Stratification of Patients with Renal Cell Carcinoma by the Abundance of Sarcomatoid Features Reveals Differences in Survival and the Underlying Pathobiology.

Sarcomatoid renal cell carcinoma (sRCC) is histologically heterogeneous, with variable sarcomatoid amounts intermixed within epithelial carcinoma. However, the current classification for this aggressive disease is homogeneous and agnostic to the sarcomatoid proportion. We investigated whether sRCC subclassification has prognostic value and can reveal the biology underlying dedifferentiation and its clinical aggressiveness. On the basis of the intratumoral abundance of sarcomatoid features, cases were classified as sarcomatoid-high (≥10% sarcomatoid features) or sarcomatoid-low (<10% sarcomatoid features) in a cohort of 104 consecutive patients with sRCC undergoing nephrectomy at a single center. In comparison to sarcomatoid-low patients (n = 52), sarcomatoid-high patients (n = 52) had significantly shorter overall survival (median 14.5 vs 62.9 mo; p < 0.001), which was confirmed on multivariable analysis, and significantly shorter median metastasis-free survival among patients with clinically localized disease (10.7 vs 39.0 mo; p = 0.043). Transcriptomic analyses of 45 sRCC tumors revealed significant upregulation of nine hallmark pathways related to cell cycle/proliferation, epithelial-to-mesenchymal transition, reactive oxidative species, and interferon-α signaling among sarcomatoid-high (n = 24) versus sarcomatoid-low (n = 21) tumors. Categorization into transcriptomic clusters revealed predominance of proliferative, inflammatory, and immune effector phenotypes among sarcomatoid-high tumors, versus a hypoxia/angiogenesis phenotype among sarcomatoid-low tumors. Overall, these findings indicate prognostic value for sRCC subclassification into high versus low sarcomatoid groups and highlight key biology underlying the differences in clinical outcomes. PATIENT SUMMARY: Sarcomatoid renal cell carcinoma (sRCC) is a highly aggressive form of kidney cancer. The percentage of sarcomatoid features varies among tumors, but sRCC is still defined as a single kidney cancer type. Our results show that grouping patients according to their percentage of sarcomatoid features improves prediction of whether their tumors will become metastatic or lethal, and reveal molecular differences that may be important for this disease. Future assignment of sRCC to high and low sarcomatoid groups may help in guiding research and patient management.

A Bacterial and Ganglioside-based Nanoparticle Initiates Reprogramming of Macrophages and Promotes Antitumor Phenotypes.

Macrophages represent the most abundant immune component of the tumor microenvironment and often exhibit protumorigenic (M2-like) phenotypes that contribute to disease progression. Despite their generally accepted protumorigenic role, macrophages can also display tumoricidal (or M1-like) behavior, revealing that macrophages can be functionally reprogrammed, depending on the cues received within the tumor microenvironment. Moreover, such plasticity may be achieved by pharmacologic or biologic interventions. To that end, we previously demonstrated that a novel immunomodulator termed the "very small size particle" (VSSP) facilitates maturation of dendritic cells and differentiation of myeloid-derived suppressor cells to APCs with reduced suppressive activity in cancer models. VSSP was further shown to act in the bone marrow to drive the differentiation of progenitors toward monocytes, macrophages, and dendritic cells during emergency myelopoiesis. However, the underlying mechanisms for VSSP-driven alterations in myeloid differentiation and function remained unclear. In this study, in mouse models, we focused on macrophages and tested the hypothesis that VSSP drives macrophages toward M1-like functional states via IRF8- and PU.1-dependent mechanisms. We further hypothesized that such VSSP-mediated actions would be accompanied by enhanced antitumor responses. Overall, we showed that (1) VSSP drives naive or M2-derived macrophages to M1-like states, (2) the M1-like state induced by VSSP occurs via IRF8- and PU.1-dependent mechanisms, and (3) single-agent VSSP induces an antitumor response that is accompanied by alterations in the intratumoral myeloid compartment. These results provide a deeper mechanistic underpinning of VSSP and strengthen its use to drive M1-like responses in host defense, including cancer.

Local Delivery of SBRT and IL12 by mRNA Technology Overcomes Immunosuppressive Barriers to Eliminate Pancreatic Cancer.

