Early Stages Of Tumor Development Research Articles

Novel mechanism of macrophage-mediated metastasis revealed in a zebrafish model of tumor development.

Cancer metastasis can occur at early stages of tumor development due to facilitative alterations in the tumor microenvironment. Although imaging techniques have considerably improved our understanding of metastasis, early events remain challenging to study due to the small numbers of malignant cells involved that are often undetectable. Using a novel zebrafish model to investigate this process, we discovered that tumor-associated macrophages (TAM) acted to facilitate metastasis by binding tumor cells and mediating their intravasation. Mechanistic investigations revealed that IL6 and TNFα promoted the ability of macrophages to mediate this step. M2 macrophages were particularly potent when induced by IL4, IL10, and TGFβ. In contrast, IFNγ-lipopolysaccharide-induced M1 macrophages lacked the capability to function in the same way in the model. Confirming these observations, we found that human TAM isolated from primary breast, lung, colorectal, and endometrial cancers exhibited a similar capability in invasion and metastasis. Taken together, our work shows how zebrafish can be used to study how host contributions can facilitate metastasis at its earliest stages, and they reveal a new macrophage-dependent mechanism of metastasis with possible prognostic implications.

Hypoxia-specific ultrasensitive detection of tumours and cancer cells in vivo

Highly sensitive and specific non-invasive molecular imaging methods are particularly desirable for the early detection of cancers. Here we report a near-infrared optical imaging probe highly specific to the hypoxic tumour microenvironment to detect tumour and cancer cells with the sensitivity to a few thousands cancer cells. This oxygen-sensitive, near-infrared emitting and water-soluble phosphorescent macromolecular probe can not only report the hypoxic tumour environment of various cancer models, including metastatic tumours in vivo, but can also detect a small amount of cancer cells before the formation of the tumour based on the increased oxygen consumption during cancer cell proliferation. Thus, the reported hypoxia-sensitive probe may offer an imaging tool for characterizing the tumour microenvironment in vivo, detecting cancer cells at a very early stage of tumour development and lymph node metastasis.

Dormant Cells: The Original Cause of Tumor Recurrence and Metastasis.

Tumor dormancy is one of the stages in tumor development without clinical symptoms. Tumor dormant cells may appear in early stages of tumor development, as well as in micrometastasis and minimal residual disease. The mechanism for the switch of dormant cells between quiescent and proliferative stages is still largely unknown. Potential mechanisms that may account for the transition between dormant tumor cells and proliferative cells include angiogenesis, immune response, cellular factors, and signaling pathways. The clinical and therapeutic importance of dormant cells requires further studies to provide therapeutic strategies for inhibition of metastasis and tumor recurrence.

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

Biological and clinical significance of epigenetic silencing of MARVELD1 gene in lung cancer.

Epigenetic silence in cancer frequently altered signal-transduction pathways during the early stages of tumor development. Recent progress in the field of cancer epigenetics has led to new opportunities for diagnosis and treatment of cancer. We previously demonstrated that novel identified nuclear factor MARVELD1 was widely expressed in human tissues, but down-regulated by promoter methylation in multiple cancers. This study was carried out to determine the biological and clinical significance of MARVELD1 gene silencing in lung cancer. Here, we found the reduced MARVELD1 expression significantly correlated with diagnostic histopathology and malignant degree of lung cancers. DNA hypermethylation and histone deacetylation synergistically inactivated MARVELD1 gene in lung cancer cells. Moreover, MARVELD1 modulated the efficiency of nonsense-mediated mRNA decay (NMD) through interaction with NMD core factor SMG1. The decreased MARVELD1 level in lung cancer reduces NMD efficiency through diminishing the association between NMD complex component UPF1/SMG1 and premature termination codons containing mRNA (PTC-mRNA). The results suggested that MARVELD1 silencing is an appealing diagnostic biomarker for lung cancer and epigenetic silencing of MARVELD1 gene links with the regulatory mechanism of NMD pathway in lung cancer, which may be required for tumorigenesis.

Generation of MHC class I diversity in primary tumors and selection of the malignant phenotype.

