Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo

Abstract Background Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, a labeled metabolite can be enzymatically changed in cellular metabolism, complicating the use of this experimental strategy to understand how a labeled metabolite moves between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (IRMS). Results We exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. IRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 μg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues. Conclusion Measuring tumor growth in a D. melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.

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Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo

10.1101/2020.09.22.307587 ◽

2020 ◽

Cited By ~ 1

Author(s):

Petter Holland ◽

William M. Hagopian ◽

A. Hope Jahren ◽

Tor Erik Rusten

Keyword(s):

Tumor Growth ◽

Low Cost ◽

Tumor Model ◽

Isotope Ratio Mass Spectrometry ◽

Isotopic Labeling ◽

Stable Isotope Ratio ◽

Carbon Transfer ◽

Stable Isotopic ◽

Robust Measurements

AbstractBackgroundRadioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, the transformation of the labeled metabolite by cellular metabolism within organs complicates the use of this experimental strategy to quantify and understand metabolite transfer between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (SIRMS).ResultsWe exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. SIRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 µg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues.ConclusionMeasuring tumor growth in a D.melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.

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Knockdown of LncRNA LINC00958 Inhibits the Proliferation and Migration of NSCLC Cells by MiR-204-3p/KIF2A Axis

Cell Transplantation ◽

10.1177/09636897211025500 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110255

Author(s):

Qing Wang ◽

Kai Li ◽

Xiaoliang Li

Keyword(s):

Lung Cancer ◽

Cell Proliferation ◽

Tumor Growth ◽

Tumor Model ◽

Luciferase Reporter ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Increasing evidence suggests that long non-coding RNAs (lncRNAs) function in the tumorigenesis of NSCLC. LINC00958, a newly identified lncRNA, has been reported to be closely linked to tumorigenesis in several cancers. However, its specific role in NSCLC remains unclear. In this study, we determined the expression of LINC00958 in NSCLC by RT-qPCR analysis and evaluated cell proliferation and migration by CCK-8 and transwell assays, respectively. We established a xenograft tumor model to examine the effect of LINC00958 on tumor growth in vivo. Luciferase reporter assays were performed to determine the interaction between LINC00958 and miR-204-3p and the interaction between miR-204-3p and KIF2A. We found that LINC00958 was up-regulated in NSCLC tissues and cell lines. Down-regulation of LINC00958 inhibited cell proliferation and migration in vitro and suppressed tumor growth in vivo. Besides, miR-204-3p was identified as a target of LINC00958 and miR-204-3p inhibitor could reverse the inhibitory effect of LINC00958 knockdown on proliferation and migration of NSCLC cells. We also validated that KIF2A, a direct target of miR-204-3p, was responsible for the biological role of LINC00958. KIF2A antagonized the effect of miR-204-3p on NSCLC cell proliferation and migration and was regulated by LINC00958/miR-204-3p. Taken together, these data indicate that the LINC00958/miR-204-3p/KIF2A axis is critical for NSCLC progression, which might provide a potential therapeutic target of NSCLC.

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Micro-MRI at 11.7 T of a Murine Brain Tumor Model Using Delayed Contrast Enhancement

Molecular Imaging ◽

10.1162/15353500200303112 ◽

2003 ◽

Vol 2 (3) ◽

pp. 153535002003031

Author(s):

Rex A. Moats ◽

Sendhil Velan-Mullan ◽

Russell Jacobs ◽

Ignacio Gonzalez-Gomez ◽

David J. Dubowitz ◽

...

Keyword(s):

Brain Tumors ◽

Tumor Growth ◽

Intraperitoneal Administration ◽

Three Dimensional ◽

Tumor Model ◽

Qualitative Assessment ◽

Response To Treatment ◽

Simple Method ◽

Serial Measurement

In vivo imaging methodologies allow for serial measurement of tumor size, circumventing the need for sacrificing mice at given time points. In orthotopically transplanted murine models of brain tumors, cross-section micro-MRI allows for visualization and measurement of the physically inaccessible tumors. To allow for long resident times of a contrast agent in the tumor, intraperitoneal administration was used as a route of injection for contrast-enhanced micro-MRI, and a simple method for relative tumor volume measurements was examined. A strategy for visualizing the variability of the delayed tumor enhancement was developed. These strategies were applied to monitor the growth of brain tumors xenotransplanted into nude mice and either treated with the antiangiogenic peptide EMD 121974 or an inactive control peptide. Each mouse was used as its own control. Serial imaging was done weekly, beginning at Day 7 after tumor cell implantation and continued for 7 weeks. Images obtained were reconstructed on the MRI instrument. The image files were transferred off line to be postprocessed to assess tumor growth (volume) and variability in enhancement (three-dimensional [3-D] intensity models). In a small study, tumor growth and response to treatment were followed using this methodology and the high-resolution images displayed in 3-D allowed for straightforward qualitative assessment of variable enhancement related to vascular factors and tumor age.

