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Licun Wu
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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: 2
- Moderators:
- Coordinates: 9/25/2018, 10:30 - 12:00, Room 202 BD
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MA12.09 - Preclinical Investigations of Folate Receptor Targeted Nanoparticles for Photodynamic Therapy of Malignant Pleural Mesothelioma (ID 11277)
11:30 - 11:35 | Author(s): Licun Wu
- Abstract
- Presentation
Background
Photodynamic therapy (PDT) following lung-sparing extended pleurectomy (EPD) for malignant pleural mesothelioma (MPM) has been investigated as a potential means to kill residual microscopic cells. High expression of folate receptor 1 (FOLR1) has been reported in MPM, and targeting the FOLR1 has been considered as a new potential strategy. We have developed FOLR1-targeting porphyrin-lipid nanoparticles (folate-porphysomes; FP) for PDT. The inhibition of survival pathways of activated epidermal growth factor (EGFR) also enhance the PDT efficacy. Here, we have combined these approaches by using FP based PDT together with an EGFR-tyrosine kinase inhibitor (EGFR-TKI).
a9ded1e5ce5d75814730bb4caaf49419 Method
The frequency of FOLR1 and EGFR expression in MPM was analyzed using tissue microarrays. Confocal microscopy and a cell viability assay were performed to confirm the specificity of FOLR1-targeting cellular uptake and photocytotoxicity in vitro. In vivo fluorescence activation and the therapeutic efficacy were then examined. The effect of EGFR-TKI was assessed in vitro. The in vivo combined anti-tumor effect of EGFR-TKI and FP-PDT was then evaluated.
4c3880bb027f159e801041b1021e88e8 Result
FOLR1 and EGFR were expressed in 79 % and 89 % of the MPM samples, respectively. The intracellular uptake of FP corresponded well with FOLR1 expression. When MPM cells were incubated in FP and then irradiated at 671 nm, there was significant in vitro cell kill, which was inhibited in the presence of free folic acid, suggesting the specificity of FPs. FOLR1 targeting resulted in disassembly of the porphysomes and subsequent fluorescence activation in intrathoracic disseminated MPM tumors, as demonstrated by ex vivo tissue imaging. FP-PDT resulted in significant cellular damage and apoptosis in vivo. Furthermore, the combination of pre-treatment with EGFR-TKI plus FP-PDT showed further marked improvement of treatment responses.
8eea62084ca7e541d918e823422bd82e Conclusion
Folate-porphysome based PDT shows selective destruction of MPM cells based on FOLR1 targeting, and pre-treatment with EGFR-TKI further enhances the therapeutic response.
6f8b794f3246b0c1e1780bb4d4d5dc53Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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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): Licun Wu
- Abstract
- Presentation
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.
6f8b794f3246b0c1e1780bb4d4d5dc53Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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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: 2
- 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): Licun Wu
- 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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P2.06-38 - Mesothelioma Stem Cells May Be the Critical Factor of Treatment Failure (ID 11344)
16:45 - 18:00 | Presenting Author(s): Licun Wu
- Abstract
Background
Cancer cell repopulation during treatments of chemotherapy or radiotherapy is a major factor resulting in treatment failure. It has been indicated that cancer stem cells (CSC) may play critical roles during this process. The goal of our study is to characterise mesothelioma stem cells (MSC) and evaluate the prognostic values in those patients with malignant pleural mesothelioma (MPM). The eventual aim would be to design specific target therapy against MSC and develop novel approaches in clinical practice.
a9ded1e5ce5d75814730bb4caaf49419 Method
We have screened a group of genes that are most likely MSC-specific. Further characterization of the selected genes will be of critical importance in tumorigenesis, progression and prognosis. Murine mesothelioma AB12 and RN5 cells treated with either chemotherapy or γ-ray irradiation in culture, were used to compare gene expression profiles. The selected genes were confirmed by real-time PCR, flow cytometry and immunostaining. In vivo models, peritoneal lavage was collected at different time points after RN5 cell injection, to perform magnetic ranking cytometry with antibody-nanoparticle conjugates, and microarray assay. The expression of Tnfsf18 and Ngfr (CD271) genes associated with prognosis was evaluated in tumor tissues from MPM patients treated with SMART vs pre-SMART protocols, as SMART protocol has already shown significant clinical benefit. Image analysis was performed using Apero Imagescope program.
4c3880bb027f159e801041b1021e88e8 Result
The proportion of MSC significantly increased after RN5 parental cells were treated with either chemotherapy, or γ-ray irradiation, or in combination, while MSC showed more resistance to the above treatments, suggesting that chemoradiation resulted in MSC enrichment. Upregulation of genes Tnfsf18, Serpinb9b, Ly6a (Sca-1), Ngf, and Nppb were confirmed. CD271, the receptor of NGF, was shown to be upregulated after chemoradiation, especially after γ-ray radiation with a dose of 10Gy. Mesothelial precursors captured with magnetic nanoparticles conjugated to anti-Msln and trapped in the microfluidic device in the presence of a magnetic field showed an increase over time from 2-8weeks. Image analysis of human section slides indicated that total positive area of CD271 staining was significantly lower in those who were treated with SMART protocol than those with pre-SMART protocol (p<0.0025). Similar results were obtained in the high, medium and low positive areas from the SMART group, and p values are 0.0013, 0.0017 and 0.0035, respectively, when compared with the pre-SMART group.
8eea62084ca7e541d918e823422bd82e Conclusion
MSC-specific genes like CD271 and Tnfsf18 might be used as potential prognostic indicators and therapeutic targets.
6f8b794f3246b0c1e1780bb4d4d5dc53
