Scientists have demonstrated that the growth of pancreatic ductal adenocarcinoma (PDAC) in mice can be suppressed by inhibiting chemokines that act to mediate interaction between the cancer cells and fibroblasts in the stroma. Their research found that invasive PDAC cells secrete much higher levels of a number of chemokines than preinvasive pancreatic intraepithelial neoplasia (PanIN) cells, and that these chemokines stimulate stromal fibroblasts to induce connective tissue growth factor (Ctgf), a profibrotic and tumor-promoting factor, which accelerates tumor growth in vivo.
In vivo studies by the team at Vanderbilt University and the University of Tokyo showed that treating a mouse model of human PDAC using an inhibitor of the interleukin 8 receptor, CXCR2, reduced Ctgf expression and angiogenesis, slowed tumor progression, and led to increased survival. Vanderbilt’s Harold L. Moses, M.D., and the University of Tokyo’s Hideaki Ijichi, M.D., and colleagues claim the findings suggest that inhibiting tumor-stromal interaction might be a promising therapeutic strategy for PDAC. They report their findings in the Journal of Clinical Investigation, in a paper titled “Inhibiting Cxcr2 disrupts tumor-stromal interactions and improves survival in a mouse model of pancreatic ductal adenocarcinoma.”
Previous studies have suggested that development of PDAC from preinvasive PanIN occurs through a multistep progression involving the accumulation of specific genetic alterations, the team notes. Indeed, at the invasive stage, mutations and deletions in KRAS, P16INK4A, P53, and SMAD4 are found in approximately 90%, 90%, 75%, and 55% of PDAC, respectively, suggesting that alteration in these signaling pathways plays a causal, or at least permissive, role that allows progression from PanIN to PDAC in vivo.
This notion has been supported by work in various mouse models, which demonstrated that inactivating a number of these genes dramatically accelerates PDAC progression through activation of Kras. The researchers led by Drs. Moses and Ijichi had previously generated a particular mouse model of human PDAC, designated Kras+Tgfbr2KO, which develops aggressive PDAC that histologically mirrors manifestations of the disease in humans. In particular, these mice demonstrate abundant stromal components in the tumor tissue that recapitulates desmoplasia, a hallmark of PDAC histology in humans that occurs as a proliferation of fibrotic and connective tissue around the invasive tumor.
Although prior studies have indicated that desmoplasia is associated with faster tumor progression and chemoresistance, little is known about tumor-stromal interactions during tumor progression. The team hypothesized that PDAC cells may produce and release certain factors into the microenvironment to which the stromal cells could in turn respond, by establishing favorable conditions for tumor growth.
They screened for secreted factors produced by the PDAC cells from Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox PDAC tissue, and from mPanIN cells from the Ptf1acre/+;LSL-KrasG12D/+pancreas tissue. The results showed that several chemokines, cytokines, and cell surface proteins were secreted from PDAC cells at much higher levels than from mPanIN cells. These included the Cxc chemokines Cxcl1, Cxcl2, Cxcl5, and Cxcl16. Further analysis showed that expression of the chemokines was upregulated in PDAC cells at the transcriptional level.
The increased expression and secretion of cytokines prompted the investigators to look at whether the interactions between chemokines and their receptors (Cxcl1, Cxcl2, and Cxcl5 bind to Cxcr2, while Cxcl16 binds to Cxcr6) might play a role in regulating PDAC progression. They found that although the receptor Cxcr2 was detected in both normal and mPanIN pancreas epithelia, its expression was relatively prominent at the invasive front of PDAC tissue, in both the stroma and epithelium. Inhibiting expression of Cxcr2 in PDAC cells using the Cxcr2 inhibitor SB225002 had no effect on PDAC cell proliferation, which led to the notion that the increased secretion of Cxc chemokines by PDAC cells might instead be acting on the stromal cells.
Previous work has suggested that the profibrotic Ctgf may have tumor-promoting activities, and the researchers' own prior studies had also found strong expression of Ctfg at the tumor-stromal border of Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox PDAC tissues. “This suggested that an active Ctgf-dependent tumor-stromal interaction was present in the PDAC tissue that could be a therapeutic target, since the interaction increased in intensity during tumor progression,” they note.
The researchers therefore looked to see whether secretion of Cxc chemokines by PDAC cells was inducing Ctgf expression in stromal fibroblasts. QRT-PCR confirmed that the fibroblasts demonstrated much higher basal expression of Ctgf mRNA compared with the PDAC cells, and that Ctgf expression was significantly upregulated when the cells were stimulated with Cxcl1, Cxcl2, and Cxcl5. When fibroblasts were incubated with culture media taken from either PDAC cells or mPanIN cells (a control media without cells was used as a comparison), PDAC cell culture medium induced significantly higher expression level of Ctgf mRNA than mPanIN medium. Furthermore, administering either of the Cxcr2 inhibitors repertaxin or SB225002 inhibited PDAC culture medium-induced Ctgf upregulation by fibroblasts in a dose-dependent manner.
