Tumor Leukocyte Infiltration Research Articles

Immunological profiles of the breast cancer microenvironment represented by tumor-infiltrating lymphocytes and PD-L1 expression

Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) are established prognostic and predictive biomarkers for certain breast cancer subsets. However, their association with the immune response complexity is not fully understood. Therefore, we analyzed the association between the immune cell fractions in breast cancer tissues and histologically assessed TIL (hTIL) and PD-L1 (hPD-L1). Forty-five tumor and eighteen blood samples were collected from patients with breast cancer. Total leukocyte counts, frequency of 11 immune cell populations, and PD-L1 expression in each cell fraction were evaluated by flow cytometry. TILs and PD-L1 were assessed by hematoxylin and eosin staining and immunohistochemistry, respectively. A higher hTIL score showed association with increased leukocyte infiltration, higher CD4+ and CD8+ T cell proportions, and lower natural killer and natural killer T cell proportions. PD-L1 was highly expressed in nonclassical monocytes, monocyte/macrophages, myeloid-derived suppressor cells, myeloid dendritic cells, dendritic cells, and other lineages in tumors. hPD-L1 positivity reflected PD-L1 expression accurately in these fractions, as well as increased leukocyte infiltration in tumors. These results indicate that hTILs reflect differences in the immune responses in the tumor microenvironment, and certain immune cell fractions are favorably expressed in the PD-L1 pathway in breast cancer microenvironments.

Breast cancer pathology patterns in Armenia.

e12586 Background: Breast cancer (BC) is the most common malignancy among the women in Armenia (AM). Currently there is a knowledge gap regarding the morphology distribution of the BC in AM. Methods: The data on patients with BC diagnosed in 2015-2016 in the pathology lab “Davidyants Labs” in AM were retrospectively reviewed. Pts with Her2+ results by IHC were excluded from the study, due to unavailability to perform FISH or CISH analyses. Overall 361 pathology reports were evaluated. Results: The median age was 54 years; range [19-82]. Histopathological subtypes were defined for 305 pts, from which lobular carcinoma 57.4% of cases (175/305), ductal carcinoma 26.9% (82/305), mucinous carcinoma 2.6% (8/305), mixed type carcinoma (lobular and ductal) 2.6% (8/305), DCIS 2% (n = 6/305), non specified carcinoma 2% (6/305), medullary carcinoma 1% (n = 3/305) and others 5.6% (17/305). Within the cohort 8.5% (23/270) were grade 1, 65.9% grade 2 (178/270); 25.6% grade 3 (n = 69/270). Vascular or lymphatic invasion was present in 59.5% (50/84) and 64.7% (55/85), respectively. Staging distribution, based on pT pN data for 92 pts who went to primary surgery, was: 0 stage 7.6% (7/92), I stage 22.8% (21/92), II stage 41.3% (38/92), III stage 28.3% (26/92). Staging distribution based on ypT ypN data for 27 pts who went to surgery after neoadjuvant chemo was 0 stage 25.9% (7/27), I stage 18.5% (5/27), II stage 29.6% (8/27), III stage 25.9% (7/27). ER and PR were defined for 244 patients. ER positive 89.8% (219/244) of cases, PR pos. 73% (178/244), ER/PR pos. 72.5% (177/244) cases. Her receptor was defined for 237 patients. Her3+ 16.9% (40/237); Her2+ 12.7% (30/237); Her1+ 38% (90/237); Her0 32.5% (n = 77/237). We could not evaluate Her2+ status by FISH or CISH, so these results were excluded from the analysis. Ki67 was low (≤20) in 42.1% (101/240) of cases and high ( > 20) in 57.9% (139/240). Within the group Luminal A type was 41.4% (84/203); Luminal B 32.5% (66/203); Her positive 19.7% (40/203) and triple negative 6.4% (13/203). p53 and perineural invasion (Pn) was present in 32% (16/50) and 52% (26/50), respectively. Tumor leukocyte infiltration was determined for 16 patients. Leukocyte infiltration was positive in 43.7% (7/16) cases, negative in 25% (4/16) cases, minimal in 31.3% cases (5/16). Conclusions: BC in Armenian women presents with different epidemiological characteristics in comparison with other ethnicities. Lobular type BC is the most frequent type among Armenian women, however, differential diagnosis between lobular/ductal carcinomas was done without IHC (E-Cadherin), which rises the need for further studies on that regard.

