Single-cell profiling of peripheral blood identifies unique signatures of extracellular matrix scaffold-assisted cancer vaccine with long-term immune memory

Introduction: : Implantable biomaterials have improved several facets of cancer care, such as enabling targeted and sustained drug delivery and promoting tissue regeneration and recovery after tumor resection surgery. The host immune response is a crucial determinant of biomaterial function, and every implanted biomaterial triggers an inflammatory reaction unique to its composition and architecture. Extracellular matrix (ECM) scaffolds derived from decellularized mammalian tissues are one of the biomaterials implanted in patients to repair damaged tissues following tumor resection surgically. These scaffolds initiate a Type 2 immune response driven by the cytokine IL-4, promoting scaffold remodeling and tissue regeneration. ECM scaffold implantation in the post-resection bed creates a unique opportunity for in situ cancer immunotherapy delivery to prevent tumor recurrence. While previous studies have explored the use of ECM scaffolds in tissue repair, cancer progression, and metastasis, a few have investigated their potential as immunomodulatory scaffolds for cancer vaccines, particularly inducing CD8 T cell-mediated cytotoxic immunity. However, there is increasing evidence that Type 2 immunity can promote anti-tumor T cell function. We therefore sought to integrate a Type 2 immune-inducing ECM scaffold with a Type I immune adjuvant to create an implantable therapeutic cancer vaccine. The objective was to enhance the recruitment and activation of antigen-presenting cells (APCs) and mediate a robust CD8 T cytotoxic immune response against the tumor. Here, we engineered an ECM scaffold-assisted therapeutic cancer vaccine that maintained an immune microenvironment consistent with tissue reconstruction, creating the beneficial pro-regenerative hallmarks of the ECM scaffold response.

Materials and

Methods: : Porcine small intestine submucosa (SIS) was decellularized, lyophilized, and cryomilled to form SIS-ECM particles, which were sterilized by irradiation. Decellularization was confirmed by the reduction in dsDNA content by PicoGreen assay. We screened cancer vaccine adjuvants for co-delivery with an ECM scaffold by characterizing the local, regional, and systemic in vivo immunological changes in C57Bl/6 mice after subcutaneously injecting SIS-ECM particulate with adjuvant via immunofluorescence, qRT-PCR, and plasma Luminex assay. The adjuvants CDA (cyclic diAMP; STING agonist), GM-CSF (granulocyte-macrophage colony-stimulating factor; cytokine), and MPLA (monophosphoryl lipid A; TLR4 agonist) were co-delivered with an ECM scaffold. In vivo cytotoxic T lymphocyte (CTL) assay was used to elucidate the optimal adjuvant when delivered with an ECM scaffold using ovalbumin as a model tumor antigen. The in vivo live animal imaging was used to determine the release profile of CDA adjuvant and ovalbumin from the ECM scaffold. Production of antigen-specific CD8 T cells after SIS-ECM scaffold (SIS-CDA-OVA) vaccination was determined by using intracellular cytokine staining. Next, we implemented the ECM scaffold-assisted cancer vaccine formulation to therapeutically treat established ovalbumin-expressing E.G7-OVA tumors (75-100 mm3) in 6-8-week-old C57BL/6 mice. Cytotoxic effector cells were depleted via intermittent systemic administration of monoclonal antibodies (CD8b, NK1.1, or isotype) to determine the cell population responsible for tumor regression. Finally, single-cell RNA sequencing was done on the peripheral blood isolated from the tumor-bearing mice on days 3 and 13 post-vaccination to identify unique immune signatures of the ECM-scaffold vaccine.

Results, Conclusions, and Discussions:: Injectable particulate SIS-ECM decellularization was confirmed by 95% reduction in double-stranded DNA content (from 6792.49 ± 184.65 to 282.83 ± 60.62 ng/mg). Quantitative histology and multiplex immunofluorescent staining showed SIS-ECM recruited a large number of immune cells comprising neutrophils (Ly6G+) on day 1 followed by antigen-presenting cells (APCs; F4/80+) by 7 days of implantation with 1314.73 ± 844 cells/mm2 which increased by the addition of adjuvants, especially, CDA which showed 38.6% increase. (Figure 1A-C). In the cytotoxic T-lymphocyte (CTL) assay, which measures response against T cell-dependent antigens, CDA showed greater than 95% specific killing with SIS more than MPLA (56%) and GM-CSF (21%), indicating that STING agonist CDA is the most potent cytotoxic T cell inducer when delivered with the SIS-ECM scaffold (Figure 1D). Further, CDA did not diminish the Type 2 immune signature of SIS-ECM driven by IL-4 cytokine, which is a hallmark of ECM immune response, as shown by the 11-fold increase in IL-4 gene expression data of the implant draining LN. The live animal imaging showed sustained release of OVA (50% at Day 4) over a period of 30 days and burst release of CDA from the SIS-ECM scaffold (Figure 1E). Finally, SIS+CDA led to a complete response to E.G7-OVA lymphoma tumor in curing 50-75% of animals and protection on tumor rechallenge (Figure 1F, G). No tumor regression was observed in the control SIS or CDA groups. Mice were protected after tumor rechallenge over 1 year later, producing 4-fold effector memory CD8+ T cells (Tem), demonstrating long-term antigen-specific memory by SIS+CDA vaccination compared to untreated controls. The preliminary single-cell RNA sequencing analysis from the peripheral blood of vaccinated mice revealed higher expression of activated antigen-presenting cells and B cells genes in vaccinated groups (Figure 1H-J) and higher levels of cytotoxic CD8 T cells in SIS-CDA-treated mice on day 13 post-vaccination.

In conclusion, ECM scaffolds are an effective immunomodulatory scaffold for delivering cancer vaccines, augmenting efficacy while preserving the distinctive characteristics of tissue regeneration and immune responses. The Type 2 ECM-scaffold-based cancer vaccine may hold clinical promise for local immunotherapy after tumor resection.

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