Extracellular matrix scaffold assisted cancer vaccine enhances anti-tumor efficacy and provides long term protection

Introduction: Implantable biomaterials have revolutionized cancer patient care in applications of drug delivery and surgical oncology. Every implanted biomaterial initiates a host immune response, which exists on a spectrum; Type 1 immunity is associated with cytotoxic anti-tumor responses, and Type 2 with wound healing. Extracellular matrix (ECM) scaffolds derived from decellularized mammalian tissues are implanted in patients to surgically reconstruct damaged tissues immediately following tumor resection where they initiate a Type 2 immune signature driven by IL-4 cytokine to promote constructive 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. Previous studies have shown that the ECM scaffold immune response sensitizes the local environment to cancer immunotherapy to restrict local tumor growth but is insufficient to generate robust CD8+ anti-tumor cytotoxic T cell responses needed to attack disseminated cancer cells. We therefore sought to combine an ECM scaffold with a cytotoxic T cell-stimulating adjuvant to create an implantable therapeutic vaccine. The objective was to generate cytotoxic tumor-specific immunity synergistically with an ECM scaffold environment. 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 (Figure 1A).

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 and nuclei by DAPI staining. 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, cytokine qRT-PCR, and plasma Luminex assay. The adjuvants CDA (cyclic diAMP; STING agonist), GM-CSF (granulocyte-macrophage colony-stimulating factor; cytokine), MPLA (monophosphoryl lipid A; TLR4 agonist), or vehicle were compared. We utilized an in vivo cytotoxic T lymphocyte (CTL) assay to elucidate an adjuvant formulation that induced antigen-specific cellular immunity when delivered with an ECM scaffold. Finally, we implemented the resultant ECM scaffold-assisted cancer vaccine formulation to therapeutically treat established ovalbumin-expressing E.G7-OVA mouse lymphoma tumors. E.G7-OVA cells were injected subcutaneously in the flanks of 8-week-old C57Bl/6 mice and treated once the tumors grew to 75-100 mm3. Cytotoxic effector cells were depleted via intermittent systemic administration of monoclonal antibodies (CD4, CD8b, NK1.1, or isotype) to determine the cell population responsible for tumor regression. OVA specific-dextramer staining was performed to determine antigen-specific immune memory, which we analyzed by FACS for memory T cells.

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). STING agonist CDA emerged as the most potent cytotoxic inducer when delivered with the SIS-ECM scaffold. Quantitative histology of the SIS-ECM implant showed that MPLA induced 5840 ± 534 cells/mm2, the greatest cellularity after 14 days, qualitatively mimicking infection, and approximately half that amount for GM-CSF or CDA adjuvants (Figure 1B, C). Multiplex immunofluorescent staining showed SIS-ECM alone was effective in attracting antigen-presenting cells (APCs; F4/80+) by 7 days of implantation with 1314.73 ± 844 cells/mm2 which increased by 38.6% with SIS+CDA (Figure 1B, D). Peripheral blood plasma evaluation using Luminex cytokine panel after adjuvant co-delivery with SIS-ECM did not show any deleterious systemic cytokine dysregulation. In the CTL assay, MPLA showed 56% specific killing with SIS and 85% without SIS whereas CDA showed greater than 95% specific killing with and without SIS indicating that CDA can be delivered locally with SIS to induce antigen-specific immunity (Figure 1E). Further, CDA did not diminish the Type 2 immune signature of SIS-ECM driven by IL-4 cytokine which is a hallmark ECM immune response as shown by il-4 gene expression data of LN (Figure 1F). 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. No tumor regression was observed in control SIS or CDA groups (Figure 1G, H). SIS-ECM scaffold-assisted vaccination extended antigen exposure was mediated by CD8+ cytotoxic T cells and generated long-term antigen-specific memory for at least 10 months post-vaccination, producing 4-fold effector memory CD8+ T cells (Tem) on tumor challenge than untreated controls.
In conclusion, ECM scaffolds are an effective delivery system for a cancer vaccine, augmenting efficacy while preserving the distinctive characteristics of tissue regeneration immune responses. The Type 2 ECM scaffold immune environment acts collaboratively with immune adjuvant and is more effective than traditional soluble cancer vaccines. ECM scaffolds enhance cytotoxic T-cell priming while preserving tissue repair hallmarks with an appropriate immune adjuvant and may hold clinical promise for local immunotherapy post-tumor resection.

Acknowledgements (Optional):