Harnessing the Histotripsy Induced Immune Response

            Histotripsy is a non-invasive, nonthermal tissue ablation modality that uses short (~5 𝜇s),  focused, high frequency ultrasound pulses to mechanically generate lesions within solid tumors. Histotripsy reduces cancerous tissue to subcellular debris that is subsequently reabsorbed by the body. We believe this subcellular debris contains the essential components necessary to induce immunogenic cell death (ICD). We aim to use histotripsy initiated ICD in conjunction with immunotherapeutics in order to achieve an enhanced, in vivo, tumor-specific immune response. 

Background

            Tumors occur when the immune system fails to recognize and/or regulate the growth and replication of tumor cells. Tumors have the ability to deploy a wide range of evasion techniques in order to evade and subvert the immune system. The immune system’s primary defense against tumorigenesis is immunogenic cell death (ICD). ICD is an immunologically driven subset of regulated cell death within immunocompetent hosts that specifically targets tumor cells. Two main components are necessary in order for the immune system to perform ICD: (1) distinguishable tumor antigens and (2) co-stimulatory molecules (a.k.a. adjuvants). Tumor antigens are molecular surface signatures that can be recognized by immune cell receptors.  Adjuvants typically come in the form of Damage Associated Molecular Patterns (DAMPs). DAMPs are intracellular molecules that serve as the immune system’s alarm bells once they are released from stressed/dead cells. These main components can be produced by either chemical or physical (i.e. histotripsy) stimuli. Histotripsy delivers intense, alternating, high-frequency, focused, acoustic pulses to an area on the order of 2-3 mm. The alternating pressure cycles (~26 MPa) induce cavitation which results in mechanical fractionation of the tumor, leaving behind a soup of subcellular debris. We hypothesize this post-treatment debris contains the essential tumor neoantigens and DAMPs necessary for initiating ICD.

            Immune checkpoint proteins, like cytotoxic T lymphocyte-associated 4 (CTLA4) and programmed cell death protein 1 (PD-1), are receptors on the surface of CD8+ T-cells that serve as failsafes against autoimmunity. Checkpoint Inhibitors prevent these proteins from binding, keeping CTLs engaged longer and essentially kicking the immune system into overdrive. Immune checkpoint therapy has been shown to perform well against immunogenic tumors like melanoma and non small cell lung cancer. However it struggles against non-immunogenic tumors like liver and glioma. We aim to use checkpoint inhibitors in conjunction with histotripsy in order to improve treatment outcome for both immunogenic and non-immunogenic tumors.

 

Objectives

  1. Characterize and quantify Immune Response due to histotripsy tumor ablation
  2. Investigate immunological mechanisms underlying histotripsy induced immune response
  3. Demonstrate the enhanced efficacy of immunotherapy in conjunction with histotripsy

 

Experiments

Melanoma Model

  • Mice (Ly5.2 C57BL/6) are injected with subcutaneous bilateral melanoma tumors (B16GP33).
  • Anti-CTLA4 is administered on days 6 and 9
  • Histotripsy is performed on day 10
  • Combination refers to the administration of Anti-CTLA4 and Histotripsy

Experiment 156 (n = 16)

Experiment 159 (n = 20)

Experiment 165 (n=12)

Group

Treatment

Group

Treatment

Group

Treatment

A

Control (no treatment)

A

Control (no treatment)

A

Control (no treatment)

B

anti-CTLA4

B

anti-CTLA4

B

anti-CTLA4

C

Histotripsy

C

Histotripsy

C

Histotripsy

D

Combination

D

Combination

D

Combination

 

 

E

Normal Tissue

 

*Survival Study*

 

Liver Model

  • 2 C57BL/6 mice (n = 16)
  • Subcutaneous bilateral Hepa 1-6 (liver tumor) inoculations
  • Histotripsy (HT) is performed at day 10
  • Mice harvested at either early (3 days post-treatment) or late timepoint (10 days) post-treatment)

Glioma Model

  • Unilateral glioma (GL261) inoculation
  • Anti PD-L1
  • Bioluminescence imaging and MRI pre and post treatment

References

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  2. G. P. Dunn, A. T. Bruce, H. Ikeda, L. J. Old, and R. D. Schreiber, “Cancer immunoediting: from immunosurveillance to tumor escape” Nature Immunology 3, 991 (2002).
  3. L. Galluzzi, A. Buqué, O. Kepp, L. Zitvogel, and G. Kroemer, “Immunogenic cell death in cancer and infectious disease”, Nature Reviews Immunology 17, 97 (2016).
  4. A. Garg, A. Dudek-Peric, E. Romano, P. Agostinis, “Immunogenic Cell Death”, International Journal of Developmental Biology 59, 131 (2015)
  5. V. A. Khokhlova, J. B. Fowlkes, W. W. Roberts, G. R. Schade, Z. Xu, T. D. Khokhlova, T. L. Hall, A. D. Maxwell, Y.-N. Wang, and C. A. Cain, “Histotripsy Methods in Mechanical Disintegration of Tissue: Toward Clinical Applications”, International Journal of Hyperthermia 31, 145 (2015).
  6. K. Lin, Y. Kim, A. Maxwell, T. Wang, T. Hall, Z. Xu, J. B. Fowlkes, C. Cain, “Histotripsy beyond the “Intrinsic” Cavitation Threshold using Very Short Ultrasound Pulses: “Microtripsy”, IEEE Trans Ultrason Ferroelectr Freq Control 61, 251 (2014)
  7. W. W. Roberts, “Development and Translation of Histotripsy: Current Status and Future Directions”, Current Opinion in Urology 24, 104 (2014).