The CXCR1 Axis: A Putative Therapeutic Cancer Stem Cell-Like Marker in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of all cancers in the United States. Not only is PDAC found at the late stages, but patients also present with or develop chemotherapy resistance at an elevated frequency. Left with limited options for treatment, researchers are investigating new options for these patients. One major area of interest is the sub-population of cells in the tumor called cancer stem cells (CSCs). These cells are known for having high resistance to chemotherapy, along with propagating and re-building the tumor after most non-CSCs have been therapeutically targeted. Previous studies have determined CXCR4, ALDH1, CD24, CD44, and CD133 as markers for CSC-like PDAC cells. In the present study, we investigate the closely related CXCR1 as another possible marker and therapeutic target for PDAC CSCs. CXCR1 is known for its role in inflammation and wound healing. The CXCR1 axis includes the ligands CXCL6 and IL-8, both of which promote the progression of cancer. Previously, Ginesteir et al. has shown targeting the CXCR1 axis in triple negative breast cancer reduced CSC-like phenotypes in vitro and in vivo. Investigations of CXCR1 in PDAC demonstrate IL-8 induces increased tumorsphere formation in vitro (Chen et al.), leading us to investigating CXCR1 in PDAC CSCs. We hypothesize that PDAC cells with high CXCR1 activity also exhibit increased CSC-like characteristics and targeting CXCR1 will reduce those characteristics. To investigate the role of CXCR1 in CSC-like phenotype of PDAC, we used the PDAC cell line CD18, along with its gemcitabine resistant (GemR) counterpart. We used the CXCR1/2 antagonist Navarixin at high enough concentrations to inhibit CXCR1. Using the previously found gemcitabine and navarixin IC50 concentrations for each parent cell line, we treated cells for 72 hours. Post-treatment, we analyzed the expression of several known CSC markers, CXCR1, and IL-8 through qRT-PCR and ELISA. We expected to see higher expression and activity of CXCR1 in cells with higher known CSC marker expression. We also anticipated that gemcitabine treatment would induce higher expression of CSC markers, whereas navarixin would exhibit lower expression. From our results, we see the beginning trends of gemcitabine treated cells having increased expression of the CSC markers and navarixin decreasing or not changing the expression levels. These results differ for IL-8, which undergoes an increase in expression when treated with both gemcitabine and navarixin, which may warrant further exploration into the role of ligands in CSC-like phenotypes. One possible explanation for this difference would be the regulation of IL-8 expression based on CXCR1 activity, as IL-8 interacts with CXCR1.https://digitalcommons.unmc.edu/surp2021/1040/thumbnail.jp

Neutrophils in Pancreatic Cancer Progression

Pancreatic cancer (PC) remains a challenge to modern-day cancer therapeutics, with a dismal five-year survival rate of 10%. Due to the pancreas\u27s location and desmoplasia surrounding it, patients receive late diagnoses and fail to respond to chemotherapy regimens. Tumor-promoting inflammation, one of the emerging hallmarks of cancer, contributes to tumor cells\u27 survival and proliferation. This inflammation is often the result of infiltrating leukocytes and pro-inflammatory cytokines released into the tumor microenvironment (TME). Neutrophils, one of the most prominent immune cells in our body, play an essential role in sustaining this smoldering inflammation observed in the TME. Previously, our group has shown that these neutrophils are complicit in breast cancer progression and even metastasis. With a similar rationale in mind, this study focuses on how neutrophils invading the TME, also known as tumor-associated neutrophils (TAN\u27s), correlate with disease progression in pancreatic cancer. Our data demonstrated that TAN infiltration is associated with disease progression. Furthermore, to understand this TAN infiltration, we theorized that the TME plays a significant role in TAN recruitment and TAN proliferation. Our previous work elucidated TAN recruitment by showing increased expression of chemokines in the TME. We also examined TAN and tumor cell interaction in vitro and observed increased tumor cell survival and decreased neutrophil survival. This is theoretically explained by the increased propensity of neutrophils to undergo NETosis and form neutrophil extracellular traps, which have also been shown to correlate with disease progression. Our data suggested neutrophil differentiation in the TME leads to the upregulation of multiple chemokines and, in theory, explains the high TAN infiltration observed in the TME. Together, these data suggest the critical role of TAN and tumor cell interaction in the TME