Smad7 Ameliorates TGF-β–Mediated Skin Inflammation and Associated Wound Healing Defects but Not Susceptibility to Experimental Skin Carcinogenesis

We assessed the roles of Smad7 in skin inflammation and wound healing using genetic and pharmacological approaches. In K5.TGFβ1/K5.Smad7 bigenic (double transgenic) mice, Smad7 transgene expression reversed transforming growth factor (TGF)-β1 transgene-induced inflammation, fibrosis, and subsequent epidermal hyperplasia and molecularly abolished TGF-β and NF-κB activation. Next, we produced recombinant human Smad7 protein with a Tat-tag (Tat-Smad7) that rapidly enters cells. Subcutaneous injection of Tat-Smad7 attenuated infiltration of F4/80+ and CD11b+ leukocytes and α-smooth muscle actin+ fibroblasts before attenuating epidermal hyperplasia in K5.TGFβ1 skin. Furthermore, topically applied Tat-Smad7 on K5.TGFβ1 skin wounds accelerated wound closure, with improved re-epithelialization and reductions in inflammation and fibrotic response. A short treatment with Tat-Smad7 was also sufficient to reduce TGF-β and NF-κB signaling in K5.TGFβ1 skin and wounds. Relevant to the clinic, we found that human diabetic wounds had elevated TGF-β and NF-κB signaling compared with normal skin. To assess the oncogenic risk of a potential Smad7-based therapy, we exposed K5.Smad7 skin to chemical carcinogenesis and found reduced myeloid leukocyte infiltration in tumors but not accelerated carcinogenesis compared with wild-type littermates. Our study suggests the feasibility of using exogenous Smad7 below an oncogenic level to alleviate skin inflammation and wound healing defects associated with excessive activation of TGF-β and NF-κB.

Evaluation of Neutrophils Immunophenotype in the Microenvironment of Malignant Pleural Effusions

Abstract The lung cancer is often associated with the development of pleural effusion. Neutrophils are the most numerous population of immune system cells which are an essential component of tumor leukocyte infiltration. These cells are engaged in the development and maintenance of the inflammation. It is indicated that neutrophils support the development of cancer. The aim of the study was the evaluation of neutrophils, regarding their presence and activity in pleural effusions. This was achieved by assessing of molecular structures, which are used by neutrophils in chemotaxis and phagocytosis. 60 pleural effusions and 34 peripheral blood samples received from patients and 15 peripheral blood samples from the control group were analyzed. Expression of CD11a, CD11b, CD11c, CD18 and CD62L molecules with use flow cytometry was evaluated. The concentration of the neutrophil elastase in pleural effusions were measured with use ELISA test. The number of neutrophils in the peripheral blood of patients with pleural effusion was lower than that observed in the control group. Neutrophils present in pleural effusions were characterized by an increased ability to chemotaxis and secrete significant amounts of neutrophil elastase. Neutrophils recruited into the pleura during the formation of the effusion are an essential element of the developing inflammatory reaction in this environment. The presence of neutrophils in pleural effusion may promote its further formation and support the development of cancer.

CD204-Expressing Tumor-Associated Macrophages Are Associated With Malignant, High-Grade, and Hormone Receptor-Negative Canine Mammary Gland Tumors.

Tumor-associated macrophages (TAMs) are an important component of leukocyte infiltration in tumors. TAMs can be classified into M1 and M2 phenotypes. In the present study, the expression of CD204, an M2-polarized macrophage receptor, was investigated by immunohistochemistry in the area surrounding TAMs in 101 cases of canine mammary gland tumor (CMT). We examined the relationship between M2-polarized TAMs and malignancy, histological subtype, histological grade, molecular subtype, hormone receptor (HR) status, and clinical obesity indices. The mean number of CD204-positive macrophages was significantly higher in malignant CMTs than in benign CMTs ( P = .000). The number of CD204-positive macrophages differed significantly between histological grades ( P = .000) and were significantly higher in grade III than in grades I and II. Moreover, the mean number of CD204-positive macrophages was significantly higher in HR-negative malignant CMTs than in HR-positive malignant CMTs ( P = .035) and in malignant CMTs with lymphatic invasion compared to malignant CMTs without lymphatic invasion ( P = .000). These findings suggest that CD204-positive macrophages might affect the development and behavior of CMTs and highlight the potential of CD204 as a prognostic factor.

Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-\u03baB activation

BackgroundWe previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces.MethodsIn this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting.ResultsDoxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1β, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80+ macrophages and CD31+ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate.ConclusionThese results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

Chemo-immunotherapy induces tumor regression in a mouse model of spontaneous mammary carcinogenesis.

Tumor-specific immune tolerance represents an obstacle for the development of effective anti-tumor immune responses through cancer vaccines. We here evaluated the efficacy of chemo-immunotherapy in breaking tumor-specific immune tolerance in an almost incurable mouse model of spontaneous carcinogenesis.Transgenic HER-2/neu mice bearing large mammary tumors received the adoptive transfer of splenocytes and serum isolated from immune donors, with or without pre-conditioning with cyclophosphamide. Treatment efficacy was assessed by monitoring tumor growth by manual inspection and by magnetic resonance imaging. The same chemo-immunotherapy protocol was tested on tumor-free HER-2/neu mice, to evaluate the effects on tumor emergence.Our data show that chemo-immunotherapy hampered carcinogenesis and caused the regression of large mammary tumor lesions in tumor-bearing HER-2/neu mice. The complete eradication of a significant number of tumor lesions occurred only in mice receiving cyclophosphamide shortly before immunotherapy, and was associated with increased serum anti HER-2/p185 antibodies and tumor leukocyte infiltration. The same protocol significantly delayed the appearance of mammary tumors when administered to tumor-free HER-2/neu mice, indicating that this chemo-immunotherapy approach acted through the elicitation of an effective anti-tumor immune response. Overall, our data support the immune-modulatory role of chemotherapy in overcoming cancer immune tolerance when administered at lymphodepleting non-myeloablative doses shortly before transfer of antigen-specific immune cells and immunoglobulins. These findings open new perspectives on combining immune-modulatory chemotherapy and immunotherapy to overcome immune tolerance in cancer patients.

Abstract LB-081: In situ vaccination of low-grade lymphoma with intratumoral Flt3L and poly-ICLC with low-dose radiotherapy

Abstract Background: Lymphomas are the 5th most common cancer in the United States, 40% of these are cases are indolent non-Hodgkin's lymphoma (iNHL) and are incurable with standard therapy. A previous trial of in situ vaccination in iNHLs, in which intratumoral CpG, the TLR9 agonist, was combined with low dose radiation to induce a systemic immune response against tumor. In this trial induction of tumor-specific CD8 T cell responses and durable clinical remissions of patients’ untreated sites of disease was seen in some patients. One limitation in this previous trial may have been the scarcity of intratumoral dendritic cells (DC) and the suppressive tumor microenvironment. DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. In this new iteration of in situ vaccine, Flt3L is added as a priming step to increase the presence of intratumoral DCs ahead of vaccination. Fms-like tyrosin kinase 3 ligand (Flt3L) induces tumor leukocyte infiltration and regression in lymphoma tumors in pre-clinical trials, and CDX-301- a formulation of Flt3L - was shown to mobilize BDCA-1 and BDCA-3 myeloid DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DCs. While pDCs are high expressors of TLR9, responsive to CpG, myeloid dendritic cells express a wider array of TLRs, including high levels of TLR3. Methods: This Phase I/II trial tests the hypothesis that this novel in situ vaccination will induce clinical remissions at distant (untreated) tumor sites in two cohorts of patients with either previously untreated or relapsed/refractory iNHL (n = 15 per group). Intratumoral CDX-301 25ug/kg is injected into a palpable lymph node for 9 days, followed 2Gy local radiotherapy on day 9 and 10 to the target lymph node. On day 10, following radiation therapy, intratumoral poly-ICLC 2mg is injected to activate local DCs. Poly-ICLC 2mg is then injected on day 14, day 17 (1 week after initial dose) and weekly thereafter for a total of 9 treatments over 8 weeks. Response is assessed with CT scans every three months as per the Revised Response Criteria for Malignant Lymphoma, also known as the Cheson criteria, and leukemic phase of lymphoma monitored by peripheral blood flow cytometry. The trial started accrual in January 2014 and is ongoing, 11 have been enrolled including 8 untreated patients and 3 with refractory disease. To perform correlative studies during the clinical trial we perform excisional biopsy of a distal lymph node before starting trial to assess baseline immunomodulatory or immunosuppressive characteristics of a patients tumor microenvironment. Fine needle aspirate (FNA) of the target tumor (site of Flt3L, Radiation and poly-ICLC administration) treatment at weeks 0, 2, 4 and 6, after which target tumor is significantly smaller and biopsy not typically possible. Blood is collected for immune monitoring at weeks 0, 2, 4, 6, 8, 12 to assess for transient enrichment of DC populations, and monitoring for tumor-specific effector T cell populations. Citation Format: Thomas U. Marron, Nina Bhardwaj, Linda Hammerich, Seunghee Kim-Schulze, Tibor Keler, Tom Davis, Elizabeth Crowley, Andres M. Salazar, Joshua Brody. In situ vaccination of low-grade lymphoma with intratumoral Flt3L and poly-ICLC with low-dose radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-081.