Intratumor heterogeneity among cancer cells is promoted by reversible or irreversible genetic alterations and by different microenvironmental factors. There is considerable experimental evidence of the presence of a variety of malignant cell clones with a wide diversity of major histocompatibility class I (MHC-I) expression during early stages of tumor development. This variety of MHC-I phenotypes may define the evolution of a particular tumor. Loss of MHC-I molecules frequently results in immune escape of MHC-negative or -deficient tumor cells from the host T cell-mediated immune response. We review here the results obtained by our group and other researchers in animal models and humans, showing how MHC-I intratumor heterogeneity may affect local oncogenicity and metastatic progression. In particular, we summarize the data obtained in an experimental mouse cancer model of a methylcholanthrene-induced fibrosarcoma (GR9), in which isolated clones with different MHC-I expression patterns demonstrated distinct local tumor growth rates and metastatic capacities. The observed "explosion of diversity" of MHC-I phenotypes in primary tumor clones and the molecular mechanism ("hard"/irreversible or "soft"/reversible) responsible for a given MHC-I alteration might determine not only the metastatic capacity of the cells but also their response to immunotherapy. We also illustrate the generation of further MHC heterogeneity during metastatic colonization and discuss different strategies to favor tumor rejection by counteracting MHC-I loss. Finally, we highlight the role of MHC-I genes in tumor dormancy and cell cycle control.

Dichotomous Roles for Hdac1 Haploinsufficiency in T Cell Acute Lymphoblastic Leukemia (T-ALL)

Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups from histones and non-histone proteins, thereby modifying the structure of chromatin and regulating gene expression. HDACs have frequently been found to be upregulated in solid tumors and in hematological malignancies, leading to clinical trials of HDAC inhibitors for the treatment of various cancers. Oncogenic effects of HDACs are thought to be mediated by the epigenetic silencing of tumor suppressors. However, understanding of the specific gene targets of individual HDACs is lacking. In order to elucidate the function of HDAC1 in a cancer model, we have performed experiments comparing T cell leukemias in hdac1 haploinsufficient zebrafish with leukemias in their wild type siblings.For our initial studies, we tested whether loss of one allele of the hdac1 gene (haploinsufficiency) would reduce the oncogenic effects of c-Myc, a potent oncogene, and protect against leukemia. We generated T cell lymphoblastic leukemia (T-ALL) in both hdac1+/- haploinsufficient and wild type zebrafish siblings by overexpressing murine c-Myc in T cells using a rag2 promoter. We then monitored the hdac1+/- and wild type fish for tumor incidence, latency, growth and overall survival.The tumor incidence rates and mean latencies were not significantly different for hdac1+/- and wild type tumors. However, we found significant differences in primary tumor growth between these genotypes. Tumor progression was significantly faster for hdac1+/- fish (p=0.001), with mean time to stage 3 tumor (>50% of the animal showing evidence of tumor dissemination) of 43.3 ± 10.1 days (mean ± S.D.) compared with 76.3 ± 9.2 days for wild type siblings. Furthermore, survival of hdac1+/- fish was significantly shorter (p<0.01) at 70.3 ± 36.2 days compared with wild type siblings at 129.2 ± 57.8 days.In contrast, progression of transplanted hdac1+/- tumors was slower compared with transplanted wild type tumors. Only 8.3% of transplanted hdac1+/- tumors had extended into the thymus and other organs by 21 days following intra-peritoneal injection into recipients. In contrast, 92% of the wild type tumors had evidence of widespread tumor dissemination by 21 days post-transplant.Several mechanisms were considered as potential contributing factors to these differences between the hdac +/- and wild type leukemias, including cell cycle differences, the effects of different tumor micro-environments, and differences in leukemia-initiating cell (LIC) frequencies. We did not find obvious differences in cell cycle parameters between genotypes. Also, we found no effect from different microenvironments of tumors following transplantation into either hdac1 +/- or wild type recipients. In contrast, we did find significant differences in leukemia-initiating cell frequencies between the genotypes (p<0.0001). The LIC frequency for hdac1 +/- tumors was measured at 1 in 410 compared with a higher LIC frequency of 1 in 30 for wild type tumors.In summary, our data provide support for dual roles of hdac1 in leukemogenesis. In this model, hdac1 has an initial protective role acting as an oncosuppressor, leading to more rapid tumor progression and decreased survival of animals with hdac1 +/- haploinsufficient tumors compared with wild type tumors. Interestingly, hdac1 also behaved as an oncogene, with decreased tumor progression following transplantation of hdac1 haploinsufficient tumors compared with wild type tumors. Our data indicate that caution is warranted regarding the use of HDAC inhibitors in treating hematologic malignancy, as this treatment could have unintended consequences, particularly during early stages of tumor development. Explaining these apparently contradictory roles of hdac1 in leukemogenesis is the focus of ongoing investigation, including gene expression studies and validation of targets. DisclosuresNo relevant conflicts of interest to declare.

