Fig 1.
(A) NHP administered 50 mg/kg anti-DLL4 and (B) CRLM patient treated with oxaliplatin both have sinusoidal dilatation in the centrilobular area near the central vein with a severity grade of 3. Thin arrows point to dilated sinusoids, and thick arrows to the central veins. The human liver (B) has blood retained within the dilated sinusoids that imparts a pink color. Scale bar = 90 μm.
Fig 2.
PCA and hierarchical clustering of NHP FFPE LCM hepatic regions and whole liver samples with or without anti-DLL4-associated SD based on their gene expression with FDR cut off at 0.25.
Results from microfluidic high-throughput RT-qPCR were normalized by calculating–ΔCt using the average of reference genes. PCA (A) and hierarchical clustering based on Pearson correlation (B) were undertaken using genes with differential expression (FDR<0.25) between the LCM hepatic regions derived from liver samples of NHPs with SD present (moderate SD severity score 2 (anti-DLL4 15 mg/kg) n = 6)) and absent (SD severity score 0 (vehicle) (n = 6)) LCM hepatic regions. PCA (C) and hierarchical clustering based on Pearson correlation (D) were undertaken using genes with differential expression (FDR<0.25) between whole liver samples of NHPs with SD present (mild to severe SD severity scores 1–3 (anti-DLL4 5–50 mg/kg) (n = 17)) and absent (SD severity score 0 (vehicle or anti-DLL4 5 mg/kg) (n = 7)). PCA data are presented as spheres in PC1, PC2 and PC3 3 dimensional (3-D) space. The axes have been rotated to highlight the separation of the distinct clusters. The blue spheres surrounded with blue line represent the samples without SD and the red spheres surrounded with red line represent the samples with SD. The silver, blue, and pink axes represent principal components 1, 2 and 3 respectively (A and C). In hierarchical clustering, each row represents a gene and each column represents a sample. Red squares indicate high gene expression; green squares indicate low gene expression (B and D).
Table 1.
List of differentially expressed genes in LCM hepatic regions-derived from NHPs with moderate anti-DLL4-associated SD (severity score 2, SD 2) (n = 6) compared to LCM hepatic regions-derived from NHPs without anti-DLL4-associated SD (severity score 0, SD 0) (n = 6).
Table 2.
List of differentially expressed genes in whole liver samples-derived from NHPs with any anti-DLL4-associated SD (severity score 1–3, SD 1–3) (n = 17) compared to whole liver samples-derived from NHPs without anti-DLL4-associated SD (severity score 0, SD 0) (n = 7).
Table 3.
Patient characteristics.
Fig 3.
Box-plot indicating gene affected by an overall SD effect among oxaliplatin (+/- bev) treated CRLM patients (SD severity score 0–1 (n = 48) vs. SD severity score 2–3 (n = 13)).
Two-way ANOVA with treatment and SD status as factors was used. P value and FDR<0.05 are indicated in each box plot graph. OX = oxaliplatin, Bev = bevacizumab.
Fig 4.
Box-plots indicating genes affected by SD effect among oxaliplatin only-treated CRML patients (SD severity score 0–1 (n = 26) vs. SD severity score 2–3 (n = 12)).
Two-way ANOVA with treatment and SD status as factors was used. P value and FDR<0.05 are indicated in each box plot graph (A-F). OX = oxaliplatin, Bev = bevacizumab.
Table 4.
List of differentially expressed genes in CRLM patients with moderate to severe oxaliplatin (+/- bev)-associated SD (severity score 2–3, SD 2–3) compared to CRLM patients without or mild oxaliplatin (+/- bev)-associated SD (severity score 0–1, SD 0–1).
Fig 5.
Box-plots indicating genes affected by an overall treatment effect (oxaliplatin (n = 38) vs. oxaliplatin + bev (n = 23)).
Two-way ANOVA with treatment and SD status as factors was used. P-value and FDR < 0.05 are indicated in each box plot graph (A-C). OX = oxaliplatin, Bev = bevacizumab.
Table 5.
List of differentially expressed genes in CRLM patients receiving oxaliplatin and bev treatment compared to CRLM patients receiving oxaliplatin alone treatment.
Fig 6.
Box-plots indicating genes affected by treatment effect among CRML patients with no or mild SD only (oxaliplatin (n = 26) vs. oxaliplatin + bev (n = 22)).
Two-way ANOVA with treatment and SD status as factors was used. P-value and FDR < 0.05 are indicated in each box plot graph (A-E). OX = oxaliplatin, Bev = bevacizumab.
Fig 7.
Schematic summary of findings on drug-induced SD based on current analyses in anti-DLL4-induced SD in NHP model and oxaliplatin +/- bev in CRLM patients.
Anti-DLL4 and oxaliplatin can lead to dysregulation of NOTCH/VEGF signaling pathways genes in the LSECs. These changes in hepatic gene expression lead to alterations in vascular homeostasis and subsequently to LSEC dysfunction. LSEC dysfunction leads to ECM remodeling, inflammation and eventually to LSEC injury. In response to this SD is developed and may progress to SOS/VOD. Bev hepato-protective effect against OX-induced SD acts through inhibition of NOTCH/VEGF signaling pathways that leads to inhibition of vascular remodeling and inflammation.