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Anti-Tumor Activity of the Aurora a Inhibitor MLN8237 in Diffuse Large B-Cell Lymphoma Preclinical Models.

Blood ◽

10.1182/blood.v112.11.1592.1592 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1592-1592 ◽

Cited By ~ 1

Author(s):

Jessica J Huck ◽

Mengkun Zhang ◽

Marc L Hyer ◽

Mark G Manfredi

Keyword(s):

Tumor Growth ◽

Growth Inhibition ◽

Tumor Model ◽

B Cell Lymphoma ◽

Tumor Growth Inhibition ◽

Aurora A ◽

Hct 116 ◽

Xenograft Models

Abstract Aurora A kinase is a serine/threonine protein kinase that is essential for normal transit of cells through mitosis. In many tumor types the Aurora A gene is amplified and/or the protein is over-expressed. The Aurora A small-molecule inhibitor MLN8237 demonstrated robust tumor growth inhibition in xenograft models of solid tumors grown subcutaneously (S.C.) in immunocompromised mice. Here we explored the antitumor activity of MLN8237 in models of diffuse large B-cell lymphoma (DLBCL) both in vitro and in vivo. In vivo three established DLBCL xenograft models (OCI-Ly7, OCI-Ly19, and WSU-DLCL2; all cells expressing luciferase) and a primary DLBCL tumor model PHTX-22-06 were tested using MLN8237 at different doses. Rituximab, an anti-CD20 monoclonal antibody that is active against CD20+ malignant B cells and is a standard of care agent was used for comparison. Using these model systems, tumor cells were injected either I.V. (to evaluate disseminated disease), or S.C. in severe combined immunodeficient mice (SCID). Animals were dosed orally for 21 days with MLN8237 (QD or BID) at various doses, or Rituximab dosed at 10mg/kg IV (once/week) and tumor growth inhibition was monitored using either bioluminescent imaging for the disseminated models or vernier calipers for the S.C. models. Tumor growth inhibition by MLN8237 was dose dependent with 20 mg/kg bid being the most efficacious dose (TGI>100% in both disseminated OCI-Ly19 and WSU models). All animals in the OCI-Ly19 disseminated model 20 mg/kg BID treatment group demonstrated regressions and remained disease free until the end of the study, day 65. In this study the Rituximab treated animals were euthanized on day 31 due to a high level of tumor burden. In the primary tumor model, PHTX-22-06, MLN8237 dosed at 20 mg/kg BID was also the most efficacious with a TGI of 95%. Moreover, tumor growth inhibition was durable as determined by prolonged tumor growth delay (>50 days). Significant efficacy was achieved in all models tested, whether grown as disseminated or subcutaneous models. A noted increase in durability of response was observed with MLN8237 treatment when compared with previous data from solid tumor models. In vitro, MLN8237 treatment increased levels of apoptosis in the OCI-Ly19 cells in comparison to the solid tumor cell line HCT-116 (colon). Greater Annexin V positive cells and greater cleaved PARP and Caspase-3 signals were detected in the MLN8237 treated OCI-Ly19 cells when compared to HCT-116 cells. The demonstration of robust and durable anti-tumor activity in preclinical models treated with MLN8237 provides the basis for its clinical evaluation as a treatment option for DLBCL. MLN8237 is currently in multiple Phase I clinical trials.

