Development of a syngeneic colon cancer tumor model with deep vein thrombosis. (A) A group of 8- to 12-week-old C57BL/6 female mice were injected at the flank with 1 million colon cancer MC38 cells or vehicle. Age-matched mice were compared between the tumor-bearing and control mice. Upon tumors reaching a certain size (typically within 2-3 weeks; see “Methods”), IVC ligation was performed, and clots were harvested 48 hours thereafter. (B) Averages of tumor growth in individual mice injected with MC38 over 3 weeks. (C) Histological analysis of MC38 cells; H&E stain was used. (D) Immunohistochemistry (IHC) of tumors using β-catenin antibody are shown at 100× original magnification (scale bar, 100 μm). (E) A schematic figure of the partial IVC ligation (see “Methods”) and an intraoperative feature of the IVC ligation model. (F) Averages of clot weights normalized to body weight from both groups are shown. Error bars represent the standard error of the mean (SEM; n = 5 mice per group; P = .0016). (G) Paraffin-embedded sections of IVC were stained using anti-TF and anti-CD31 antibodies. Alexa Fluor secondary antibodies and DAPI were used. Images are representative of IVCs from 5 mice in each group. Arrowheads point to endothelial cells. White arrowheads point to the TF expression on endothelial cells, and asterisk points to the subendothelial cells (scale bar, 100 μm). (H) Three images per mouse were analyzed, and a region of interest was marked corresponding to the endothelial cells. ID was normalized to the surface area of the region of interest measured in microns by using ImageJ. Averages of ID normalized to surface area are shown. Error bars represent SEM (P = .0154). (I) IVCs were stained by using anti–PAI-1 and anti-CD31 antibodies. Alexa Fluor secondary antibodies and DAPI were used. Representative images from 5 mice in each group (scale bar, 100 μm). (J) PAI-1 expression was quantified as previously described. Averages of ID normalized to the surface area are shown. Error bars represent SEM (P = .0056). DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; ID, integrated density; K, kidney; L, ligated IVC. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001, ∗∗∗∗P < 0.0001.
Figure 1.

Development of a syngeneic colon cancer tumor model with deep vein thrombosis. (A) A group of 8- to 12-week-old C57BL/6 female mice were injected at the flank with 1 million colon cancer MC38 cells or vehicle. Age-matched mice were compared between the tumor-bearing and control mice. Upon tumors reaching a certain size (typically within 2-3 weeks; see “Methods”), IVC ligation was performed, and clots were harvested 48 hours thereafter. (B) Averages of tumor growth in individual mice injected with MC38 over 3 weeks. (C) Histological analysis of MC38 cells; H&E stain was used. (D) Immunohistochemistry (IHC) of tumors using β-catenin antibody are shown at 100× original magnification (scale bar, 100 μm). (E) A schematic figure of the partial IVC ligation (see “Methods”) and an intraoperative feature of the IVC ligation model. (F) Averages of clot weights normalized to body weight from both groups are shown. Error bars represent the standard error of the mean (SEM; n = 5 mice per group; P = .0016). (G) Paraffin-embedded sections of IVC were stained using anti-TF and anti-CD31 antibodies. Alexa Fluor secondary antibodies and DAPI were used. Images are representative of IVCs from 5 mice in each group. Arrowheads point to endothelial cells. White arrowheads point to the TF expression on endothelial cells, and asterisk points to the subendothelial cells (scale bar, 100 μm). (H) Three images per mouse were analyzed, and a region of interest was marked corresponding to the endothelial cells. ID was normalized to the surface area of the region of interest measured in microns by using ImageJ. Averages of ID normalized to surface area are shown. Error bars represent SEM (P = .0154). (I) IVCs were stained by using anti–PAI-1 and anti-CD31 antibodies. Alexa Fluor secondary antibodies and DAPI were used. Representative images from 5 mice in each group (scale bar, 100 μm). (J) PAI-1 expression was quantified as previously described. Averages of ID normalized to the surface area are shown. Error bars represent SEM (P = .0056). DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; ID, integrated density; K, kidney; L, ligated IVC. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001, ∗∗∗∗P < 0.0001.

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