene through homologous recombination. Flow cytometry analysis showed human VSIG4 was detectable in peritoneal exudate macrophages (PEMs) in homozygous B-hVSIG4 mice compared to wild type mice. Additionally, percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, macrophages, CD8+ T cells, CD4+ T cells and Tregs in homozygous B-hVSIG4 mice were similar to those in wild type mice, demonstrating that introduction of human VSIG4 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. For in vivo efficacy evaluation of an anti-human VSIG4 antibody (Ab-A, synthesized in house) using B-hVSIG4 mice, mouse colon adenocarcinoma MC38 cells were subcutaneously implanted into homozygous B-hVSIG4 mice (female, 7-week-old, n=5). Mice were grouped and treated when the tumor volume reached approximately 100 mm3. Results showed that an anti-hVSIG4 antibody was efficient at inhibiting tumor growth in B-hVSIG4 mice. In conclusion, B-hVSIG4 mice are as a promising model for preclinical in vivo studies to assess pharmacodynamic of anti-hVSIG4 antibodies. Keywords: VSIG4, humanized mice, immune checkpoint molecule Citation Format: Ruili Lyu, Chengzhang Shang, Leila Kokabee, Yanhui Nie, Yi Yang. Evaluating in vivo efficacy of anti-VSIG4 antibodies in humanized B-hVSIG4 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1642.

Preclinical anti-tumor activity of small-molecule oral PD-L1 checkpoint inhibitors.

e14558 Background: The PD-1/PD-L1 checkpoint axis is a validated target in oncology, and immunotherapy with antibody approaches have proven efficacy across various tumor types. Oral small-molecule inhibitors of PD-L1 may offer advantages over antibody approaches by enabling tunable on-target engagement and the potential for better tissue penetration and improved efficacy. Here we report the preclinical in vitro activity of small-molecule inhibitors of PD-L1 possessing a novel mechanism of action, favorable pharmacokinetics, and demonstrated in vivo efficacy in a mouse colon adenocarcinoma model. Methods: In vitro activity was assessed in a Jurkat T cell NFAT reporter assay and PD-L1 reduction was confirmed in CHO-K1 cells expressing human PD-L1 (CHO-K1-hPD-L1) and in peripheral blood mononuclear cells (PBMCs) from healthy donors. T cell activation assays were conducted with PBMCs from healthy donors. Non-specific cytokine release was evaluated in human whole blood. Pharmacokinetic (PK) evaluations were conducted in rodents and non-human primates (NHP). In vivo efficacy was evaluated in an MC38 tumor humanized PD-L1 and PD-1 mouse model. Results: Small-molecule inhibitors of PD-L1 were able to disrupt PD-1:PD-L1 and PD-L1:CD80 interactions through the reduction of PD-L1 expression on the cell surface via a novel internalization mechanism, resulting in PD-L1 degradation (EC50s ranged from 1.9 – 24 nM in CHO-K1-hPD-L1 and primary human myeloid cells). Lead PD-L1 inhibitors mediated potent activation of T cells in a NFAT reporter assay (EC50s of 13 and 18 nM) as well as dose responsive elevations in IL-2 production in human PBMCs stimulated with staphylococcal enterotoxin B. Compound treatment did not elicit non-specific cytokine release in human whole blood, supportive of favorable immune safety. PK profiles showed low systemic clearance in rodents and NHP. In an MC38 tumor model, once daily oral administration of compounds at 3, 10, and 30 mg/kg resulted in dose responsive tumor reduction that was associated with T cell activation and infiltration into tumors. Conclusions: Oral small-molecule PD-L1 inhibitors possessing a novel mechanism of action and the ability to mediate T cell activation in primary human immune cell types were identified. These compounds display in vivo anti-tumor efficacy comparable to anti-PD-L1 antibody and possess favorable preclinical profiles for further development, with the potential for all-oral treatment regimens for oncology.

In silico and In vitro Investigation of a Likely Pathway for Anti-Cancerous Effect of Thrombocidin-1 as a Novel Anticancer Peptide.