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Kruppel-like factor 6 suppresses growth and invasion of hepatocellular carcinoma cells in vitro and in vivo

International Journal of Immunopathology and Pharmacology ◽

10.1177/0394632016655171 ◽

2016 ◽

Vol 29 (4) ◽

pp. 666-675 ◽

Cited By ~ 2

Author(s):

Pei-Hao Wen ◽

Dong-Yu Wang ◽

Jia-Kai Zhang ◽

Zhi-Hui Wang ◽

Jie Pan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Tumor Growth ◽

Tumor Model ◽

Carcinoma Cells ◽

Tumor Suppressive Function ◽

Xenograft Tumor Growth ◽

Hcc Cells

Kruppel-like factor 6 (KLF6) as a novel tumor suppressive gene participates in multiple biological behaviors and plays an important role in regulating tumor cell growth and invasion. However, the functions of KLF6 in hepatocellular carcinoma (HCC) remain poorly understood. The expression level of KLF6 was examined by immunohistochemical assay in human HCC tissues, and KLF6-overexpressed HCC cells (SMCC-7721 and HepG2) were used for evaluating cell proliferation and invasion by MTT and Transwell assays. A subcutaneous HCC tumor model was established for assessing tumor growth in vivo. Our results showed that the expression of KLF6 was significantly downregulated in HCC tissues compared with the adjacent non-cancerous tissues (50.0% vs. 72.0%, P = 0.034) and negatively associated with the lymph-vascular space invasion (LVSI) in HCC patients ( P = 0.003). Furthermore, overexpression of KLF6 reduced cell proliferation and weakened the cell invasive potential followed with the decreased expression of PCNA and MMP-9 in HCC cells. The in vivo experiment indicated that KLF6 overexpression suppressed the xenograft tumor growth. Therefore, our findings show that KLF6 suppresses growth and invasion of HCC cells in vitro and in vivo, suggesting a tumor suppressive function in HCC and provides the potential therapeutic target for the treatment of HCC.

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Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells Proliferation In Vitro and In Vivo Xenograft Tumor Growth

10.5772/intechopen.96021 ◽

2021 ◽

Author(s):

Nagindra Prashad

Keyword(s):

Cell Proliferation ◽

Tumor Growth ◽

Cellular Differentiation ◽

Tumor Model ◽

Significant Inhibition ◽

Luciferase Reporter Plasmid ◽

Combination Of Sirnas ◽

Proliferation In Vitro

HDAC8, c MYC and MYCN are involved in the tumorigenesis of neuroblastoma. A mouse Neuroblastoma (NB) tumor model was used to understand the role of miRNA, miR-665 in NB tumorigenesis and cellular differentiation. During cellular differentiation of NB cells there is an up regulated miRNA-665. We found that HDAC 8, c MYC and MYCN are the direct targets of mimic miR-665 which was validated by luciferase reporter plasmid with 3’ UTR and ELISA. Mimic miR-665 inhibited cell proliferation, arrested cells in G1 stage and decreased S Phase in cell cycle. miR-665 increased the acetylation of histones and activated Caspase 3. This is the first report to recognize miRNA 665 as a suppressor miRNA of NB. The effects of miR-665 were confirmed with the transfection of siRNA for HDAC8 and siRNA for MYC. Individual siRNA- HDAC8 or siRNA-MYC inhibited 40–50% of cell proliferation in vitro, however, the treatment with the combination of both siRNA-MYC + siRNA- HDAC8 inhibited 86% of cell proliferation. Indicating that both the targets c MYC and HDAC 8 should be reduced to obtain a significant inhibition of cell proliferation. Intratumoral treatment of xenograft tumors in mice with the combination of siRNA-MYC + siRNA- HDAC8 reduced the levels of target c-MYC protein by 64% and target HDAC 8 protein by 85% and the average tumor growth reduced by 80% compared to control tumors treated with NC-siRNA. Our results suggest the potential therapeutic effect of suppressor miR-665 and the combination of siRNA-MYC + siRNA-HDAC8 for neuroblastoma treatment.

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Adhesion of Platelets to Colon Cancer Cells Is Necessary to Promote Tumor Development in Xenograft, Genetic and Inflammation Models

Cancers ◽

10.3390/cancers13164243 ◽

2021 ◽

Vol 13 (16) ◽

pp. 4243

Author(s):

Marica Cariello ◽

Elena Piccinin ◽

Roberta Zerlotin ◽

Marilidia Piglionica ◽

Claudia Peres ◽

...

