Anti-VEGF treatment-resistant pancreatic cancers secrete proinflammatory factors that contribute to malignant progression by inducing an EMT cell phenotype

Abstract

Purpose: The resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. There are currently no validated predictive biomarkers for selecting which cancer patients will benefit from antiangiogenic therapy. Also lacking are resistance biomarkers that can identify which escape pathways should be targeted after tumors develop resistance to VEGF treatment. Recent studies showed that anti-VEGF treatment can make tumor cells more aggressive and metastatic. However, the mechanisms and mediators of this are unidentified. Therefore, we aimed this study at directly identifying the tumor cell-initiated mechanisms responsible for the resistance of pancreatic cancer to anti-VEGF treatment.

Experimental design: We established and validated two murine models of human pancreatic cancer resistant to the VEGF-specific antibody bevacizumab in vivo. We used a genome-wide analysis to directly identify which tumor-secreted factors were overexpressed by pancreatic cancer cells that were resistant to anti-VEGF treatment.

Results: Rather than direct proangiogenic factors, we identified several proinflammatory factors that were expressed at higher levels in cells resistant to anti-VEGF treatment than in treatment-sensitive control cells. These proinflammatory factors acted in a paracrine manner to stimulate the recruitment of CD11b(+) proangiogenic myeloid cells. Also, we found that secreted factors overexpressed by anti-VEGF treatment-resistant pancreatic cancer cells acted in an autocrine manner to induce epithelial-to-mesenchymal transition (EMT) and were thus responsible for increased aggressiveness of bevacizumab-resistant pancreatic tumors.

Conclusions: Our results identified proinflammatory factors and EMT markers as potential biomarkers for selecting patients with pancreatic cancer for antiangiogenic therapy.

Figure 1. In vivo selection of two pancreatic cancer models with evasive resistance to anti-VEGF treatment

(A) Green fluorescent protein⁺/luciferase⁺ PANC-1 or COLO357FG cells were orthotopically injected into nude mice. When the resulting tumors became detectable, the mice were given 100 µg of bevacizumab intraperitoneally (ip) twice a week (biw). The tumor growth was quantified weekly based on the bioluminescence emitted by the tumor cells as the sum of all detected photons within the region of the tumor per second. A digital grayscale image was acquired, followed by the acquisition and overlay of a pseudocolor image representing the spatial distribution of detected photons emerging from the active luciferase within the mouse. (B) Light-microscopic phenotypes of the anti-VEGF treatment-sensitive PANC-1 and COLO357FG cells, and anti-VEGF treatment-resistant P1BR and FGBR pancreatic cancer cells. (C) Thirty-two athymic nude mice bearing orthotopic COLO357FG, FGBR, PANC-1, or P1BR pancreatic tumors were randomly assigned to eight groups (n= 4 per group) to receive 100 µg of either bevacizumab or saline (control) ip twice a week. Mice were sacrificed by carbon dioxide inhalation when evidence of advanced bulky disease developed. The day of sacrifice was considered the day of death from disease for the purpose of survival evaluation.

Figure 2. Selection of relevant biological processes and secreted protein genes using global transcript profiling

(A) Disease, disorder, or physiological systems or functions enriched among genes differentially expressed in anti-VEGF treatment-resistant pancreatic cancer cell lines vs. their respective treatment-sensitive control cell lines. The X-axis represents the −log(10) P value for enrichment, with the threshold drawn at P=0.05. (B) Interaction network derived from genes upregulated in anti-VEGF treatment-resistant pancreatic cancer cell lines vs. their respective treatment-sensitive control cell lines. Each interaction is supported by at least one literature reference identified in the Ingenuity Pathway Knowledge Base, with solid lines representing direct interactions, and dashed lines representing indirect interactions. (C) Gene expression levels and unsupervised hierarchical clustering of differentially secreted genes in treatment-resistant FGBR versus treatment-sensitive COLO357FG and treatment-resistant P1BR versus treatment-sensitive PANC-1 cells. In the heat map shown in this figure, the logarithms of the gene expression levels are shown in colors (green = decreased expression, red = increased expression). (D) Serial paraffin-embedded pancreatic tumor sections stained immunohistochemically with antibodies against CD11b⁺ cells. (E) Anti-VEGF treatment-resistant FGBR and P1BR tumors demonstrating significantly greater infiltration by CD11b⁺ cells than do that in anti-VEGF treatment-sensitive COLO357FG and PANC-1 tumors. ***P < 0.0001 (unpaired Student t-test).

