Angiopoietin1 Deficiency in Hepatocytes Affects the Growth of Colorectal Cancer Liver Metastases (CRCLM)

Figures

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  Figure 1. IHC staining of Ang1 in RHGP and DHGP chemonaïve lesions (A–F). Positivity representing positive pixels staining quantified with Aperio software (G). T-Tumor, L-liver, BV-Blood vessel, CT-Center of the tumor, PT-Periphery of the tumor, ADN-Adjacent normal, DN-Distal normal, DHGP ring-DR, dashed-lines represents the border between tumor region and livers data are represented as the mean +/− SEM, significant * p-value < 0.005 and *** p-value < 0.00005.

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  Figure 2. Ang1 was highly expressed in the hepatocytes of the adjacent normal of RHGP chemonaïve lesion (A) FISH double staining indicates higher Ang1 mRNA (yellow signal) in hepatocyte region compared to tumor region stained by tumor marker CK20 protein (green signal). Immunofluorescent staining showed (B) localization of Ang1 (red signal) and hepatocyte marker HSA (green signal), and (C) high expression of Ang1 (red signal) adjacent to vascular marker CD31 (green signal). T-tumor, L-liver.

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  Figure 3. IHC staining of (A) Ang2 and (B) Tie2 in RHGP and DHGP chemonaïve lesions. (C) Positivity representing positive pixels staining quantified with Aperio software. RT- replacement tumor, DT- desmoplastic tumor. T- Tumor, L-liver, BV-Blood vessel, CT-Center of the tumor PT-Periphery of the tumor, DHGP ring-DR, dashed-lines represents the border between tumor region and livers, data are represented as the mean +/− SEM, and * significant p-value < 0.005, ** p-value < 0.0005 and *** p-value < 0.00005.

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  Figure 4. Liver metastatic lesions and H&E staining of metastatic MC-38 mouse colon cancer cells. (A) Representative images of control, non-induced mice and Ang1 KO mice that received intra-splenic injections of MC-38 cells. Control mice developed large and multi-focal RHGP lesions whereas the Ang1KO mice had few or no lesions. (B) Quantitation of the number of lesions formed in the livers of control vs Ang1 KO mice. A significant difference (p-value < 0.005) was observed between the two groups. (C) H&E staining of metastatic lesions in control mice demonstrate Replacement HGP phenotype and (D) H&E staining of metastatic lesions in Ang1 KO mice demonstrate desmoplastic HGP phenotype. T-Tumor, L-Liver, DR-DHGP ring, dashed-lines represent the border between tumor region and liver, data are represented as the mean +/− SEM, and * significant p-value < 0.005.

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  Figure 5. Expression of Ang1 and CD31 in control and Ang1 KO mouse livers. (A) IHC staining of Ang1 in control mouse and (B) Ang1 KO mouse that developed DHGP tumor. (C) qPCR for Ang1 gene expression in control and Ang1 KO mice. qPCR data was first normalized to b-actin and results presented as log2 fold change of control liver tissues with or without tumors for both groups relative to control livers for mice without injection. IHC staining of CD31 in (D) control liver and (E) Ang1 KO liver. (F) Blood vessel counts in control and Ang1 KO mouse tumors. It is important to note that only 2/9 mice developed lesions and therefore the blood vessels counts were performed on the two mice that formed tumors. T-Tumor, L-Liver, DR-DHGP ring, dashed-lines presented the DR the border between tumor region and livers, data are represented as the mean +/− SEM, and * significant p-value < 0.0005.

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  Figure 6. Expression of Ang1 in isolated hepatocytes and MC-38 cell viability. (A) qPCR of Ang1 gene expression in isolated Ang1 KO hepatocytes, data is presented as log2 fold change of Ang1 KO hepatocytes relative to Ang1 control hepatocytes. Data was normalized to b-actin. Western blot of Ang1 expression in (B) isolated hepatocytes, and (C) hepatocytes cultured in serum free medium alone or cultured with MC-38 cells using insert. (D) Phase contrast microscopy of MC-38 cells cultured in Ang1 WT and Ang1 KO hepatocyte conditioned media (yellow floating cells represent dead cells). (E) MTT assay for MC-38 cells cultured in control or Ang1 KO hepatocyte media. Data are represented as the mean +/− SEM, and ** significant p-value < 0.0005.

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  Figure 7. Invasion and Migration assays of MC-38 cells cultured in hepatocytes Ang1 WT and Ang1 KO conditioned media. Ang1 KO hepatocyte media (A,B) reduced the invasive ability and decreased the number of migrated MC-38 cells through the Matrigel. (C) wound healing assay showed inhibition in migration ability of M-38 cells cultured in Ang1 KO hepatocyte media, images from the same area were captured at 0 and 24 h. Data are represented as the mean +/− SEM, and * significant p-value < 0.005.

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  Figure 8. Proliferation and migration assay assessing the effect of recombinant Ang1 on SW620 and MC-38 cells. (A) cell proliferation assay of SW620, human colon cancer cells, incubated with vehicle alone (blue line) and 0.1 μM (red line) and 0.05 μM (green line) recombinant Ang1. (B) cell proliferation assay of MC-38, mouse colon cancer cells, incubated with vehicle alone (blue line) and 0.1 μM (red line) and 0.05 μM (green line) recombinant Ang1. Scratch assays using (C) SW620 and (D) MC-38 cells incubated with vehicle alone (0), 0.05 and 0.1 μM of recombinant Ang1. After 24h of culture a wound was generated and pictures taken at time 0 and 24 h after incubation. All experiments were performed in triplicate (n = 3).