Breast Cancer Cell–Neutrophil Interactions Enhance Neutrophil Survival and Pro-Tumorigenic Activities

Breast cancer remains the most prevalent cancer in women with limited treatment options for patients suffering from therapy-resistance and metastatic disease. Neutrophils play an important role in breast cancer progression and metastasis. We examined the pro-tumorigenic nature of the breast cancer cell–neutrophil interactions and delineated the differences in neutrophil properties between the chemotherapy-resistant and the parent tumor microenvironment. Our data demonstrated that high neutrophil infiltration is associated with disease aggressiveness and therapy resistance. In the human breast cancer dataset, expression of neutrophil-related signature gene expression was higher in tumors from therapy-resistant patients than therapy-sensitive patients. We observed that breast cancer-derived factors significantly enhanced neutrophil survival, polarization, and pro-inflammatory cytokine expression. Breast cancer cell-derived supernatant treated neutrophils significantly expressed high levels of interleukin-1β (IL-1β), CC-chemokine ligand-2-4 (CCL2, CCL3, CCL4), inducible nitric oxide synthase (iNOS), and matrix metallopeptidase-9 (MMP9), and formed extracellular traps (NETs). Moreover, neutrophils showed increased secretion of MMP9 when cultured with the supernatant of chemotherapy-resistant Cl66-Doxorubicin (Cl66-Dox) and Cl66-Paclitaxel (Cl66-Pac) cells in comparison with the supernatant of Cl66-parent cells. Together, these data suggest an important role of breast cancer cell–neutrophil interactions in regulating pro-tumor characteristics in neutrophils and its modulation by therapy resistance

Plexin-B3 Regulates Cellular Motility, Invasiveness, and Metastasis in Pancreatic Cancer

The Plexins family of proteins are well-characterized transmembrane receptors of semaphorins, axon guidance cue molecules, that mediate the cell attraction or repelling effects for such cues. Plexins and their ligands are involved in numerous cellular activities, such as motility, invasion, and adhesion to the basement membrane. The detachment of cells and the gain in motility and invasion are hallmarks of the cancer metastasis cascade, thus generating interest in exploring the role of plexins in cancer metastasis. Semaphorin–plexin complexes can act as tumor promoters or suppressors, depending upon the cancer type, and are under investigation for therapeutic purposes. Our group has identified Semaphorin-5A (SEMA5A)/Plexin-B3 as an attractive targetable complex for pancreatic cancer (PC) metastasis. However, our understanding of the Plexin-B3 function and pathological expression in PC is limited, and our present study delineates the role of Plexin-B3 in PC malignancy. We examined the pathological expression of Plexin-B3 in PC tumors and metastasis using a human tissue microarray, disease progression model of PDX-Cre-Kras(G12D) (KC) mice, and different metastatic sites obtained from the KrasG12D; Trp53R172H; Pdx1-Cre (KPC) mice model. We observed a higher Plexin-B3 expression in PC tumor cores than the normal pancreas, and different metastatic sites were positive for Plexin-B3 expression. However, in the KC mice model, the Plexin-B3 expression increased initially and then decreased with the disease progression. Next, to evaluate the functional role of Plexin-B3, we utilized T3M-4- and CD18/HPAF-Control and -Plexin B3 knockdown cells for different in vivo and in vitro studies. The knockdown of Plexin-B3 enhanced the in vitro cellular migration, invasiveness, and impaired colony formation in three-dimensional culture, along with an increase in cellular spread and remodeling of the actin filaments. We also observed a higher metastasis in nude mice injected with T3M-4- and CD18/HPAF-shPlexin-B3 cells compared to their respective control cells. Furthermore, we observed a lower number of proliferating Ki-67-positive cells and higher ALDH1-A1-positive cells in the tumors formed by Plexin-B3 knockdown cells compared to tumors formed by the control cells. Together, our data suggest that the loss of Plexin-B3 is associated with the interference of cell division machinery and the induction of stem cell-like characteristics in PC cells