Modification of topoisomerases in mammospheres derived from breast cancer cell line: clinical implications for combined treatments with tyrosine kinase inhibitors.

BackgroundAccumulating evidences suggest that tumors are driven by a small population of cells, termed “cancer stem cells” (CSCs), which may be resistant to current therapeutic approaches. In breast carcinoma, the CSCs have been identified as a CD44+/CD24− cell population. These rare cells are able to grow as non-adherent sphere-like structures, termed “mammospheres”, which enables their isolation and expansion in culture. To design efficient strategies for the complete eradication of CSCs, it is important to identify enzymes and proteins that are known as anti-cancer targets, and differ in their properties from those present in the none CSCs. Here we investigated the activity and expression of type I and type II DNA topoisomerases (topo I and topo II) in CSCs and their response to anti-topoisomerase inhibitors.MethodsMCF7 breast cancer cells, PC3 prostate cancer cells and 4 T1-Luc-Oct3/4pG mouse mammary carcinoma cells were grown on low-attachment dishes in specific medium and allowed to form spheres. Enrichment of CSC population was verified by immunostaining, flow cytometry or fluorescent microscopy imaging. Nuclear protein extracts were prepared and topoisomerases activity and protein levels were determined. Cell viability was examined by the MTT and Neutral Red assays.ResultsUnlike the adherent MCF7 cell line, topo I activity is decreased and topo II activity is increased in the CSCs. However, the relative levels of the enzyme proteins were similar in both mammospheres and adherent cells. Topo I activity in mammospheres is regulated, at least in part, by PARP-1, as observed by the recovery of topo I activity after treatment with PARP-1 inhibitor 3-Aminobenzamide. Mammosphere-derived cells show reduced sensitivity to topo I inhibitor, camptothecin, and increased sensitivity to topo II inhibitor etoposide. Intact mammospheres show increased resistance to both drugs. A combined treatment of intact mammospheres with either CPT and gefitinib, or etoposide and erlotinib, increased the anti-cancer effect of both drugs.ConclusionsThe data of this study suggest that the understanding of biological behavior of essential enzymes such as topoisomerases, in CSCs’ progression and early stages of tumor development, is important for developing new strategies for cancer treatment as well as new therapies for advanced disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-910) contains supplementary material, which is available to authorized users.

Abstract IA18: Pathogenesis of gastric cancer: Lessons and therapeutic targets from mouse models

Abstract Many if not most gastrointestinal cancers arise in the setting of chronic inflammation, and a prime example is H. pylori-associated gastric cancer. We have utilized murine models of both proximal and distal gastric neoplasia to investigate the role of chronic inflammation in the development of malignancy. The development of gastric cancer in the mouse is dependent upon commensal flora and activation of both immature myeloid cells and cancer-associated fibroblasts. Gastric cancer arises in highly innervated portions of the stomach, and can be suppressed by surgical or chemical denervation of the stomach. Neurons and neurotransmitters promote the growth of gastric stem cells in organoid culture in vitro. Carcinogenesis of the stomach is associated with activation and early activation and amplification of epithelial stem or progenitor cells appears to be a requirement for the development of neoplasia. Further, conditional loss in gastric stem cells of tumor suppressor genes in vivo may not be sufficient for tumorigenesis but instead may require inflammatory signals in order to maintain self-renewal. In summary, the early stages of tumor development are often niche-dependent, and targeting the cancer niche represents an attractive strategy for cancer prevention and cancer therapy. Citation Format: Timothy C. Wang. Pathogenesis of gastric cancer: Lessons and therapeutic targets from mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA18.

