Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-‘quiescent’ microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.
IntroductionSoft tissue sarcomas (STS) are highly metastatic, connective-tissue lineage solid cancers. Immunologically, sarcomas are frequently characterized by a paucity of tumor infiltrating lymphocytes and an immune suppressive microenvironment. Activation of the STING pathway can induce potent immune-driven anti-tumor responses within immunogenic solid tumors; however, this strategy has not been evaluated in immunologically cold sarcomas. Herein, we assessed the therapeutic response of intratumoral STING activation in an immunologically cold murine model of undifferentiated pleomorphic sarcoma (UPS).Materials and ResultsA single intratumoral injection of the murine STING agonist, DMXAA resulted in durable cure in up to 60% of UPS-bearing mice. In mice with synchronous lung metastases, STING activation within hindlimb tumors resulted in 50% cure in both anatomic sites. Surviving mice all rejected UPS re-challenge in the hindlimb and lung. Therapeutic efficacy of STING was inhibited by lymphocyte deficiency but unaffected by macrophage deficiency. Immune phenotyping demonstrated enrichment of lymphocytic responses in tumors at multiple timepoints following treatment. Immune checkpoint blockade enhanced survival following STING activation.DiscussionThese data suggest intratumoral activation of the STING pathway elicits local and systemic anti-tumor immune responses in a lymphocyte poor sarcoma model and deserves further evaluation as an adjunctive local and systemic treatment for sarcomas.
Background Soft tissue sarcomas are rare connective tissue malignancies that are highly resistant to traditional systemic therapies. 1 Sarcomas are relatively common in dogs, yet few studies have previously investigated anti-sarcoma immunotherapies in this species. 2,3 In this study, we sought to investigate several small molecule STING (STimulator of INterferon Genes) agonists' dose toxicities and pathway inductions via canonical cytokine responses in canine macrophage and sarcoma lines, as compared to murine and human macrophage and sarcoma lines. We further aimed to assess the efficacy of a selected small molecule STING agonist without species specificity (ADU-S100) as an intra-tumourally administered drug in two canine patients with soft tissue sarcoma. Methods To assess the cytotoxicity of STING agonists DMXAA, ADU-S100, and MSA-2, an in-vitro MTT cell viability assay was used. Murine, canine, and human macrophages and sarcoma cells were exposed to the following conditions: media, vehicle control, or 0.1, 1, 10, and 100mg/mL of treatment (up to 200mg/mL of treatment for ADU-S100). 6, 12, and 24-hours post-exposure to the conditions, cell viability was assessed via formazan absorbance values. STING-pathway induced interferon-dependent cytokine production (IFN-b, TNF-?, CXCL-10) in cells was assessed via the Luminex cytokine assay. All cells were treated with 177nmol/L of the STING agonists, and cytokine release 2-and 6-hours postexposure were quantified. Clinical efficacy of ADU-S100 was further evaluated in vivo for two canine STS patients (1 hindlimb, 1 forelimb). Serial intra-tumoural doses further ranged from 200mg to 2.0mg of ADU-S100. Tumour volumes were calculated from caliper measurements of tumour length, width, and depth. Results DMXAA and ADU-S100 were not cytotoxic below 100 and 200mg/mL, respectively, and MSA-2 was cytotoxic above 10mg/mL. All STING agonists effectively stimulated the interferon-dependent STING pathway in murine macrophage cells. In addition, ADU-S100, MSA-2, and E7766 stimulated the canine and human interferon-dependent STING pathways. Our subsequent in vivo pilot study demonstrated that the intra-tumoural administration of ADU-S100 led to a 3.5-fold and 2.3-fold reduction of canine patients' tumour volumes over the course of 6-weeks, respectively (from 594cm^3 to 172cm^3 for patient 1, and from 420cm^3 to 180cm^3 for patient 2). Conclusions Overall, our findings suggest that STING agonists in particular, ADU-S100possess potential as a novel and effective therapeutic approach for canine STS. As sarcomas are highly metastatic and commonly fatal in dogs, further evaluating STING agonist therapy in canines may provide therapeutic insights into similar challenges for treating human disease using a comparative biology approach.
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