Osteosarcoma Cells Secrete CXCL14 That Activates Integrin α11ß1 on Fibroblasts to Form a Lung Metastatic Niche.

Cooperation between primary malignant cells and stromal cells can mediate the establishment of lung metastatic niches. Here, we characterized the landscape of cell populations in the tumor microenvironment in treatment-naïve osteosarcoma using single-cell RNA sequencing and identified a stem cell-like cluster with tumor cell-initiating properties and prometastatic traits. CXCL14 was specifically enriched in the stem cell-like cluster and was also significantly upregulated in lung metastases compared with primary tumors. CXCL14 induced stromal reprogramming and evoked a malignant phenotype in fibroblasts to form a supportive lung metastatic niche. Binding of CXCL14 to heterodimeric integrin α11ß1 on fibroblasts activated actomyosin contractility and matrix remodeling properties. CXCL14-stimulated fibroblasts produced TGFß and increased osteosarcoma invasion and migration. mAbs targeting the CXCL14-integrin α11ß1 axis inhibited fibroblast TGFß production, enhanced CD8+ T cell-mediated antitumor immunity, and suppressed osteosarcoma lung metastasis. Taken together, these findings identify cross-talk between osteosarcoma cells and fibroblasts that promotes metastasis and demonstrate that targeting the CXCL14-integrin α11ß1 axis is a potential strategy to inhibit osteosarcoma lung metastasis. SIGNIFICANCE: Cooperation between stem-like osteosarcoma cells and fibroblasts mediated by a CXCL14-integrin α11ß1 axis creates a tumor-supportive lung metastatic niche and represents a therapeutic target to suppress osteosarcoma metastasis.

Mass Spectrometry-Based Proteomics Identifies Serpin B9 as a Key Protein in Promoting Bone Metastases in Lung Cancer.

Bone metastasis (BM) is one of the most common complications of advanced cancer. Immunotherapy for bone metastasis of lung cancer (LCBM) is not so promising and the immune mechanisms are still unknown. Here, we utilized a model of BM by injecting cancer cells through caudal artery (CA) to screen out a highly bone metastatic derivative (LLC1-BM3) from a murine lung cancer cell line LLC1. Mass spectrometry-based proteomics was performed in LLC1-parental and LLC1-BM3 cells. Combining with prognostic survival information from patients with lung cancer, we identified serpin B9 (SB9) as a key factor in BM. Molecular characterization showed that SB9 overexpression was associated with poor prognosis and high bone metastatic burden in lung cancer. Moreover, SB9 could increase the ability of lung cancer cells to metastasize to the bone. The mechanistic studies revealed that tumor-derived SB9 promoted BM through an immune cell-dependent way by inactivating granzyme B, manifesting with the decreased infiltration of cytotoxic T cells and increased expression level of exhausted markers. A specific SB9-targeting inhibitor [1,3-benzoxazole-6-carboxylic acid (BTCA)] significantly suppressed LCBM in the CA mouse model. This study reveals that SB9 may serve as a therapeutic target and potential prognostic marker for patients with LCBM. IMPLICATIONS: SB9 as a therapeutic target for LCBM.

A nanodrug combining CD47 and sonodynamic therapy efficiently inhibits osteosarcoma deterioration.

Treatments for osteosarcoma (OS) with pulmonary metastases reach a bottleneck with a survival rate of 10-20%. The suppressive tumor associated macrophages(TAMs) and CD47 over-expression greatly lead to the treatment failure. Sonodynamic therapy (SDT) can generate ROS with deep tumor penetration to induce tumor cell apoptosis, which is reported to further induce M1 macrophage polarization. CD47 inhibition combined with SDT to synergistically modulate TAMs may induce superior effects for OS treatment. In this work, for the first time, a biomimetic nanodrug named MPIRx was deveploped by loading IR780 (a sonosensitizer) and RRx-001 (a CD47 inhibitor) in PEG-PCL nanomicelles and then coating with OS cell membranes. After ultrasound activation, the nanodrug significantly inhibited OS proliferation and migration, induced apoptosis and immunogenic cell death in OS cells. Furthermore, MPIRx could guide macrophage migrating towards tumor cells and promote M1-type polarization while increasing the phagocytosis activity of macrophages on OS cells. Ultimately, MPIRx showed good tumor accumulation in vivo and successfully inhibited subcutaneous OS and orthotopic tumor with deterioration of pulmonary metastasis. Overall, by creating a local oxidative microenvironment and modulating the TAMs/CD47 in tumor tissue, the MPIRx nanodrug presents a novel strategy for macrophage-related immunotherapy to successfully eliminate OS and inhibit the intractable pulmonary metastasis.

