Cytotoxic antimelanoma drugs suppress the activation of M2 macrophages.

Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Although cytotoxic antimelanoma drugs such as dacarbazine (DTIC), nimustine hydrochloride (ACNU) and vincristine (VCR) have been used for the treatment of malignant melanoma as adjuvant therapy in Japan, the detailed mechanisms of their immunomodulatory effects are not fully understood. As the majority of TAMs are alternatively activated M2 macrophages that favour tumor development, the aim of this study was to elucidate the immunomodulatory effects of these reagents on human monocyte-derived M2 macrophages. First, mRNA expressions and protein production of immune checkpoint molecules, PD-L1 and chemokines by CD163+ CD206+ M2 macrophages derived from peripheral blood mononuclear cells were investigated to determine the immunomodulatory effects of DTIC, ACNU, and VCR. DTIC and VCR significantly decreased PD-L1 mRNA expression, which was confirmed by flow cytometry. Moreover, the mRNA expression and production of CCL22 were significantly decreased by DTIC, which suggested that DTIC might suppress the recruitment of Tregs in the tumor site. Furthermore, the decreased expression of PD-L1 and production of CCL22 were validated in vivo, using the B16F10 mouse melanoma model, leading to abrogation of the suppressive function of T-cell proliferation. The present report suggests one of the possible antimelanoma mechanisms of DAV combination chemotherapy for melanoma patients.

A possible interaction between periostin and CD163+ skin-resident macrophages in pemphigus vulgaris and bullous pemphigoid.

Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are autoimmune blistering diseases, and substantial numbers of CD163+ tissue-associated macrophages (TAMs) are detected in both diseases. PV and BP possess different subsets of helper T cells, suggesting that the cytokine profiles of PV and BP might be different. The purpose of this study was to investigate the microenvironment of lesional skin and serum of PV and BP patients, focusing on the immunomodulatory factors related to TAMs, such as periostin (POSTN), chemokines, cytokines and matrix metalloproteinases (MMPs). We first performed immunohistological staining of POSTN in PV and BP lesions. POSTN was prominent in the superficial dermis in both PV and BP lesions. Next, to validate the activation of CD163+ TAMs in PV and BP patients, we examined the serum levels of soluble (s)CD163. The serum sCD163 levels in PV and BP patients are significantly higher than in healthy controls. To further elucidate the molecular mechanisms of the effects of POSTN on CD163+ TAMs in PV and BP, we examined chemokines, MMPs and cytokines selected by DNA microarray database. The serum CXCL5 levels from PV patients are significantly higher than those in BP patients and healthy controls. The IL-36γ expression on infiltrating macrophages was prominent only in the lesional skin of PV, while the MMP12 deposition was detected in both PV and BP lesions. Our results shed light on the novel pathogenesis of PV through CD163+ TAMs.

The possible interaction between periostin expressed by cancer stroma and tumor-associated macrophages in developing mycosis fungoides.

Mycosis fungoides (MF) starts as an indolent disease, progresses from a patch stage to confluent plaques and ultimately develops skin tumors. Tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment in MF. The purpose of this study was to elucidate the involvement of TAMs in the lesional skin of different stages of MF. First, we immunohistologically examined the percentage of CD163+ macrophages and CD206+ cells, as well as the levels of periostin and IL-4 in cancer stroma. The percentage of CD206+ cells increased in parallel with tumor progression, while there was no significant difference in the percentage of CD163+ cells. Periostin was prominent in the stromal area at the patch and plaque stages but decreased at the tumor stage. In contrast, IL-4 was prominently stained at both plaque and tumor stages. To further elucidate the molecular mechanisms of the effects of these stromal factors on TAMs, we examined their effects on mRNA expression in monocyte-derived macrophages in vitro. Based on microarray analysis and gene ontology, we examined a series of chemokines and MMPs whose expression was strongly connected with periostin stimulation. The DNA microarray results were verified in M2 macrophages using real-time PCR. We further examined the mRNA expression of these chemokines and MMPs in the presence of periostin and IL-4 to simulate the advanced stages of MF and validated their protein expression by ELISA. Our present report suggests possible roles of periostin on TAMs in establishing the tumor microenvironment in MF.

RANKL expression is a useful marker for differentiation of pagetoid squamous cell carcinoma in situ from extramammary Paget disease.

