1408 Histotripsy induced necroptosis is critical for antitumor immune infiltration and ferroptosis

🔗 Access full article via Journal for ImmunoTherapy of Cancer

Background

Histotripsy is a non-invasive, non-ionizing focused ultrasound-based tumor ablation therapy that has recently been FDA approved for the use in liver tumors. We have previously observed that histotripsy treatment causes transient hypoxia inducible factor 1 alpha (HIF-1α) abrogation, early time point necroptotic immunological cell death (ICD) and late time point ferroptotic ICD in the remaining tumor, and makes the tumor more susceptible to CD8 T cell infiltration.¹ Currently the role of HIF-1α in histotripsy-induced necroptosis and downstream immune infiltration is unknown, and further studies to elucidate their connection is critical to strengthen the immunostimulatory effects of histotripsy.

Methods

For in vitro experiments, we used cobalt chloride (CoCl₂) to induce HIF-1α in parental YUMM1.7 cell line. For in vivo experiments, we genetically modified YUMM1.7 to overexpress HIF-1α (YUMM1.7-H). C57BL/6NTac mice received subcutaneous bilateral flank B16F10, YUMM1.7, or YUMM1.7-H tumor. One flank tumor per mouse was treated with either sham or subtotal histotripsy when the tumors reached 6 – 10 mm maximal diameter. Tumors were harvested 2 days and 7 days after histotripsy treatment for immunofluorescence and flow cytometry analysis. A necroptosis inhibitor, necrostatin-1, was administered one day before and after histotripsy treatment.

Results

Histotripsy treatment of YUMM1.7 cells in vitro resulted in necroptosis measured by pMLKL and pRIPK3 expression, which were significantly inhibited by CoCl₂. Similarly, treating YUMM1.7 cells in vivo resulted in significantly increased pMLKL and pRIPK3 expression, while YUMM1.7-H tumors did not exhibit a significant increase in necroptosis. At late time points, the YUMM1.7-H tumors reveal similar levels of CD8 T cell infiltration, but less ferroptosis, measured by HNEJ2 and BODIPY 581/591 C11. Phenotypic characterization of the tumor infiltrating CD8 T cells shows increased exhaustion in YUMM1.7-H compared to YUMM1.7 tumors. Remarkably, inhibiting histotripsy-induced necroptosis with necrostatin-1 led to decreased CD8 T cell infiltration and ferroptosis.

Conclusions

Modulation of HIF-1α signaling regulates the ability of histotripsy to induce necroptosis, and thus late immune infiltration and ferroptosis. This observation highlights the critical role that hypoxia abrogation and necroptosis play in the immunostimulatory effect of histotripsy, which may help identify therapeutic strategies to enhance the clinical benefit of histotripsy tumor ablation. Further studies will examine the effect of therapeutic manipulation of HIF-1 α and necroptosis to promote a stronger immune response following Ht.

Acknowledgements

I would like to acknowledge the following grants: NIH R01CA269394 NIH R01CA211217 VA I01BX005267.

Reference