Antimicrobial and antifungal activities of Thrombocidin-1 (TC-1) is shown previously, however,.the anti-cancerous feature of this peptide is still uncovered. The objective is to evaluate anti-cancerous feature of recombinant TC-1. In this study, based on the significant similarity of rTC-1 and IL-8 in case of coding sequence, tertiary structure, and also docking and molecular dynamic simulation (MD) results with CXCR1, a receptor which has positive correlation with different cancers, a likely pathway for anticancerous effect of rTC-1 was proposed. In addition, the coding sequence of TC-1+6xhistidine (rTC-1) was inserted into the pET22b(+) vector and cloned and expressed by E. coli BL21 and finally purified through nickel affinity column. Afterward, the retrieved rTC-1 was used in MTT assay against mouse colon adenocarcinoma, hepatocellular carcinoma, chondrosarcoma, mouse melanoma, and breast adenocarcinoma cell lines to investigate its probable anticancer application. Docking and MD simulation results showed that rTC-1 and IL-8 share almost the same residues in the interaction with CXCR1 receptor. Besides, the stability of the rTC-1_CXCR11-38 complex was shown during 100ns MD simulation. In addition, the successful expression and purification of rTC-1 depict an 8kD peptide. The IC50 results of MTT assay revealed that rTC-1 has cytotoxic effect on C26-A and SW1353 cancerous cell lines. Therefore, apart from probable anti-cancerous effect of rTC-1 on C26-A and SW1353 cell lines, this peptide may be able to mimic the anti-cancerous pathway of IL-8.

Targeted doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting against colorectal cancer

Exosomes hold great potential for cancer treatment to deliver therapeutics due to its inherent low immunogenicity. Exosomes are biocompatible cell-exocytosed secreted vesicles by most cell types, which can be used to construct novel biomanufacturing platform for drug delivery and cancer therapy. In this study, we implemented nano-sized vesicles which were secreted by mesenchymal stem cell (MSC), to encapsulate doxorubicin (DOX) through electroporation method (DOX@exosome). DOX was loaded into exosomes, with an encapsulation efficiency of up to 35% and separated by ultracentrifugation.Subsequently, carboxylic acid-end MUC1 aptamer was used to covalently decorate the surface amine groups of the exosomes via amide bond formation to provide selective guided drug delivery (DOX@exosome-apt).The data showed that the DOX@exosome-apt provided highly efficient DOX transportation to MUC1-positive cancer cells in vitro as confirmed by MTT and flow cytometry experiments. Moreover, in vivo study on ectopic model of C26 (mouse colon adenocarcinoma) in BALB/c mice indicated that the single dose intravenous injection of DOX@exosome-apt significantly suppress tumor growth in comparison with free DOX. Ex vivo fluorescent imaging also verified the desirable biodistribution of DOX@exosome-apt by exhibiting higher tumor accumulation and faster liver clearance in comparison with DOX@exosome and free DOX. It could be concluded that MUC1 aptamer-decorated exosomes can be implemented therapeutically for the safe and versatile delivery of DOX to colon adenocarcinoma, thus offering valuable platform for clinical applications.

Experimental model for the irradiation-mediated abscopal effect and factors influencing this effect.

Radiotherapy (RT) is the primary treatment for cancer. Ionizing radiation from RT induces tumor damage at the irradiated site, and, although clinically infrequent, may cause regression of tumors distant from the irradiated site-a phenomenon known as the abscopal effect. Recently, the abscopal effect has been related to prolongation of overall survival time in cancer patients, though the factors that influence the abscopal effect are not well understood. The aim of this study is to clarify the factors influencing on abscopal effect. Here, we established a mouse model in which we induced the abscopal effect. We injected MC38 (mouse colon adenocarcinoma) cells subcutaneously into C57BL/6 mice at two sites. Only one tumor was irradiated and the sizes of both tumors were measured over time. The non-irradiated-site tumor showed regression, demonstrating the abscopal effect. This effect was enhanced by an increase in the irradiated-tumor volume and by administration of anti-PD1 antibody. When the abscopal effect was induced by a combination of RT and anti-PD1 antibody, it was also influenced by radiation dose and irradiated-tumor volume. These phenomena were also verified in other cell line, B16F10 cells (mouse melanoma cells). These findings provide further evidence of the mechanism for, and factors that influence, the abscopal effect in RT.

Drag reducing polymers attenuate adverse effects of ischemia-reperfusion upon resection of liver metastases modeled by MC38 mouse colon adenocarcinomama.