The Mycotoxin Beauvericin Exhibits Immunostimulatory Effects on Dendritic Cells via Activating the TLR4 Signaling Pathway.

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNß production in Toll-like receptor 4 (Tlr4)-deficient BMDCs, whereas induction of these cytokines was not compromised in Tlr3/7/9 deficient BMDCs. This suggests that TLR4 might be the functional target of BEA on BMDCs. Consistently, in luciferase reporter assays BEA stimulation significantly promotes NF-κB activation in mTLR4/CD14/MD2 overexpressing but not control HEK-293 cells. RNA-sequencing analyses further confirmed that BEA induces transcriptional changes associated with the TLR4 signaling pathway. Together, these results identify TLR4 as a cellular BEA sensor and define BEA as a potent activator of BMDCs, implying that this compound can be exploited as a promising candidate structure for vaccine adjuvants or cancer immunotherapies.

Minimally invasive separation surgery for the treatment of spinal metastases with small incision and freehand pedicle screw fixation: the surgical learning curve.

BACKGROUND: Minimally invasive separation surgery (MISS) is a safe and effective surgical technique, the current optimal treatment for spinal metastases. However, the learning curve for this technique has not been analyzed. This study aimed to define and analyze the surgical learning curve of MISS for the treatment of spinal metastases with small incision and freehand pedicle screw fixation. METHODS: A continuous series of 62 patients with spinal metastases who underwent MISS were included. Each patient's operative data were accurately counted. The improvement of the patients' neurological function was followed up after surgery to evaluate the surgical treatment effect. Logarithmic curve-fit regression was used to analyze the surgical learning curve of MISS. The number of cases needed to achieve proficiency was analyzed. Based on this cut-off point, this series of cases was divided into the early phase and later phase groups. The influence of the time sequence of MISS on surgical data and surgical efficacy was analyzed. RESULTS: The operative time decreased gradually with the number of surgical cases increasing and stabilized after the 20th patient. There was no statistical difference in demographic characteristics and preoperative characteristics between the two groups. The mean operative time in the later phase group was about 39 min shorter than that in the early phase group (mean 227.95 vs. 189.02 min, P = 0.027). However, it did not affect other operative data or the surgical treatment effect. CONCLUSION: The learning curve of MISS for spinal metastases is not steep. With the increase of surgeons' experience, the operative time drops rapidly and stabilizes within a certain range. MISS can be safely and effectively performed at the beginning of a surgeon's caree.

BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment.

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease. SIGNIFICANCE: The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.

Slow viral propagation during initial phase of infection leads to viral persistence in mice.

Immune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

Treatment of Large and Complicated Scalp Defects with Free Flap Transfer.

BACKGROUND: Large scalp defects, especially those complicated by calvarial defects, titanium mesh exposure, or cerebrospinal fluid (CSF) leak, pose a challenge for the neurosurgeon and plastic surgeon. Here, we describe our experience of reconstructing the complex scalp defect with free flap transfer. METHODS: From October 2012 to September 2017, 8 patients underwent free flap transfer for the reconstruction of the scalp or complicated scalp and calvarial defects. Five patients presented with scalp tumor and the other 3 patients with scalp necrosis or ulceration (2 patients with titanium plate exposure). Seven anterolateral thigh flaps and one radial forearm flap were harvested and employed. The clinical data, including defect characteristics, flap type, complications, and outcomes, were recorded and analyzed. RESULTS: Five patients were pathologically diagnosed with malignant tumor, and 3 of them were given further radiotherapy. For the 2 patients with exposure of titanium plate, no titanium plate was removed. For the patient with scalp necrosis after decompressive craniectomy accompanied by CSF leakage, the CSF leak was stopped after reconstruction. The size of the flaps ranged from 3 to 14 cm in width and 4 to 18 cm in length. No flap failure occurred in these cases. From follow-up to the present, no ulceration or necrosis occurred. CONCLUSIONS: Free flap transfer is an ideal method for the reconstruction of large, complicated scalp defects with a one-stage operation. The anterolateral thigh flap is favored because of its durability, adjustability, water tightness, and infection prevention.

Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.

Dead Cells Induce Innate Anergy via Mertk after Acute Viral Infection.

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor ß (TGF-ß). Lack of Mertk in Mertk-/- mice prevents induction of IL-10 and TGF-ß, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV.

