Anti-Complement Immunotherapy for Pancreatic Cancer

PROJECT SUMMARY / ABSTRACT
Pancreatic Ductal Adenocarcinoma (PDAC) is an almost uniformly lethal disease, with an overall survival under
10%. Despite therapeutic advances in all arenas of cancer treatment, including immunotherapy, overall survival
has not significantly improved in PDAC, representing a critical need for the development of novel therapeutic
strategies for this disease. The tumor microenvironment (TME) in PDAC is characterized by an
immunosuppressive infiltrate causing T-cell exclusion, and dense stromal desmoplasia. The complement
cascade is activated in the PDAC TME and may promote this uniquely challenging TME by signaling to immune
cells and fibroblasts expressing complement receptors. Complement activation has previously been
demonstrated to promote infiltration of immune cells which contribute to T-cell exclusion in multiple tumor types
in a context-dependent fashion. The long-term objectives of this project are to understand microenvironmental
mechanisms by which complement promotes oncogenesis in PDAC, and to elucidate the interactions between
radiotherapy (RT) and complement blockade in PDAC. A novel autochthonous mouse model of PDAC was
generated and crossed with complement deficient C3 knockout (KO) mice to allow for studies which define the
role of complement in PDAC. Tumor growth in complement-proficient wild type (WT) PDAC mice and C3 KO
PDAC mice will be tracked using serial, PET/CT imaging. Tumors will be assessed histologically from 3-6 months
after induction with tamoxifen to determine histologic grade, normal acinar area, and fibrotic area. Flow cytometry
will be used to determine the impact of complement on infiltrating immune cells while single-cell RNA-sequencing
will provide insight into the development and activation of cancer-associated fibroblasts. We will also assess the
role of tumor cell vs. stromally derived C3 with shRNA depletion of C3 in YFP-expressing KPC cells (KPCY) in
flank tumor growth studies in WT and C3 KO mice as a second model to confirm these results. To assess the
impact of photon, proton, and carbon ion RT on complement activation and immune tolerance we will irradiate
KPCY using each radiation modality. Complement deposition assays will be performed by culturing irradiated
and non-treated tumor cells in the presence of serum as a complement source, before measuring activation by
flow cytometry. Next, dendritic cells will be co-cultured with irradiated and non-irradiated KPCY cells with serum
to determine if complement deposition on irradiated tumor cells reduces dendritic cell activation markers
measured by flow cytometry. Translational studies will be performed using neutralizing antibodies and small
molecule inhibitors to blockade complement signaling alone and with RT in an orthotopic KPCY model. Mice will
be treated with neutralizing antibodies and inhibitors to blockade complement signaling and irradiated with an X-
strahl Small Animal Radiation Research Platform or a Varian ProBeam proton therapy machine and growth will
be tracked by IVIS. Complement activation will be assessed by western blot and immunofluorescence, and flow
cytometry will assess differences in infiltrating immune cells post-irradiation.

Grant Number: 5F30CA278322-02
NIH Institute/Center: NIH
Principal Investigator: Brett Bell