Figure 3

Oncomine bar charts of the expression levels of the indicated proinflammatory factors between human normal pancreatic tissues (N) (n= 16) and human pancreatic cancer (T) (n= 36) in the dataset by Pei et al. (19). P values were determined by t-Student test.

Figure 4. Anti-VEGF treatment-resistant pancreatic cancer cells showing features of malignant progression and EMT

(A) Levels of cancer cell migration between anti-VEGF treatment-resistant and treatment-sensitive pancreatic cancer cells. Results are presented as percentages of the total distances between the wound edges enclosed by cancer cells. The mean values and 95% confidence intervals from three independent experiments performed in quadruplicate are shown. ***P < 0.001 (unpaired Student t-test).(B) Photographs of the wound area were taken using phase-contrast microscopy immediately and 24 h after the incision. (C) Cell invasion assays. Representative photographs of Matrigel-coated transwell traversing activity and branching morphogenesis in 3D Matrigel of pancreatic cancer cells; (D) to quantify invasiveness, the stained invading cells were lysed and their absorbance measured. The mean values and 95% confidence intervals are shown. **P < 0.01; *P < 0.05 (unpaired Student t-test). (E) Representative photograph of hemorrhagic ascites in an orthotopic treatment-resistant P1BR tumor-bearing mouse. (F) Amount of ascites drained from pancreatic tumor-bearing mice. The mean values and 95% confidence intervals are shown. ***P < 0.001 (unpaired Student t-test). (G) Heat map showing EMT gene expression values in treatment-resistant FGBR versus treatment-sensitive COLO357FG and treatment-resistant P1BR versus treatment-sensitive PANC-1 cells. (H) Results of quantitative real-time polymerase chain reaction analysis of E-cadherin (CDH1) and vimentin (VIM) genes presented as the fold change in RNA expression between the gene of interest and β-actin. The mean values and 95% confidence intervals from three independent experiments performed in quadruplicate are shown. ***P < 0.001 (unpaired Student t-test). (I) Expression of E-cadherin and vimentin proteins.

Figure 5

Serial paraffin-embedded tumor sections stained immunohistochemically with antibodies against E-cadherin and vimentin proteins. Masson’s trichrome staining protocol was used to visualize the extracellular matrix (blue).

Figure 6. Induction of EMT by secreted proteins in anti-VEGF treatment-resistant pancreatic cancer cells

(A) Schematic representation of the co-culture technique used to culture anti-VEGF treatment-resistant and treatment-sensitive pancreatic cancer cells physically separated by a hydrophobic barrier but in the same culture medium. Samples of different cell lines cultured under different conditions are indicated as follows: 1a = empty, 1b = PANC-1, 2a = P1BR cocultured with PANC-1, 2b = PANC-1 cocultured with P1BR, 3a = empty, 3b = P1BR, 4a = empty, 4b = COLO357FG, 5a = FGBR cocultured with COLO357FG, 5b = COLO357FG cocultured with FGBR, 6a = empty, and 6b = FGBR. (B) Results of quantitative real-time polymerase chain reaction analysis of the E-cadherin (CDH1) gene presented as the fold change in RNA expression between the gene of interest and β-actin. The mean values and 95% confidence intervals from three independent experiments performed in quadruplicate are shown. (C) Expression of E-cadherin and vimentin proteins. The results shown are representative of three independent experiments performed. (D) Pancreatic cancer cell migration. The results are presented as the percentages of the total distances between the wound edges enclosed by cancer cells. The mean values and 95% confidence intervals from three independent experiments performed in quadruplicate are shown. (E) Photographs of the wound area were taken immediately and 24 h after the incision. (F) Cell invasion assays. Representative photographs of branching morphogenesis in 3D Matrigel of pancreatic cancer cells.