Associate Professor Worcester Polytechnic Institute, United States
Introduction: : Lymphatic capillary function is characterized by loose cell-cell junctions that enable tissue drainage and facilitate immune cell travel to lymph nodes. When tissue stiffness increases due to fibrosis, lymphatic capillary sprouting (i.e., growth) decreases and cell-cell junctions tighten to form zipper-like junctions that inhibit normal entry of immune cells into the vasculature. Soluble factors like tumor necrosis factor (TNF)-α have been shown to disrupt tight junctions in lymphatics in edema, restoring function to lymphatic vessels in stiff tissue. Pancreatic ductile adenocarcinoma (PDAC) has a stiff, cytokine-rich stroma that produces an avascular tumor with a dense network of peritumoral lymphatics through which cancer cells metastasize. Current PDAC metastasis research focuses on the effects of stiffness and cytokines on the movement of immune and PDAC cells out of the tumor. Meanwhile, mechanisms behind the entry of immune and PDAC cells into lymphatic vessels are understudied. To address this gap, we performed experiments to investigate individual components of immune and PDAC entry into lymphatic vessels, including migration towards lymphatic endothelial cells (LEC) in co-culture and LEC response to stiffness and inflammatory signals. These study results provide a foundation for future development of a 3D microfluidic model to study mechanisms of entry for lymphatic function during PDAC metastasis and immune cell clearance under fibrotic conditions. Future work using this model will test therapeutic approaches to reduce metastasis and encourage immune response through increased vascular entry.
Materials and
Methods: : To assess LEC response to TNF-α, primary human dermal LEC (HDLEC) (PromoCell) were seeded in a 6-well plate on tissue culture plastic at 30,000 cells/cm2. HDLECs were cultured in MV2 media (PromoCell) supplemented with 5% fetal calf serum without Vitamin C or hydrocortisone. HDLEC were exposed to TNF-α (20 ng/mL) for 24 hours. Media was collected for chemoattractant analysis using a human chemokine array kit (R&D Systems). HDLEC were then lysed for western blot quantification of VCAM and E-Selectin expression.
To study immune and cancer migration, green fluorescent protein (GFP) expressing mature dendritic cell (mDC) derived THP-1 cells (Gift from J. Coburn, WPI) and PANC-1 pancreatic ductal adenocarcinoma cells (ATCC) were used. Two polydimethylsiloxane (PDMS) blocks with 1 cm diameter holes were added to each well of a 6-well plate. HDLEC were added to one block (30,000 cells/cm2) and either THP-1 monocytes (500,000 cells) in 10% fetal bovine serum (FBS) supplemented RPMI Glutamax® or PANC-1 cells (50,000 cells/cm2) in 10% FBS supplemented DMEM were added to the other PDMS block and cultured for 3 days. THP-1 cells were allowed to differentiate into mDC using TNF-α (20 ng/mL), IL-4 (200 ng/mL), GM-CSF (100 ng/mL), and ionomycin (200 ng/mL). PANC-1 cells were labeled with CellTracker® CMFDA (Invitrogen) before PDMS blocks were removed allowing for HDLEC direct co-culture with either mDC or PANC-1 cells at 1:1 media ratio. Co-cultures were then imaged using fluorescence microscopy on Day 2 for mDC and PANC-1 cultures.
Results, Conclusions, and Discussions:: The current study modeled individual steps leading to immune and cancer cell entry and migration through lymphatic vessels. First, HDLEC response to inflammatory signaling using TNF-α showed increases in chemoattractants, upregulation of cell adhesion proteins (Figure 1). Altered chemokine production and increased expression of trafficking proteins indicate increased crosstalk between LEC and both immune and cancer cells, leading to lymphatic entry. Subsequent migration studies of mDC or PANC-1 cells co-cultured with HDLEC showed increased migration toward HDLECs with groups dosed with TNF-α (Figure 2). Both results show that not only do LEC release relevant chemoattractants that encourage immune and cancer cell interactions, but that immune and cancer cells are responsive to LECs.
Results from these studies, combined with prior work from our research group, provide a solid foundation to develop a microfluidic model of cellular entry into lymphatic capillaries in the future. We previously showed that ColMA stiffness alters junction formation (i.e., VE-Cadherin) in HDLECs with increased zippering observed in cells on stiff collagen (~6 kPa) in a well plate and lining an ECM channel within a microfluidic chip. Through sprouting studies within a microfluidic chip, soft (~1 kPa) and stiff (~6 kPa) ColMA replicated in vivo sprouting patterns observed in acute and chronic fibrotic conditions. Collectively, these studies provide the framework for next steps to integrate relevant PDAC tumor signals—ECM stiffness and TNF-α exposure—with our microfluidic lymphatic capillary model to replicate how stiffness and inflammatory signaling regulate lymphatic entry of mDC and PDAC cells during metastasis. By developing this model, mechanisms behind cancer metastasis through lymphatics can be better understood. The proposed model can also be used in future work as a therapeutic screening platform for targeting lymphatic metastasis, as well as serve as a modeling platform for other lymphatic conditions such as lymphedema.
