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Israel Canadas



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    ES11 - Lung Cancer Plasticity and Drug Resistance (ID 14)

    • Event: WCLC 2019
    • Type: Educational Session
    • Track: Biology
    • Presentations: 1
    • Now Available
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      ES11.03 - Immunotherapy and Endogenous Retroviruses in Small-Cell Lung Cancer (Now Available) (ID 3213)

      15:15 - 16:45  |  Presenting Author(s): Israel Canadas

      • Abstract
      • Presentation
      • Slides

      Abstract

      Introduction: Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by

      mesenchymal cell subpopulations. While these subclones can secrete growth factors, chemokines and cytokines, the immune signaling

      networks that fuel this pro-tumorigenic state remain incompletely defined. Elucidating what underlies this state would provide insights into

      tumor biology and inform clinical strategies to improve anti-cancer therapies.

      Methods: Because of their well-defined nature, we used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC)

      mesenchymal subclones to uncover a novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine

      H69 cells. Analysis of gene signatures across TCGA and CCLE databases, functional studies in additional cell lines, and ex vivo testing of

      patient-derived organotypic tumor spheroids (PDOTS) were conducted to determine the broader relevance across human cancers.

      Results: We discovered a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in

      mesenchymal cancer subpopulations. Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3’UTRs of

      certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNγ exposure

      due to bi-directional transcription from the STAT1-activated gene promoter and the 5’ LTR of the antisense ERV. We found that dsRNA sensing

      preferentially by MAVS fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction across

      specific human tumors and cell lines is tightly associated with downregulation of chromatin modifying enzymes, including EZH2, a

      mesenchymal AXL positive cell state, and B2M and MHC class 1 antigen expression. SPARCS high tumors were marked by immune infiltration,

      but also exhibited multiple features of tumor

      immune suppression. IFNγ treatment of PDOTS with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to

      PD-1 blockade.

      Conclusions: Together, these data unveil a novel subclass of ERVs whose de-repression triggers pathologic innate immune signaling in cancer,

      with potentially important implications for cancer immunotherapy.

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    P2.04 - Immuno-oncology (ID 167)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Immuno-oncology
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.04-23 - Tumor-Stroma Interactions Promote cGAS-STING Driven Inflammation in Lung Tumor Microenvironment (Now Available) (ID 2351)

      10:15 - 18:15  |  Author(s): Israel Canadas

      • Abstract
      • Slides

      Background

      The tumor stroma is an essential component of the tumor microenvironment (TME) and has critical roles in promoting resistance to immunotherapies. Most anticancer therapies target cancer cells specifically, however, it is important to also study signaling contributions from the TME. The recruitment of immune cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the TME is a critical event in the cGAS-STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. Towards this, the infiltration of immune cells rely on functional vasculature to infiltrate into the tumor tissue. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels due to mitochondrial dysfunction and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistently, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma.

      Method

      3-D microfluidic device was fabricated using cyclic olefin polymer (COP) at AIM BIOTECH. NCI-H1355 cells were cultured for 24h in ultra low-attachment culture plates for spheroid formation. To form the 3D tumor microvascular model, cancer spheroids, human lung fibroblasts (hLFBs) and human umbilical vein endothelial cells (HUVECs) were resuspended in an extracellular matrix-like fibrin/collagen gel and loaded into the central channel and cultured for 7 days and hydrated with culture medium (Vasculife, lifeline). Cytokine profiling (Human Cytokine 40-plex panel) was performed with media collected from 3D culture.

      Result

      To examine how LKB1 alters immune cell recruitment, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. Co-culturing tumor spheroids and vasculature, we identified changes in morphology, cytokine production, and gene expression that occur during the co-culture. We found that co-culture induced cooperative production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF (Fig.1b,c). Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, knocking down STING in the LKB1-reconstituted cancer cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vasculature interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature.

      Conclusion

      Developing these more complex models that incorporate 3-dimensional tumor and self-assembled microvasculature may elucidate important aspects of cGAS-STING biology in KL lung cancer microenvironment, and may ultimately aid further development of effective immunotherapies targeting this signaling pathway.

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