The resection of metastases within healthy parenchyma improves significantly the long-term outcome in metastatic colorectal cancer. Until now, the resection technique involves Pringle maneuver, which allows reducing blood loss during transsection of liver parenchyma. However, the classical Pringle maneuver has restrictions due to ischemia/reperfusion (I/R) effect, in particular increasing risk of tumor recurrence after liver surgery. To study the pathological impact of surgical intervention and I/R effect on healthy liver tissue in the experimental setting by evaluating the markers of redox-homeostasis and oxidatively induced mutage-nesis, and also to assess the current possibilities of their correction by application of drag-reducing polymers (DRPs). MC38mouse colon adenocarcinoma cells were transplanted intrahepatically to C57Bl/6mice. The influence of warm ischemia on metastatic potential of MC38cells, the speed of superoxide radicals (SR) generation and 8-hydroxydeoxyguanosine content were studied. In case of modeled liver metastases, the surgery initiates an increase in the rate of SR generation into the remaining liver tissue and, consequently, provokes its ischemic injury. The application of DRPs protects liver tissue under I/R conditions. The warm I/R can promotes metastatic lesions in the healthy part of the organ with underlying increase in the rate of SR generation and oxidatively induced damage of guanine in DNA. The hemorheological effects of DRPs ensure increase of microcirculatory perfusion and oxygenation of liver tissues with the reduction of the rate of SR generation and decrease of 8-hydroxydeoxyguanosine as a marker of oxidatively induced mutations in DNA of hepatocytes. The intraperitoneal administration of nanomolar doses of DRPs prevents the activation of the growth of dormant metastatic MC38 cellsin the liver. Further experimental and clinical study of these substances will allow reducing the risks of activation of uncontrolled tumor growth in the liver due to the pathological effect of post-operative I/R.

Dendritic cells loaded with CD44+ CT-26 colon cell lysate evoke potent antitumor immune responses.

Increasing evidence supports the concept that cancer stem cells (CSCs) are responsible for cancer progression and metastasis, therapy resistance and relapse. In addition to conventional therapies for colon cancer, the development of immunotherapies targeting cancer stem cells appears to be a promising strategy to suppress tumor recurrence and metastasis. In the present study, dendritic cells (DCs) were pulsed with whole-tumor cell lysates or total RNA of CD44+ colon cancer stem cells (CCSCs) isolated from mouse colon adenocarcinoma CT-26 cell cultures and investigated for their antitumor immunity against CCSCs in vivo and in vitro. In a model of colon adenocarcinoma using BALB/c mice, a sequential reduction in tumor volume and weight was associated with an extended survival in tumor-bearing mice vaccinated with DCs pulsed with RNA or CCSC lysate. In addition, a lactate dehydrogenase assay indicated that cytotoxic T-cells derived from the treated mice exhibited strong cytotoxic activity. Additionally, an enzyme-linked immunosorbent assay revealed that the cytotoxic T-cells of the treated mice released higher levels of interferon-γ against CCSCs compared with those of the control group. In all experiments, the antitumor efficacy of the lysate-pulsed DC-treated and RNA-pulsed DC-treated groups were significantly higher compared with that of the DC-treated and control groups. The results of the present study indicated the potential use of DCs pulsed with cancer stem cell lysates as a potent therapeutic antigen to target CSCs in colon cancer. Additionally, the results provided a rationale for using lysate-pulsed DCs in vivo to eliminate residual tumor deposits in post-operative patients.

The Antitumor Efficacy of a Complex Based on Two-Vector System for Coexpression of the Suicide Gene Fcu1 and Cre Recombinase.

In this study, we evaluated the antitumor activity of a gene therapy complex in which the tumor-specific control of the expression of the effector suicide gene FCU1 was performed using a two-vector system based on the site-specific Cre-LoxP recombinase system. The complex of interest showed a high therapeutic potential in a mouse colon adenocarcinoma model.

SPECT/CT imaging of HER2 expression in colon cancer-bearing nude mice using 125I-Herceptin

An accurate assessment of HER2 status in patients with colorectal cancer is very important, because only the patients overexpressing HER2 can benefit most from the anti-HER2 targeted therapy. In this study, we investigated the feasibility of detecting HER2 expression in colon cancer by SPECT imaging using 125I-Herceptin, which showed high labeling rate, good in vitro stability and high binding specificity for HER2. HER2-positive mouse colon adenocarcinoma cell line (MC 38) was chosen as the colon caner cell model, and used for the establishment of colon cancer-bearing nude mice model. SPECT/CT imaging suggested that the tumors can be visualized at 12 h after the injection of 125I-Herceptin, and the uptake of tracer in tumors reached the peak at 24 h after injection, and can be attenuated significantly by pretreatment with an excess of nonlabeled Herceptin. These results indicates that 125I-Herceptin can be considered as an effective SPECT probe for the non-invasive detection of HER2 expression in colon cancer.