TGF-β signaling is also well known to induce Ctgf expression, and the researchers provided confirmation of this separately, by demonstrating that the Tgfbr1 inhibitor SB431542 dramatically suppressed Ctgf induction. “Taken together, the data suggest that Cxc chemokines produced by PDAC cells can stimulate pancreatic fibroblasts to express Ctgf in a Cxcr2 signal-and TGF-β signal-dependent manner,” the authors write.
To corroborate their in vitro findings in vivo, the researchers compared tumor development in nude mice injected with either PDAC cells taken from a KRAS-activated mouse tissue, or with a mixture of PDAC cells and pancreatic fibroblasts. Although animals receiving both cell types were effectively given just half the number of PDAC cells than animals receiving PDAC cells alone (all animals received the same total number of cells), the mixed-cell injection resulted in faster subcutaneous tumor growth, indicating a tumor-promoting effect of the tumor-stromal interaction. When the resulting mixed-cell tumors were treated with the Cxcr2 inhibitor repertaxin, a significant growth-inhibitory effect was observed within weeks, suggesting that the tumor-promoting effect of the observed tumor-stromal interaction was Cxcr2 dependent.
Cxcr2 was then knocked out in PDAC cells and fibroblasts, and the experiments repeated. This time animals received either Cxcr2 wild-type PDAC cells and Cxcr2-knockdown fibroblasts, or Cxcr2-knockdown PDAC cells and Cxcr2 wild-type fibroblasts. In this case it was the combination of Cxcr2 wild-type PDAC cells and Cxcr2-knockdown fibroblasts that led to slower tumor growth.
To more closely mimic a clinical situation, the researchers evaluated the antitumor effect of Cxcl-Cxcr2 axis inhibition by treating the Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox mice with repertaxin or SB225002. Another cohort of the same mouse model was treated using gemcitabine, with or without repertaxin.
While dissected tumors were generally large enough to take up most of the pancreas in control, repertaxin-treated, and SB225002-treated mice, these animals did often demonstrate areas of morphologically normal pancreatic tissue, which suggested that the inhibitor delayed the tumor development, they remark. Among gemcitabine-treated mice, tumor formation was obviously inhibited, as there was well-retained normal pancreas structure as well as focal tumor areas. Importantly, however, “morphologically normal pancreatic tissue was more frequently observed in the combination treatment with repertaxin and gemcitabine,” the authors stress.
Tumor volume measurements also showed that repertaxin or SB225002 treatment alone significantly decreased the tumor volume, and adding gemcitabine resulted in further suppression. Interestingly, the microvessel density (MVD) in the Cxcr2 inhibitor-treated tumors was significantly lower than that of the control group, while the gemcitabine-treated group did not show obvious inhibition of angiogenesis. Encouragingly, mice treated using SB225002 demonstrated statistically significant increases in overall survival.
Immunohistochemistry confirmed that Ctgf expression had been inhibited in the treatment groups, with both Cxcr2 inhibitors decreasing Ctgf expression in the remaining stroma of PDAC tissue. Gemcitabine-treated tissues, meanwhile, showed a decrease in Ctgf expression that appeared to correlate with the observed decrease of stromal volume.
“The antitumor mechanisms of gemcitabine and the Cxcr2 inhibitor were obviously different,” the authors state. “Pancreas of the mice treated with gemcitabine still retained normal structure with multiple tumor foci, while they did not show a decrease in MVD. In contrast, mice treated with Cxcr2 inhibitor showed diffuse tumor formation in the pancreas with minimal normal area remaining and significantly decreased MVD.
These apparent different mechanisms of action hinted that combining the two forms of antitumor might be a promising therapeutic strategy for PDAC. To test this, the researchers carried out a survival study in which mice were treated using either a combination of gemcitabine plus SB225002, or gemcitabine alone. The combination and gemcitabine-alone treatments showed significant survival extension compared with the control group, although unfortunately, “the combination treatment did not show an advantage in the survival data compared with the single treatment," the authors admit. Observations in some of the treated mice indicated that this may have related at least in part to dose-related drug toxicities.
Nevertheless, they conclude, “These results indicate that blockade of the Cxcl-Cxcr2 axis may be an effective adjuvant therapeutic strategy for PDAC…The combination of gemcitabine, which inhibits DNA synthesis, mainly targeting tumor cells, and Cxcr2 inhibitor, which mainly modulates tumor microenvironment and inhibits angiogenesis, might be a synergistic therapeutic strategy and may prevent excessive toxicities by allowing dose reduction of each drug.”