Dataset Information

Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

ABSTRACT: Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 104 tissue cells, which corresponds to a metastatic burden of 1.8 × 104 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.

SUBMITTER: Deng W

PROVIDER: S-EPMC5847376 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

Deng Wentao W McLaughlin Sarah L SL Klinke David J DJ

The Analyst 20170801 16

Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers ...[more]

PMID: 28725894

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Project description:Purpose: Transvaginal meshes for the treatment of Pelvic Organ Prolapse (POP) have been associated with severe adverse events and have been banned for clinical use in many countries. We recently reported the design of degradable poly (L-lactic acid)-co-poly (e-caprolactone) nanofibrous mesh (P nanomesh) bioengineered with endometrial mesenchymal stem/stromal cells (eMSC) for POP repair. We showed that such bioengineered meshes had high tissue integration as well as immunomodulatory effects in vivo. This study aimed to determine the key molecular players enabling eMSC- based foreign body response modulation. Methods: SUSD2+ eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting. Electrospun P nanomeshes with and without eMSC were implanted in a NSG mouse skin wound repair model for 1 and 6 weeks. Quantitative PCR was used to assess the expression of extracellular matrix (ECM), cell adhesion, angiogenesis and inflammation genes as log2 fold changes compared to sham controls. Histology and immunostaining was used to visualize the ECM, blood vessels and multinucleated foreign body giant cells around implants. Results: Bioengineered P nanomesh/ eMSC constructs explanted after 6 weeks showed significant increase in 35 genes associated with ECM, ECM regulation, cell adhesion angiogenesis and immune response in comparison to P nanomesh alone. In the absence of eMSC, acute inflammatory genes were significantly elevated at 1 week. However, in presence of eMSC, there was an increased expression of anti-inflammatory genes including Mrc1 and Arg1 by 6 weeks. There was formation of multinucleated foreign body giant cells around both implants at 6 weeks that expressed CD206, a M2 macrophage marker. Conclusion: This study reveals that eMSC modulate the foreign body response to degradable P nanomeshes in vivo by altering the expression profile of mouse genes. eMSC reduce acute inflammatory and increase ECM synthesis, angiogenesis and anti-inflammatory gene expression at 6 weeks while forming newly synthesized collagen within the nanomeshes and neo-vasculature in close proximity. From a tissue engineering perspective, this is a hallmark of a highly successful implant, suggesting significant potential as alternative surgical constructs for the treatment of POP.

Dataset Information

Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

Publications

Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

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