Abstract IA03: Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma

Abstract Background: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients' non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. Methods: Flt3L – the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of: intratumoral Flt3L administration to increase DC within the tumorlow-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, andintratumoral poly-ICLC administration to activate tumor antigen-loaded DC. Results: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Conclusions: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. Research supported by: Damon Runyon Cancer Research Foundation Clinical Investigator Award Lymphoma Research Foundation Clinical Research Mentorship Program Citation Format: Thomas Marron, Nina Bhardwaj, Elizabeth Crowley, Tibor Keler, Thomas Davis, Andres Salazar, Joshua Brody. Turning a tumor into a vaccine factory: In situ vaccination for low-grade lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA03.

Turning a Tumor into a Vaccine Factory: In Situ Vaccination for Low-Grade Lymphoma

BACKGROUND: Lymphomas are the 5th most common cancer in the U.S. and most are incurable with standard therapy. Previously, we completed three trials of ‘in situ vaccination’ - combining low-dose radiotherapy (XRT) with intratumoral administration of TLR9 agonist (CpG). We demonstrated induction of anti-tumor CD8 T cell responses and clinical remissions of patients’ non-irradiated sites of disease, lasting up to 4+ years. One limitation may have been the paucity of intratumoral dendritic cells (DC). DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells.METHODS: Flt3L– the predominant DC differentiation factor– induces tumor leukocyte infiltration and regression of lymphoma tumors pre-clinically and a new formulation of this cytokine -CDX-301- was shown to mobilize BDCA-1 and BDCA-3 DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DC (the CpG-responsive DC subset). We initiated a phase I/II study of a new iteration of the in situ vaccine, adding Flt3L-priming and replacing the prior TLR9 agonist with the TLR3 agonist poly-ICLC (Fig 1A). The vaccine consists of:-intratumoral Flt3L administration to increase DC within the tumor-low-dose XRT to induce immunogenic tumor cell death and release tumor-associated antigens, and-intratumoral poly-ICLC administration to activate tumor antigen-loaded DC.RESULTS: Six patients have been enrolled, two patients have completed therapy. Treated patients had 2-200-fold increases in BDCA1 and BDCA3 intratumoral DC after Flt3L administration and marked DC activation after XRT and poly-ICLC. Both treated patients have had partial remissions of untreated sites per Cheson criteria, persisting or improving for >6 months after vaccination. These include regressions of bulky lymph nodes (Fig 1B), as well as peripheral blood (Fig1C) and bone marrow disease. A patient with significant peripheral blood tumor burden experienced >10-fold decrease in malignant B cells with concurrent increase in non-tumor B cells, suggesting a degree of cell specificity in the tumor-killing mechanism. Adverse effects have been mild.CONCLUSIONS: Preliminary results suggest that the Flt3L-primed in situ vaccine is feasible, safe and immunologically and clinically effective, warranting further study. [Display omitted] DisclosuresOff Label Use: CDX-301 - the purpose of which is to mobilize dendritic cells to an injected tumor site.. Crowley:Celldex Therapeutics Inc: Employment. Keler:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics Inc: Employment.

