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Supplementary Figure 7 - PDF file 111K, Characterization of myeloid cells in lung, tumor, spleen, and lymph nodes

Supplementary Figure 1 - PDF file 183K, Overproduction of immature myeloid cells in both tumor bearing mice and cancer patients, flow cytometry analysis and sorting

Supplementary Figure 4 - PDF file 129K, Validation of over expression of TGFbeta1, IL-4, IL10, as well as Arginase1 and iNOS in Tgfbr2

Supplementary Figure 3 - PDF file 76K, IFN-gamma production by T cells and its effect on tumor growth in Tgfbr2MyeKOand Tgfbr2flox/flox control mice

Supplementary Figure 8 - PDF file 84K, Immune cell profiling and IFN-gamma production in CD8 T cells in tumor tissues between Tgfbr2flox/flox and Tgfbr2MyeKO mice bearing 4T1 tumors

Supplementary Figure 9 - PDF file 150K, Gating strategy, sorting efficiency, and TbetaRII expression in myeloid cell subsets

TGF-β is overexpressed in advanced human cancers. It correlates with metastasis and poor prognosis. However, TGF-β functions as both a tumor suppressor and a tumor promoter. Here, we report for the first time that genetic deletion of Tgfbr2 specifically in myeloid cells (Tgfbr2MyeKO) significantly inhibited tumor metastasis. Reconstitution of tumor-bearing mice with Tgfbr2MyeKO bone marrow recapitulated the inhibited metastasis phenotype. This effect is mediated through decreased production of type II cytokines, TGF-β1, arginase 1, and inducible nitric oxide synthase, which promoted IFN-γ production and improved systemic immunity. Depletion of CD8 T cells diminished the metastasis defect in the Tgfbr2MyeKO mice. Consistent with animal studies, myeloid cells from patients with advanced-stage cancer showed increased TGF-β receptor II expression. Our studies show that myeloid-specific TGF-β signaling is an essential component of the metastasis-promoting puzzle of TGF-β. This is in contrast to the previously reported tumor-suppressing phenotypes in fibroblasts, epithelial cells, and T cells.Significance: Our study identifies myeloid-specific TGF-β signaling as a critical mediator in tumor metastasis, distinct from the tumor-suppressive effect of TGF-β signaling in epithelial cells, fibroblasts, and T cells. We further provide mechanistic insight into host antitumor immunity and suggest a cell type–specific cancer-targeting strategy. Cancer Discov; 3(8); 936–51. ©2013 AACR.See related commentary by Souza-Fonseca-Guimaraes and Smyth, p. 846This article is highlighted in the In This Issue feature, p. 826

Supplementary Figure 5 - PDF file 382K, Analysis of percentage or number in B cells, T cells, or myeloid cell subsets in the lungs between Tgfbr2MyeKO and control littermates under normal or tumor conditions

Supplementary Figure 2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Table 1. Th1 vs. Th2 Cytokine Trend. Supplementary Table 2. Effect of L-NAME on Radiation-induced Cytokine Expression.

Supplementary Figure 3 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 4 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 1 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 1 from mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

Supplementary Figure 4 from mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

Supplementary Figure 5 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 3 from mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

Supplementary Figure Legends, Supplementary Tables 1-2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 6 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 5 from mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

Supplementary Figure 2 from mTOR Kinase Inhibitor AZD8055 Enhances the Immunotherapeutic Activity of an Agonist CD40 Antibody in Cancer Treatment

Background Acne is a chronic disease with a varying presentation that requires long-term management. Despite this, the clinical guidelines for acne offer limited guidance to facilitate personalized or longitudinal management of patients. Objectives To generate recommendations to support comprehensive, personalized, long-term patient management that address all presentations of acne and its current and potential future burden. Methods The Personalising Acne: Consensus of Experts panel consisted of 13 dermatologists who used a modified Delphi approach to reach consensus on statements related to longitudinal acne management. The consensus was defined as ≥75% voting "agree" or "strongly agree." All voting was electronic and blinded. Results Key management domains, consisting of distinct considerations, points to discuss with patients, and "pivot points" were identified and incorporated into the Personalised Acne Care Pathway. Long-term treatment goals and expectations and risk of (or fears about) sequelae are highlighted as particularly important to discuss frequently with patients. Limitations Recommendations are based on expert opinion, which could potentially differ from patients' perspectives. Regional variations in health care systems may not have been captured. Conclusions The Personalised Acne Care Pathway provides practical recommendations to facilitate the longitudinal management of acne, which can be used by health care professionals to optimize and personalize care throughout the patient journey.

