“Heating Up Tumor Microenvironment with Cold”: Engineering Cold-Responsive Nanoparticles-Potentiated Cryoimmunotherapy for Fighting Against Cancer Metastasis

“Heating Up Tumor Microenvironment with Cold”: Engineering Cold-Responsive Nanoparticles-Potentiated Cryoimmunotherapy for Fighting Against Cancer Metastasis

The advent of checkpoint immunotherapy has revolutionized cancer treatments. Checkpoint immunotherapy effectively takes the brakes off the body’s immune system to destroy tumors1. The immunosuppressive tumor microenvironment (TME), which is shaped by the interaction of cancer cells, deactivated or compromised immune contents, and soluble factors, however, significantly reduces the efficacy of immunotherapy2. One promising strategy to modulate TME is cryosurgery, a minimally invasive surgical technique that uses “frostbite” temperatures to kill cancer cells and can be done in an outpatient setting with excellent cosmetic and analgesic effect. Cryosurgery has been shown to induce immunogenic cell death through the release of damage-associated molecular patterns (DAMPs), signals that mobilize immune cells into action3,4. However, contemporary approaches for utilizing the cryoimmune effect work effectively only for localized tumors. As a result, cryosurgery is limited for treating localized small tumor in the clinic and patients with advanced/metastatic cancer can’t benefit from the many advantages of cryosurgery.  Here, we develop an in-situ cryo-immune engineering (ICIE) strategy for effective destruction of not only localized but also distant/metastatic tumors, by leveraging cold-responsive nanoparticles (CRNPs) for co-delivering chemotherapy and immunotherapy agents for combining with cryosurgery to turn the TME from immunologically “cold” into “hot”.

In this work, we synthesized polymeric CRNPs carrying a chemotherapy agent (irinotecan, CPT) and an immunotherapy agent (PD-L1 silencing siRNA) that are precisely tuned to release their cargo at the “frostbite” temperatures produced by cryosurgery. The thermo-responsive behavior of our nano-carriers is determined by a co-polymer of poly (N-isopropylacrylamide) and butyl acrylate, pNIPAAm-BA, which exhibits a phase change at a certain temperature, known as the lower critical solution temperature (LCST). We tuned the LCST of the polymer to match the “frostbite” temperatures (~ -4° or lower) by controlling the ratio of the two polymers during synthesis, choosing the LCST of −4.4 ± 0.6 °C for nanoparticle synthesis. The CRNPs for co-delivering CPT and siRNA (CPT&siR CRNPs) disassemble after cooling below the LCST, confirmed by transmission and scanning electron microscopy. The CPT&siR CRNPs also preferentially accumulate in cancer cells over normal cells. After the cancer cells take up the CRNPs, the cells can be cooled below the LCST to rupture the CRNPs and release drug and siR. Importantly, this cold-triggered release allows siR to escape from endo/lysosomes and perform its silencing effect in the cytosol, as we demonstrated with the silencing of green fluorescence protein and PD-L1 in cancer cells.

We confirmed that, indeed, “frostbite” temperatures induced by cryosurgery can increase the release of DAMPs like high mobility group box protein 1, calreticulin, heat shock protein-70, and heat shock protein-90 in vitro. Importantly, the combination of cryosurgery and the CPT&siR CRNPs causes a huge surge of these DAMPs. More DAMPs translate into a more robust and deadly anti-tumor immune response, which we see manifest through higher antigen presenting cell maturation, cytotoxic T-cell activation, and T-cell tumor attacking capability. Before testing our combination therapy in vivo, we traced the distribution of our CRNPs in mice, observing that the CRNPs accumulate more in tumor than the free, unencapsulated chemo-/gene therapy drugs.

Encouraged by the results, we developed primary, distant, and metastatic tumor models to evaluate the potential of the ICIE strategy as a cancer immunotherapy treatment in vivo. Our data show that ICIE can transform the primary tumor site from immunologically “cold” into “hot”. Before ICIE, the “cold” TME is rife with suppressive immune cells, like M2 macrophages, myeloid-derived suppressor cells (M-MDSCs), and regulatory T-cells (Treg), which allow cancer cells to thrive undisturbed. When ICIE is deployed and DAMPs are released in the primary TME, things start to heat up. We show the suppressive immune cells are greatly reduced by ICIE at the primary tumor site, and immunologically “hot” cells like M1 macrophages and cytotoxic T-cells (CD8+GZMB+) flood the TME to kill cancer cells. ICIE is not only effective in shrinking the primary tumor, but also in sparking immune cells to attack distant and metastatic tumors. We found that the hot response in the primary tumor triggered by ICIE leaves its mark on the immune system, generating effector and central memory T cells that can fight cancer cells that have migrated from the primary tumor to other sites in the body. ICIE increases these memory cells, which we see can help inhibit growth of distant tumors and reduce cancerous nodes on lungs in a metastasis model.

We leveraged cold-responsive nanotechnology to bolster the combined effects of cryosurgery and immunotherapy on cancer treatment. Because the cold-responsive nanoparticles release both a chemotherapeutic and immune -checkpoint silencing siRNA only in the “frostbite” areas of cryosurgery, we achieve precision targeting of the tumor and minimize system toxicity.

Taking together, the ICIE introduced in this work is a cryoimmunotherapy strategy that uses cryosurgery and cold-responsive nanomaterials loaded with CPT and PD-L1 silencing siRNA to achieve cold-triggered drug release, reverse the immunosuppressive TME, generates a robust in-situ and long-term antitumor memory immune response for eradicating both primary and distant as well as metastatic tumors. ICIE could be a valuable strategy of combating cancer metastasis, the major cause of mortality for most cancers.



  1. Versluis, J. M., Long, G. V. & Blank, C. U. Learning from clinical trials of neoadjuvant checkpoint blockade. Nat Med. 26, 475-484 (2020).
  2. Binnewies, M. et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 24, 541-550 (2018).
  3. Regen-Tuero, H. C., Ward, R. C., Sikov, W. M. & Littrup, P. J. Cryoablation and Immunotherapy for Breast Cancer: Overview and Rationale for Combined Therapy. Radiol Imaging Cancer. 3, e200134 (2021).
  4. Kwak, K., Yu, B., Lewandowski, R. J. & Kim, D. H. Recent progress in cryoablation cancer therapy and nanoparticles mediated cryoablation. Theranostics. 12, 2175-2204 (2022).

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