Epigenetic Changes in Basal Cell Carcinoma Affect SHH and WNT Signaling Components

Background

The genetic background of Basal Cell Carcinoma (BCC) has been studied extensively, while its epigenetic makeup has received comparatively little attention. Epigenetic alterations such as promoter hypermethylation silence tumor suppressor genes (TSG) in several malignancies.

Objective

We sought to analyze the promoter methylation status of ten putative (tumor suppressor) genes that are associated with Sonic Hedgehog (SHH), WNT signaling and (hair follicle) tumors in a large series of 112 BCC and 124 healthy control samples by methylation-specific PCR.

Results

Gene promoters of SHH (P = 0.016), adenomatous polyposis coli (APC) (P = 0.003), secreted frizzled-related protein 5 (SFRP5) (P = 0.004) and Ras association domain family 1A (RASSF1A) (P = 0.023) showed significantly more methylation in BCC versus normal skin. mRNA levels of these four genes were reduced for APC and SFRP5 in BCC (n = 6) vs normal skin (n = 6). Down regulation of SHH, APC and RASSF1A could be confirmed on protein level as well (P<0.001 for all genes) by immunohistochemical staining. Increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β-catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β-catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples).

Conclusions

We provide evidence that promoter hypermethylation of key players within the SHH and WNT pathways is frequent in BCC, consistent with their known constitutive activation in BCC. Epigenetic gene silencing putatively contributes to BCC tumorigenesis, indicating new venues for treatment.     

Monocyte-Derived Dendritic Cells from Patients with Dermatophytosis Restrict the Growth of Trichophyton rubrum and Induce CD4-T Cell Activation

Dermatophytes are the most common agents of superficial mycoses that are caused by mold fungi. Trichophyton rubrum is the most common pathogen causing dermatophytosis. The immunology of dermatophytosis is currently poorly understood. Recently, our group investigated the interaction of T. rubrum conidia with peritoneal mouse macrophages. We found that macrophages phagocytose T. rubrum conidia resulted in a down-modulation of class II major histocompatibility complex (MHC) antigens and in the expression of co-stimulatory molecules. Furthermore, it induced the production of IL-10, and T. rubrum conidia differentiated into hyphae that grew and killed the macrophages after 8 hrs of culture. This work demonstrated that dendritic cells (DCs) and macrophages, from patients or normal individuals, avidly interact with pathogenic fungus T. rubrum. The dermatophyte has two major receptors on human monocyte-derived DC: DC-SIGN and mannose receptor. In contrast macrophage has only mannose receptor that participates in the phagocytosis or bound process. Another striking aspect of this study is that unlike macrophages that permit rapid growth of T. rubrum, human DC inhibited the growth and induces Th activation. The ability of DC from patients to interact and kill T. rubrum and to present Ags to T cells suggests that DC may play an important role in the host response to T. rubrum infection by coordinating the development of cellular immune response.     

Diclofenac Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer by Modulation of VEGF Levels and Arginase Activity

Background

Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A₂ (PLA₂), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically.

Methodology/Principal Findings

We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development.

Conclusion/Significance

In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.     

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4⁺ and/or CD8⁺ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4⁺ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8⁺ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4⁺ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8⁺ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4⁺ T cell-mediated antitumor activity in vivo.     

Ovarian Tumor-Induced T Cell Suppression Is Alleviated by Vascular Leukocyte Depletion

The ovarian cancer microenvironment recruits an array of immune cells to the site of tumor growth. Within the peritoneal ascites of both humans and mice, the predominant population of tumor-infiltrating leukocytes is a CD11c⁺CD11b⁺ population variably referred to as vascular leukocytes (VLCs), tumor-associated macrophages, and immature dendritic cells. We have previously shown that these cells are critical for tumor growth because their selective elimination from the peritoneal tumor microenvironment inhibited tumor progression. However, the underlying mechanism by which this therapy was efficacious is poorly understood. Here, we use the murine ID8 ovarian tumor model to demonstrate that the tumor microenvironment induces in vivo immunosuppression of T cells and that the SR-A⁺ VLCs mediate this suppression. Importantly, the elimination of SR-A⁺ VLCs from the peritoneum of tumor-bearing mice relieves the T cell suppression. Moreover, the profound changes that VLC elimination has on the immune system are T cell-dependent because the protective antitumor effect of VLC elimination does not occur when CD8 T cells are concomitantly depleted. These results were confirmed and extended with the use of a genetic model for VLC depletion, which demonstrated that short-term therapeutic depletion of VLCs alleviates immunosuppression and allows for efficacious vaccination against model tumor antigens in tumor-bearing mice. These studies provide a mechanistic explanation for how leukocytes contribute to ovarian tumor progression and, correspondingly, how leukocyte depletion inhibits tumor growth.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Socioeconomic status and non-melanoma skin cancer: A nationwide cohort study of incidence and survival in Denmark