Alginate hydrogel co-loaded with cisplatin and gold nanoparticles for computed tomography image-guided chemotherapy.

The biomedical applications of gold nanoparticles (AuNPs) have experienced rapid growth in recent years, due to their expected benefits in medical imaging and therapy. In this work, we report the development of a theranostic nanocomplex constructed from alginate hydrogel co-loaded with cisplatin and AuNPs (abbreviated as ACA) for simultaneous drug delivery and computed tomography imaging. CT26 cells derived from mouse colon adenocarcinoma were exposed to various concentrations of ACA nanocomplex (for 24 h) and the cytotoxicity was measured using MTT assay. Moreover, the cells treated with ACA nanocomplex were imaged in a computed tomography scanner and the contrast enhancement due to the presence of nanocomplex was assessed. The cytotoxicity results showed that ACA nanocomplex had a more potent chemotherapy efficacy than free cisplatin, so that ACA nanocomplex at the concentration of 5 µg/ml (per cisplatin) and 20 µg/ml of free cisplatin resulted in the same cytotoxicity (survival rate: 66%). The computed tomography imaging study revealed that ACA nanocomplex increased the brightness of computed tomography images, the computed tomography number value, and contrast-to-noise ratio (CNR). ACA nanocomplex can be presented as a computed tomography-traceable nanocarrier that allows to monitor the delivery of therapeutics by assessing their localized accumulation and in vivo biodistribution.

Localized immune transcriptional response to intratumorally administrated TLR7 agonist conjugates

Induced or circumstantial immunosuppression present opportunities for cancer development, and progression. Recently, multiple therapies have been developed to harness the immune system's ability to selectively eradicate cancer cells. These approaches have primarily targeted features of the adaptive immune response such as checkpoint blockade and vaccination of tumorigenic peptides. Toll‐Like receptors (TLRs) are innate immune receptors that recognize molecules broadly shared by various pathogens. TLR pathways are increasingly targeted in cancer immunotherapy approaches seeking to overcome immune‐evasion by cancer cells. Here, we hypothesize that activation of TLR7, an endosomal receptor which recognizes pathogen associated nucleic acids, can mitigate tumor immunosuppression. TLR7 agonist (TLR7‐A) conjugated to silica nanoparticles will be used as an adjuvant in a mouse colon adenocarcinoma (CT26 cell line derived) in vivo tumor model. Transcriptional targets of adaptive immune cells (CD3, CD4, CD8, Granzyme B and Perforin), antigen presenting cells (MHCII, CD80, CD86 and CD40) and inflammatory cytokine response (TNFα, IL‐6, IP‐10, MCP1, MIP1α and IFNγ) were measured by qPCR in tumor tissue following 21 days of treatment. We found that treatment with TLR7‐A unconjugated and conjugated increased localized transcript levels of inflammatory cytokines and chemokines: IL‐6, IP‐10, MCP1, MIP1α and IFNγ. This project will provide valuable insights on the distinct transcriptional immune interplay in response to TLR7‐A treatment (localized vs systemic), support the decision‐making process of subsequent experimental approaches and evaluate TLR7‐A conjugate's potential for treatment.Support or Funding InformationThis research was supported by the National Cancer Institute of the National Institutes of Health under award numbers:U54CA132384 & U54CA132379This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Can "Indirect" Contact Laser Surgery be Used for Fluorescence-Image Guided Tumor Resections? Preliminary Results.