Macrophage-secreted IL-8 induces epithelial-mesenchymal transition in hepatocellular carcinoma cells by activating the JAK2/STAT3/Snail pathway.

Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.

In vitro and clinical study of association between macrophage and epithelial-mesenchymal transition in hepatocellular carcinoma cells.

e15149 Background: Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mecha...

The Inflammatory Microenvironment in Hepatocellular Carcinoma: A Pivotal Role for Tumor-Associated Macrophages

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human cancers worldwide. HCC is an example of inflammation-related cancer and represents a paradigm of the relation occurring between tumor microenvironment and tumor development. Tumor-associated macrophages (TAMs) are a major component of leukocyte infiltrate of tumors and play a pivotal role in tumor progression of inflammation-related cancer, including HCC. Several studies indicate that, in the tumor microenvironment, TAMs acquire an M2-polarized phenotype and promote angiogenesis, metastasis, and suppression of adaptive immunity through the expression of cytokines, chemokines, growth factors, and matrix metalloproteases. Indeed, an established M2 macrophage population has been associated with poor prognosis in HCC. The molecular links that connect cancer cells and TAMs are not completely known, but recent studies have demonstrated that NF-κB, STAT-3, and HIF-1 signaling pathways play key roles in this crosstalk. In this paper, we discuss the current knowledge about the role of TAMs in HCC development, highlighting the role of TAM-derived cytokines, chemokines, and growth factors in the initiation and progression of liver cancer and outlining the signaling pathways involved in the interplay between cancer cells and TAMs.

Dynamic Education of Macrophages in Different Areas of Human Tumors

Human tumor tissues can often be anatomically classified into areas of cancer nest, invading edge, and peritumoral stroma, each with distinct compositions and functional properties. Macrophages (Mφ) constitute a major component of the leukocyte infiltrate in tumors. These cells are derived from circulating monocytes, and in response to environmental signals, they exhibit distinct phenotypes with diverse functions. Soluble factors derived from cancer cells can alter the normal developmental process of Mφ that is intended to trigger transient early activation of monocytes in the peritumoral region, which in turn induces formation of suppressive Mφ in cancer nests. The activated monocytes in the peritumoral region attenuated the T-cell response by expressing B7-H1, and were superior to the suppressive tumor Mφ in inducing Th17 expansion, and thus repurpose the inflammatory response away from anti-tumor immunity (the sword) and towards tissue remodeling and proangiogenic pathways (a plowshare). In contrast, the suppressive Mφ can induce the production of Tregs in cancer nest. Accordingly, angiogenesis was most active at the invading edge, which was situated close to the peritumoral stroma with activated Mφ and the density of these activated monocytes is selectively associated with vascular invasion and metastasis in patients with hepatocellular carcinoma. These data reveal an intriguing mechanism in which human Th17 cells are generated and regulated by a fine-tuned collaborative action between different types of immune cells in distinct tumor microenvironments. These results give important new insights into the distinct role of macrophages in human tumor progression which would be helpful for the rational design of novel immune-based anticancer therapies.

