![]() Here we extend this method to culture clinical tumor samples as Patient-Derived Organoids (PDOs) ( Neal and Kuo, 2016 Vlachogiannis et al., 2018), in distinction to in vivo patient-derived xenograft tumors (PDX). ![]() We previously reported an air-liquid interface (ALI) murine organoid model containing tightly integrated epithelial and stromal compartments that recapitulates multi-hit tumorigenesis within normal stomach, pancreas and colon organoids ( Li et al., 2014 Ootani et al., 2009). Despite shortcomings, these studies increasingly support the use of 3-dimensional organoid models for holistic study of the immune TME. Alternatively, custom microfluidic devices with human tumor suspension-derived microspheroids containing immune cells exhibit response to immunotherapeutics, but without tumor-immune specificity ( Deng et al., 2018 Jenkins et al., 2018). Short-term preservation of murine macrophages ( Chen et al., 2018) and several human immune cell types ( Finnberg et al., 2017) have not evidenced presence or functionality of T cells. However, such in vitro models of tumor immunity do not robustly retain the complex full diversity and physical architecture of the TME and particularly do not allow the co-culture of primary tumor epithelium with their native infiltrating immune populations en bloc without reconstitution. Immune cells from blood or patient tumors have been reconstituted with heterologous established cancer cell lines in traditional monolayer, spheroid ( Feder-Mengus et al., 2008) ( Hirt et al., 2014) or primary organoid cultures ( Dijkstra et al., 2018). There is, however, a dearth of models, 2D or 3D, that represent the in vivo interaction of tumor and immune cells in the TME. The recent promise of therapies manipulating tumor-infiltrating immune cells has created a particular exigency for human cancer models that recapitulate this TME diversity. The vast heterogeneity within cell types of the tumor microenvironment (TME) crucially impact treatment responses ( Junttila and de Sauvage, 2013 Klemm and Joyce, 2015 Palucka and Coussens, 2016). Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immunooncology investigations within the TME and facilitate personalized immunotherapy testing. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Robust droplet-based, single cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Here, an air-liquid interface (ALI) method propagated Patient-Derived Organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. In vitro cancer cultures, including 3-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). (D) Expanded y-axis from (C) showing nivolumab expansion of organoid TIL populations without anti-CD3/CD28. (C) Anti-CD3 plus anti-CD28 augments nivolumab expansion of organoid TIL populations in the ccRCC PDO of Figure 7D– E. Nivolumab decreased viable Annexin-V(−)/7-AAD(−) cells and increased Annexin-V(+)/7-AAD(−) early apoptotic (orange) and Annexin-V(+)/7-AAD(+) late apoptotic/necrotic cells (pink). (B) Dual Annexin V-FITC/7-AAD FACS analysis of the human kidney ccRCC PDO from Figure 7D– E following treatment with nivolumab or control IgG4 for 7 days. At both 7 and 14 days there is a decrease in viable Annexin-V(−)/7-AAD(−) cells and increased Annexin-V(+)/7-AAD(−) early apoptotic (orange) and Annexin-V(+)/7-AAD(+) late apoptotic/necrotic cells (pink). Pre-gating for tumor epithelium was performed by scatter properties. tumors were cultured for 7 or 14 days in the presence of IL-2 and either anti-mouse anti-PD-1, anti-PD-L1 or control IgG followed by FACS analysis with Annexin V-FITC and AAD. (A) Murine B16-SIY organoids prepared from syngeneic s.c. Anti-PD-1 or anti-PD-L1 treatment of ALI tumor organoids induces tumor cell killing, related to Figures 6 and 7.
0 Comments
Leave a Reply. |