BACKGROUND: Pagetoid squamous cell carcinoma in situ (SCCIS) is a histopathologic variant of SCCIS composed of cells that display an abundant, pale-staining cytoplasm in a pagetoid distribution within the epidermis. As pagetoid SCCIS is sometimes difficult to differentiate from extramammary Paget disease (EMPD) histopathologically, specific markers for pagetoid SCCIS or EMPD are needed by dermatopathologists. METHODS: In this report, we employed immunohistochemical staining for receptor of activated nuclear factor kappa ligand (RANKL) and programmed death-ligand 1 (PD-L1) in six cases each of pagetoid SCCIS and EMPD. RESULTS: The Paget cells strongly expressed RANKL in EMPD, whereas the atypical keratinocytes did not express RANKL in any of the six cases of pagetoid SCCIS. In all cases of pagetoid SCCIS, atypical keratinocytes expressed PD-L1. In EMPD, Paget cells expressed PD-L1 in half of the cases at a lower level of expression than was seen in the surrounding keratinocytes. CONCLUSION: This study suggested that RANKL, but not PD-L1, could be a marker to differentiate between pagetoid SCCIS and EMPD.

Immunomodulatory effects of peplomycin on immunosuppressive and cytotoxic cells in the lesional skin of cutaneous squamous cell carcinoma.

BACKGROUND: Continuous intra-arterial administration of peplomycin (PEP) through a tumor-feeding artery using an intravascular indwelling catheter is one of the best treatments for cutaneous squamous cell carcinoma (SCC) on cosmetic areas. Although this reagent is useful for the treatment of SCC, its immunomodulatory effect on the tumor microenvironment is still unknown. OBJECTIVE/METHODS: In this study, we investigated the immunomodulatory effects of PEP on the tumor-infiltrating regulatory T cells and tumor-associated macrophages as well as CD8(+)TIA-1(+) cytotoxic T cells in the lesional skin of 5 patients with SCC on the lips. RESULTS: Our data suggest that, in addition to the direct antitumor effects, PEP decreased immunosuppressive cells and increased cytotoxic T lymphocytes at the tumor sites, which might maintain antitumor immune response against SCC.

Comparison of CD163+ CD206+ M2 macrophages in the lesional skin of bullous pemphigoid and pemphigus vulgaris: the possible pathogenesis of bullous pemphigoid.

BACKGROUND: M2 macrophages play a critical role in the induction of T helper 2 (Th2) polarization. METHODS: To investigate the contribution of M2 macrophages to the pathogenesis of bullous pemphigoid (BP) and pemphigus vulgaris (PV), we employed immunohistochemical staining for CD163 and CD206, as well as pSTAT1, pSTAT6, interleukin (IL)-4, IL-13, CCL17, CCL18 and Foxp3 in the lesional skin of 10 cases of BP and PV. RESULTS: The numbers of CD163+ CD206+ M2 macrophages were higher in BP than in PV. Moreover, pSTAT6+ cells, CCL17+ cells, CCL18+ cells and Foxp3+ regulatory T cells were prominent only in the lesional skin of BP. To further investigate the function of M2 macrophages, we examined the mRNA expression and production of Th2-related chemokines from M2 macrophages in vitro, which showed a significant increase in the mRNA expression and production of CCL18 when stimulated by IL-4 or IL-13. CONCLUSION: Our study sheds light onto one of the possible immunological mechanisms of BP and PV.

Immunomodulatory effect of bisphosphonate risedronate sodium on CD163+ arginase 1+ M2 macrophages: the development of a possible supportive therapy for angiosarcoma.

An imbalance of immunosuppressive cells and cytotoxic cells plays an important role in inhibiting the antitumor immune response of the tumor-bearing host. We previously reported the profiles of tumor infiltrating leukocytes in cutaneous angiosarcoma (AS) and suggested that a combination of docetaxel (DTX) with bisphosphonate risedronate sodium (RS) might be effective for MMP9-expressing AS by targeting immunosuppressive cells such as M2 macrophages. To further confirm the effect of this combination therapy, in this report we investigated the immunomodulatory effect of DTX and RS on CD163(+) arginase 1 (Arg1)(+) M2 macrophages in vitro. Interestingly, our present study demonstrated that DTX in combination with RS significantly upregulated the mRNA expression of CXCL10 on M2 macrophages and significantly decreased the mRNA expression of CCL17 and Arg1. Moreover, the production of CXCL10 and CXCL11 from M2 macrophages was significantly increased by DTX with RS though there was no effect of DTX with RS on the production of CCL5 and CCL17. Furthermore, DTX with RS significantly decreased the production of CCL18, which was previously reported to correlate with the severity and prognosis in cancer patients. Our present report suggests one of the possible mechanisms of DTX with RS in the supportive therapy for angiosarcoma.

Successful treatment of syringotropic CD8+ mycosis fungoides accompanied by hypohidrosis with vorinostat and retinoids.