Background The physical sequelae of acne include erythema, hyperpigmentation, and scarring, which are highly burdensome for patients. Early, effective treatment can potentially limit and prevent sequelae development, but there is a need for guidance for and evidence of prevention-oriented management to improve patient outcomes. Objective To identify unmet needs of acne sequelae and generate expert recommendations to address gaps in clinical guidance. Methods The Personalizing Acne: Consensus of Experts panel of 13 dermatologists used a modified Delphi approach to achieve a consensus on the clinical aspects of acne sequelae. A consensus was defined as ≥75% of the dermatologists voting "agree" or "strongly agree." All voting was electronic and blinded. Results The panel identified gaps in current guidance and made recommendations related to acne sequelae. These included identification and classification of sequelae, pertinent points to consider for patient consultations, and management aimed at reducing the development of sequelae. Limitations The recommendations are based on expert opinion and made in the absence of high-quality evidence. Conclusions The identified gaps should help inform future research and guideline development for acne sequelae. The consensus-based recommendations should also support the process of consultations throughout the patient journey, helping to reduce the development and burden of acne sequelae through improved risk factor recognition, early discussion, and appropriate management.

PURPOSE The rho-associated coiled-coil–containing protein kinase-2 (ROCK2) signaling pathway regulates the Th17/regulatory T cells balance and controls profibrotic pathways. Selective ROCK2 inhibition with belumosudil (KD025) may offer a novel approach to the management of chronic graft-versus-host disease (cGVHD). PATIENTS AND METHODS A phase IIa, open-label, dose-finding study of belumosudil enrolled 54 patients with cGVHD who had received one to three prior lines of therapy (LOTs). The primary end point was overall response rate (ORR). RESULTS The median time from cGVHD diagnosis to enrollment was 20 months. Seventy-eight percent of patients had severe cGVHD, 50% had ≥ 4 organs involved, 73% had cGVHD refractory to their last LOT, and 50% had received ≥ 3 prior LOTs. With an overall median follow-up of 29 months, the ORR (95% CI) with belumosudil 200 mg once daily, 200 mg twice daily, and 400 mg once daily was 65% (38% to 86%), 69% (41% to 89%), and 62% (38% to 82%), respectively. Responses were clinically meaningful, with a median duration of response of 35 weeks, and were associated with quality-of-life improvements and corticosteroid (CS) dose reductions. CS treatment was discontinued in 19% of patients. The failure-free survival rate was 76% (62% to 85%) and 47% (33% to 60%) at 6 and 12 months, respectively. The 2-year overall survival rate was 82% (69% to 90%). Belumosudil was well-tolerated, with low rates of cytopenia. There were no unexpected adverse events and no apparent increased risk of infection, including cytomegalovirus infection and reactivation. CONCLUSION Belumosudil treatment resulted in a high ORR and overall survival rate and demonstrated quality-of-life improvements, CS dose reductions, and limited toxicity. Data from the study indicated that belumosudil may prove to be an effective therapy for patients with treatment-refractory cGVHD.

Modern advances in technology such as next-generation sequencing and digital PCR make detection of minor circulating cell-free tumor DNA amounts in blood from cancer patients possible. Samples can be obtained minimal-invasively, tested for treatment-determining genetic alterations and are considered to reflect the genetic constitution of the whole tumor mass. Furthermore, tumor development can be determined by a time course of the quantified circulating cell-free tumor DNA. However, systematic studies which prove the clinical relevance of monitoring patients using liquid biopsies are still lacking. In this study, we collected 115 samples from 47 late stage melanoma patients over 1.5 years alongside therapy-associated clinical routine monitoring. Mutation status was confirmed by molecular analysis of primary tumor material. We can show that detectable levels of circulating cell-free tumor DNA correlate with clinical development over time. Increasing levels of circulating cell-free tumor DNA during melanoma treatment with either targeted therapy (BRAF/MEK inhibitors) or immunotherapy, during recovery time or the intervals between last treatment cycle and second-line treatment point towards clinical progression before the progression becomes obvious in imaging. Therefore, this is a further possibility to closely screen our patients for tumor progression during therapy, in therapy-free phases and in earlier stages before therapy initiation.

Background Truncal acne is common and burdensome for patients; however, there is paucity of evidence and guidance for the management of truncal acne. Currently, clinical practice guidelines provide very little guidance on the assessment or management of truncal acne. Objectives To identify unmet needs in truncal acne and make recommendations to address clinical and management gaps using an international consensus. Methods The Personalising Acne: Consensus of Experts panel consisted of 13 dermatologists, who used a modified Delphi approach to reach a consensus on statements related to clinically relevant aspects of truncal acne evaluation and management. A consensus was defined as ≥75% of the panelists voting "agree" or "strongly agree." The voting was electronic and blinded. Results The panel identified gaps and made recommendations related to truncal acne identification, assessment, and grading; the evaluation of the impact on patients; and treatment goals and factors to be considered for its management. Limitations The recommendations are based on expert opinion, in the absence of high-quality evidence. Conclusions We highlighted addressing not just facial acne but also truncal acne during patient consultations. The recommendations made herein may help facilitate the care of patients who present with truncal acne, with or without facial acne.