Cutaneous HPV and skin cancer

Papillomaviruses (HPVs) are small non-enveloped icosahedral viruses that infect the keratinocytes of skin and mucosa. The cutaneous HPV types are represented mainly by the beta and gamma genera, which are widely present in the skin of normal individuals. More than 40 beta-HPV types and 50 gamma-HPV types have been isolated, and these numbers are continuously growing. The main cause of non-melanoma skin cancer is exposure to ultraviolet radiation (UVR). However, cutaneous HPVs that belong to the beta genus may act as a co-carcinogen with UVR. The association between beta-HPVs and skin cancer was first reported in patients with epidermodysplasia verruciformis (EV), who frequently develop cutaneous squamous cell carcinoma (SCC) on sun-exposed areas. Isolation of HPVs from the lesions suggested that HPVs might act as a co-carcinogen with UVR in EV patients. Beta-HPVs may also play a role in cutaneous SCC in immunocompromised non-EV and in immunocompetent individuals. Several studies have reported an association of viral DNA and/or antibodies to beta HPV types with SCC. Interestingly, HPV prevalence and viral load decrease during skin carcinogenesis, being significantly higher in actinic keratosis than in SCC, suggesting that the virus may play a role in the early stages of tumour development (the "hit-and-run" hypothesis). Concordantly, in vivo and in vitro studies have shown that E6 and E7 from certain cutaneous HPV types display transforming activities, further confirming their potential role in carcinogenesis.

CYP24A1 expression inversely correlates with melanoma progression: clinic-pathological studies.

The major role of 24-hydroxylase (CYP24A1) is to maintain 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) homeostasis. Recently, it has been discovered that CYP24A1 also catalyses the hydroxylation of 20(OH)D3, producing dihydroxy-derivatives that show very effective antitumorigenic activities. Previously we showed a negative correlation of vitamin D receptor (VDR) and CYP27B1 expression with progression, aggressiveness and overall or disease-free survivals of skin melanomas. Therefore, we analyzed CYP24A1 expression in relation to clinicopathomorphological features of nevi, skin melanomas and metastases. In melanocytic tumors, the level of CYP24A1 was higher than in the normal epidermis. The statistically highest mean CYP24A1 level was found in nevi and early stage melanomas. With melanoma progression, CYP24A1 levels decreased and in advanced stages were comparable to the normal epidermis and metastases. Furthermore, the CYP24A1 expression positively correlated with VDR and CYP27B1, and negatively correlated with mitotic activity. Lower CYP24A1 levels correlated with the presence of ulceration, necrosis, nodular type and amelanotic phenotypes. Moreover, a lack of detectable CYP24A1 expression was related to shorter overall and disease-free survival. In conclusion, the local vitamin D endocrine system affects melanoma behavior and an elevated level of CYP24A1 appears to have an important impact on the formation of melanocytic nevi and melanomagenesis, or progression, at early stages of tumor development.

Tissue-Infiltrating Neutrophils Constitute the Major In Vivo Source of Angiogenesis-Inducing MMP-9 in the Tumor Microenvironment

According to established notion, one of the major angiogenesis-inducing factors, pro-matrix metalloproteinase-9 (proMMP-9), is supplied to the tumor microenvironment by tumor-associated macrophages (TAMs). Accumulated evidence, however, indicates that tumor-associated neutrophils (TANs) are also critically important for proMMP-9 delivery, especially at early stages of tumor development. To clarify how much angiogenic proMMP-9 is actually contributed by TAMs and TANs, we quantitatively evaluated TAMs and TANs from different tumor types, including human xenografts and syngeneic murine tumors grown in wild-type and Mmp9-knockout mice. Whereas host MMP-9 competence was required for full angiogenic potential of both normal and tumor-associated leukocytes, direct comparisons of neutrophils versus macrophages and TANs versus TAMs demonstrated that macrophages and TAMs secrete 40- to 50-fold less proMMP-9 than the same numbers of neutrophils or TANs. Correspondingly, the levels of MMP-9–mediated in vivo angiogenesis induced by neutrophils and TANs substantially exceeded those induced by macrophages and TAMs. MMP-9–delivering TANs were also required for development of metastasis-supporting intratumoral vasculature, characterized by ≥11-μm size lumens and partial coverage with stabilizing pericytes. Importantly, MMP-9–producing TAMs exhibit M2-skewed phenotype but do not express tissue inhibitor of metalloproteinases-1 (TIMP-1), a novel characteristic allowing them to secrete TIMP-1–free, neutrophil-like MMP-9 zymogen unencumbered by its natural inhibitor. Together, our findings support the notion whereby TANs, capable of immediate release of their pre-stored cargo, are the major contributors of highly angiogenic MMP-9, whereas tumor-influxing precursors of macrophages require time to differentiate, polarize into M2-skewed TAMs, shut down their TIMP-1 expression, and only then, initiate relatively low-level production of TIMP-free MMP-9 zymogen.

