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David Gerard



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    PD01 - Poster Discussion Session (ID 4)

    • Event: NACLC 2019
    • Type: Poster Discussion Session
    • Track:
    • Presentations: 1
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      PD01.03 - Changes in Immune Checkpoints Landscape Associated with TGF-β Induced EMT in Lung Adenocarcinoma (ID 94)

      15:45 - 16:45  |  Author(s): David Gerard

      • Abstract

      Background:
      Immune checkpoint inhibitors have advanced the therapeutic approach for metastatic lung cancer with durable responses. Most patients, however, do not respond or develop secondary resistance and experience relapse. Alterations in multiple signaling pathways are involved in resistance to checkpoint inhibition. Dynamic changes in the immune checkpoints landscape in the tumor cell and its surrounding environment might be associated with invasive phenotype, which may also influence the outcome of cancer immunotherapy. This study demonstrates the impact of microenvironmental TGF-? on the expression and/or shedding patterns of immune checkpoint molecules in lung adenocarcinoma cells.


      Method:
      A549 and H358 cells were grown in RPMI-1640 containing 2.5% FBS at two treatment conditions for five days: control (no treatment) versus 20ng/mL TGF-?. Proteins from cell lysates and conditioned media were collected and were tested with the Human Immuno-Oncology Checkpoint Protein Panel (MilliporeSigma). This panel consists of BTLA, CD27, CD28, TIM-3, HVEM, CD40, GITR, GITRL, LAG-3, TLR-2, PD-1, PD-L1, CTLA-4, CD80, CD86, and ICOS. All primary data points were collected via Luminex FLEXMAP 3D system, and concentrations were calculated using a five-parametric fit algorithm (xPONENT v4.0.3 Luminex Corp Austin, TX). Identical cell cultures were performed previously by our group to profile the mRNA using paired-end sequencing on a HiSeq2000 sequencer (Illumina). Analysis to correlate RNA seq data with the proteomics pattern of immune checkpoints was done using R program version 3.5.


      Results:
      BTLA; CD28; HVEM; CD40; GITR; GITRL; LAG-3; TLR-2; PD-1; PD-L1; CD86 were measurable in cell lysates and conditioned media, whereas, CD27, TIM-3, CTLA-4, and CD80 were either untranslated or were below the level of detection. TGF-? induced significantly elevated CD28 total level in both cell lines (p0.02). PD-L1 and CD86 total levels were higher in A549 cells only (all p0.002) which is opposite to the RNA seq changes. HVEM was diminished inboth cell lines (all p0.002). BTLA and LAG-3 decreased in A549 cells; GITR, PD-1, and CD86 were reduced in H358 cels (all p0.02). Cellular GITR and GITRL ere augmented because of a lowered soluble format although the total evel was not significantly changed. CD40 and ICOS total levels were unchanged. These changes were associated with induction of protein activation cascade, humoral and cellular immune response, and extracellular matrix organization pathways (FDR< 0.002) according to RNA seq analysis.


      Conclusion:
      Invasive phenotype induced by TGF-? changes the immune checkpoints dynamics in lung adenocarcinoma cells which might affect the tumor-immune interaction and the outcome of immune checkpoint blockade.