Rho-associated coiled-coil kinase (ROCK)2 targeting down-regulates autoimmune responses in animal models and patients, however the underlying molecular mechanism is still an enigma. We report that ROCK2 binds phosphorylated-STAT3 and its kinase activity controls the formation of ROCK2/STAT3/JAK2 complex and optimal STAT3 phosphorylation in human CD4+ T cells during T helper 17 (TH17)-skewing. Moreover, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis revealed that, genome-wide, about 70% of ROCK2 and STAT3 peaks overlapped and co-localized to several key genes controlling TH17 and T follicular helper (TFH) cell functions. Specifically, the co-occupancy of ROCK2 and STAT3 on the Irf4 and Bcl6 genes was validated by ChIP-qPCR analysis. Furthermore, the binding of ROCK2 to both the Irf4 and Bcl6 promoters was attenuated by STAT3 silencing as well as by selective ROCK2 inhibitor. Thus, the present study demonstrated previously unidentified evidence that ROCK2-mediated signaling in the cytosol provides a positive feed-forward signal for nuclear ROCK2 to be recruited to the chromatin by STAT3 and potentially regulates TH17/TFH gene transcription.

Background Acne confers an increased risk of physical, psychiatric, and psychosocial sequelae, potentially affecting multiple dimensions of health-related quality of life (HRQoL). Morbidity associated with truncal acne is poorly understood. Objective To determine how severity and location of acne lesions impact the HRQoL of those who suffer from it. Methods A total of 694 subjects with combined facial and truncal acne (F+T) and 615 with facial acne only (F) participated in an online, international survey. Participants self-graded the severity of their acne at different anatomical locations and completed the dermatology life quality index (DLQI). Results The F+T participants were twice as likely to report "very large" to "extremely large" impact on HRQoL (ie, DLQI > 10 and children's DLQI [CDLQI] > 12) as compared with the F participants (DLQI: odds ratio [OR] 1.61 [95% confidence interval {CI} 1.02-2.54]; CDLQI: OR 1.86 [95% CI 1.10-3.14]). The impact of acne on HRQoL increased with increasing acne severity on the face (DLQI and CDLQI P values = .001 and .017, respectively), chest (P = .003; P = .008), and back (P = .001; P = .028). Limitations Temporal evaluation of acne impact was not estimated. Conclusions Facial and truncal acne was associated with a greater impact on HRQoL than facial acne alone. Increasing severity of truncal acne increases the adverse impact on HRQoL irrespective of the severity of facial acne.

Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression—for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and Trem2 expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that TREM1 expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.

Contains fulltext : 196332.pdf (Publisher’s version ) (Closed access) Reducing the activities of the pro-inflammatory cytokine IL-17 is an effective treatment strategy for several chronic autoimmune disorders. Rho-associated coiled-coil containing kinase 2 (ROCK2) is a member of the serine-threonine protein kinase family that regulates IL-17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL-17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin-1 agonist Candida albicans. C. albicans induced IL-17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll-like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL-1beta, IL-6 or IL-1alpha levels; in contrast, a 1.5 fold increase in the level of IL-1 receptor antagonist (IL-1Ra) was observed (p < 0.001). KD025 down-regulated C. albicans-induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti-CD3/CD28 activation of the TCR, KD025 similarly suppressed IL-17 independent of a reduction in IL-1beta. Thus, ROCK2 directly regulates IL-17 secretion independent of endogenous IL-1 and IL-6 supporting development of selective ROCK2 inhibitors for treatment of IL-17-driven inflammatory diseases.

A major challenge of cancer immunotherapy is the potential for undesirable effects on bystander cells and tumor-associated immune cells. Fundamentally, we need to understand what effect targeting tumor metabolism has upon the metabolism and phenotype of tumor-associated leukocytes, whose function can be critical for effective cancer therapeutic strategies. Undesirable effects of cancer therapeutics are a major reason for drug-associated toxicity, which confounds drug dosing and efficacy. As with any chemotherapeutic agent, drugs targeting tumor metabolism will exert potent effects on host stromal cells and tumor-associated leukocytes. Any drug targeting glycolysis, for example, could metabolically starve tumor-infiltrating T cells, inhibit their effector function and enable tumor progression. The targeting of oxidative phosphorylation in tumors will have complex effects on the polarization and function of tumor-associated macrophages. In short, we need to improve our understanding of tumor and immune cell metabolism and devise ways to specifically target tumors without compromising necessary host metabolism. Exploiting cell-specific metabolic pathways to directly target tumor cells may minimize detrimental effects on tumor-associated leukocytes.