Background: The two main types of non-melanoma skin cancer differ with the pattern of exposure to ultraviolet radiation (UVR): basal cell carcinoma (BCC) appears to be more closely related to intermittent solar exposure and sunburn, while the risk for squamous cell carcinoma (SCC) is a result of lifetime cumulated exposure to UVR. As these exposures may differ by social position, we investigated its role in the risk for and survival after BCC and SCC diagnosed in Denmark in 1994–2006 with follow-up through 2006. Methods: The analyses were based on 52,166 cases of BCC and 5033 cases of SCC in a cohort of 3.7 million people born between 1925 and 1976 and residing in Denmark in 1992–2006. Information on cancer cases and socioeconomic indicators were obtained from population-based registries. We used log-linear Poisson regression models to estimate incidence rate ratios and cumulative relative survival to estimate survival up to 10 years after the first incident cases of BCC and SCC. Results: High socioeconomic status, measured by both education and disposable income, was strongly associated with a higher risk for BCC, whereas there was no association between SCC and educational level and only a weak association with income. In general, relative survival after BCC was better than after SCC; the pattern of survival was not affected by socioeconomic indicators. Conclusions: The observed pattern of social status and risk for non-melanoma skin cancer differed substantially for the two cancer types, supporting the hypothesis that they may have different aetiologies.     

Genetic determinants of UV-susceptibility in non-melanoma skin cancer

A milieu of cytokines and signaling molecules are involved in the induction of UV-induced immune suppression and thus the etiology of non-melanoma skin cancer (NMSC). Targeting the UV-induced immunosuppression pathway, and using a large population based study of NMSC, we have investigated the risk associated with functional variants in 10 genes (IL10, IL4, IL4R, TNF, TNFR2, HTR2A, HRH2, IL12B, PTGS2, and HAL). The most prominent single genetic effect was observed for IL10. There was increasing risk for both basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) with increasing number of variant IL10 haplotypes (BCC: p_trend = 0.0048; SCC: p_trend = 0.031). Having two IL10 GC haplotypes was associated with increased odds ratios of BCC and SCC (OR_BCC = 1.5, 95% CI 1.1–1.9; OR_SCC = 1.4, 95% CI 1.0–1.9), and these associations were largely confined to women (OR_BCC = 2.2, 95% CI 1.4–3.4; SCC: OR_SCC = 1.8, 95% CI 1.1–3.0). To examine how combinations of these variants contribute to risk of BCC and SCC, we used multifactor dimensionality reduction (MDR) and classification and regression trees (CART). Results from both of these methods found that in men, a combination of skin type, burns, IL10, IL4R, and possibly TNFR2 were important in both BCC and SCC. In women, skin type, burns, and IL10 were the most critical risk factors in SCC, with risk of BCC involving these same factors plus genetic variants in HTR2A, IL12B and IL4R. These data suggest differential genetic susceptibility to UV-induced immune suppression and skin cancer risk by gender.     

Basal Cell Carcinoma in Gorlin’s Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients.     

Bone marrow-independent adventitial macrophage progenitor cells contribute to angiogenesis

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c⁺/Sca-1⁺ adventitial progenitor cells. Analysis of the NCX^−/− mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34⁺ progenitor cells within the adventitial vasculogenic zone to differentiate into CD31⁺ endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders.Subject terms: Stem-cell differentiation, Stem-cell niche, Adult stem cells, Cell signalling     

Enhancement of phagocytosis and cytotoxicity in macrophages by tumor-derived IL-18 stimulation

Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b⁺ cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells. [BMB Reports 2014; 47(5): 286-291]     

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK4 (BLP)

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam₃CSK₄ (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.     