We describe a 34-year-old Japanese man with syringotropic CD8+ mycosis fungoides (MF) accompanied by hypohidrosis who was treated with vorinostat and retinoids. Interestingly, immunohistochemical staining for dermcidin revealed a decrease of sweat in the eccrine glands, and a sweat test by the iodine starch method proved hypohidrosis in the MF-affected areas. Six months after treatment with this combination therapy, the patient's advanced MF was under control.

The Expression of Matrix Metalloproteinases in Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-expressing Cancer of Apocrine Origin.

Primary cutaneous apocrine carcinoma (PCAC) is a rare and highly aggressive tumor entity. Since there is no conventional therapy for advanced PCAC, exploratory treatments are sometimes used. As we previously reported, receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL)/RANK signaling on M2 macrophages promotes the production of chemokines and proinflammatory cytokines to maintain the immunosuppressive tumor environment of extramammary Paget's disease (EMPD). Since EMPD is a skin adenocarcinoma of apocrine gland origin that expresses high levels of RANKL and matrix metalloproteinase (MMP) 7, and EMPD is associated with the presence of RANK+ M2 macrophages, we hypothesized that tumor-associated macrophages (TAMs) in adenocarcinomas such as PCAC might also express RANKL and MMP7. MATERIALS AND METHODS: We employed immunohistochemical staining of RANKL and MMP7 in the lesional skin from five patients with PCAC, and microarray analysis of MMPs using human monocyte-derived macrophages. RESULTS: According to DNA microarray analysis, the expression of MMP1 and MMP25 was augmented. The DNA microarray results were verified by using real-time polymerase chain reaction (RT-PCR). Immunohistochemical staining of MMP1 and MMP25 as well as chemokine (C-C motif) ligand (CCL) 5 in the lesional skin from five patients with PCAC showed a substantial number of MMP1-bearing cells and MMP25-bearing cells, as well as CCL5-producing cells, that were distributed in the lesional skin. CONCLUSION: Our study suggests that the RANKL/RANK pathway contributes to the development and maintenance of the immunosuppressive tumor microenvironment and denosumab may be a promising adjuvant therapy targeting TAMs in cancer of apocrine origin.

Increased serum production of soluble CD163 and CXCL5 in patients with moyamoya disease: Involvement of intrinsic immune reaction in its pathogenesis.

Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by a progressive stenosis at the terminal portion of the internal carotid artery and an abnormal vascular network at the base of the brain. Although its etiology is still unknown, intrinsic immune reactions such as autoimmune response has been implicated in the pathogenesis of MMD. Recently, the RING finger protein 213 (RNF213) was found to be an important risk gene for MMD, and is predominantly expressed in blood cells and the spleen. Thus, we hypothesized that patients with MMD represent an intrinsic autoimmune status mediated by M2-polarized macrophages, which play an important role in tissue remodeling and angiogenesis. We compared the serum level of soluble (s)CD163, an activating marker for CD163+ M2-polarized macrophages that has been implicated in a variety of autoimmune disorders, between MMD patients and healthy controls. We also analyzed serum levels of CXCL5, an augmented cytokines that has been correlated with the severity of autoimmune diseases. As a result, the serum sCD163 levels of MMD patients (281,465 pg/ml) were significantly higher than those of healthy controls (174,842 pg/ml) (p = .004). The serum CXCL5 levels of MMD patients (679.02 pg/ml) were significantly higher than those of healthy controls (401.79 pg/ml) (p = .046). There were no differences in the serum sCD163 and CXCL5 levels between each genotype of the RNF213 polymorphism (wild-type or variant) among MMD patients. Although this is a pilot study and further validation with larger number of samples is necessary, our results indicate that patients with MMD may have increased autoimmune activity, and our results shed light on the pathogenesis of MMD via CD163+ M2-polarized macrophages.

Efficacy of oral cholecalciferol on rhododendrol-induced vitiligo: A blinded randomized clinical trial.

Rhododendrol (RD), 4-(4-hydroxyphenyl)-2-butanol, inhibits melanin synthesis and has been used for skin-whitening cosmetic products. RD has been very effective in lightening skin pigmentation, but some persons have developed so-called RD vitiligo, in which vitiligo starts on the face, neck and hands where topical RD has been applied and even extended over skin areas where RD has not been applied. RD vitiligo lesions in some patients have lasted for years and have been resistant to conventional vitiligo treatments. We examined the effects of cholecalciferol on RD vitiligo in a blinded randomized clinical trial. Forty-eight female RD vitiligo patients were recruited for the trial and were randomized into two groups: the vitamin D (VD)-intervention group that received daily 5000 IU cholecalciferol for 5 months and the control group. Three blinded investigators scored vitiligo improvement by comparing photographic images of baseline and at 5-month observation. Serum 25(OH)D3 of RD vitiligo patients was not significantly different from age-matched healthy volunteers. Twenty-two in the VD-intervention group and 23 in the control group completed the 5-month observation. Serum 25(OH)D3 levels were significantly increased after the 5-month VD intervention, while the control group did not change. The improvement scores were significantly higher in the VD-intervention group than the control group. The improvement scores were positively correlated with the serum 25(OH)D3 levels after the 5-month intervention period but not before the treatment. This blinded randomized clinical trial showed favor in administrating 5000 IU cholecalciferol daily to RD vitiligo patients.