The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC. Statement of significance: This study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.

Tumor and immune remodeling following radiotherapy in human renal cell carcinoma.

BACKGROUND: Studies evaluating peripheral patient samples show radiation can modulate immune responses, yet the biological changes in human tumors particularly at the cellular level remain largely unknown. Here, we address how radiation treatment shapes the immune compartment and interactions with cancer cells within renal cell carcinoma (RCC) patient tumors. METHODS: To identify how radiation shaped the immune compartment and potential immune interactions with tumor cells we evaluated RCC tumors from patients treated only with nephrectomy or with radiation followed by nephrectomy. Spectral flow cytometry using a 35-marker panel was performed on cell suspensions to evaluate protein expression within immune subsets. To reveal how radiation alters programming of immune populations and interactions with tumor cells, we examined transcriptional changes by single-cell RNA sequencing (scRNAseq). RESULTS: Spectral flow cytometry analysis revealed increased levels of early-activated as well as effector programmed cell death protein-1 (PD-1)+ CD8 T-cell subsets within irradiated tumors. Following quality control, scRNAseq of tumor samples from nephrectomy-only or radiation followed by nephrectomy-treated patients generated an atlas containing 34,626 total cells. Transcriptional analysis revealed increased transition from stem-like T-cell populations to effector T cells in irradiated tumors. Interferon (IFN) pathways, that are central to radiation-induced immunogenicity, were enriched in irradiated lymphoid, myeloid, and cancer cell populations. Focused cancer cell analysis showed enhanced antigen presentation and increased predicted TRAIL-mediated and IFN-mediated interactions between tumor cells and the same effector T-cell subsets increased by radiation. TRAIL and IFN pathways enriched in irradiated tumors were associated with survival in patients treated with immunotherapy. CONCLUSIONS: These findings identify the source of IFN enrichment within irradiated RCC and reveal heightened levels of PD-1+ CD8+ T-cell subsets and increased probability of interactions with tumor cells following standalone radiation treatment. This study provides a window into the irradiated tumor-immune microenvironment of patients and rationale for treatment combinations.

Mitigating the prevalence and function of myeloid-derived suppressor cells by redirecting myeloid differentiation using a novel immune modulator.

BACKGROUND: Immune suppression is common in neoplasia and a major driver is tumor-induced myeloid dysfunction. Yet, overcoming such myeloid cell defects remains an untapped strategy to reverse suppression and improve host defense. Exposure of bone marrow progenitors to heightened levels of myeloid growth factors in cancer or following certain systemic treatments promote abnormal myelopoiesis characterized by the production of myeloid-derived suppressor cells (MDSCs) and a deficiency in antigen-presenting cell function. We previously showed that a novel immune modulator, termed 'very small size particle' (VSSP), attenuates MDSC function in tumor-bearing mice, which was accompanied by an increase in dendritic cells (DCs) suggesting that VSSP exhibits myeloid differentiating properties. Therefore, here, we addressed two unresolved aspects of the mechanism of action of this unique immunomodulatory agent: (1) does VSSP alter myelopoiesis in the bone marrow to redirect MDSC differentiation toward a monocyte/macrophage or DC fate? and (2) does VSSP mitigate the frequency and suppressive function of human tumor-induced MDSCs? METHODS: To address the first question, we first used a murine model of granulocyte-colony stimulating factor-driven emergency myelopoiesis following chemotherapy-induced myeloablation, which skews myeloid output toward MDSCs, especially the polymorphonuclear (PMN)-MDSC subset. Following VSSP treatment, progenitors and their myeloid progeny were analyzed by immunophenotyping and MDSC function was evaluated by suppression assays. To strengthen rigor, we validated our findings in tumor-bearing mouse models. To address the second question, we conducted a clinical trial in patients with metastatic renal cell carcinoma, wherein 15 patients were treated with VSSP. Endpoints in this study included safety and impact on PMN-MDSC frequency and function. RESULTS: We demonstrated that VSSP diminished PMN-MDSCs by shunting granulocyte-monocyte progenitor differentiation toward monocytes/macrophages and DCs with heightened expression of the myeloid-dependent transcription factors interferon regulatory factor-8 and PU.1. This skewing was at the expense of expansion of granulocytic progenitors and rendered the remaining MDSCs less suppressive. Importantly, these effects were also demonstrated in a clinical setting wherein VSSP monotherapy significantly reduced circulating PMN-MDSCs, and their suppressive function. CONCLUSIONS: Altogether, these data revealed VSSP as a novel regulator of myeloid biology that mitigates MDSCs in cancer patients and reinstates a more normal myeloid phenotype that potentially favors immune activation over immune suppression.

