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Mikihiro Kohno



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    MA12 - Mesothelioma Surgery and Novel Targets for Prognosis and Therapy (ID 913)

    • Event: WCLC 2018
    • Type: Mini Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/25/2018, 10:30 - 12:00, Room 202 BD
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      MA12.10 - Long-Term Impact of Radiotherapy Before Surgery for Mesothelioma on the Distribution of Memory T Cell Subsets (ID 12728)

      11:35 - 11:40  |  Author(s): Mikihiro Kohno

      • Abstract
      • Presentation
      • Slides

      Background

      Postoperative recurrence remains one of the critical issues in treatments for mesothelioma. We previously reported that non-ablative, hypo-fractionated radiation before surgery generated an antigen-specific activation of the immune system and could provide an in situ vaccination with long-term protection against mesothelioma in our murine model. An effective immunological protection depends on memory T cell subset diversification. However, limited work has been done to address the distribution of memory T cell subsets and its effects on the immune system after radiotherapy followed by surgery for mesothelioma.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      C57BL/6 mice bearing AE17-OVA tumor were treated with local radiotherapy (LRT). LRT 5Gy was delivered on days 10, 11 and 12. We performed radical tumor resection 7 days after LRT. The mice were re-challenged under the skin or into thoracic cavity with AE17-OVA 28 days after surgery and defined as immunological protective memory model if the tumors were completely rejected. Memory model received subcutaneous tumor inoculation once again (second rechallenge), samples were harvested on day 0, 3, 10. We investigated memory T cell subsets using flow cytometry. In addition, the harvested total splenocytes (effector) were co-cultured with CFSE-labeled AE17-OVA (target) for three days. Each of their cytotoxic potential was analyzed by evaluating a number of AE17-OVA and its early or late apoptosis.

      4c3880bb027f159e801041b1021e88e8 Result

      8 out of 10 mice completely rejected the subcutaneous tumor in mice treated with LRT and surgery after re-challenged. We observed significantly better survival in the memory model re-challenged into the thoracic cavity compared with no treatment mice. After subcutaneous tumor inoculation, central memory T cells (CD44[+]CD62L[+]KLRG1[-]) on day 0, effector memory T cells (CD44[+]CD62L[-]KLRG1[-]) and terminal effector T cells (CD44[+]CD62L[-]KLRG1[+]) on day 0, 3, 10 increased significantly in CD8[+] splenocytes of memory model compared with no treatment mice. This observation was also seen in draining and non-draining lymph nodes. The MFI of CFSE reflecting a number of AE17-OVA cells decreased, whereas the proportion of early (Annexin V[+]FVD[low]) or late (Annexin V[+]FVD[high]) apoptotic cells in CFSE[+] cells increased, depending on time passage and effector/target ratio after tumor inoculation in both memory model and naïve mice. However, during time passage, memory model always had a stronger cytotoxicity (even at Day 0) as compared to naïve mice.

      8eea62084ca7e541d918e823422bd82e Conclusion

      Our data raise an important possibility that non-ablative, hypo-fractionated radiotherapy followed by surgery for mesothelioma contributes to the development and long-term maintenance of memory T cell subsets, which could remain poised to rapidly recall effector functions upon antigen re-exposure.

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    P2.06 - Mesothelioma (Not CME Accredited Session) (ID 955)

    • Event: WCLC 2018
    • Type: Poster Viewing in the Exhibit Hall
    • Track:
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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      P2.06-06 - Role of GITRL-GITR System in Promoting Proliferation of Malignant Mesothelioma (ID 12852)

      16:45 - 18:00  |  Author(s): Mikihiro Kohno

      • Abstract

      Background

      Using microarray analysis to compare the gene expression profile of untreated murine mesothelioma cell line (RN5), RN5 treated with cisplatin, RN5 treated with radiation, and enriched mesothelioma stem cell (RN5-EOS-Puro2), we found 41 genes potentially linked to cell stemness. Among those 41 genes, Tnfsf18(GITRL) was one of the cell surface markers which is likely related to tumor proliferation. We therefore decided to analyze the role of this ligand and it’s receptor (GITRL-GITR system) in proliferation of human mesothelioma cancer cells.

      a9ded1e5ce5d75814730bb4caaf49419 Method

      Three human mesothelioma cell lines (CRL5820, CRL5915, CRL5946) were used in this study. They were treated with cisplatin and Cs-137 irradiator respectively. The rt-PCR and Western Blot were used to evaluate the GITRL and GITR expression level at different time points. To evaluate the effect of GITRL-GITR system in mesothelioma cell lines we design in vitro and in vivo model for it. A neutralizing monoclonal antibody (mAb) was used to break the GITRL-GITR system in vitro and in vivo. In the in vitro model, mesothelioma cells were seeded into 96 well plates and the MTT test was used to compare cell proliferating rate 4 days after seeding. In the in vivo model we injected the CRL5946 cells into the peritoneal cavity and implanted patient-derived xenograft subcutaneously of the NOD/SCID mice. We sacrificed mice 4 weeks later to evaluate tumor spheres formation number and draw tumor growing curve.

      4c3880bb027f159e801041b1021e88e8 Result

      All the three mesothelioma cell lines demonstrated increased expression of Tnfsf18 (GITRL) and Tnfrsf18(GITR) at an mRNA and protein levels after treatment with chemotherapy or radiothreapy. Breaking the GITRL-GITR system with neutralizing mAb decreased cell growth and survival rate in mesothelioma cell lines after chemotherapy or radiotherapy. In vitro cell viability test, the MTT test results showed in cisplatin-treated or radiation-treated cell lines with adding mAb to break GITRL-GITR system, the cell viability decreased. In vivo xenografting model of CRL5946 cell line which is treated in advance by chemotherapy or radiotherapy, the average tumor sphere number (>100um) also decreased after using mAb intraperitoneally. The inhibiting effect of GITR neutralizing monoclonal antibody could be demonstrated in vitro and in our in vivo model. In patient-derived xenografting subcutaneous model, the mAb could also delay cell growth.

      8eea62084ca7e541d918e823422bd82e Conclusion

      The results of our study demonstrate that the GITRL-GITR system could play an important role in mesothelioma cells growth and survival especially after chemotherapy or radiotherapy.

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