The Immunological Roles of Periostin/Tumor-Associated Macrophage Axis in Development of Dermatofibrosarcoma Protuberans.

BACKGROUND/AIM: Dermatofibrosarcoma protuberance (DFSP) is a fibrohistiocytic tumor of intermediate malignancy characterized by slow infiltrative growth and a high tendency to recur locally. Periostin is involved in modulating cell function and inducing the production of proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs) from tumor-associated macrophages (TAMs) to promote fibrosis and tumor growth. This study aimed to examine the cancer stroma of DFSP, focusing on TAMs-related proteins and MMPs. PATIENTS AND METHODS: Using immunohistochemical staining and DNA microarray database, we evaluated periostin, CD163, CD206, MMP1 and MMP12 in 10 cases of DFSP and dermatofibroma. RESULTS: Dense deposits of periostin as well as a substantial number of CD163+ TAMs were detected at the peripheral areas of DFSP. Moreover, MMP1 and MMP12, that were selected by using a DNA microarray database of monocyte-derived macrophages, were observed in the TAMs-detected area. CONCLUSION: Increased levels of MMP1 and MMP12 on TAMs in the peripheral areas of DFSP might contribute to local invasion.

Immunomodulatory Effect of Imiquimod Through CCL22 Produced by Tumor-associated Macrophages in B16F10 Melanomas.

BACKGROUND/AIM: Tumor-associated macrophages (TAMs), together with splenic CD11b+ cells, help maintain the tumor microenvironment. The immunomodulatory compound imiquimod (IQM) stimulates innate immune cells, including macrophages, to induce antitumor effects. In order to elucidate the effects of IQM on the tumor microenvironment, we investigated the immunomodulatory effect of IQM during melanoma growth by using the B16F10 melanoma model. MATERIALS AND METHODS: To elucidate the immunomodulatory effects of IQM on the tumor microenvironment, we isolated CD11b+ TAMs and splenic CD11b+ cells and evaluated the immunomodulatory effects of IQM, using the B16F10 melanoma model. RESULTS: IQM suppressed B16F10 melanoma growth in parallel with reduction of Foxp3+ regulatory T cells (Tregs) at the tumor site, caused by the down-regulation of CCL22 production by tumor-derived and splenic CD11b+ cells. Subsequently, we investigated the antitumor or tumor-loading effects of splenic CD11b+ cells on B16F10 melanoma growth in vivo. B16F10 melanoma growth was accelerated by splenic CD11b+ cells from untreated mice, but was inhibited by splenic CD11b+ cells from IQM-treated mice. Consistent with these results, Foxp3+ Tregs were significantly decreased in tumors of mice implanted with both melanoma and splenic CD11b+ cells from topical IQM-treated mice. Furthermore, intratumoral administration of anti-CCL22 antibody inhibited B16F10 melanoma growth by decreasing Treg recruitment at the tumor site. CONCLUSION: Our results suggest a possible mechanism for the antitumor immune response induced by IQM through tumor-associated macrophages.

Phospho-STAT5B Expression Is a Prognostic Marker for Merkel Cell Carcinoma.

BACKGROUND/AIM: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma. Although recent reports suggest that tumor-infiltrating leukocytes (TILs), especially CD8+ T-cells, contribute to the pathogenesis of MCC, it is difficult for a single Institute with a small number of patients with MCC to determine the threshold number of CD8+ cells. Therefore, clearer and easier methods of evaluating prognostic factors of MCC are needed. PATIENTS AND METHODS: In order to identify the prognostic factors of 24 cases of MCC, we employed immuno histochemical staining of phospho-signal transducer and activator of transcription 5B (pSTAT5B), which has been reported to be a prognostic marker for several types of cancers. RESULTS: All MCC cases with a good outcome (n=16) expressed pSTAT5B, whereas all MCC cases with a poor outcome (n=8) did not express pSTAT5B. Moreover, we additionally employed immunohistochemical staining of periostin (POSTN) and interleukin-4, as well as sub-populations of TILs (granulysin-bearing cells, regulatory T-cells, CD163+ cells, and CD206+ cells), and the deposition of matrix metalloproteinase 12 in the lesional skin of patients with MCC. The results suggested that there is no significant difference in stromal factors between MCC cases with a good and those with a poor outcome. CONCLUSION: pSTAT5B expression may be an indicator of positive prognosis in patients with MCC.