Modified cannulated screw fixation in the treatment of Pauwels type III femoral neck fractures: A biomechanical study.

BACKGROUND: Achieving satisfactory internal fixation for patients with Pauwels type III femoral neck fractures has become a critical problem. The purpose of this study was to compare a common standard internal reduction and fixation method for femoral neck fractures to the modified fixation methods. METHODS: A computed tomography scan of the femur was performed to make a Three-dimensional (3D) model, and a fracture line was simulated in the femoral neck. 3Dfinite element analysis was carried out for different insertion methods of cannulated tension screws. Six healthy femur specimens were harvested from three formalin-fixed cadavers, and Pauwels type III femoral neck fracture was artificially created in bilateral femurs. The right side was treated with the inverted triangle construct method and the left side by the modified screw fixation method. After fixation, uniaxial compression and maximum load experiments on the bilateral femoral necks were carried out using the non-contact full-field dynamic strain measurement system (VIC-3D) on a pressure testing machine. FINDINGS: Both 3D finite element analysis and biomechanical study showed that the modified screw fixation method(group D) provided better anti-shearing and anti-rotation properties for Pauwels type III femoral neck fractures, and offered better interfragmentary compression. Therefore, this modified screw fixation method can offer patients a better option for treatment of Pauwels type III femoral neck fractures. INTERPRETATION: Changing the placement of the anterosuperior screw in the inverted triangle construct as perpendicular to the fracture line has the advantages in anti-shearing, anti-rotation and increasing interfragmentary compression.

Laminin-modified gellan gum hydrogels loaded with the nerve growth factor to enhance the proliferation and differentiation of neuronal stem cells.

The reconstruction of peripheral nerves has lately received great attention as many patients suffer from peripheral nerve injury every year around the world. However, the damage to human nerve cells has different degrees of irreversibility due to a slow growth speed and low adhesion with the surrounding tissues. In an effort to overcome this challenge, we applied novel laminin (LN)-modified thiolated gellan gum (TGG) and loaded the nerve growth factor (NGF) as a tissue engineering scaffold for facilitating neuronal stem cell proliferation via a synergy effect for the ERK-MAPK pathway. TGG was characterized by 1H NMR spectroscopy and scanning electron microscopy, and its rheological behavior was also studied. The NGF release curve fitted the Korsmeyer-Peppas model and belonged to a Fickian diffusion-controlled release mechanism. The neuronal stem cells from newborn SD rats could adhere tightly and proliferate at a relatively rapid speed, showing excellent biocompatibility and the ability to promote growth in the modified TGG. LN and NGF could decrease the apoptosis effects of neuronal stem cells, as shown via the flow cytometry results. In a three-dimensional culture environment, LN and NGF could facilitate neuronal stem cells to differentiate into neurons, as proved by immunofluorescence, q-PCR, and western blot analyses. Therefore, the rational design of the TGG gel loaded with NGF has promising applications in the reconstruction of peripheral nerves.

Biomechanical effect of anterior talofibular ligament injury in Weber B lateral malleolus fractures after lateral plate fixation: A finite element analysis.

OBJECTIVE: To determine the biomechanical effect of anterior talofibular ligament injury in Weber B lateral malleolus fractures after lateral plate fixation. METHOD: A three-dimensional model was established based on CT images from a healthy volunteer. The simulation of lateral malleolus fracture, and the modeling and assembly of plate were completed by referring to characteristics of Weber B lateral malleolus fractures, as well as the technical characteristics of open reduction and internal fixation of lateral plate. Operating conditions were set up for groups A-D. The proximal end of the model was restrained in all four groups, 200N of upward force and 100N of backward force were applied at anterior of talus head in order to simulate the dorsiflexion of ankle joint. Biomechanical differences of the lateral plate were observed under various conditions of different ligament ruptures. RESULTS: The maximum stress value of group A was the smallest, approximately 78.47N, while that of group C was the largest, approximately 238.83N. The maximum stress value of group B was about 91.69N; and that of group D was about 184.08N. Importantly, location of the maximum stress in group D (CUT ATaF) was displaced from the posterior edge to the anterior edge of the plate, which was different from those of the other three groups. CONCLUSIONS: The anterior talofibular ligament injury may be a major contributing factor to the stress of lateral plate fixation following Weber B lateral malleolus fracture. It should be considered as an essential risk factor for evaluation of the stability in these fractures.

Progranulin prevents regulatory NK cell cytotoxicity against antiviral T cells.