High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection

Macrophages constitute a major component of the leukocyte infiltrate of tumors and perform distinct roles in different tumor microenvironments. This study attempted to investigate the prognostic values of tumor-infiltrating macrophages in patients with hepatocellular carcinoma after resection, paying particular attention to their tissue microlocalization. The CD68(+) macrophages were assessed by immunohistochemistry in tissues from 137 patients with hepatocellular carcinoma. Prognostic value of intratumoral, marginal, and peritumoral macrophage densities was evaluated by Kaplan-Meier analysis and Cox regression. Both intratumoral and marginal macrophage densities were associated inversely with overall survival (P = .034 and .004, respectively) and disease-free survival (P = .006 and .008, respectively). In contrast, peritumoral macrophage density was associated with neither overall survival nor disease-free survival. Intratumoral macrophage density emerged as an independent prognosticator of overall survival (hazard ratio = 1.721, P = .049) and disease-free survival (hazard ratio = 2.165, P = .007). Marginal macrophage density, but not intratumoral macrophage density, was associated with vascular invasion, tumor multiplicity, and fibrous capsule formation. Our results demonstrate that high macrophage infiltration predicts poor prognosis in patients with hepatocellular carcinoma. These results, together with our previous report showing the distinct activation patterns of macrophages in different areas of tumor tissue, implies that macrophages in those areas may use different strategies to promote the tumor progression.

Effect of Tumor Microenvironment Modulation on the Efficacy of Oncolytic Virus Therapy

The tumor microenvironment is being increasingly recognized as an important determinant of tumor progression as well as of therapeutic response. We investigated oncolytic virus (OV) therapy-induced changes in tumor blood vessels and the impact of modulating tumor vasculature on the efficacy of oncolytic virus therapy. Rat glioma cells (D74/HveC) were implanted intracranially in immune-competent rats. Seven days later, the rats (groups of 3-7 rats) were treated with oncolytic virus (hrR3), and, 3 days later, brains were harvested for evaluation. Some rats were treated with angiostatic cRGD peptide 4 days before oncolytic virus treatment. Some rats were treated with cyclophosphamide (CPA), an immunosuppressant, 2 days before oncolytic virus treatment. Changes in tumor vascular perfusion were evaluated by magnetic resonance imaging of live rats and by fluorescence microscopy of tumor sections from rats perfused with Texas red-conjugated lectin immediately before euthanasia. Leukocyte infiltration in tumors was evaluated by anti-CD45 immunohistochemistry, and the presence of oncolytic virus in tumors was evaluated by viral titration. Changes in cytokine gene expression in tumors were measured by quantitative real-time polymerase chain reaction-based microarrays. Survival was analyzed by the Kaplan-Meier method. All statistical tests were two-sided. Oncolytic virus treatment of experimental rat gliomas increased tumor vascular permeability, host leukocyte infiltration into tumors, and intratumoral expression of inflammatory cytokine genes, including interferon gamma (IFN-gamma). The increase in vascular permeability was suppressed in rats pretreated with cyclophosphamide. Compared with rats treated with hrR3 alone, rats pretreated with a single dose of cRGD peptide before hrR3 treatment had reduced tumor vascular permeability, leukocyte infiltration, and IFN-gamma protein levels (mean IFN-gamma level for hrR3 versus hrR3 + cRGD = 203 versus 65.6 microg/mg, difference = 137 microg/mg, 95% confidence interval = 72.7 to 202.9 microg/mg, P = .006); increased viral titers in tumor tissue; and longer median survival (21 days versus 17 days, P<.001). A single dose of angiostatic cRGD peptide treatment before oncolytic virus treatment enhanced the antitumor efficacy of oncolytic virus.

Proliferating Endothelial Cells and Leukocyte Infiltration as Prognostic Markers in Colorectal Cancer

Leukocyte infiltration in tumors is dependent on angiogenic potential. In this study we aimed to retrospectively investigate the angiogenic potential in archival colorectal cancer (CRC) tissues and its relationship to amount and composition of the inflammatory infiltrate. In tumor tissues of 117 CRC patients with a 12-year follow-up, microvessel density (MVD) and proliferating endothelial cells (ECs) were assessed by CD31/CD34 double staining with the proliferation marker Ki-67. Leukocyte infiltration was determined by using CD45, CD3, CD8, CD16, CD20, and CD68 antibodies in peritumoral, tumor stroma, and intratumoral areas. Proliferating ECs, but not MVD, are correlated to Dukes' stage and survival in CRC (P < .05). This parameter correlated significantly with the expression of vascular endothelial growth factor (r = 0.82; P < .012). The number of inflammatory cells in the tumor stroma and cells infiltrated into the tumor cell nests, but not of peritumoral leukocytes, predicted patient survival. This was most obvious for T lymphocytes (CD3; P < .05) and polymorphonuclear cells (CD16; P < .04). We found a significant relationship between angiogenesis parameters and infiltrated leukocytes (r = -0.70; P < .02). Combination of high numbers of infiltrated leukocytes and low amounts of proliferating ECs demonstrated to be an improved prognostic value compared with either parameter alone (P < .006). We found a correlation between the intrinsic tumor parameters of ongoing angiogenesis and leukocyte infiltration with prognosis and survival in CRC. These findings have a potential impact on therapeutic applications for both antiangiogenesis as well as immunotherapy.