Abstract LB-247: Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer

Abstract In theory, any or all of tumor-secreted proteins can serve as cancer biomarkers. However, the reality is challenging to monitor because of the large degree of fluctuation in abundance and localization of these tumor-secreted proteins, especially in the early stage of tumor development and/or metastasis. As such, it seems feasible that we might take advantage of the fact that secreted proteases/peptidases in the tumor microenvironment generate proteolytic products, also referred to as “circulating peptides”, which are detectable in bloodstream and provide ample information about the body, “coded” in the patterns and quantity of these peptides. Herein we clearly link the catalytic activity of Carboxypeptidase N (CPN) to its proteolytic products during breast tumor progression in mouse model and clinical samples. CPN plays important roles in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Circulating fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood taken from the tumor-bearing mice, healthy women and breast cancer patients. We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both mouse and clinical patient samples, the amount of CPN was increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from both the mice at 2 weeks after orthotropic implantation and the patients’ plasma as early as the first pathologic stage of breast cancer. In conclusion, the circulating level of the selected 6 CPN-catalyzed proteolytic products reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive early diagnosis of breast cancer. Citation Format: Ye Hu. Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2014-LB-247

Folate ligand anchored liquid crystal microdroplets emulsion for in vitro detection of KB cancer cells.

A KB cancer cell-selective, liquid crystal microdroplets emulsion is prepared using folic acid-conjugated block copolymers (PS-b-PAA-FA) and sodium dodecyl sulfate (SDS) as a mediator to induce configurational transitions in 4-cyano-4'-pentylbiphenyl (5CB) liquid crystal microdroplets emulsion. The prepared liquid crystal microdroplets emulsion has shown a configurational transition from radial to bipolar on interacting with KB cancer cells, but no transition from radial to bipolar configuration is observed when liquid crystal microdroplets emulsion was allowed to interact with other normal cells such as fibroblast and osteoblast. The KB cancer cell selectivity of liquid crystal microdroplets emulsion has been considered due to the presence of KB cancer cell folate receptor-specific ligand (FA) at the surface of liquid crystal microdroplets, which allowed liquid crystal microdroplets to interact specifically with KB cancer cells. The ligand-receptor interactions have been considered responsible for triggering the configurational transitions from radial to bipolar in liquid crystal microdroplets emulsion. Thus, folate ligand anchored liquid crystal microdroplets emulsion has shown a potential to be used for in vitro detection of KB cancer cells in the early stage of tumor development.

Heparanase cooperates with Ras to drive breast and skin tumorigenesis.

Heparanase has been implicated in cancer but its contribution to the early stages of cancer development is uncertain. In this study, we utilized nontransformed human MCF10A mammary epithelial cells and two genetic mouse models [Hpa-transgenic (Hpa-Tg) and knockout mice] to explore heparanase function at early stages of tumor development. Heparanase overexpression resulted in significantly enlarged asymmetrical acinar structures, indicating increased cell proliferation and decreased organization. This phenotype was enhanced by coexpression of heparanase variants with a mutant H-Ras gene, which was sufficient to enable growth of invasive carcinoma in vivo. These observations were extended in vivo by comparing the response of Hpa-Tg mice to a classical two-stage 12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) protocol for skin carcinogenesis. Hpa-Tg mice overexpressing heparanase were far more sensitive than control mice to DMBA/TPA treatment, exhibiting a 10-fold increase in the number and size of tumor lesions. Conversely, DMBA/TPA-induced tumor formation was greatly attenuated in Hpa-KO mice lacking heparanase, pointing to a critical role of heparanase in skin tumorigenesis. In support of these observations, the heparanase inhibitor PG545 potently suppressed tumor progression in this model system. Taken together, our findings establish that heparanase exerts protumorigenic properties at early stages of tumor initiation, cooperating with Ras to dramatically promote malignant development.

