Poster X15 - Cryoshocked T Lymphocytes (CSTLs) for Enhanced Delivery of Chemotherapeutics to the Lymph Node

Introduction:

Introduction: Cancer metastasis is responsible for a large majority of cancer-related deaths1,2. The spread of metastatic cells to local draining lymph nodes (LNs) is a common and threatening progression of the disease that can result in priming of distal sites for further spread and significant dysregulation of systemic immune responses. The LN is an innately isolated organ which is conducive to efficient immune responses, however this isolation also results in extremely poor penetration of most systemically administered therapeutics3-5. Disease progression coupled to our inability to deliver sufficient drug payloads to the LN results in a lack of viable treatment strategies for LN metastasis. To overcome this gap, we have developed a T-cell mimetic delivery vehicle to deliver chemotherapeutic drug payloads to the LN using a Trojan-horse strategy. When compared to free drug administration, our platform resulted in enhanced LN targeting, increased retention time, and local sustained release.

Materials and

Methods: Materials and

Methods: T lymphocytes used to produce CSTLs were isolated from mice (CD-1) via magnetic-activated cell sorting (MACS), and isolation purity was validated via flow cytometry. Following isolation, CSTLs are produced through cryoinjury via liquid nitrogen, and subsequently labeled with NHS Cy5 for visualization. For in vivo targeting assessment, labeled CSTLs were administered intravenously to syngenic mice at doses of 10x106 CSTLs. Following a range of time points, tissues (LNs, blood, clearance organs, etc.) were collected, sectioned via vibratome, and imaged via confocal microscopy. Next, cisplatin was used as the chemotherapeutic payload for assessment of delivery efficiency. Drug loading and release were assessed via in vitro assays, and cisplatin (platinum) concentrations were measured using inductively coupled plasma mass spectrometry (ICP-MS). After equivalent free doses were determined, mice were administered cisplatin-loaded CSTLs (cisCSTLs) and compared to free cisplatin administration. Following a range of end timepoints, tissues were collected to measure cisplatin concentration. Samples were lyophilized, digested via aqua regia, and similarly evaluated using ICP-MS.

Results, Conclusions, and Discussions: Results and

Discussion: Primary T lymphocytes isolated via MACS were assessed and cell attenuation was confirmed via Live/Dead viability assay at >>99% (Figure 1A). To evaluate the ability for the CSTLs to enter the LN, labeled CSTLs were administered intravenously to mice at a dose of 10x106 CSTLs and tissues were collected at endpoint. In the LN, sections were stained with phalloidin to confirm CSTLs interaction with local vasculature and presence in the lobule. Vehicles were clearly visualized via confocal microscopy (Figure 1B). Next, cisplatin, a common chemotherapeutic, loading and release was evaluated in vitro. Using ICP-MS to measure cisplatin concentration it was determined that cisplatin was initially released rapidly, ~50% released after 4 hours, and reached a plateau of ~75% after 12 hours (Figure 1C). Finally, to confirm CSTLs resulted in improved delivery efficiency of cisplatin to the LN, cisCSTLs were administered to mice and compared to free drug equivalents over time. cisCSTLs outperformed free cisplatin at all timepoints tested, with a maximum increase of 700% at 8 hrs (Figure 1D). Additionally, while free drug concentrations in the LN peaked at 4 hrs and decreased at the 8 hr timepoint, cisCSTL concentrations continue to rise over time. Taken together, this data suggests that CSTLs retain functional targeting and LN uptake, can be loaded with and release small molecule drugs, and can deliver drug to the LN approximately 7-times more effectively than free drug.

Conclusion: We have developed a novel delivery system for enhanced delivery of chemotherapeutic payloads to the LN. Using this approach, cisplatin was delivered at a 7-fold higher concentration to the tissue compared to free drug. In future works, this technology will be assessed for therapeutic effect using a murine metastatic cancer model.

Acknowledgements (Optional):

References: 1. Guan, Acta Pharmaceuitica. (2015), 2. Dillekås, Cancer Med. (2019), 3. Willard-Mack, Tox. Path. (2006), 4. Scholz +, Clinical Pharmacology (2021), 5. Meijer, Microcirculation (2017)