I15 - Engineering Cyborg Cells as Cancer Therapeutics

Introduction: : Whole tumor cell (WTC) vaccines are promising approaches in cancer immunotherapy by presenting the immune system with a comprehensive repertoire of tumor-associated antigens, enabling broad polyclonal responses capable of targeting heterogeneous tumors. Common formulations include irradiated tumor cells, heat-killed cells, or tumor lysates, all of which preserve antigenic content while preventing proliferation. To enhance their modest immunogenicity, state-of-the-art strategies involve engineering tumor cells to secrete immunostimulatory factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), fusing tumor cells with dendritic cells, or co-administering adjuvants like Toll-like receptor agonists, STING activators, or cytokine cocktails. However, despite their antigenic breadth, traditional WTC vaccines have key limitations: immunostimulatory effects are often transient, spatially uncoupled from the antigen source, and insufficient to drive durable immune memory. Platforms like GVAX utilize tumor cells engineered to secrete GM-CSF but require irradiation, rendering the cells metabolically inactive and reliant solely on pre-loaded cytokine release upon degradation. The disconnect between antigen presentation and immune stimulation limits the potency of such vaccines. To address these challenges, we introduce the Cyborg Cell vaccine platform, engineered via intracellular hydrogelation of cancer cells. Cyborg Cells are replication-incompetent yet metabolically active for an extended time. Unlike fixed or lysed cells, Cyborg Cells retain the ability to secrete cytokines and checkpoint blockade mimetics while preserving antigen processing, enabling sustained immune stimulation and boosting in vivo. By coupling metabolic activity with broad-spectrum antigenicity, this Cyborg Cell-based platform offers a unified, living chassis for potent cancer vaccination and durable immune priming without exogenous adjuvants.

Materials and

Methods: : Cyborg 4T1 mouse mammary carcinoma cells were engineered by incubating 4T1 cells with PEG-based monomers, followed by UV-induced intracellular hydrogel crosslinking to arrest replication while preserving metabolic activity. Replication arrest was confirmed via cell counting over a four-week period, and sustained metabolic function was assessed using PrestoBlue staining. Endogenous cytokine secretion from Cyborg cells was evaluated using a GM-CSF ELISA. To assess in vivo safety, 1 × 10⁶ Cyborg 4T1 cells were subcutaneously injected into three female BALB/c mice and monitored for signs of tumor growth over one month. Following safety validation, female BALB/c mice (n = 4 per group, 10 weeks old) were vaccinated subcutaneously on the right flank with two doses (day 0 and day 7) of either Cyborg 4T1 cells, heat-killed 4T1 cells, or PBS. On day 14, all mice were challenged with 1 × 10⁵ live 4T1 cells. Tumor volume and body weight were measured twice weekly post-challenge. To assess humoral immune responses, blood samples were collected to measure IgG1 and IgG2a levels via ELISA. Serum cytokine concentrations of IL-4 and IFN-γ were also quantified to evaluate Th2 and Th1 immune responses, respectively.

Results, Conclusions, and Discussions:: We successfully engineered Cyborg 4T1 mouse mammary carcinoma cells using intracellular PEG-based hydrogelation, producing replication-incompetent yet metabolically active WTC. Cyborg 4T1 cells remained non-proliferative for at least four weeks in vitro, as confirmed by longitudinal cell counting. Despite their replication arrest, the cells maintained metabolic activity for over a week post-gelation, as assessed by PrestoBlue staining. Moreover, GM-CSF ELISA revealed that Cyborg 4T1 cells secreted detectable levels of GM-CSF at 48-, 96-, and 144-hours post-fabrication, indicating sustained immunomodulatory functionality. To assess in vivo safety, mice injected subcutaneously with 1 × 10⁶ Cyborg 4T1 cells showed no tumor growth over 30 days, in stark contrast to the aggressive tumor formation observed following injection of live 4T1 cells. These results confirmed that intracellular hydrogelation effectively eliminated tumorigenic potential without compromising the cells’ metabolic or immunological competence. In vaccination studies, BALB/c mice were immunized subcutaneously with either 1 × 10⁶ or 1 × 10⁵ Cyborg 4T1 cells, heat-killed 4T1 cells, or PBS. Following live tumor challenge on day 14, >50% of mice in both Cyborg-vaccinated groups remained completely tumor-free, whereas all mice in the heat-killed or PBS control groups showed tumor growth. These findings suggest that Cyborg cells elicit protective immunity superior to conventional heat-killed vaccines, likely due to their ability to secrete endogenous cytokines and maintain antigen presentation functions in vivo. Blood samples collected from all groups showed elevated levels of IgG1 and IgG2a antibodies and moderate levels of IL-4, and IFN-γ, suggesting polarization of the humoral responses. Cyborg Cells were also modified to secrete biomolecules that inhibit tumor-immune suppression and stimulate immune systems to enhance vaccine potency.
In conclusion, Cyborg 4T1 cells represent a safe, biocompatible, and metabolically active whole tumor cell vaccine platform that can initiate protective antitumor immunity while boosting the immune system. These results highlight the potential of our Cyborg WTC as next-generation cancer vaccines for broadening vaccine approaches to cancers of urgent need.

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