Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism

Figures & data

Figure 1. Tumor-infiltrating lymphocytes in cultured tumor fragments from established mouse tumors are activated by anti-PD-1 plus anti-CD137 mAb combinations.

a) Experimental schematic representation of tumor fragment cultures in 96× well plates, seeding one fragment per well and culturing for 72 h to retrieve supernatants and prepare cell suspensions. b and c) IFNγ concentrations in the supernatants following cultures of MC38 (b) and CT26 (c) fragments stimulated with the indicated antibodies or concanavalin A as a positive control. Dots represent single wells and colors individual mice. d) Flow cytometry dot-plots showing a representative case of MC38-derived cell suspensions in which live CD4⁺ and CD8⁺ cells can be observed and electronically gated by FACS (upper panels). Lower panels show percentages of Ki67⁺ CD8⁺ and CD4⁺ cells and percentages of CD137⁺ cells by surface staining from the different conditions tested. For these flow cytometry experiments, cell suspensions from the fragments were pooled and dots represent cultures from individual mice bearing bilateral tumors. MDTFs: mouse-derived tumor fragments.

Figure 2. Tumor-derived fragments can be frozen and thawed showing comparable results with freshly excised fragments.

a) Scheme of the experiments including the freezing and thawing procedure. b and c) Comparative results following anti-PD-1 plus anti-CD137 stimulation of the fresh and frozen/thawed fragments. d) Flow cytometry results similar to Figure 1 comparing the frozen/thawed MC38-derived samples with those freshly excised. MDTFs: mouse-derived tumor fragments.

Figure 3. IFNγ release into the supernatant does not predict the therapeutic response to anti-PD1 plus anti-CD137 mAb treatment in vivo.

a) Experiments in which mice bearing bilateral MC38-derived tumors were subjected to unilateral tumorectomy and subsequently treated with control mAb or the anti-PD-1 plus anti-CD137 combination. Excised and fragmented tumors were subjected to culture. b) Individual tumor follow-up classifying tumors as responding or not to treatment. c) IFNγ concentrations in the supernatants of the fragments stimulated ex vivo comparing responding and non-responding contralateral tumors in the mice. d) Serum concentrations of IFNγ at day 18, comparing in-vivo responding and non-responding tumors. e) Results from a multiplex array measuring 12 cytokines in the pooled supernatants of the fragments cultured in the presence of the indicated stimuli. Error bars in (e) indicate standard deviations. R: Responder. NR: non-responder.

Figure 4. TGF-β blockade enhances the activation of TILs in the fragments as induced by anti-CD137 in combination with anti-PD-1 plus anti-CD137 agents.

a) Scheme of the experiments in which fragments were cultured under the indicated conditions using the 1D11 antibody to neutralize TGF-β. b) Levels of TGF-β as detectable in the supernatants of the tumor-derived fragments in culture (left) that correlate positively with IFNγ concentrations in pooled supernatants from the same mice (right). c) Levels of IFNγ in the supernatants of the fragments cultured under the indicated color-coded conditions. d) (Left panel) Mean fluorescence intensity of Ki67 as a marker of CD8⁺ cell proliferation in gated permeabilized cell suspensions of the fragments cultured under the indicated color-coded conditions. (Right panel) Percentage of granzyme B⁺ cells among CD8⁺ T cells in the fragments at the end of the culture under the indicated color-coded conditions. MDTFs: mouse-derived tumor fragments. MFI: mean fluorescence intensity.

Figure 5. Human tumor-derived cultured fragments in response to nivolumab (anti-PD-1) plus urelumab (anti-CD137 agonist).

a) Representation of the experiments with a series of surgical samples of endometrial, ovarian, and renal tumors (Table S1). PMA plus ionomycin was used as a positive control. b) Concentrations of IFNγ in the indicated individual fragments are represented by dots and grouped according to each experimental condition. c) Flow cytometry dot-plots indicating the recovery of viable CD4⁺ and CD8⁺ T lymphocytes following 48 h ex-vivo cultures of the fragments. d) Percentage of Ki67⁺ cells on gated CD4⁺ and CD8⁺ lymphocytes from the fragments of an IFNγ-responsive endometrial carcinoma case (END23). Nivo: nivolumab. Ure: urelumab. PDTFs: patient-derived tumor fragments.

Figure 6. Nivolumab plus urelumab plus anti-TGF-β stimulate IFNγ secretion in a fraction of tumor-derived fragments from cancer patients.

a) Experimental design with fragments that were cryopreserved prior to the cultures under the influence of the indicated color-coded stimuli. A series of six cases of endometrial, ovarian and renal tumors were tested (Table S1). b) IFNγ release into the supernatants by the fragments under the indicated color-coded tissue culture conditions. PDTFs: patient-derived tumor fragments.

Data availability statement

Data are available upon reasonable request to corresponding authors.