Myeloid cell–targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell–mediated immunity
Dayson Moreira, … , Erminia Massarelli, Marcin Kortylewski
Dayson Moreira, … , Erminia Massarelli, Marcin Kortylewski
Published November 24, 2020
Citation Information: J Clin Invest. 2021;131(2):e137001. https://doi.org/10.1172/JCI137001.
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Research Article Immunology Oncology

Myeloid cell–targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell–mediated immunity

Abstract

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8+ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage–related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell–targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8+ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV– MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.

Authors

Dayson Moreira, Sagus Sampath, Haejung Won, Seok Voon White, Yu-Lin Su, Marice Alcantara, Chongkai Wang, Peter Lee, Ellie Maghami, Erminia Massarelli, Marcin Kortylewski

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Figure 3

CpG-STAT3ASO stimulates human T cell activity and inhibits growth of xenotransplanted head and neck tumors in humanized mice.

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CpG-STAT3ASO stimulates human T cell activity and inhibits growth of xen...
(A) CD15+ granulocytic myeloid cells were enriched from PBMCs of HNSCC patients. Cells were treated using CpG-STAT3ASO, STAT3ASO alone, or control CpG-scrON (0.5 μM) for 72 hours and then cocultured with allogeneic CD3+ T cells at a 3:1 ratio with anti-CD3/anti-CD28 costimulation. T cell proliferation was determined using CFSE dilution assay and flow cytometry (n = 7). (B) STAT3 knockdown in HNSCC CD15+ PMN-MDSCs after treatment using CpG-STAT3ASO, STAT3ASO alone, or control CpG-scrON (0.5 μM) for 24 hours. Shown is STAT3 expression as measured using real-time qPCR (n = 7). UBQ, ubiquitin. (C–G) Immunodeficient NSG-SGM3 mice were humanized using i.v. injection of human CD34+ cells (C) or left untreated for control experiments (D). After 6 weeks, successfully humanized mice were injected s.c. with UM-SCC1 cells. Established UM-SCC1 tumors (150–200 mm3) were intratumorally treated with 5 mg/kg CpG-STAT3ASO (n = 10), CpG-scrON (n = 6), STAT3ASO (n = 6), or PBS (n = 6) every other day. In C and D, tumor growth was measured every other day for the duration of the experiment. At the experiment completion, tumors were collected to assess immune cell infiltration using flow cytometry. (E) Percentage of activated dendritic cells (CD33+CD11c+HLA-DR+CD86+) and (F) M1 macrophages (CD33+CD68+HLA-DR+CD86+) in UM-SSC1 tumors after treatments. (G) Percentages of human T cells infiltrating the tumor after treatment. Shown are percentage of total T (CD45+CD3+), CD4+ T (CD3+CD4+CD8-), CD8+ T (CD3+CD4–CD8+), Treg (CD3+CD4+CD8–FoxP3+), and CD8+ T/Treg cell ratio as measured with flow cytometry (n = 6–10). (H) Representative images from multiplex immunohistochemical staining of UM-SCC1 tumor sections for CD8+ and Treg (CD4+FoxP3+) cells (left) and for activated CD8+ T cells together with granzyme B and PD-1 (right). Scale bar: 50 μm. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison post hoc test (B and E–G) or 2-way ANOVA with Tukey’s multiple-comparison post hoc test (C and D).