NMI and EMT.

NMI and EMT.

The role of neutrophil myeloperoxidase in models of lung tumor development.

Chronic inflammation plays a key tumor-promoting role in lung cancer. Our previous studies in mice demonstrated that neutrophils are critical mediators of tumor promotion in methylcholanthrene (MCA)-initiated, butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated the role of neutrophil myeloperoxidase (MPO) activity in this inflammation promoted model. Increased levels of MPO protein and activity were present in the lungs of mice administered BHT. Treatment of mice with N-acetyl lysyltyrosylcysteine amide (KYC), a novel tripeptide inhibitor of MPO, during the inflammatory stage reduced tumor burden. In a separate tumor model, KYC treatment of a Lewis Lung Carcinoma (LLC) tumor graft in mice had no effect on tumor growth, however, mice genetically deficient in MPO had significantly reduced LLC tumor growth. Our observations suggest that MPO catalytic activity is critical during the early stages of tumor development. However, during the later stages of tumor progression, MPO expression independent of catalytic activity appears to be required. Our studies advocate for the use of MPO inhibitors in a lung cancer prevention setting.

Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APC(Min/+) mice.

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC(Min/+) mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4(+)FoxP3(+) putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC(Min/+) adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3(+) Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.

Is there any role of mast cell density and microvessel density in cervical squamous cell carcinoma? A histologic study with special reference to CD-34 immunomarker staining.

Background:Mast cells are involved in induction of angiogenesis in the early-stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.Aims and Objectives:This study was carried out to evaluate the association between mast cell density (MCD) and microvessel density (MVD) in carcinoma in situ (CIS), microinvasive carcinoma (CA) and invasive squamous cell CA of cervix.Materials and Methods:Six cases of CIS, four cases of microinvasive CA and 38 cases of invasive CA were studied over a period of 2 years from August, 2011 to June, 2013. Ten control samples were included in the study. Routine histologic examination was done. Toluidine blue stain was used for MCD determination. Immunohistochemical analysis with CD-34 was done for assessing MVD. Student's t-test was used to calculate the statistical significance of MCD and MVD.Results:Both MCD and MVD increased from normal samples through CIS to invasive cervical CA. In the four cases of microinvasive CA, the MCD and MVD were more than that of the control samples, but less than that of the six cases of CIS.Conclusion:There is a correlation between mast cell accumulation and angiogenesis in CIS, microinvasive CA and invasive cervical squamous cell CA. MCD and MVD in invasive CA exceed those in CIS and microinvasive CA. It gives us an opportunity to postulate that therapeutic strategies against mast cell mediators and angiogenesis may be of benefit in patients of early-stage cervical CA.

A Drosophila immune response against Ras-induced overgrowth

ABSTRACTOur goal is to characterize the innate immune response against the early stage of tumor development. For this, animal models where genetic changes in specific cells and tissues can be performed in a controlled way have become increasingly important, including the fruitfly Drosophila melanogaster. Many tumor mutants in Drosophila affect the germline and, as a consequence, also the immune system itself, making it difficult to ascribe their phenotype to a specific tissue. Only during the past decade, mutations have been induced systematically in somatic cells to study the control of tumorous growth by neighboring cells and by immune cells. Here we show that upon ectopic expression of a dominant-active form of the Ras oncogene (RasV12), both imaginal discs and salivary glands are affected. Particularly, the glands increase in size, express metalloproteinases and display apoptotic markers. This leads to a strong cellular response, which has many hallmarks of the granuloma-like encapsulation reaction, usually mounted by the insect against larger foreign objects. RNA sequencing of the fat body reveals a characteristic humoral immune response. In addition we also identify genes that are specifically induced upon expression of RasV12. As a proof-of-principle, we show that one of the induced genes (santa-maria), which encodes a scavenger receptor, modulates damage to the salivary glands. The list of genes we have identified provides a rich source for further functional characterization. Our hope is that this will lead to a better understanding of the earliest stage of innate immune responses against tumors with implications for mammalian immunity.