Given the increasing incidence, very low five-year survival rates [1], and often late-stage diagnosis[2], it is crucial to gain a more comprehensive understanding of the initiation and progression of PDA. PSC not only play a role in maintaining a healthy pancreatic stroma [4] but also serve as a source of CAF within the PDA stroma [6]. Therefore, this work aimed to examine interactions between PSCs and cancer cells under various conditions to better understand their paracrine crosstalk. It has been shown previously that CAF adopt different phenotypes influenced by whether they have direct contact with cancer cells or only via paracrine effects [10]. In this work, the proximity of both cell types during paracrine communication was highlighted as a new parameter of this complex relation. Therefore, two different paracrine co-culture models with PSC and KPC cells, standard co-culture and inverse co-culture, differing by the distance of cells, were established.
RNA sequencing of PSC after 24 h and 72 h revealed that changes in PSC gene expression profiles occur after 24 h and increase over time in both co-cultures. A reduced distance between PSC and KPC cells led to a 40 higher number of differentially expressed genes in PSC. Among relevant enriched signaling pathways such as cytokine interaction, cell growth, and proteoglycan metabolism in inverse co-culture, the pathway Proteoglycans in cancer was mutually enriched at 24 h and 72 h. The proteoglycan VCAN and the enzyme CHST11 could be identified as potential key genes as two of six mutually differentially expressed genes in PSC in both co-cultures at both time points.
VCAN is a chondroitin sulfate proteoglycan and a component of the extracellular matrix involved in various physiological and pathological biological processes. Expression and modification of VCAN are regulated by different pathways such as Wnt/ß-catenin and multiple factors such as interferons, growth factors, and miRNAs [23]. CHST11 is an enzyme of the CS proteoglycan biosynthesis, which can be found in the Golgi apparatus and facilitates the sulfation of chondroitin at the C4 position of N-acetylgalactosamine (C4-GalNAc), leading to the formation of the CS subtype CS-A, one of six different CS subtypes [24]. The sulfation pattern of VCAN in PDA differs from normal pancreas tissue. In PDA, 6-sulfated disaccharides are predominant, whereas in a normal pancreas, 4-sulfated disaccharides build most of the sulfation pattern [25]. In this study, CHST11, which catalyzes the creation of 4-sulfated disaccharides, was up-regulated. CHST1, which catalyses the creation of 6-sulfated disaccharides [25], was not significantly altered. Chemical analyses of PDA tissue revealed an up to 27-fold increase of VCAN with an altered sulfation pattern, shifting towards a richer chondroitin sulfate (CS) content of side chains. These changes could create a tumor-permissive environment, as CS chains are less likely to decelerate tumor cell migration than glycosaminoglycans like dermatan sulfate [25]. The abundance of CS chains in PDA compared to normal pancreas could also explain the elevated levels of CHST11, as the overall content of 4-sulfated disaccharides was much higher in PDA than in normal pancreatic tissue due to a higher CS content. Furthermore, the temporal dynamic of changes in the VCAN PDA sulfation pattern has not been investigated in detail yet and could play a role in the elevated CHST11 expression observed in this work.
Carbohydrate sulfotransferases, such as CHST7, CHST11-13, or CHST15, have been suggested to be relevant in developing and progressing multiple types of cancer, as in PDA. In contrast, the exact roles of the enzymes in the PDA context have yet to be determined [26]. CHST11 may be important in tumor-associated VCAN sulfation patterns, especially in early stromal changes. In agreement with this hypothesis, CHST11 was recently found to be involved in a CAF signature of 12 genes that correlates with the overall survival of PDA patients and predicts a poor response to chemotherapy [27]. Further investigation is required to explore how and whether the upregulation of CHST11 is attributed to specific post-translational tumorigenic modifications of VCAN.
Fn1, as part of the VCAN PPI-network and up-regulated at 24 h in inverse co-culture, has been identified in a bioinformatic analysis of PDA pathogenesis as one of the potential hub genes [28]. Fn1 is a glycoprotein of the ECM which plays a role in cell migration and adhesion processes [29]. In human PDA, Fn1 is the primary element of the tumour matrix [30].
SDC4, as another up-regulated member of the VCAN-PPI network, has already been described in VCAN-PPI networks of breast cancer [33]. SDC4 is a transmembrane heparan sulfate proteoglycan that plays a role in extracellular matrix processes such as cell adhesion, organization of cell cytoskeleton, and cell motility. While Sdc4 is not present in PDA cells, upregulation in activated PSC was described. Syndecans also can influence tissue stiffness [34], a notable concern in PDA, given the characteristic dense stroma associated with the disease.
The elevated levels of VCAN in this work are consistent with previous work from Rainiero et al., where even in the earliest stages of PDA, VCAN production was detected by immunohistochemistry in epithelial and stromal cells. Elevated levels of VCAN could also be detected in PSC derived from human PDA, but interestingly, PDA-conditioned media did not significantly influence the expression in PSC [35]. This emphasizes the potential requirement for cellular crosstalk, rather than solely secreted factors from cancer cells, in specific processes such as VCAN upregulation. Corresponding to these findings, in this work, the immunohistochemistry of human PDA samples showed an enhanced VCAN expression compared to a healthy pancreas. Both epithelial and stromal cells can be sources of VCAN. VCAN derived from myeloid cells acts anti-inflammatory, whereas VCAN from stromal cells is part of the inflammatory response that occurs in most cancers by interacting with immune cells, chemokines, and growth factors [23]. This study does not provide evidence of whether the increased VCAN gene expression in PSC has a protective or cancer-permissive effect; hence, obtaining a better understanding of the pro- and anti-tumorigenic functions of VCAN is essential. Despite the abundance of identified potential key genes in PDA bioinformatic studies, VCAN was consistently described as one of the relevant hub genes in PDA [28],[36], which emphasizes the possible key role of VCAN.
As VCAN is part of various structures in the human body, it would be interesting if specific PDA-modified VCAN variants exist, if there is shedding into the bloodstream, and how it could be detected and even be used as a biomarker. For a more detailed understanding of PSC-KPC crosstalk, it would have been beneficial to conduct RNA sequencing on PSC and KPC cells. The analysis of VCAN expression and VCAN PPI networks in KPC cells would have helped to gain a deeper insight into the complex interactions of cancer and stromal cells.
In conclusion, this study shows the importance and influence of the distance of cells to each other regarding changes in gene expression profiles. VCAN was identified as one of the potential key genes altered through early PSC cancer cell crosstalk, and further studies investigating the role of VCAN in PDA initiation and progression are needed.