Research ArticleCancer

Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases

See allHide authors and affiliations

Science Translational Medicine  21 Mar 2018:
Vol. 10, Issue 433, eaar1916
DOI: 10.1126/scitranslmed.aar1916
  • Fig. 1 A biodegradable hydrogel scaffold extends local release of payloads in situ.

    (A) Picture of a representative scaffold loaded with R848. (B) A hydrogel scaffold to which Alexa Fluor 750 was conjugated was implanted into the resection site of a mouse after tumor removal, and fluorescence IVIS imaging was performed at the indicated time points. The lone mouse to survive surgery in the absence of additional therapy is shown. (C) Quantification of fluorescence IVIS imaging (shown in fig. S1, E and F) of non–tumor-bearing mice to whom dye was administered in solution or conjugated to a hydrogel placed by the fourth mammary fat pad after incision. (D) Fluorescence IVIS imaging depicting the in vivo release profile of a model small-molecule payload (Cy7-CA), administered locally either in solution or after loading into a hydrogel. (E) Quantification of the in vivo release profile of Cy7-CA. The experiment was performed once with n = 5 biological replicates. Fold difference is indicated for each time point. Statistics were calculated using a two-sided unpaired t test. Data are means ± SD. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 2 Extended local release of agonists of innate immunity prevents tumor recurrence and eliminates distal metastases.

    Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and hydrogels loaded with the following payloads were evaluated: anti–PD-1, anti-CTLA-4, IL-15sa, lenalidomide, celecoxib, STING-RR, or R848. Mice that did not receive a hydrogel were examined as a negative control. (A) IVIS imaging of 4T1-Luc2 cells is shown for all groups described and illustrates tumor burden. (B) Kaplan-Meier curves are shown comparing hydrogels loaded with antibodies that induce immune checkpoint blockade to no hydrogel. (C) Kaplan-Meier curves are shown comparing a hydrogel loaded with the potent cytokine IL-15sa to no hydrogel. (D) Kaplan-Meier curves are shown comparing hydrogels loaded with various immunomodulatory small molecules to no hydrogel. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to the group treated with no hydrogel using the log-rank (Mantel-Cox) test. **P ≤ 0.01, ***P ≤ 0.001. d, days; n/a, not achieved.

  • Fig. 3 Agonists of innate immunity are effective only when released locally from the hydrogel.

    (A and B) Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells. (A) Kaplan-Meier curves are shown for the following groups: no hydrogel, an empty hydrogel, a hydrogel loaded with R848, weekly intravenous (IV) injection of R848, weekly intraperitoneal (IP) injection of R848, or an empty hydrogel plus local administration of R848 in solution. (B) Kaplan-Meier curves are shown for the following groups: no hydrogel, an empty hydrogel, a hydrogel loaded with STING-RR, or an empty hydrogel plus local administration of STING-RR in solution. (C and D) Tumors were not resected but were instead injected intratumorally (IT) with PBS, a single dose of R848, or a single dose of STING-RR 10 days after orthotopic inoculation of 4T1-Luc2 cells. Tumor volume (C) and mouse survival (D) were measured. (E and F) Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells. (E) Kaplan-Meier curves are shown for the following groups: no hydrogel, a hydrogel loaded with CCL4, a hydrogel loaded with CCL5, or a hydrogel loaded with CXCL10. (F) Kaplan-Meier curves are shown for all groups described: no hydrogel, a hydrogel loaded with paclitaxel, or a hydrogel loaded with doxorubicin. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. For (A) and (B), statistics were calculated relative to the group treated with hydrogel containing R848 or STING-RR, respectively, using the log-rank (Mantel-Cox) test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For (C) to (F), statistics were calculated relative to the no hydrogel control using the log-rank (Mantel-Cox) test, and no differences between groups were statistically significant.

  • Fig. 4 Both the innate and adaptive arms of the immune system are critical to the observed efficacy.

    Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and scaffolds containing (A) R848 or (B) STING-RR were placed in the resection site. Specific immune cell subsets (NK cells, CD8+ T cells, or CD4+ T cells) were depleted, or innate immune signaling (IFNAR1) was inhibited to explore their relative contribution to the observed efficacy. Kaplan-Meier curves are shown for all groups described. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to the group treated with hydrogel containing the indicated agonist of innate immunity and treated with PBS (no depletion control) using the log-rank (Mantel-Cox) test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 5 Extended local release of R848 increases the number of innate and adaptive antitumor immune cells and cytokines.

    Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and scaffolds were placed in the resection site. Spleens were recovered from mice 3 and 14 days after surgery for flow cytometric analysis, and blood was recovered from mice 1.5 hours, 6 hours, 3 days, and 14 days after surgery for cytokine analysis. (A to C) Increased numbers of leukocytes with activated and effector phenotypes are observed. Quantitation of flow cytometry gating of subsets of (A) NK cells (day 3), (B) dendritic cells (day 3), and (C) CD4+ T cells and CD8+ T cells (day 14) is shown. (D) Increased numbers of central memory–like CD8+ T cells are observed. (E) Increased numbers of T cells producing proinflammatory cytokines and cytolytic molecules are observed. Quantitation of flow cytometry gating of CD4+ T cells and CD8+ T cells (day 14) is shown. Splenocytes were cultured for 6 hours in the presence of a specific immunodominant peptide expressed by 4T1 cells (gp70423–431) and brefeldin A before flow cytometry was performed. (F) Increased concentrations of type I IFNs are observed in plasma collected at various time points after surgery. Data were generated by multiplexing laser bead technology. The experiment was performed once with n = 5 biological replicates. Statistics were calculated using a two-sided unpaired t test. Data are means ± SEM. *P ≤0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 6 Localized release of perioperative immunotherapy is efficacious in an additional model of spontaneous metastasis.

    Tumors were resected from mice when tumor volumes reached ~500 mm3 after subcutaneous inoculation of LLC cells. (A and B) Kaplan-Meier curves are shown for no hydrogel or a hydrogel loaded with either (A) R848 or (B) STING-RR. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to the group treated with hydrogel containing the indicated agonist of innate immunity using the log-rank (Mantel-Cox) test. *P ≤ 0.05.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/433/eaar1916/DC1

    Fig. S1. A hydrogel composed of cross-linked hyaluronic acid is stable but biodegradable in vivo.

    Fig. S2. The hydrogel scaffold extends the release of biologics and small molecules in vitro.

    Fig. S3. The hydrogel scaffold extends the local release of biologics and small molecules in vivo.

    Fig. S4. The activity of a biologic released from the hydrogel is preserved completely.

    Fig. S5. Multiple systemic administrations of R848 fail to confer survival benefit and are less well tolerated than R848 released from a hydrogel.

    Fig. S6. STING-RR confers superior efficacy to 2′3′-cGAMP upon extended release from a hydrogel in the perioperative setting.

    Fig. S7. IVIS imaging confirms that STING-RR must be released from a hydrogel to protect against tumor recurrence and eliminate metastases.

    Fig. S8. Efficacy of STING-RR loaded in a hydrogel is retained after storage for 1 week at 4°C.

    Fig. S9. Intraoperative placement of the immunotherapy-loaded hydrogel into the tumor resection site is required for therapeutic benefit.

    Fig. S10. Extended local release of combination immune checkpoint blockade confers limited survival benefit.

    Fig. S11. Flow cytometric analysis confirms that NK cells, CD8+ T cells, and CD4+ T cells are depleted after administration of appropriate antibodies.

    Fig. S12. Extended local release of STING-RR increases the number of activated innate immune cells.

    Fig. S13. Extended local release of R848 increases the numbers of several leukocyte subsets in the lung.

    Fig. S14. Increased concentrations of cytokines are observed in plasma collected at various time points after surgery.

