The adsorption of cationic peptide JM21 onto different mesoporous silica nanoparticles (MSNs) from an aqueous solution was studied as a function of pH. In agreement with the literature, the highest loading degrees could be achieved at pH close to the isoelectric point of the peptide where the peptide-peptide repulsion is minimum. However, mesopore size, mesopore geometry, and surface polarity all had an influence on the peptide adsorption in terms of both affinity and maximum loading at a given pH. This adsorption behavior could largely be explained by a combination of pH-dependent electrostatic interactions and confinement effects. It is demonstrated that hydrophobic interactions enhance the degree of peptide adsorption under pH conditions where the electrostatic attraction was absent in the case of mesoporous organosilica nanoparticles (MONs). The lower surface concentration of silanol groups for MON led to a lower level of peptide adsorption under optimum pH conditions compared to all-silica particles. Finally, the study confirmed the protective role of MSNs in preserving the biological activity of JM#21 against enzymatic degradation, even for large-pore MSNs, emphasizing their potential as nanocarriers for therapeutic peptides. By integrating experimental findings with theoretical modeling, this research elucidates the complex interplay of factors that influence peptide-silica interactions, providing vital insights for optimizing peptide loading and stabilization in biomedical applications.

Pancreatic cancer is a fatal disease and is one of the most aggressive and metastatic malignancies worldwide. The dissemination of tumor cells is the prerequisite of metastases and correlates with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumor progression. Migrating cancer stem cells (miCSCs) characterized by CD133+ and CXCR4+ expression play a pivotal role in malignant tumor formation, have been reported to form the invasive front of the metastasis and in silico analysis showed that both CSCs and miCSCs are significantly upregulated in PDAC. However, the regulatory pattern maintaining these CSCs and especially miCSCs in PDAC remains widely elusive. Thus, this study further helps to unravel pathways responsible for maintenance of CSC (CD133+) and miCSC (CD133+ CXCR4+) population. To identify key signaling pathways responsible for both CSCs and miCSCs maintenance, we first generated a protein-protein interaction network using STRING database. Afterwards, we validated these signaling pathway(s) involved in aiding CSC and miCSC population by performing shRNA mediated knockdown of key signaling proteins in different patient-derived pancreatic cancer cell lines. Moreover, we interrogated the involvement of the tumor-stroma crosstalk in the regulation of these pathways by co-culturing tumor cells with pancreatic stellate cells. Protein-protein interaction network incorporating relevant factors involved in EMT, stemness, as well as SHH, NF-kB and AKT signaling pathway identified a strong link between the CXCL12/CXCR4 signaling axis and BMI1. Migration assay, sphere formation assay and western blot upon shRNA mediated knockdown of either CXCR4 and/or BMI1 ascertained BMI1 as a key player downstream of the CXCL12/CXCR4 axis to mechanistically effect both EMT and stemness. Pathway focused gene expression analysis as well as ELISA and immunofluorescence for CXCL12 and actin filaments, respectively, revealed the indispensable nature of tumor-stroma crosstalk on promoting CSC and miCSC population through the chemokine CXCL12. In addition, co-culture systems revealed that particularly pancreatic stellate cells play a significant role in maintaining both CSCs and miCSCs population as well as their characteristic phenotype including but not limited to chemotherapy resistance. Taken together, our results obtained in this study established mechanistically that the CXCL12/CXCR4 signaling pathway driven by tumor-stroma crosstalk not only enhances but also maintains both CSCs and miCSCs in pancreatic ductal adenocarcinomas through BMI1 ultimately promoting metastases and therapeutic resistance. Citation Format: Kanishka Tiwary, Anton Lahusen, Syeda Inaas, Stefanie Hauff, Karolin Walter, Alexander Kleger, Thomas Seufferlein, Bruno Sainz Jr., Patrick Christian Hermann. CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B035.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the “stemness” maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) remains a largely incurable cancer type. Its high mortality is attributed to the lack of efficient biomarkers for early detection combined with its high metastatic properties. The aim of our study was to investigate the role of NF-κB signaling in the development and metastasis of PDAC. We used the well-established KPC mouse model, and, through genetic manipulation, we deleted NF-κB essential modulator (NEMO) in the pancreata of KPC mice. Interestingly, NEMO deletion altered the differentiation status of the primary tumor but did not significantly affect its development. However, in the absence of NEMO, the median survival of the mice was prolonged by 13.5 days (16%). In addition, examination of the liver demonstrated that, whereas KPC mice occasionally developed liver macro-metastasis, NEMO deletion completely abrogated this outcome. Further analysis of the tumor revealed that the expression of epithelial–mesenchymal transition (EMT) transcription factors was diminished in the absence of NEMO. Conclusively, our study provides evidence that NF-κB is dispensable for the progression of high-grade PanINs towards PDAC. In contrast, NF-κB signaling is essential for the development of metastasis by regulating the gene expression program of EMT.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with a very poor prognosis. At the same time, its incidence is on the rise, and PDAC is expected to become the second leading cause of cancer-related death by 2030. Despite extensive work on new therapeutic approaches, the median overall survival is only 6-12 months after diagnosis and the 5-year survival is less than 7%. While pancreatic cancer is particularly difficult to treat, patients usually succumb not to the growth of the primary tumor, but to extensive metastasis; therefore, strategies to reduce the migratory and metastatic capacity of pancreatic cancer cells merit close attention. The vast majority of pancreatic cancers harbor RAS mutations. The outstanding relevance of the RAS/MEK/ERK pathway in pancreatic cancer biology has been extensively shown previously. Due to their high dependency on Ras mutations, pancreatic cancers might be particularly sensitive to inhibitors acting downstream of Ras. Herein, we use a genetically engineered mouse model of pancreatic cancer and primary pancreatic cancer cells were derived from this model to demonstrate that small-molecule MEK inhibitors functionally abrogate cancer stem cell populations as demonstrated by reduced sphere and organoid formation capacity. Furthermore, we demonstrate that MEK inhibition suppresses TGFβ-induced epithelial-to-mesenchymal transition and migration in vitro and ultimately results in a highly significant reduction in circulating tumor cells in mice.

Little is known about the mechanisms that enable organisms to cope with unpredictable environments. To address this issue, we used replicate populations of Escherichia coli selected under complex, randomly changing environments. Under four novel stresses that had no known correlation with the selection environments, individual cells of the selected populations had significantly lower lag and greater yield compared to the controls. More importantly, there were no outliers in terms of growth, thus ruling out the evolution of population-based resistance. We also assayed the standing phenotypic variation of the selected populations, in terms of their growth on 94 different substrates. Contrary to expectations, there was no increase in the standing variation of the selected populations, nor was there any significant divergence from the ancestors. This suggested that the greater fitness in novel environments is brought about by selection at the level of the individuals, which restricts the suite of traits that can potentially evolve through this mechanism. Given that day-to-day climatic variability of the world is rising, these results have potential public health implications. Our results also underline the need for a very different kind of theoretical approach to study the effects of fluctuating environments.