Ensuring the complete removal of tumor tissue is the main challenge during resection operations. Recently, a technique of “indirect” contact laser surgery has been developed. In this study we assess the possibility of using such surgery for fluorescence image-guided tumor resection. Mouse colon adenocarcinoma CT-26 cells stably expressing the fluorescent protein mKate-2 was used as the tumor model. Resections of the tumor nodes were performed with either a scalpel blade, a laser scalpel with a bare tip, or a laser scalpel with a strongly absorbing coating on the fiber tip. Tumor-positive resection margins were detected using an IVIS Spectrum fluorescence imaging system. After tumor resection with the scalpel blade over half of the animals needed one additional resection to remove residual tumor cells. Animals in this group showed tumor recurrence within 7 days. Fluorescence imaging of the tumor bed, performed after resection to assess the presence of tumor cell clusters, was sufficiently effective only with a bloodless resection. The laser scalpels both with the bare tip and with the strongly absorbing coating on the tip provided such bloodless tumor resection in contact mode. Fewer animals required additional resections when the bare tipped scalpel was used and this also resulted in a reduction in tumor recurrence. After resections were carried out with the laser scalpel with the strongly absorbing coating on the tip, fluorescence was detected in the operative field and this led to undertaking additional resections, although subsequent investigation suggested that this was “false” fluorescence, resulting from the effects of the scalpel rather than the presence of residual tumor cells. The method of laser resection with a strongly absorbing coating on the tip therefore did not appear to demonstrate definite advantages over laser resection with a bare tip when removing tumors.

Pantoprazole blocks the JAK2/STAT3 pathway to alleviate skeletal muscle wasting in cancer cachexia by inhibiting inflammatory response.

ObjectiveCancer cachexia is often present in patients with advanced malignant tumors, and the subsequent body weight reduction results in poor quality of life. However, there has been no progress in developing effective clinical therapeutic strategies for skeletal muscle wasting in cancer cachexia. Herein, we explored the functions of pantoprazole on cancer cachexia skeletal muscle wasting.MethodsThe mouse colon adenocarcinoma cell line C26 was inoculated in the right forelimb of male BALB/C mice to establish a cancer cachexia model. The animals were treated with or without different concentrations of pantoprazole orally, and the body weight, tumor growth, spontaneous activity, and muscle functions were determined at various time points. Two weeks later, the levels of serum IL-6 and TNF-α, the mRNA levels of gastrocnemius JAK2 and STAT3, and the expression levels of p-JAK2, p-STAT3, Fbx32, and MuRF1 were examined with ELISA assay, qRT-PCR assay, and Western blotting, respectively. Further studies were performed to assess the levels of Fbx32 and MuRF1 expression and morphological changes.ResultsPantoprazole can alleviate cancer cachexia-induced body weight reduction and inhibit skeletal muscle wasting in a dose-dependent manner. Our results indicated that pantoprazole treatment can decrease the levels of serum IL-6 and TNF-α (56.3% and 67.6%, respectively), and inhibit the activation of the JAK2/STAT3 signaling pathway. Moreover, the expression levels of MuRF1 and Fbx32 were also suppressed after pantoprazole treatment.ConclusionOur findings suggested that pantoprazole can alleviate cancer cachexia skeletal muscle wasting by inhibiting the inflammatory response and blocking the JAK2/STAT3 or ubiquitin proteasome pathway.

Six1 promotes colorectal cancer growth and metastasis by stimulating angiogenesis and recruiting tumor-associated macrophages.

The homeoprotein Six1 is overexpressed in many human cancers and is associated with increased tumor progression and metastasis. Recent studies have shown that Six1 is associated with poorer overall survival in advanced-stage colorectal cancer (CRC). In the current study, we explored the functional changes and molecular events associated with Six1 overexpression in a mouse model of CRC. An orthotopic model and a splenic injection metastasis model were used to investigate the role of Six1 in CRC tumor growth and metastasis using mouse colon adenocarcinoma MC38 cells overexpressing Six1. We found that overexpression of Six1 dramatically promotes CRC tumor growth and metastasis in vivo. Six1 overexpression in MC38 increased protein levels of aldehyde dehydrogenase-1 and expanded CD44+/CD166+ populations, indicating Six1 increased features of cancer stem cells. In addition, Six1 overexpression stimulated angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF). Six1-overexpressing tumor cells recruited tumor-associated macrophages (TAM) by increasing the expression of macrophage-specific colony stimulating factor, chemokine (C-C motif) ligand 2/5 and VEGF, further facilitating CRC tumor growth and metastasis. Furthermore, we determined that Six1 activated mitogen-activated protein kinase (MAPK) signaling in CRC cells. In summary, our studies strongly suggest that Six1 overexpression promotes CRC growth and metastasis and remodels tumor stroma by stimulating angiogenesis and recruiting TAM. MAPK activation may be a pivotal event in Six1-associated tumor progression, which may provide opportunities for pharmacologic intervention.