Significance Nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) are inflammation-associated enzymes with oncogenic function in breast cancer. We show that crosstalk between NOS2/COX2 promotes aggressive phenotypes and that elevated coexpression of NOS2/COX2 in tumors predict significantly reduced patient survival (33%) when compared with 95% survival of estrogen receptor-negative patients with low NOS2/COX2 tumor expression. In addition, we have identified a tumor subtype-specific mechanism showing involvement of TNFα and/or endoplasmic reticulum stress as key players in this autocrine loop. Importantly, the simultaneous inhibition of NOS2/COX2 significantly reduced tumor growth in a xenograft murine model, suggesting that targeted inhibition of NOS2/COX2 may be therapeutically beneficial., Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER−) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER− patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER− and TNBC disease.

Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, “alternatively activated” macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes., Graphical abstract fx1, Highlights • Optimal macrophage IL-10 production requires un-restricted utilization of glucose. • IL-10 production is limited by AMP Kinase activation and subsequent TORC suppression. • Macrophage glycolytic commitment is wholly dependent on nitric oxide. • Autocrine IL-10 inhibits NO production, limiting macrophage glycolytic commitment. • Autocrine IL-10 is a rheostat for glycolytic commitment of macrophages.

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// Jian Wang 1 , Peijun He 1 , Matthias Gaida 2 , Shouhui Yang 1 Aaron J. Schetter 3 , Jochen Gaedcke 4 , B. Michael Ghadimi 4 , Thomas Ried 5 , Harris Yfantis 6 , Dong Lee 6 , Jonathan M. Weiss 7 , Jimmy Stauffer 8 , Nader Hanna 9 , H. Richard Alexander 9 , S. Perwez Hussain 1 1 Pancreatic Cancer Unit, Laboratory of Human Carcinogenesis, CCR, NCI, Bethesda, MD, USA 2 Institute of Pathology, University Hospital of Heidelberg, Heidelberg, Germany 3 US Food and Drug Administration, Silver spring, MD, USA 4 Department of General, Visceral and Pediatric Surgery, University Medicine, Gottingen, Germany 5 Genetics Branch, CCR, NCI, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA 6 Pathology and Laboratory Medicine, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA 7 Cancer and Inflammation Program, CCR, NCI Frederick, MD, USA 8 Laboratory of Cell and Developmental Signaling, NCI Frederick, MD, USA 9 Division of Surgical Oncology, University of Maryland School of Medicine, Baltimore, MD, USA Correspondence to: S. Perwez Hussain, email: hussainp@mail.nih.gov Keywords: NOS2, NO, PDAC, KPC mouse model Received: February 12, 2016 Accepted: June 12, 2016 Published: June 29, 2016 ABSTRACT Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients ( N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.

Background & Aims Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. Methods Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. Results Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8 + T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL + hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. Conclusions Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.

Chemokines and chemokine receptors have critical roles in cancer metastasis and have emerged as one of the targeting options in cancer therapy. However, the treatment efficacy on both tumor and host compartments needs to be carefully evaluated. Here we report that targeting CXCR3 decreased tumor cell migration and at the same time improved host anti-tumor immunity. We observed an increased expression of CXCR3 in metastatic tumor cells compared to those from non-metastatic tumor cells. Knockdown (KD) of CXCR3 in metastatic tumor cells suppressed tumor cell migration and metastasis. Importantly, CXCR3 expression in clinical breast cancer samples correlated with progression and metastasis. For the host compartment, deletion of CXCR3 in all host cells in 4T1 mammary tumor model significantly decreased metastasis. The underlying mechanisms involve a decreased expression of IL-4, IL-10, iNOs, and Arg-1 in myeloid cells and an increased T cell response. IFN-γ neutralization diminished the metastasis inhibition in the CXCR3 knockout (KO) mice bearing 4T1 tumors, suggesting a critical role of host CXCR3 in immune suppression. Consistently, targeting CXCR3 using a small molecular inhibitor (AMG487) significantly suppressed metastasis and improved host anti-tumor immunity. Our findings demonstrate that targeting CXCR3 is effective in both tumor and host compartments, and suggest that CXCR3 inhibition is likely to avoid adverse effects on host cells.

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