Figure 2. Tumor cells surviving immunotherapy organize in a transient cell-in-cell formation.

(A) Tumor size (in mm²) of B16F10 control (WT) and cell-lines established from relapsed B16F10 tumors following re-treatments with DC adjuvant and anti-TRP1 tumor-binding antibodies (n=5). (B) Mean percentages of apoptotic tumor cells after incubation overnight with gp100-reactive CD8⁺ T cells (n=4). (C) Tumor size (in mm²) of B16F10 control (WT) and cell-lines established from relapsed B16 tumors following additional treatment with gp100-reactive T cell (n=4). Arrowheads indicate treatment. (D) Illustration of experimental design. (E) Confluence percentage of B16F10 following one or two incubations with gp100-reactive or TRP2-reactive CD8⁺ T cells. (F) Principal Component Analysis (PCA) of B16F10 tumor cell lines freshly isolated from treated mice. (G) Representative images of B16F10 tumor cells sorted from tumors relapsed after immunotherapy. (H) Transmitting electron microscopy images of immunotherapy-relapsed B16F10 tumor cells. (I) Representative histological sections of untreated B16F10 tumors, and 5 days following immunotherapy. Arrowheads indicate cell-in-cell formation. (J) Mean percentage of cell-in-cell and single cells in B16F10 tumor-bearing mice, left untreated, treated with DC adjuvant and anti-TRP1 (Ab-IT), and treated with gp100-reactive T cells (gp100 ACT) (n=4). (K) Representative images of B16F10 labeled with H2B-tdTomato and H2B-GFP immediately after isolation from tumor-bearing mice treated with DC adjuvant and anti-TRP1, and after 4 days in culture. Experiments were repeated independently at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (* denotes p<0.05, *** denotes p<0.001, and **** denotes p<0.0001). Error bars represent standard error. Scale bars = 20 μm (G, I), 5 μm (H left), 500 nm (H right).

Figure 2—figure supplement 1. The immunogenicity of tumor cell that relapsed following immunotherapy does not inherently alter, compared to their parental cells.

Relates to Figure 2. (A). Individual mice tumor growth curves from Figure 2A (n=5). (B) Mean fluorescence intensity of MHCI, TRP2 antigen and gp100 antigen on B16F10 and cell-lines established from relapsed B16 tumors. (C) Extended confocal section and 3D projection of histological sections of B16F10 tumors 5 days after immunotherapy.

Figure 2—video 1. Spontaneous induction of cell-in-cell formation following sorting of single cells from tumors treated with immunotherapy, related to Figure 2.

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Figure 2—video 2. Spontaneous dissemination of cell-in-cell sorted directly from tumors following treatment with immunotherapy, related to Figure 2.

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