Immunotherapy response modeling by ex-vivo organ culture for lung cancer

Abstract

One of the major hurdles for the advancement of cancer immunotherapy is lack of robust, accessible experimental models. We aimed to produce an ex-vivo organ culture (EVOC) model of immunotherapy for non-small cell lung cancer (NSCLC). Freshly resected early stage tumors were collected from the operating room, fragmented to clusters < 450 µm and cultured with fetal calf serum and human autologous serum. The resulting EVOC includes cancer epithelial cells within tumor tissue clusters and immune cells. Original tissue features are reflected in the EVOCs. The response to immune checkpoint inhibitors (ICI) was assessed by IFNγ gene induction. Interestingly, IFNγ EVOC induction was numerically higher when anti-CTLA4 was added to anti-PD-L1 treatment, supporting the notion that anti-CTLA4 impacts cancer partly through tumor-resident immune cells. In parallel, immunohistochemistry (IHC) for key immune-related proteins was performed on the formalin-fixed paraffin embedded (FFPE) corresponding tumors. EVOC IFNγ induction by ICI correlated with basal non-induced IFNγ, CD8, CD4 and FOXP3 mRNA levels within EVOCs and with tumor-FFPE-IHC for CD8 and granzyme B. A weaker correlation was seen with tumor-FFPE-IHC for CD3, CD4, CD68, FOXP3 and tumor-PD-L1. Tertiary lymphoid structure density was also correlated with the ICI response. Our study provides novel data about biomarkers that correlate with ICI-induced response of early stage NSCLC. Retention of the microenvironment and minimal addition of exogenous factors suggest this model to reliably represent the original tumor. The cluster-based EVOC model we describe can provide a valuable, yet simple and widely applicable tool for the study of immunotherapy in NSCLC.

Keywords: Anti-CTLA4; Anti-PD-L1; Early-stage NSCLC; IFNγ; Tumor-microenvironment.

Fig. 1

EVOC preparation and component analysis. a Tumor tissue is collected directly from the operating room, rapidly dissected with surgical razor blades, followed by gentleMACS dissociation. Released cells and clusters are washed and cultured in medium supplemented with autologous serum and fetal calf serum. Drugs are added to the culture medium and culture is incubated for four days (see materials and methods for details). b Bright field microscopic images of: single cells fraction (< 30 μm; left panel), clusters fraction (> 30 μm; < 450 μm, middle panel) and mixed clusters and single cells (as used in all experiments; right panel). c Gene expression in single-cells fraction (S) and clusters fraction (C) determined by real-time PCR (normalized to GAPDH; single cells fraction was used as the reference sample): CK7 (representing adenocarcinoma epithelial cells), PD-L1, α-SMA (fibroblasts), CD163 (macrophages), CD8 (cytotoxic T cells), CD4 (helper T cells). *p < 0.05, student’s t-test. d IHC of a representative EVOC; upper panel—microscopic images of 10 × magnification, lower panels—enlargement of the cluster area. IHC of CK7 (left panel), CD8 (middle panel) and PD-L1 (right panel)

Fig. 2

Tumor morphological features and EVOC cluster formation. Adenocarcinoma FFPE specimens (of standard pathologic processing) with a single dominant adenocarcinoma subtype and their corresponding EVOCs were analyzed (n = 21). EVOCs analyzed were either cluster-rich (≥ 10 clusters/1.85 mm²) or cluster-poor (< 10 clusters/1.85 mm²). a Two upper panels: microscopic-images of representative FFPE adenocarcinoma specimens with a dominant subtype of acinar, papillary or solid. IHC of H&E and cytokeratin 7 epithelial cell marker (CK7-tumor); Two bottom panels—microscopic images of FFPE Bio-Agar embedded cell block of morphologically similar EVOCs stained with epithelial cells marker (CK7-EVOC) and light microscopy images of the EVOCs (EVOC culture). b Analysis of the number of cluster-rich and cluster-poor EVOCs, within each adenocarcinoma subtype group (acinar, solid or papillary). Correlation between the acinar and solid predominant morphology subtypes of resected tumors and the corresponding cases of cluster-poor or cluster-rich EVOCs; *p = 0.022, Fisher's exact test

Fig. 3

EVOC responsiveness to immune signals. a EVOCs were treated (+ IL2) or not (NT) with IL-2 for 4 days. Representative experiment showing average and standard deviation of IFNγ and CD8 gene expression (qRT-PCR; each condition was tested in triplicate). b EVOCs were treated for four days with either durvalumab (Durva; n = 17) or combination of durvalumab and ipilimumab (Durva + Ipi; n = 15; different numbers due to limited material, thus not all EVOC experiments included both treatment types). Immunotherapy-induced IFNγ fold-induction was calculated as the ratio of treated to non-treated IFNγ expression (measured as in A) for each EVOC/treatment. Each point represents one EVOC/treatment. IFNγ fold-induction is shown for “responders” (R), and “non-responders” (N). Centre line is the median, the box limits are the lower and upper quartiles, and the whiskers extend to the most extreme values within 1.5 × the interquartile range. The differences between responders and non-responders group in each treatment group are statistically significant at p < 0.0014 (Mann–Whitney test). c Basal (non-induced) IFNγ expression levels determined in non-treated EVOCs, four days after EVOC initiation, in non-responder EVOCs (N) and responders (R; represented as -∆CT; normalized to GAPDH). Box plot representation as in B. The differences between the groups are statistically significant (p < 0.003, Mann–Whitney test)

Fig. 4

Correlation between key immune proteins’ expression in tumors and immunotherapy response in the matched EVOCs. a Representative microscopic images of IHC conducted for the indicated proteins on FFPE specimens of the tumors from which the EVOCs were produced. CK7 or CK5/6 were stained to identify epithelial cancer cells. Left—tumor producing a responsive EVOC, right—tumor producing a non- responsive EVOC. Black scale bars represent 100 μm, red scale bars—50 μm. b Heat map of 12 proteins expression in immune-responsive and non-responsive EVOCs with hierarchical clustering. Rows correspond to IHC quantification of individual proteins, columns correspond to EVOC samples. Besides the proteins depicted in A, IHC results for CD21 and CD23, identifying follicular dendritic cells, and KI67, indicative of cell proliferation rate, were also included. IHC of PD-L1, quantified separately for immune and tumor cells was incorporated. At the bottom: immunotherapy response of the corresponding EVOCs, coded as indicated. H&E: Hematoxylin and eosin. Granz.B: granzyme B