Keyword(s):

Colon Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Cancer Progression ◽

Tumor Development ◽

Tumor Model ◽

Cell Interaction ◽

Colorectal Carcinogenesis ◽

Intestinal Tumorigenesis

Platelets represent the linkage between tissue damage and inflammatory response with a putative role in tumorigenesis. Given the importance of the microenvironment in colon cancer development, we elucidated the eventual role of platelets-cancer cells crosstalk in in vivo colon cancer models. To evaluate the involvement of platelets in intestinal tumorigenesis, we first analyzed if the ablation of β-integrin P-selectin that drives platelets-cell adhesion, would contribute to platelets-colon cancer cell interaction and drive cancer progression. In a xenograft tumor model, we observed that when tumors are inoculated with platelets, the ablation of P-selectin significantly reduced tumor growth compared to control platelets. Furthermore, in genetic models, as well as in chronic colitis-associated colorectal carcinogenesis, P-selectin ablated mice displayed a significant reduction in tumor number and size compared to control mice. Taken together, our data highlights the importance of platelets in the tumor microenvironment for intestinal tumorigenesis. These results support the hypothesis that a strategy aimed to inhibit platelets adhesion to tumor cells are able to block tumor growth and could represent a novel therapeutic approach to colon cancer treatment.

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AVIDIO as a novel oncolytic immunotherapy platform for the treatment of colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15210 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15210-e15210

Author(s):

Bijan Almassian ◽

Bhaskara R Madina ◽

Ju Chen ◽

Xiaoyang Ye ◽

Marie M Krady ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Tumor Growth ◽

Semliki Forest Virus ◽

Tumor Model ◽

Oncolytic Viruses ◽

Immunomodulatory Agents ◽

Oncolytic Activity

e15210 Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.

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Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Enhances Anti-Tumor Activity After Transplant

Blood ◽

10.1182/blood.v120.21.1897.1897 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1897-1897

Author(s):

Tulin Budak-Alpdogan ◽

Cavan P Bailey ◽

Michelle Panis ◽

Christopher Sauter ◽

Vikas Agrawal ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Tumor Growth ◽

Low Dose ◽

Tumor Model ◽

Tumor Escape ◽

Tumor Bearing ◽

Mhc Haplotype ◽

Anti Tumor Activity

Abstract Abstract 1897 We have previously shown that haploidentical (HI) HSCT with low dose donor T-cell infusion provides a survival advantage in tumor bearing mice when compared to parent F1 or MHC-matched transplant models. We suggest that MHC difference in HI-HSCT generates early T-cell clonal activation against the unshared MHC haplotype, which eliminates residual tumor cells that express the unshared MHC haplotype. However, alteration in MHC antigen expression is a significant contributor to tumor escape from graft-versus-tumor (GVT) activity. Recent haploidentical transplant data revealed that uniparental disomy, the loss of the HLA haplotype, is a clinically relevant mechanism of tumor escape that leads to post-transplant leukemia relapse. Murine renal cell carcinoma, RENCA-TGL, cell line normally expresses only H2Kd as a MHC molecule. Therefore, in our haploidentical transplant model, T cell clonal activity is usually restricted against H2Kd molecule only. For circumventing the single haplotype expression of tumor model, we transfected this cell line with a H2Kb expressing vector, pAcGFP-NeoR-H2Kb, and generated stable clones with G418 selection. The clone that has more than 95% H2Kb expression used for in vivo experiments. Both tumor cell lines, i.e. parental and transfected clone, had similar in vivo tumor growth acceptance and growth rate. We then used two different haploidentical donors that were targeting different MHC haplotypes. Lethally irradiated B6D2F1 (H2Kb/d) mice were transplanted with T cell depleted bone marrow (TCD-BM) from either B6C3F1 (H-2Kb/k) (single haplo-1; SH1), or C3D2F1 (H2Kk/d) (single haplo 2; SH2) or both donor mice with low-dose (1×105) T-cells. In some experiments, animals were also injected either H2Kd or H2Kb/d expressing RENCA-TGL cells for the evaluation of GVT activity. Bone marrow (BM), spleens and thymi were harvested from recipients of single and double HI-HSCT at day 35 and showed similar cellularities. Interestingly, spleen and bone marrow had similar chimerism from both donors in DH-HSCT. There were no early transplant mortality, graft failure, weight loss and GVHD scoring difference among the double or single-haploidentical transplant recipients. In two other sets of experiments, we followed the tumor growth and the survival of tumor bearing mice after transplant. The recipients of DH-HSCT showed a better survival and GVT activity than the recipients of SH-HSCT in RENCA-TGL (H2Kb/d) bearing tumor model. These observations confirmed that MHC targeting plays a prominent role in tumor surveillance, and immune targeting the unshared MHC haplotype with haploidentical transplant induce remarkable survival advantage. Double HI-HSCT provides an unique anti-tumor activity that continues to exert GVT effect, even in case of MHC haplotype loss. Disclosures: No relevant conflicts of interest to declare.

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