Augmenting antibody response to EGF-depleting immunotherapy: Findings from a phase I trial of CIMAvax-EGF in combination with nivolumab in advanced stage NSCLC.

Background: CIMAvax-EGF is an epidermal growth factor (EGF)-depleting immunotherapy which has shown survival benefit as a switch maintenance treatment after platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). The primary objective of this trial is to establish the safety and recommended phase II dose (RP2D) of CIMAvax-EGF in combination with nivolumab as second-line therapy for NSCLC. Methods: Patients with immune checkpoint inhibitor-naive metastatic NSCLC were enrolled using a "3+3" dose-escalation design. Toxicities were graded according to CTCAE V4.03. Thirteen patients (one unevaluable), the majority with PD-L1 0%, were enrolled into two dose levels of CIMAvax-EGF. Findings: The combination was determined to be safe and tolerable. The recommended phase 2 dose of CIMAvax-EGF was 2.4 mg. Humoral response to CIMAvax-EGF was achieved earlier and in a greater number of patients with the combination compared to historical control. Four out of 12 evaluable patients had an objective response.

Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis.

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

Phase Ib/II Study of Cetuximab plus Pembrolizumab in Patients with Advanced RAS Wild-Type Colorectal Cancer.

PURPOSE: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC). PATIENTS AND METHODS: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence. RESULTS: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9-5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01). CONCLUSIONS: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.

Radiation induces dynamic changes to the T cell repertoire in renal cell carcinoma patients.

Clinical studies combining radiation and immunotherapy have shown promising response rates, strengthening efforts to sensitize tumors to immune-mediated attack. Thus, there is an ongoing surge in trials using preconditioning regimens with immunotherapy. Yet, due to the scarcity of resected tumors treated in situ with radiotherapy, there has been little investigation of radiation's sole contributions to local and systemic antitumor immunity in patients. Without this access, translational studies have been limited to evaluating circulating immune subsets and systemic remodeling of peripheral T cell receptor repertoires. This constraint has left gaps in how radiation impacts intratumoral responses and whether tumor-resident T cell clones are amplified following treatment. Therefore, to interrogate the immune impact of radiation on the tumor microenvironment and test the hypothesis that radiation initiates local and systemic expansion of tumor-resident clones, we analyzed renal cell carcinomas from patients treated with stereotactic body radiation therapy. Transcriptomic comparisons were evaluated by bulk RNA sequencing. T cell receptor sequencing monitored repertoires during treatment. Pathway analysis showed radiation-specific enrichment of immune-related processes, and T cell receptor sequencing revealed increased clonality in radiation-treated tumors. The frequency of identified, tumor-enriched clonotypes was tracked across serial blood samples. We observed increased abundance of tumor-enriched clonotypes at 2 wk postradiation compared with pretreatment levels; however, this expansion was not sustained, and levels contracted toward baseline by 4 wk posttreatment. Taken together, these results indicate robust intratumoral immune remodeling and a window of tumor-resident T cell expansion following radiation that may be leveraged for the rational design of combinatorial strategies.

In situ thermal ablation augments antitumor efficacy of adoptive T cell therapy.

