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.
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.
Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of cancers, with a five-year relative survival of 9%. The significant factor contributing to low survivorship is the intrinsic resistance to chemotherapy. In the present study, we elucidate a targetable pathway that facilitates resistance in these cells. One inflammatory pathway used in cancer progression is CXCR2, which binds chemokines CXCL 1-3 and 5-8. Our group and others have shown an association of CXCR2 with tumor progression, angiogenesis and metastasis in PDAC, and chemotherapy resistance in other cancers. In this study, we test the hypothesis that CXCR2 is integral in PDAC chemotherapy resistance. We generated gemcitabine resistant cell lines (GemR) from the parent cell lines of T3M4 (WT KRAS) and CD18/HPAF (Mutant KRAS) with 40- and 67-fold higher resistance in comparison to the parent cell lines, respectively. Using qRT-PCR and ELISA, we profiled chemokines' expression, CXCL1, 5, and 8, on parent- and GemR-cell lines. We observed a higher basal level expression of CXCL1, 5, and 8 ligands in GemR cells than the parent cell lines. Further treatment with gemcitabine, the parent cell lines had a higher increase in CXCL1 and 8 ligand expression in comparison with GemR cell lines. With further investigation of T3M4, we found a dose-dependent and time-dependent increase for CXCL1 and CXCL8. CXCL5 had a constant high expression with all doses of gemcitabine. With our initial observations of gemcitabine treatment enhancing CXCR2 ligands in PDAC cells, we evaluated the effects of the small molecule CXCR2 antagonist SCH 479833 (SCH 47) in combination with gemcitabine treatment on the parent and GemR cell lines. We used the relative IC50 and half the relative IC50 for both the gemcitabine and SCH 47 treatment doses. We observed that a lower concentration of gemcitabine in the presence of the SCH 47 was more effective than the higher concentration of gemcitabine without SCH 47 for the GemR-cell lines. Together, our observations show chemotherapy-resistant PDAC cell lines express higher CXCR2 ligand levels at baseline, with parent cell lines increasing expression with chemotherapy treatment. We also show an advantageous combination treatment of CXCR2 antagonist with lower gemcitabine concentration. Overall, these results show a promising future for CXCR2 antagonism in the treatment of resistant PDAC tumors. Citation Format: Caitlin Molczyk, Dipakkumar R. Prajapati, Sugandha Saxena, Paran Goel, Michelle Varney, Rakesh K. Singh. CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1087.
Pancreatic cancer (PC) remains a challenge to modern-day cancer therapeutics, with a dismal five-year survival rate of 10%. Due to the pancreas' tricky location and the dense stroma surrounding it, patients have late diagnosis and fail to respond to chemotherapy regimens. The genetic stability of the tumor microenvironment (TME) in comparison with rapidly evolving tumor cells makes it an attractive target for the development of cancer therapeutics. Neutrophils, the most abundant immune cell, is an essential player in the TME. These neutrophils can become pro-tumorigenic based on signals they receive in the TME by regulating immune suppression, tumor metastasis, and angiogenesis. Previous studies from our laboratory demonstrate an increase in the number of neutrophils in the pancreatic tumor with the PC disease's progression in the KPC Model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). Based on our previous observation and accumulating literature, we hypothesize that pancreatic tumor cells can increase neutrophil proliferation and/or survival in circulation and tumor in situ. We investigated the effect of soluble factors released by pancreatic tumor cells on neutrophil proliferation/survival with this rationale. For this objective, we utilized murine promyelocytes cells (MPRO), a mouse neutrophil cell line, and a human leukemia cell line (HL60), a human neutrophil cell line. We treated MPRO and HL60 cells with a gradient of supernatants collected from T3M-4- and CD18/HPAF-parent and gemcitabine-resistant cells. By utilizing the WST assay, we observed an increase in MRPO and HL60 cells' viability with an increasing gradient of supernatant collected from both T3M-4, CD18/HPAF-parent gemcitabine-resistant cells with serum-free media as a control. We also observed a slight increase in neutrophil viability when treated with the supernatant collected from parent cell-lines supernatant compared to supernatant collected from the gemcitabine resistant cell-lines. Next, to delineate the effect of direct interaction between pancreatic tumor cells and neutrophils, we utilized a co-culture assay between MPRO cells and CD18/HPAF cells. We observed an increase in the viability of CD18/HPAF cells when placed in contact with the neutrophils; however, we observed inhibition in the viability of neutrophils placed in contact with the tumor cells. Our results showing positive viability of neutrophils with the treatment of soluble tumor factors explain higher neutrophil to lymphocyte ratio in PC patients' blood circulation. Neutrophils in the TME release pro-tumorigenic cytokines that increase tumor cell proliferation, albeit resulting in neutrophil death as observed by neutrophil growth inhibition. Along with neutrophil survival, a more mechanistic understanding of neutrophil recruitment and polarization in TME will facilitate designing novel therapeutics for PC. Citation Format: Paran Goel, Sugandha Saxena, Caitlin Molczyk, Rakesh Singh. Soluble factors released by pancreatic cancer cells enhance neutrophil survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3196.
Pancreatic cancer (PC) continues to be a challenge for today’s therapeutics, with a meager 5-year survival rate of 11%. Due to the pancreas’ location, patients often receive diagnosis late, leaving them with limited treatment options. Often, they fail to respond to chemotherapy regimens or develop resistance to those therapies. Tumor-promoting inflammation is a hallmark of cancer that contributes to tumor cells’ survival and proliferation. Infiltrating leukocytes and pro-inflammatory cytokines released into the tumor microenvironment (TME) often cause this inflammation. Previously we have determined that inflammation and CXCR2 ligand expression increases with pancreatic ductal adenocarcinoma (PDAC) progression. In the present study, we examined the role of neutrophil recruitment in PDAC progression using Kras-dependent KC and KPC murine models. Our data suggest a positive association between tumor-associated neutrophils (TANs) and PDAC progression. Furthermore, the frequency of neutrophil extracellular traps (NETs) also increased as the disease progressed. In vitro, we observed PDAC cell condition-media enhanced survival of neutrophils in a concentration-dependent manner. Moreover, increased survival was associated with disease aggressiveness and therapy resistance. Additionally, we observed that PDAC-conditioned media modulated the expression of immunoregulatory cytokines and chemokines in neutrophils. Together, our data suggest that increased recruitment of TANs supports PDAC progression, and PDAC cell-neutrophil interaction modulates neutrophil survival and immunoregulatory phenotype. Citation Format: Reegan Sturgeon, Paran Goel, Caitlin Molczyk, Surindar K. Batra, Rakesh Singh. Neutrophil pro-tumorigenic role in progression and therapy resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1348.
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