Soft tissue sarcomas (STS) are heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have previously been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cell subsets, including NKp46 and CD56bright/dim, has yet to be investigated in detail. Despite the known role of MHC-I on immunoregulation of NK and T cells, limited data exist characterizing the spatial relationship of NK cells to MHC-I+/- cells and T cells in the STS tumor microenvironment (TME). Using STS specimens from 130 patients, we evaluated intratumoral NK cell subsets by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF) to assess their impact on overall survival (OS) and metastasis-free survival (MFS). We also assessed the spatial localization of NK and T cells by multiplex IF in the TME, specifically analyzing the effects of MHC-I expression status on NK and T cell clustering. High intratumoral NKp46 expression was associated with improved OS by IHC (P=0.04) and IF (P=0.02). CD56dim NK cells were associated with a survival benefit (P=0.05), while higher infiltrates of CD56bright NK cells predicted worse prognosis (P=0.05). CD3-CD56+ NK cells demonstrated a significant inverse relationship with CD3+ T cells by both flow cytometry and IF. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NKp46+ NK cells (P<0.0001). Intratumoral NK cell subsets, including NKp46+ and CD56bright/dim NK cells, are prognostic in STS and localize closer to MHC-I- cells than they do to T cells or MHC-I+ cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status reinforces inherent opposite but interconnected roles for these cells in anti-tumor surveillance.

Purpose

Given the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.

Experimental design

Using prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.

Results

TILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.

Conclusion

Intratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

Background

Groundbreaking studies have linked the gut microbiome with immune homeostasis and antitumor immune responses. Mounting evidence has also demonstrated an intratumoral microbiome, including in soft tissue sarcomas (STS), although detailed characterization of the STS intratumoral microbiome is limited. We sought to characterize the intratumoral microbiome in patients with STS undergoing preoperative radiotherapy and surgery, hypothesizing the presence of a distinct intratumoral microbiome with potentially clinically significant microbial signatures.

Methods

We prospectively obtained tumor and stool samples from adult patients with non-metastatic STS using a strict sterile collection protocol to minimize contamination. Metagenomic classification was used to estimate abundance using genus and species taxonomic levels across all classified organisms, and data were analyzed with respect to clinicopathologic factors.

Results

Fifteen patients were enrolled. Most tumors were located at an extremity (67%) and were histologic grade 3 (87%). 40% were well-differentiated/dedifferentiated liposarcoma histology. With a median follow-up of 24 months, 4 (27%) patients developed metastases, and 3 (20%) died. Despite overwhelming human DNA (>99%) intratumorally, we detected a small but consistent proportion of bacterial DNA (0.02-0.03%) in all tumors, including Proteobacteria, Bacteroidetes, and Firmicutes, as well as viral species. In the tumor microenvironment, we observed a strong positive correlation between viral relative abundance and natural killer (NK) infiltration, and higher NK infiltration was associated with superior metastasis-free and overall survival by immunohistochemical, flow cytometry, and multiplex immunofluorescence analyses.

Conclusions

We prospectively demonstrate the presence of a distinct and measurable intratumoral microbiome in patients with STS at multiple time points. Our data suggest that the STS tumor microbiome has prognostic significance with viral relative abundance associated with NK infiltration and oncologic outcome. Additional studies are warranted to further assess the clinical impact of these findings.