Introduction: Tertiary lymphoid structures (TLSs), lymphoid formations present in tissues outside of the lymphatic system, often form in tumors to facilitate anticancer immunity by inducing an influx of immune cells into the tumor site[1]. As such, the presence of TLSs often correlates to not only more positive prognoses in most solid tumor types, but also better responses to cancer immunotherapies[3,4], because of their ability to draw lymphocytes into tumors and foster the production of circulating central-memory T and B cells[5]. TLS presence has also been reported to be independent of tumor mutational burden, which is typically a limiting factor in the ability of tumor infiltrating lymphocytes (TIL) to initiate an anti-tumor immune response[3].
The increase in effectiveness of the anti-cancer immune response in the presence of TLSs introduces the potential of generating artificial TLSs at the tumor site to prime and enhance clinical responses to cancer immunotherapies. We are therefore developing a novel injectable hydrogel system to locally induce TLS formation in vivo. We have formulated a diverse library of polymer-nanoparticle (PNP) hydrogels which are shear thinning and self-healing, allowing them to re-assemble into solid gels after injection into tissues, and have demonstrated tunable stiffness and other mechanical and biochemical properties. We have additionally shown T cell viability and motility in our hydrogel formulations after injection. By utilizing these materials and incorporating cellular adhesion motifs and cytokines crucial for governing TLS progenitor cells, we aim to recruit and organize immune cells to induce TLSs in tumors in vivo.
Materials and
Methods: Dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12) was prepared by dissolving hypromellose (HPMC) in N-methylpyrrolidone (NMP), heating, and adding dodecyl isocyanate and Hunig’s catalyst dropwise, after which the heat was shut off and the reaction allowed to continue overnight while stirring. Polymer was precipitated in acetone, dissolved in Millipore water and then purified via dialysis, lyophilized, and dissolved in sterile PBS.
Hydrogels were formulated by mixture of polystyrene (carboxylate modified (CML) latex) nanoparticles (NPs) and HPMC-C12 using a dual-syringe mixer. Desired amounts of NPs and HPMC-C12 solution were loaded into two separate 1-mL luer lock syringes. The two syringes were connected via a luer-lock elbow connector, and the solutions were mixed by alternating depression on the connected syringes until the solution was homogeneous and a hydrogel was formed. Hydrogel formulations consisted of the following: 2 wt % HPMC-C12 and varying weight percentages of polystyrene NPs (either 5% or 10%).
Rheological characterization of hydrogels was performed using a 20-mm-diameter serrated parallel plate at a 500-mm gap on a stress-controlled TA Instruments DHR-2 rheometer at 25C.
For cell viability experiments, the polymer and NPs were dissolved in RPMI 1640 media (supplemented with FBS, L-glutamine, and penicillin-streptomycin) instead of PBS. Jurkat T cells were stained with CellTracker Green BODIPY and mixed with the polystyrene NPs prior to syringe loading. Hydrogels were otherwise formulated as previously described. Hydrogels (~1mm thickness) were then seeded into a black, clear-bottom 96 well plate with RPMI 1640 media. Imaging was performed on a Nikon Ti Eclipse scanning confocal microscope at 10x magnification.
Results, Conclusions, and Discussions: We have demonstrated that polystyrene NPs generate shear-thinning, self-healing, and injectable hydrogels when mixed with HPMC-C12 (Figure 1). These shear-thinning and self-healing properties allow the hydrogels to re-assemble into solid gels after injection into tissues. Furthermore, the mechanical properties of this system can be tuned by altering the concentrations (weight percentage, 5%-10%) or sizes (20 nm-100nm) of NPs, which act as crosslinkers in the hydrogel system.
We utilized the 2 wt% HPMC-C12 and 5 wt% 100 nm polystyrene NP hydrogel formulation to mix Jurkat T cells into the hydrogels as mentioned above. Imaging of the PNP hydrogels at 0 and 48 hours (2d) shows T cell viability in our hydrogel system, as we observe cell proliferation with increasing time (Figure 2). Additionally, we observed T cell motility in our hydrogels when imaging the hydrogels every 10 minutes during the first hour immediately after seeding.
The motility and proliferation of T cells are crucial in our PNP hydrogel formulations, as our aim to generate artificial TLSs requires for the hydrogel to be an environment suitable for immune cells to proliferate and divide. This will in turn promote the recruitment of TLS-inducing cells and facilitate effective immune responses. In addition, T lymphocyte and other immune cell motility is integral for effective scanning of antigen-presenting cells (APCs) within the artificial TLS. Additional work with other types of cells known to initiate TLSs is underway.
These data indicate that our PNP hydrogel platform is highly tunable to mimic the mechanical properties of a biological environment and can support the proliferation of T cells. We aim to further optimize our system by incorporating chemotaxis-regulating molecules and extracellular matrix (ECM) adhesion motifs to further support the formation of an immune cell niche.
Acknowledgements (Optional):
References: 1. Schumacher, T, Thommen, D. Tertiary lymphoid structures in cancer. Science 2022, 375, eabf9419. doi:10.1126/science.abf9419 2. Narvaez, D.; Nadal, J.; Nervo, A.; Costanzo, M.V.; Paletta, C.; Petracci, F.E.; Rivero, S.; Ostinelli, A.; Freile, B.; Enrico, D.; et al. The Emerging Role of Tertiary Lymphoid Structures in Breast Cancer: A Narrative Review. Cancers 2024, 16, 396. https://doi.org/10.3390/cancers16020396 3. Trüb M, Zippelius A. Tertiary Lymphoid Structures as a Predictive Biomarker of Response to Cancer Immunotherapies. Front. Immunol. 2021, 12:674565. doi: 10.3389/fimmu.2021.674565 4. Sautès-Fridman C, Lawand M, Giraldo NA, Kaplon H, Germain C, Fridman WH and Dieu-Nosjean M-C. Tertiary Lymphoid Structures in Cancers: Prognostic Value, Regulation, and Manipulation for Therapeutic Intervention. Front. Immunol 2016. 7:407. doi: 10.3389/fimmu.2016.00407 5. Luc de Chaisemartin, Jérémy Goc, Diane Damotte, Pierre Validire, Pierre Magdeleinat, Marco Alifano, Isabelle Cremer, Wolf-Herman Fridman, Catherine Sautès-Fridman, Marie-Caroline Dieu-Nosjean; Characterization of Chemokines and Adhesion Molecules Associated with T cell Presence in Tertiary Lymphoid Structures in Human Lung Cancer. Cancer Res 2011; 71 (20): 6391–6399. https://doi.org/10.1158/0008-5472.CAN-11-0952
.jpg "Santiago Correa, PhD (he/him/his) photo")