Purpose: The aim of this study is to investigate whether radiofrequency ablation (RFA) improves the efficacy of adoptive T cell immunotherapy in preclinical mouse cancer models.Method: Mice implanted subcutaneously (sc) with syngeneic colon adenocarcinoma or melanoma were treated with sub-curative in situ RFA (90 °C, 1 min). Trafficking of T cells to lymph nodes (LN) or tumors was quantified by homing assays and intravital microscopy (IVM) after sham procedure or RFA. Expression of trafficking molecules (CCL21 and intercellular adhesion molecule-1 [ICAM-1]) on high endothelial venules (HEV) in LN and tumor vessels was evaluated by immunofluorescence microscopy. Tumor-bearing mice were pretreated with RFA to investigate the therapeutic benefit when combined with adoptive transfer of in vitro-activated tumor-specific CD8+ T cells.Results: RFA increased trafficking of naïve CD8+ T cells to tumor-draining LN (TdLN). A corresponding increase in expression of ICAM-1 and CCL21 was detected on HEV in TdLN but not in contralateral (c)LN. IVM revealed that RFA substantially enhanced secondary firm arrest of lymphocytes selectively in HEV in TdLN. Furthermore, strong induction of ICAM-1 in tumor vessels was associated with significantly augmented trafficking of adoptively transferred in vitro-activated CD8+ T cells to tumors after RFA. Finally, preconditioning tumors with RFA augmented CD8+ T cell-mediated apoptosis of tumor targets and delayed growth of established tumors when combined with adoptive T cell transfer immunotherapy.Conclusions: These studies suggest that in addition to its role as a palliative therapeutic modality, RFA may have clinical potential as an immune-adjuvant therapy by augmenting the efficacy of adoptive T cell therapy.

Tumor-associated macrophage expression of interferon regulatory Factor-8 (IRF8) is a predictor of progression and patient survival in renal cell carcinoma.

Tumor-associated macrophages have been well-characterized in solid malignancies, including renal cell carcinoma and generally correlate with poor prognosis. However, the molecular mechanisms which govern intratumoral macrophage behavior and patient outcome are unclear. Here, we investigated whether alterations in macrophage expression of the transcriptional regulator for myeloid commitment and function, interferon regulatory factor-8 (IRF8), could predict survival of clear cell renal cell carcinoma patients. Transcriptional analysis of publicly available data revealed elevated IRF8 expression was associated with prolonged disease-free survival. Evaluation of protein expression within histologic sections of primary clear cell renal cell carcinoma patient samples showed intensity of IRF8 by CD68+ macrophages correlated inversely with stage. Survival outcomes of patients with primary or metastatic disease could be stratified on the basis of IRF8 levels by macrophages. Patients with high levels of IRF8 expression within metastatic sites had prolonged overall survival (log-rank P < 0.01, HR = 0.44, 95% C.I.: 0.23-0.84) compared to patients with low levels of IRF8 expression. When patient cohorts were further separated based on macrophage infiltration within metastatic lesions, patients with a macrophagelo IRF8hi profile had a more than 10 year increase in median overall survival compared to patients with a macrophagelo IRF8lo profile (log-rank, P < 0.001). In summary, we report that macrophage expression of IRF8 is inversely correlated with tumor mass and directly related to survival outcome. These findings support the utilization of IRF8 expression by macrophages to predict patient outcome, which may have important implications for guiding treatment decisions for renal cell carcinoma patients with metastatic disease.

A Pilot Study of Stereotactic Body Radiation Therapy Combined with Cytoreductive Nephrectomy for Metastatic Renal Cell Carcinoma.

Purpose: While stereotactic body radiotherapy (SBRT) can reduce tumor volumes in patients with metastatic renal cell carcinoma (mRCC), little is known regarding the immunomodulatory effects of high-dose radiation in the tumor microenvironment. The main objectives of this pilot study were to assess the safety and feasibility of nephrectomy following SBRT treatment of patients with mRCC and analyze the immunological impact of high-dose radiation.Experimental Design: Human RCC cell lines were irradiated and evaluated for immunomodulation. In a single-arm feasibility study, patients with mRCC were treated with 15 Gray SBRT at the primary lesion in a single fraction followed 4 weeks later by cytoreductive nephrectomy. RCC specimens were analyzed for tumor-associated antigen (TAA) expression and T-cell infiltration. The trial has reached accrual (ClinicalTrials.gov identifier: NCT01892930).Results: RCC cells treated in vitro with radiation had increased TAA expression compared with untreated tumor cells. Fourteen patients received SBRT followed by surgery, and treatment was well-tolerated. SBRT-treated tumors had increased expression of the immunomodulatory molecule calreticulin and TAA (CA9, 5T4, NY-ESO-1, and MUC-1). Ki67+ -proliferating CD8+ T cells and FOXP3+ cells were increased in SBRT-treated patient specimens in tumors and at the tumor-stromal interface compared with archived patient specimens.Conclusions: It is feasible to perform nephrectomy following SBRT with acceptable toxicity. Following SBRT, patient RCC tumors have increased expression of calreticulin, TAA, as well as a higher percentage of proliferating T cells compared with archived RCC tumors. Collectively, these studies provide evidence of immunomodulation following SBRT in mRCC. Clin Cancer Res; 23(17); 5055-65. ©2017 AACR.

Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence.

The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater effect on the IRF8 nuclear localization versus total fluorescence assessment. The data thus indicate that IRF8 can have cytoplasmic presence and that during disease its nuclear-cytoplasmic distribution may be altered, which may provide a basis for potential myeloid defects during certain pathologies.

Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes.

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity.

Unmasking targets of antitumor immunity via high-throughput antigen profiling.

More than three decades of evidence has established that antitumor immune responses, initially shown with IL-2 treatment, can result in complete, durable eradication of malignant disease in metastatic patients. Recent studies have demonstrated that immune checkpoint blockade as well as cellular therapies, including dendritic cell activation of T cells and adoptive T cell transfer, can induce long-lasting responses. To elicit cytolysis of tumor cells, effector T cells rely on tumor expression of target antigens. However, the antigens targeted during antitumor responses are largely unknown. Technological advancements and availability of sequencing data have paved the way for more efficient screening and validation of tumor-associated antigens and neoantigens derived from non-synonymous mutations targeted by T cells under baseline conditions and in the context of immunotherapy.

Intraoperative intravital microscopy permits the study of human tumour vessels.

Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer.

PURPOSE: While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. EXPERIMENTAL DESIGN: Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. RESULTS: Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. CONCLUSION: Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.

IL-17 promotes neutrophil entry into tumor-draining lymph nodes following induction of sterile inflammation.

Blood-borne neutrophils are excluded from entering lymph nodes across vascular portals termed high endothelial venules (HEVs) because of lack of expression of the CCR7 homeostatic chemokine receptor. Induction of sterile inflammation increases neutrophil entry into tumor-draining lymph nodes (TDLNs), which is critical for induction of antitumor adaptive immunity following treatments such as photodynamic therapy (PDT). However, the mechanisms controlling neutrophil entry into TDLNs remain unclear. Prior evidence that IL-17 promotes neutrophil emigration to sites of infection via induction of CXCL2 and CXCL1 inflammatory chemokines raised the question of whether IL-17 contributes to chemokine-dependent trafficking in TDLNs. In this article, we demonstrate rapid accumulation of IL-17-producing Th17 cells in the TDLNs following induction of sterile inflammation by PDT. We further report that nonhematopoietic expression of IL-17RA regulates neutrophil accumulation in TDLNs following induction of sterile inflammation by PDT. We show that HEVs are the major route of entry of blood-borne neutrophils into TDLNs through interactions of l-selectin with HEV-expressed peripheral lymph node addressin and by preferential interactions between CXCR2 and CXCL2 but not CXCL1. CXCL2 induction in TDLNs was mapped in a linear pathway downstream of IL-17RA-dependent induction of IL-1ß. These results define a novel IL-17-dependent mechanism promoting neutrophil delivery across HEVs in TDLNs during acute inflammatory responses.

Preconditioning thermal therapy: flipping the switch on IL-6 for anti-tumour immunity.

Cancer immunotherapy aims to generate long-lived, tumour-specific adaptive immunity to limit dysregulated tumour progression and metastasis. Tumour vasculature has emerged as a critical checkpoint controlling the efficacy of immunotherapy since it is the main access point for cytotoxic T cells to reach tumour cell targets. Therapeutic success has been particularly challenging to achieve because of the local, cytokine-rich inflammatory milieu that drives a pro-tumourigenic programme supporting the growth and survival of malignant cells. Here, we focus on recent evidence that systemic thermal therapy can switch the activities of the inflammatory cytokine, interleukin-6 (IL-6), to a predominantly anti-tumourigenic function that promotes anti-tumour immunity by mobilising T cell trafficking in the recalcitrant tumour microenvironment.