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In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, Amigorena S - J. Exp. Med. (2007)

Bottom Line: We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs.We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities.CTLs migrating along blood vessels preferentially adopt an elongated morphology.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, F-75245 Paris Cedex 05, France.

ABSTRACT
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.

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Tumor cell clearance during early and late phase. (A) Representative TPLSM images (160 × 160 μm) of GFP-EG7 or GFP-EL4 tumors during early (day 3) and late phase (day 6) of tumor rejection after adoptive transfer of 107 OT1 cells. Vessels (red) are imaged by intravenous injection of 70 kD rhodamine-dextran (2.5 μg/ml) and collagen fibers (blue) using SHG signals. Capture parameters were identical for all images. Bar, 44 μm. (B) TPLSM images (150 × 150 μm) of GFP-EG7 (green) during early (day 4) and late (day 6) phase of rejection after intratumoral injection of propidium iodide (blue). Bar, 41 μm.
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fig3: Tumor cell clearance during early and late phase. (A) Representative TPLSM images (160 × 160 μm) of GFP-EG7 or GFP-EL4 tumors during early (day 3) and late phase (day 6) of tumor rejection after adoptive transfer of 107 OT1 cells. Vessels (red) are imaged by intravenous injection of 70 kD rhodamine-dextran (2.5 μg/ml) and collagen fibers (blue) using SHG signals. Capture parameters were identical for all images. Bar, 44 μm. (B) TPLSM images (150 × 150 μm) of GFP-EG7 (green) during early (day 4) and late (day 6) phase of rejection after intratumoral injection of propidium iodide (blue). Bar, 41 μm.

Mentions: To investigate if this transient arrest of migration in the antigen-expressing tumors is related to the killing of tumor cells by the CTLs, EG7 and EL4 tumor cells were transfected with GFP-encoding plasmid. The GFP-expressing tumors behaved like their GFP-negative counterparts in terms of tumor growth and rejection after transfer of OT1 cells. Tumor cell viability in vivo was determined at different time points by the level and the distribution of intracellular GFP using two-photon microscopy. During the early phase of tumor rejection, tumor cells formed a dense network of bright living cells in both EL4-GFP and EG7-GFP tumors, similar to that observed in the absence of OT1 cells (Fig. 3 A). In the late phases of rejection, in contrast, EG7 tumors, but not EL4 tumors, were mainly composed of residual dead tumor cells— i.e., cells displaying lower GFP levels and a GFP-negative, compact nucleus. In EG7 tumors during the late phase, the enhanced second harmonic generation (SHG) signal reflected an increase in the density of collagen fibers (Fig. 3 A), suggesting increased fibrosis in this region of the tumor.


In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, Amigorena S - J. Exp. Med. (2007)

Tumor cell clearance during early and late phase. (A) Representative TPLSM images (160 × 160 μm) of GFP-EG7 or GFP-EL4 tumors during early (day 3) and late phase (day 6) of tumor rejection after adoptive transfer of 107 OT1 cells. Vessels (red) are imaged by intravenous injection of 70 kD rhodamine-dextran (2.5 μg/ml) and collagen fibers (blue) using SHG signals. Capture parameters were identical for all images. Bar, 44 μm. (B) TPLSM images (150 × 150 μm) of GFP-EG7 (green) during early (day 4) and late (day 6) phase of rejection after intratumoral injection of propidium iodide (blue). Bar, 41 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2118741&req=5

fig3: Tumor cell clearance during early and late phase. (A) Representative TPLSM images (160 × 160 μm) of GFP-EG7 or GFP-EL4 tumors during early (day 3) and late phase (day 6) of tumor rejection after adoptive transfer of 107 OT1 cells. Vessels (red) are imaged by intravenous injection of 70 kD rhodamine-dextran (2.5 μg/ml) and collagen fibers (blue) using SHG signals. Capture parameters were identical for all images. Bar, 44 μm. (B) TPLSM images (150 × 150 μm) of GFP-EG7 (green) during early (day 4) and late (day 6) phase of rejection after intratumoral injection of propidium iodide (blue). Bar, 41 μm.
Mentions: To investigate if this transient arrest of migration in the antigen-expressing tumors is related to the killing of tumor cells by the CTLs, EG7 and EL4 tumor cells were transfected with GFP-encoding plasmid. The GFP-expressing tumors behaved like their GFP-negative counterparts in terms of tumor growth and rejection after transfer of OT1 cells. Tumor cell viability in vivo was determined at different time points by the level and the distribution of intracellular GFP using two-photon microscopy. During the early phase of tumor rejection, tumor cells formed a dense network of bright living cells in both EL4-GFP and EG7-GFP tumors, similar to that observed in the absence of OT1 cells (Fig. 3 A). In the late phases of rejection, in contrast, EG7 tumors, but not EL4 tumors, were mainly composed of residual dead tumor cells— i.e., cells displaying lower GFP levels and a GFP-negative, compact nucleus. In EG7 tumors during the late phase, the enhanced second harmonic generation (SHG) signal reflected an increase in the density of collagen fibers (Fig. 3 A), suggesting increased fibrosis in this region of the tumor.

Bottom Line: We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs.We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities.CTLs migrating along blood vessels preferentially adopt an elongated morphology.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale U653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, F-75245 Paris Cedex 05, France.

ABSTRACT
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.

Show MeSH
Related in: MedlinePlus