`NK cell-mediated regulation of antigen-specific T cells can contribute to and exacerbate chronic viral infection, but the protective mechanisms against NK cell-mediated attack on T cell immunity are poorly understood. Here, we show that progranulin (PGRN) can reduce NK cell cytotoxicity through reduction of NK cell expansion, granzyme B transcription, and NK cell-mediated lysis of target cells. Following infection with the lymphocytic choriomeningitis virus (LCMV), PGRN levels increased - a phenomenon dependent on the presence of macrophages and type I IFN signaling. Absence of PGRN in mice (Grn-/-) resulted in enhanced NK cell activity, increased NK cell-mediated killing of antiviral T cells, reduced antiviral T cell immunity, and increased viral burden, culminating in increased liver immunopathology. Depletion of NK cells restored antiviral immunity and alleviated pathology during infection in Grn-/- mice. In turn, PGRN treatment improved antiviral T cell immunity. Taken together, we identified PGRN as a critical factor capable of reducing NK cell-mediated attack of antiviral T cells.

CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.

seq-ImmuCC: Cell-Centric View of Tissue Transcriptome Measuring Cellular Compositions of Immune Microenvironment From Mouse RNA-Seq Data.

The RNA sequencing approach has been broadly used to provide gene-, pathway-, and network-centric analyses for various cell and tissue samples. However, thus far, rich cellular information carried in tissue samples has not been thoroughly characterized from RNA-Seq data. Therefore, it would expand our horizons to better understand the biological processes of the body by incorporating a cell-centric view of tissue transcriptome. Here, a computational model named seq-ImmuCC was developed to infer the relative proportions of 10 major immune cells in mouse tissues from RNA-Seq data. The performance of seq-ImmuCC was evaluated among multiple computational algorithms, transcriptional platforms, and simulated and experimental datasets. The test results showed its stable performance and superb consistency with experimental observations under different conditions. With seq-ImmuCC, we generated the comprehensive landscape of immune cell compositions in 27 normal mouse tissues and extracted the distinct signatures of immune cell proportion among various tissue types. Furthermore, we quantitatively characterized and compared 18 different types of mouse tumor tissues of distinct cell origins with their immune cell compositions, which provided a comprehensive and informative measurement for the immune microenvironment inside tumor tissues. The online server of seq-ImmuCC are freely available at http://wap-lab.org:3200/immune/.

MiR-34a promotes DCs development and inhibits their function on T cell activation by targeting WNT1.

MicroRNAs serve important functions in numerous biological processes. Whether microRNAs also act on dendritic cell (DC) differentiation and function remains unclear. In this study, both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were increased in miR-34a overexpressing bone marrow chimeric and transgenic (TG) mice. Further experiments showed that miR-34a promoted preDC differentiated into cDCs and pDCs without affecting the proliferation and apoptosis of DCs. Luciferase report assay and Western blot experiments demonstrated that WNT1 is the direct target of miR-34a in DCs. Interestingly, miR-34a overexpressing cDCs also produced a large amount of IL-17a and suppressed T cell activation because of the inhibition of TCF1 expression, thus increasing RORγT expression. Taken together, miR-34a promotes preDC to differentiate into cDCs and pDCs, as well as inhibits the function of cDCs on the activation of CD4+ T cells by producing IL-17a.

A human programmed death-ligand 1-expressing mouse tumor model for evaluating the therapeutic efficacy of anti-human PD-L1 antibodies.

Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8+T cells inside tumor tissues. Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.

Inference of immune cell composition on the expression profiles of mouse tissue.

Mice are some of the widely used experimental animal models for studying human diseases. Defining the compositions of immune cell populations in various tissues from experimental mouse models is critical to understanding the involvement of immune responses in various physiological and patho-physiological conditions. However, non-lymphoid tissues are normally composed of vast and diverse cellular components, which make it difficult to quantify the relative proportions of immune cell types. Here we report the development of a computational algorithm, ImmuCC, to infer the relative compositions of 25 immune cell types in mouse tissues using microarray-based mRNA expression data. The ImmuCC algorithm showed good performance and robustness in many simulated datasets. Remarkable concordances were observed when ImmuCC was used on three public datasets, one including enriched immune cells, one with normal single positive T cells, and one with leukemia cell samples. To validate the performance of ImmuCC objectively, thorough cross-comparison of ImmuCC predicted compositions and flow cytometry results was done with in-house generated datasets collected from four distinct mouse lymphoid tissues and three different types of tumor tissues. The good correlation and biologically meaningful results demonstrate the broad utility of ImmuCC for assessing immune cell composition in diverse mouse tissues under various conditions.