Enumeration of leukocyte infiltration in solid tumors by confocal laser scanning microscopy

BackgroundLeukocytes commonly infiltrate solid tumors, and have been implicated in the mechanism of spontaneous regression in some cancers. Conventional techniques for the quantitative estimation of leukocyte infiltrates in tumors rely on light microscopy of immunostained thin tissue sections, in which an arbitrary assessment (based on low, medium or high levels of infiltration) of antigen density is made by the pathologist. These estimates are relatively subjective and often require the opinion of a second pathologist. In addition, since thin tissue sections are cut, no data regarding the three-dimensional distribution of antigen can be obtained.ResultsTo overcome these problems, we have designed a method to enumerate leukocyte infiltration into tumors, using confocal laser scanning microscopy of fluorescently immunostained leukocytes in thick tissue sections. Using image analysis software, a threshold was applied to eliminate unstained tissue and residual noise. The total antigen volume in the scanned tissue was calculated and divided by the mean cell volume (calculated by "seeding" ten individual cells) to obtain the cell count. Using this method, we compared the calculated leukocyte counts with those obtained manually by ten laboratory personnel. There was no significant difference (P > 0.05) between the cell counts obtained by either method.We then compared leukocyte infiltration into seven tumors and matched non-malignant tissue obtained from the periphery of the resected tissue. There was a significant increase in the infiltration of all leukocyte subsets into the tumors compared to minimal numbers in the non-malignant tissue.ConclusionFrom these results we conclude that this method may be of considerable use for the enumeration of cells in tissues. Furthermore, since it can be performed by laboratory technical staff, less time input is required by the pathologist in assessing the degree of leukocyte infiltration into tumors.

Angiogenic Profile of Breast Carcinoma Determines Leukocyte Infiltration

To study the relationship between the angiogenic profile and leukocyte infiltration of tumors, single cell suspensions of archival frozen medullary and ductal breast cancer tissues were analyzed by flow cytometry. The amount of leukocytes and endothelial cells was measured, as well as the expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell fraction. A significantly higher number (3.2-fold) of infiltrating leukocytes was observed in medullary carcinoma. The composition of this infiltrate was similar to that seen in ductal carcinomas. The more intense infiltrate was explained by the approximately 3-fold enhanced endothelial ICAM-1 expression in medullary carcinoma. The angiogenic profile of all tumors was assessed by quantitative real-time reverse transcription-PCR analysis. Vascular endothelial growth factor (VEGF)-C and VEGF-D, but not VEGF-A, basic fibroblast growth factor, placental growth factor, and angiopoietins 1, 2, and 3 showed a relatively higher level of expression in ductal carcinoma than in medullary carcinoma. In vitro, both VEGF-C and VEGF-D were found to decrease endothelial ICAM-1 expression in the presence of basic fibroblast growth factor. These data suggest that in vivo angiogenic stimuli prevent the formation of an effective leukocyte infiltrate in tumors by suppressing endothelial ICAM-1 expression.

Chemokines in the recruitment and shaping of the leukocyte infiltrate of tumors

Leukocytes, and macrophages in particular, are an important component of the stroma of neoplastic tissues. Tumor-associated macrophages (TAM) have the properties of a polarized M2 population and are a key component of inflammatory circuits which promote tumor growth and progression. Chemokines play a key role in the recruitment and positioning of TAM and dendritic cells in tumors and contribute to shaping their functional properties. Chemokine-recruited and positioned tumor infiltrating leukocytes are a key component of inflammatory circuits which promote tumor progression.