Low-Dose Sorafenib Promotes Cancer Stem Cell Expansion and Accelerated Tumor Progression in Soft Tissue Sarcomas.

The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients.

Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. METHODS: We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complex class I [MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). RESULTS: Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. CONCLUSIONS: Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Experimental design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Results: Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. 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 NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T 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 may serve as a biomarker for improved immunotherapy treatment selection.

Human soft tissue sarcomas harbor an intratumoral viral microbiome which is linked with natural killer cell infiltrate and prognosis.

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.

Using the canine microbiome to bridge translation of cancer immunotherapy from pre-clinical murine models to human clinical trials.

The microbiome has clearly been established as a cutting-edge field in tumor immunology and immunotherapy. Growing evidence supports the role of the microbiome in immune surveillance, self-tolerance, and response to immune checkpoint inhibitors such as anti PD-L1 and CTLA-4 blockade (1-6). Moreover, recent studies including those using fecal microbial transplantation (FMT) have demonstrated that response to checkpoint immunotherapies may be conferred or eliminated through gut microbiome modulation (7, 8). Consequently, studies evaluating microbiota-host immune and metabolic interactions remain an area of high impact research. While observations in murine models have highlighted the importance of the microbiome in response to therapy, we lack sufficient understanding of the exact mechanisms underlying these interactions. Furthermore, mouse and human gut microbiome composition may be too dissimilar for discovery of all relevant gut microbial biomarkers. Multiple cancers in dogs, including lymphoma, high grade gliomas, melanomas and osteosarcoma (OSA) closely resemble their human analogues, particularly in regard to metastasis, disease recurrence and response to treatment. Importantly, dogs with these spontaneous cancers also have intact immune systems, suggesting that microbiome analyses in these subjects may provide high yield information, especially in the setting of novel immunotherapy regimens which are currently expanding rapidly in canine comparative oncology (9, 10). Additionally, as onco-microbiotic therapies are developed to modify gut microbiomes for maximal responsiveness, large animal models with intact immune systems will be useful for trialing interventions and monitoring adverse events. Together, pre-clinical mechanistic studies and large animal trials can help fully unlock the potential of the microbiome as a diagnostic and therapeutic target in cancer.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Results: Sorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival. Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Natural Killer and T Cell Infiltration in Canine Osteosarcoma: Clinical Implications and Translational Relevance.

Metastatic osteosarcoma has a bleak prognosis in both humans and dogs, and there have been minimal therapeutic advances in recent decades to improve outcomes. Naturally occurring osteosarcoma in dogs is shown to be a highly suitable model for human osteosarcoma, and limited data suggest the similarities between species extend into immune responses to cancer. Studies show that immune infiltrates in canine osteosarcoma resemble those of human osteosarcoma, and the analysis of tumor immune constituents as predictors of therapeutic response is a promising direction for future research. Additionally, clinical studies in dogs have piloted the use of NK transfer to treat osteosarcoma and can serve as valuable precursors to clinical trials in humans. Cytotoxic lymphocytes in dogs and humans with osteosarcoma have increased activation and exhaustion markers within tumors compared with blood. Accordingly, NK and T cells have complex interactions among cancer cells and other immune cells, which can lead to changes in pathways that work both for and against the tumor. Studies focused on NK and T cell interactions within the tumor microenvironment can open the door to targeted therapies, such as checkpoint inhibitors. Specifically, PD-1/PD-L1 checkpoint expression is conserved across tumors in both species, but further characterization of PD-L1 in canine osteosarcoma is needed to assess its prognostic significance compared with humans. Ultimately, a comparative understanding of T and NK cells in the osteosarcoma tumor microenvironment in both dogs and humans can be a platform for translational studies that improve outcomes in both dogs and humans with this frequently aggressive disease.

Surgical and oncologic outcomes following arterial resection and reconstruction for advanced solid tumors.

BACKGROUND AND OBJECTIVES: Although arterial involvement for advanced tumors is rare, vascular resection may be indicated to achieve complete tumor resection. Given the potential morbidity of this approach, we sought to evaluate perioperative outcomes, vascular graft patency, and survival among patients undergoing tumor excision with en bloc arterial resection and reconstruction. METHODS: From 2010 to 2020, we identified nine patients with tumors encasing or extensively abutting major arterial structures for whom en bloc arterial resection and reconstruction was performed. RESULTS: Mean age was 53 ± 20 years, and 89% were females. Diagnoses were primary sarcomas (5), recurrent gynecologic carcinomas (3), and benign retroperitoneal fibrosis (1). Tumors involved the infrarenal aorta (2), iliac arteries (6), and superficial femoral artery (1). Three patients (33%) had severe perioperative morbidity (Grade III + ) with no mortality. At a median follow-up of 23 months, eight patients (89%) had primary graft patency, and five patients (56%) had no evidence of disease. CONCLUSIONS: Arterial resection and reconstruction as part of the multimodality treatment of regionally advanced tumors is associated with acceptable short- and long-term outcomes, including excellent graft patency. In appropriately selected patients, involvement of major arterial structures should not be viewed as a contraindication to attempted curative surgery.