Retrospective Investigation of Cutaneous Squamous Cell Carcinoma on the Lip Treated with Peplomycin Administered Through a Superficial Temporal Artery.

BACKGROUND: Continuous intra-arterial (IA) administration of peplomycin (PEP) through a tumor-feeding artery is one of the most effective treatments for cutaneous squamous cell carcinoma (cSCC) in cosmetic areas. PATIENTS AND METHODS: In order to determine the effective and safe dose of PEP and the curative rate of IA-PEP, we retrospectively investigated a case series of 24 patients with cSCC on the lips who were treated with IA-PEP. RESULTS: IA-PEP reduced the tumor mass in all 24 cases (100%). A complete response occurred in 17 patients (70.8%), and a partial response occurred in seven (29.2%). Moreover, 17 patients (70.8%) were cured, three patients developed cervical lymph node metastasis (12.5%), and four developed local recurrence (16.7%). Three out of the 24 patients developed interstitial pneumonia (12.5%). CONCLUSION: Low-dose IA-PEP administered through a superficial temporal artery was a highly effective treatment that achieved a curative response for 70.8% of patients with cSCC on the lips.

Phase I study of nivolumab combined with IFN-ß for patients with advanced melanoma.

The efficacy of nivolumab is greater than that of other anti-melanoma drugs, so nivolumab-based combined therapies that enhance anti-tumor immune responses in patients with metastatic melanoma are of great interest to dermato-oncologists. As we have previously reported, IFN-ß enhances the anti-tumor immune response of anti-PD-1 antibodies against B16F10 melanoma in vivo. To explore the potential of this property of IFN-ß as part of a combination therapy for the treatment of metastatic melanoma patients, we performed a phase 1 trial, using a traditional rule-based 3 + 3 design, on patients with advanced melanoma. The nivolumab dose was fixed at 2 mg/kg, every 3 weeks. IFN-ß was administered to three groups at doses of 1 million, 2 million, and 3 million units, respectively. Dose-limiting toxicities were defined as any grade 3-5 adverse events occurring between day 0 and day 42 that might possibly be related to nivolumab and IFN-ß. Of the nine patients who received this combined therapy, none experienced dose-limiting toxicities, and all completed the treatment phase of the study. Patient follow-up continued for 6 months following the final treatment. There were two complete responses (22%) and one partial response (11%), all of which occurred in patients who had received monthly IFN-ß immediately prior to the study. In this study, we determined the safe dose of IFN-ß, when combined with nivolumab, to be 3 million units. To determine the efficacy of this combination therapy, further phase II trials are required.

RANKL-Expressing Ectopic Extramammary Paget's Disease on the Lower Abdomen.

Ectopic extramammary Paget's disease (EMPD) is a rare variant of EMPD that develops in nonapocrine regions. Since reports about ectopic EMPD are limited, little is known about the biological and immunological background of ectopic EMPD. In this report, we present a case of ectopic EMPD on the lower abdomen that expressed RANKL but lacked the expression of MMP7. As we previously reported, Paget's cells express RANKL and MMP7, release soluble RANKL in the tumor microenvironment, and stimulate tumor-associated macrophages to produce tumor-loading factors in conventional EMPD. In our present case, both CCL5-expressing cells and MMP25-bearing cells were lacking, whereas substantial numbers of CCL5-expressing cells and MMP25-bearing cells were found in conventional EMPD. Our case suggested that the lack of MMP7 on Paget's cells might be one of the possible explanations for the biology of ectopic EMPD.

Merkel Cell Carcinoma with Spontaneous Regression: A Case Report and Immunohistochemical Study.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma that only rarely regresses spontaneously. Since little is known about the immunological mechanisms involved in the spontaneous regression of MCC, we describe a case of MCC with spontaneous regression and employed immunohistochemical staining for cytotoxic and immunosuppressive molecules to investigate possible mechanisms involved in the spontaneous regression of MCC. Interestingly, compared to conventional MCC, tumor-infiltrating lymphocytes in MCC with spontaneous regression contained higher numbers of CD8(+) cells and granulysin-bearing cells and lower numbers of CD206(+) cells. Our present study suggests one of the possible reasons for the spontaneous regression of MCC.