YAP and TAZ are essential for basal and squamous cell carcinoma initiation

YAP and TAZ are key downstream regulators of the Hippo pathway, regulating cell proliferation and differentiation. YAP and TAZ activation has been reported in different cancer types. However, it remains unclear whether they are required for the initiation of major skin malignancies like basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Here, we analyze the expression of YAP and TAZ in these skin cancers and evaluate cancer initiation in knockout mouse models. We show that YAP and TAZ are nuclear and highly expressed in different BCC types in both human and mice. Further, we find that cells with nuclear YAP and TAZ localize to the invasive front in well‐differentiated SCC, whereas nuclear YAP is homogeneously expressed in spindle cell carcinoma undergoing EMT. We also show that mouse BCC and SCC are enriched for YAP gene signatures. Finally, we find that the conditional deletion of YAP and TAZ in mouse models of BCC and SCC prevents tumor formation. Thus, YAP and TAZ are key determinants of skin cancer initiation, suggesting that targeting the YAP and TAZ signaling pathway might be beneficial for the treatment of skin cancers.     

Electrical fusion of cells and nucleiin vitro: A preliminary note

The electrical fusion of male skin fibroblasts of the Indian muntjac (Muntiacus muntjak)in vitro is described. Fusion of cell nuclei in the resulting multinucleated giant cells was observed after application of up to ten additional DC pulses. Electrical fusion of bovine erythrocytes and alveolar macrophages is also described. Cell fusion can be obtained without the use of protease pretreatment, but the yield of fused cells is about 20 times lower than with prior use of proteases.     

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).     

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.     

Stimulation and inhibition of neoplastic cell growth by tumor promoter-treated macrophages

Macrophages treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent inflammatory and tumor-promoting agent, can have the diametrically opposed functions of contact-mediated tumor cytotoxicity and release of soluble clonal proliferation factor(s) for tumor cells. In vitro TPA treatment of macrophages at 1.0 ng/ml induced prostaglandin E₂ release and morphological changes analogous to cell activation. In addition, conditioned medium from macrophages pulsed with TPA enhanced M109 carcinoma colony formation in vitro. Although macrophages were not rendered tumoricidal by TPA in vitro, cytotoxic macrophages were recovered from mice following ip treatment with TPA at 1–100 μg/kg. This indicated an indirect pathway for the activation of macrophages by TPA. The very weak tumor promoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate lacked effects on macrophages at all doses tested. The possibility that macrophage secretions (e.g., prostaglandin E₂, angiogenesis-stimulating factor(s), and clonal proliferation factor(s) for carcinogen-triggered cells) may be involved in the tumor promotion process is discussed.     

NK cell activation and tumor infiltration are involved in the antitumor mechanism of Virulizin

Previous studies have demonstrated antitumor efficacy of Virulizin in several human tumor xenograft models and a critical role for macrophages in the antitumor mechanism of Virulizin. Although there is growing support for an immune stimulatory mechanism of action for Virulizin, the details remain to be elucidated. The aim of this study was to determine whether infiltration of natural killer (NK) cells into xenografted tumors is altered by Virulizin treatment, and whether such alterations contribute to the antitumor activity of Virulizin. Immunohistochemical analysis demonstrated that xenografted tumors from Virulizin-treated mice had an increase in infiltration of F4/80⁺ (macrophages) and NK1.1⁺ (NK) cells. The increase in NK1.1⁺ cell infiltration occurred at an early stage of Virulizin treatment, which correlated with an early sign of apoptosis. In addition, Virulizin resulted in an increase in the number of NK cells in the spleens, and NK cells isolated from the spleen exhibited increased cytotoxicity to tumor cells in vitro. In NK cell–deficient SCID-beige mice, the antitumor activity of Virulizin was compromised, providing additional support to the hypothesis that NK cells are necessary for inhibition of tumor growth by Virulizin. Finally, depletion of macrophages resulted in the loss of Virulizin-induced increase in NK1.1⁺ cell infiltration into xenografted tumors, suggesting the involvement of macrophages in NK cell infiltration into tumors. Taken together, these results strongly support a mechanism in which Virulizin stimulates a sustained expansion and infiltration of NK cells and macrophages into tumors with subsequent activation of NK cells that is responsible for the observed antitumor activity.