    Fig. S15. Induction of an adaptive antitumor memory response is confirmed by rejection of 4T1-Luc2 cells inoculated as rechallenge.

    Fig. S16. Extended local release of agonists of innate immunity does not alter the composition of the blood.

    Fig. S17. Extended local release of agonists of innate immunity is safe.

    Fig. S18. Extended local release of agonists of innate immunity is well tolerated.

    Fig. S19. The response of parental 4T1 cells to R848 released locally from a hydrogel is similar to that of 4T1 cells expressing Luc2.

    Fig. S20. Localized release of perioperative immunotherapy is efficacious in the B16-BL6 model of spontaneous metastasis.

    Fig. S21. Extended release of R848 from a scaffold derived from alginate confers survival benefit.

    Table S1. Quantitation and statistics for a panel of cytokines measured in plasma recovered 3 and 14 days after surgery.

    Table S2. A table of the antibodies used for flow cytometry experiments.

  • Supplementary Material for:

    Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases

    Chun Gwon Park, Christina A. Hartl, Daniela Schmid, Ellese M. Carmona, Hye-Jung Kim, Michael S. Goldberg*

    *Corresponding author. Email: michael_goldberg1{at}dfci.harvard.edu

    Published 21 March 2018, Sci. Transl. Med. 10, eaar1916 (2018)
    DOI: 10.1126/scitranslmed.aar1916

    This PDF file includes:

    • Fig. S1. A hydrogel composed of cross-linked hyaluronic acid is stable but biodegradable in vivo.
    • Fig. S2. The hydrogel scaffold extends the release of biologics and small molecules in vitro.
    • Fig. S3. The hydrogel scaffold extends the local release of biologics and small molecules in vivo.
    • Fig. S4. The activity of a biologic released from the hydrogel is preserved completely.
    • Fig. S5. Multiple systemic administrations of R848 fail to confer survival benefit and are less well tolerated than R848 released from a hydrogel.
    • Fig. S6. STING-RR confers superior efficacy to 2′3′-cGAMP upon extended release from a hydrogel in the perioperative setting.
    • Fig. S7. IVIS imaging confirms that STING-RR must be released from a hydrogel to protect against tumor recurrence and eliminate metastases.
    • Fig. S8. Efficacy of STING-RR loaded in a hydrogel is retained after storage for 1 week at 4°C.
    • Fig. S9. Intraoperative placement of the immunotherapy-loaded hydrogel into the tumor resection site is required for therapeutic benefit.
    • Fig. S10. Extended local release of combination immune checkpoint blockade confers limited survival benefit.
    • Fig. S11. Flow cytometric analysis confirms that NK cells, CD8+ T cells, and CD4+ T cells are depleted after administration of appropriate antibodies.
    • Fig. S12. Extended local release of STING-RR increases the number of activated innate immune cells.
    • Fig. S13. Extended local release of R848 increases the numbers of several leukocyte subsets in the lung.
    • Fig. S14. Increased concentrations of cytokines are observed in plasma collected at various time points after surgery.
    • Fig. S15. Induction of an adaptive antitumor memory response is confirmed by rejection of 4T1-Luc2 cells inoculated as rechallenge.
    • Fig. S16. Extended local release of agonists of innate immunity does not alter the composition of the blood.
    • Fig. S17. Extended local release of agonists of innate immunity is safe.
    • Fig. S18. Extended local release of agonists of innate immunity is well tolerated.
    • Fig. S19. The response of parental 4T1 cells to R848 released locally from a hydrogel is similar to that of 4T1 cells expressing Luc2.
    • Fig. S20. Localized release of perioperative immunotherapy is efficacious in the B16-BL6 model of spontaneous metastasis.
    • Fig. S21. Extended release of R848 from a scaffold derived from alginate confers survival benefit.
    • Table S1. Quantitation and statistics for a panel of cytokines measured in plasma recovered 3 and 14 days after surgery.
    • Table S2. A table of the antibodies used for flow cytometry experiments.

    [Download PDF]

Navigate This Article