(L-443) Prolactin Protein Essential for Mammary Gland Production in Females Sensitizes Colorectal Cancer Cells to TRAIL-mediated Apoptosis

The 5-year relative survival rate for localized colorectal cancer (CRC) is 91%, whereas patients with metastatic disease have a reduced rate of 13% [1]. During the metastatic cascade, cancerous cells migrate from the primary tumor, enter the bloodstream, and travel as circulating tumor cells (CTCs) until finding a secondary site for tumor formation . Previously, the King Lab designed a liposomal system which binds to the surface of healthy leukocytes in the circulation by tethering to E-selectin (ES) ligands, greatly increasing circulation time. These liposomes, also conjugated with TNF-related apoptosis inducing ligand (TRAIL), successfully killed CTCs in vitro and in vivo [2]. Since then, this technology has been adapted for a dual affinity (DA) approach where liposomes are decorated with an anti-cell surface vimentin (CSV) antibody fragment allowing nanoparticles to transfer from the leukocyte surface to the CTCs during a collision, increasing therapeutic efficacy.

While this approach effectively treats CTC models in vitro and in vivo, it relies on TRAIL-mediated apoptosis which some CRC cell lines are resistant to, such as HT29 [3]. Hyperprolactinemia is a state of increased prolactin (PRL), a protein usually found to assist in lactation during pregnancy; however, PRL has also been found to consistently coincide with disease aggression in most CRC cases [4].  While prolactin presence has previously been seen as an indicator of poor prognosis, this study reveals the ability of PRL to increase TRAIL treatment efficacy, therefore lowering the incidence of metastasis through the bloodstream.  

First, PRL sensitization to escalating TRAIL dosages was tested. 25,000 cells were plated into each well of a 24-well plate and treated with 120 ng/mL of PRL for 24 hr. TRAIL treatment was introduced at t = 48 hr (0.1, 1, 10, 100, and 1000 ng/mL).  At 72 hr, cell viability was quantified using an Annexin V and propidium iodide assay via flow cytometry. Liposomes were synthesized following our  previous protocol [2]. Half-antibodies were produced by incubating an anti-CSV antibody with reducing agent 2-MEA. Liposomes were incubated overnight at 4°C to achieve a final concentration per liposome of 6 half-antibodies, 2 his-tagged ES, and 20 TRAIL. Control groups consisted of a buffer only group and a soluble TRAIL group (590 ng/mL). HT29 cells were treated with 120 ng/mL of PRL for 4 days prior to treatments. Cone-and-plate viscometers (Brookfield) equipped with CP-41 spindles were used to expose the samples to FSS. 20mL of each liposome group was placed in 2 mL of whole blood (collected from healthy volunteers after informed consent) and sheared for 30 min.  The samples were spiked with 500,000 HT29 cells/mL as a CTC model and sheared for an additional 2 hr. The buffy coat was isolated by gradient centrifugation and samples were incubated for 24 hr. Magnetic bead separation was performed to remove immune cells and samples were stained with anti-CD45 antibody tagged with EFluor450 to gate out any remaining blood cells.  Cell viability was quantified by Annexin V and propidium iodide assay via flow cytometry.

In this study, we investigated the sensitization of the TRAIL-resistant CRC cell line HT29 to TRAIL-mediated apoptosis via PRL treatments. HT29 cells were treated with 5 concentrations of TRAIL (0.1, 1, 10, 100, and 1000 ng/mL) for 24 hr and then tested for cell viability via an Annexin V/propidium iodide assay. HT29 cells treated solely with TRAIL showed no changes in cell viability (Figure 1A) regardless of dosage. This result is consistent with findings in the literature which confirm HT29 cell resistance to TRAIL. In comparison, when HT29 cells were treated with 120 ng/mL of PRL, they showed a significant decrease in cell viability after treatment with 100 ng/mL and 1000 ng/mL of TRAIL. PRL was found to reliably sensitize the HT29 cells to TRAIL by 10% with each incremental dose.

An in vitro model for CTC targeting was then utilized to investigate the ability of DA liposomes developed by the King Lab to treat PRL-sensitized HT29 cells under physiological FSS conditions. The total TRAIL concentration in the liposomal treatment was 590 ng/mL (~20 TRAIL molecules per liposome). Liposomes were exposed to FSS in whole blood for 30 min and subsequently spiked with HT29 cells and sheared for 2 hr. As seen in Figure 1B, no change in cell viability, apoptosis, or necrosis was observed between the treatment groups compared to the controls in which blood samples were spiked with HT29 cells that had not been pretreated with PRL. When the HT29 cells were pretreated with 120 ng/mL of PRL for 4 days, they showed a significant decrease in cell viability following treatments that included TRAIL compared to the buffer control, as seen in Figure 1C. This trend was especially pronounced in the DA liposome group in which the mean cell viability of the PRL-treated HT29 cells decreased by 40%. This study indicates that doses of TRAIL delivered via DA liposomes in the presence of prolactin may finally present a solution to previously drug-resistant metastatic cases of CRC.

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