The metastatic microenvironment: Brain-derived soluble factors alter the malignant phenotype of cutaneous and brain-metastasizing melanoma cells

The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential prerequisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors upregulated the expression of the chemokine receptor CCR4 in both cutaneous and brain-metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain-metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain.     

The metastatic microenvironment: Claudin‐1 suppresses the malignant phenotype of melanoma brain metastasis

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain‐metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain‐metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1‐overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1‐overexpressing cells to form lung micrometastases was not impaired. CLDN1‐mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.     

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.     

T cells in the human metastatic melanoma microenvironment express site‐specific homing receptors and retention integrins

T‐cell infiltration into the metastatic melanoma microenvironment (MME) correlates with improved patient survival. However, diffuse infiltration into tumor occurs in only 8% of melanoma metastases. Little is known about mechanisms governing T‐cell infiltration into human melanoma metastases or about how those mechanisms may be altered therapeutically. We hypothesized that T cells in the MME would be enriched for chemokine receptors CCR4, CCR5, CXCR3 and homing receptors relevant to the tissue site. Viably cryopreserved single cell suspensions from nineteen melanoma metastases representing three metastatic sites (tumor‐infiltrated lymph node, skin and small bowel) were evaluated by multiparameter flow cytometry and compared to benign lymph nodes and peripheral blood mononuclear cells from patients with Stage IIB–IV melanoma. T cells in the melanoma metastases contained large effector memory populations, high proportions of activated, moderately differentiated cells and few regulatory T cells. Site‐specific homing was suggested in bowel, with high expression of CCR9. We neither encounter the anticipated enrichment of integrin α4β7 in bowel, cutaneous leukocyte antigen (CLA) in skin, nor integrin α4β1 or receptor CXCR3 in metastatic sites. Retention integrins αEβ7, α1β1 and α2β1 were significantly elevated in metastases. These data suggest limited tissue site‐specific homing to human melanoma metastases, but a significant role for retention integrins in maintaining intratumoral T cells. Our findings also raise the possibility that T‐cell homing, infiltration, and retention in melanoma metastases may be increased by increasing expression of ligands for CLA, α4β1 and CXCR3 on intratumoral endothelium.     

肿瘤微环境在肿瘤淋巴管生成中的作用

恶性肿瘤淋巴结转移是一个复杂的病理过程,与肿瘤淋巴管生成密切相关。肿瘤生长到一定程度,分泌某些淋巴管生长因子,在肿瘤周围或内部形成新生淋巴管,肿瘤细胞侵入管腔并通过淋巴液顺次流入各站淋巴结,逐渐形成淋巴结转移病灶。正常细胞处于一个相对稳定的内环境,按正常的程序进行着增殖、分化、凋亡以及相关因子的分泌和表达,而肿瘤发生、发展则不断打破这一平衡,逐渐形成一个适于自己生长的组织外环境,即肿瘤微环境。而肿瘤微环境中的众多促淋巴管生成因子、炎性条件、组织缺氧、酸性微环境以及间质高压形成等病理生理特性能促进肿瘤淋巴管生成,进而促进肿瘤淋巴结转移病灶的形成。      

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.     

The importance of microenvironment: the role of CCL8 in metastasis formation of melanoma

We have attempted to characterize the changes occurring on the host side during the progression of human melanoma. To investigate the role of tumor microenvironment, we set up such an animal model, which was able to isolate the host related factors playing central role in metastasis formation. One of these ‘factors’, CCL12, was consequently selected and its behavior was examined alongside its human homologue (CCL8). In our animal model, metastasis forming primary melanoma in the host exhibited increased level of CCL12 mRNA expression. In clinical samples, when examining the tumor and the host together, the cumulative (tumor and host) CCL8 expression was lower in the group in which human primary melanoma formed lung metastasis compared to non-metastatic primary tumors. We could not detect significant difference in CCL8 receptor (CCR1) expression between the two groups. Increased migration of the examined tumor cell lines was observed when CCL8 was applied as a chemoattractant. The tumor cells and their interactions can be influenced the expression of CCL8 by dermal fibroblasts, as a significant change in the metastatic microenvironment. Furthermore, we examined changes in miRNA profile resulted by CCL8 and miR146a appears to be a promising prognostic marker for following this process.     

Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment

Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alphaviruses in B16 tumor cells under varying growth conditions. We hypothesized that the tumor microenvironment might alter gene expression in B16 cells, leading to an up-regulation of the expression of virus-binding receptors or factors associated with virus entry and replication. To test our hypothesis, we performed a proteomics analysis of B16 cells cultured in vitro and of B16 cells isolated from tumors, and we identified 277 differentially regulated proteins. A further in-depth analysis to identify the biological and molecular functions of the detected proteins revealed a set of candidate genes that could affect virus infectivity. Importantly, we observed a decrease in the expression of interferon α (IFN-α) in tumor-isolated cells that resulted in the suppression of several IFN-regulated genes, thereby abrogating host cell antiviral defense. Additionally, differences in the expression of genes that regulate cytoskeletal organization caused significant alterations in cell membrane elasticity. Taken together, our findings demonstrated favorable intracellular conditions for alphavirus transduction/replication that occurred during tumor transformation. These results pave the way for optimizing the development of strategies for the application of alphaviral vectors as a potent cancer therapy.     

The metastatic microenvironment: Lung‐derived factors control the viability of neuroblastoma lung metastasis

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60–70% of children with high‐risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations—lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung‐derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro‐apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung‐derived factors affected various additional progression‐linked cellular characteristics of neuroblastoma cells, such as the expression of stem‐cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.     

Glutamate blunts cell‐killing effects of neutrophils in tumor microenvironment

Neutrophils are the first defenders of the innate system for injury and infection. They have gradually been recognized as important participants in tumor initiation and development due to their heterogeneity and plasticity. In the tumor microenvironment (TME), neutrophils can exert antitumor and protumor functions, depending on the surroundings. Tumor cells systemically alter intracellular amino acid (AA) metabolism and extracellular AA distribution to meet their proliferation need, leading to metabolic reprogramming and TME reshaping. However, the underlying mechanisms that determine how altered AAs affect neutrophils in TME are less‐explored. Here, we identified that abundant glutamate releasing from tumor cells blunted neutrophils’ cell‐killing effects toward tumor cells in vitro and in vivo. Mass spectrometric detection, flow cytometry, and western blot experiments proved that increased levels of pSTAT3/RAB10/ARF4, mediated by glutamate, were accompanied with immunosuppressive phenotypes of neutrophils in TME. We also discovered that riluzole, an FDA‐approved glutamate release inhibitor, significantly inhibited tumor growth by restoring neutrophils’ cell‐killing effects and decreasing glutamate secretion from tumor cells. These findings highlight the importance of tumor‐released glutamate on neutrophil transformation in TME, providing new possible cancer treatments targeting altered glutamate metabolism.     

The role of melanoma tumor-derived nitric oxide in the tumor inflammatory microenvironment: its impact on the chemokine expression profile, including suppression of CXCL10

Melanoma appears to be heterogeneous in terms of its molecular biology, etiology and epidemiology. We previously reported that the expression of inducible nitric-oxide synthase (iNOS) in melanoma tumor cells is strongly correlated with poor patient survival. Therefore, we hypothesized that nitric oxide (NO) produced by iNOS promotes the melanoma inflammatory tumor microenvironment associated with poor outcome. To understand the role of NO and iNOS in the melanoma inflammatory tumor microenvironment, polymerase chain reaction arrays of inflammatory and autoimmunity genes were performed on a series of stage III melanoma lymph node metastasis samples to compare the gene expression profiles of iNOS-expressing and nonexpressing tumor samples. The results indicate that expression of CXC chemokine ligand 10 (CXCL10) was inversely correlated with iNOS expression, and the high CXCL10-expressing cases had more favorable prognoses than the low CXCL10-expressing cases. Functional studies revealed that treating iNOS-negative/CXCL10-positive melanoma cell lines with a NO donor suppressed the expression of CXCL10. Furthermore, scavenging NO from iNOS-expressing cell lines significantly affected the chemokine expression profile. Culture supernatants from NO scavenger-treated melanoma cells promoted the migration of plasmacytoid dendritic cells, which was diminished when the cells were treated with a CXCL10-neutralizing antibody. CXCL10 has been reported to be an antitumorigenic chemokine. Our study suggests that the production of NO by iNOS inhibits the expression of CXCL10 in melanoma cells and leads to a protumorigenic tumor microenvironment. Inhibiting NO induces an antitumorigenic environment, and thus, iNOS should be considered to be an important therapeutic target in melanoma.     

Myeloid-derived suppressor cells: Roles in the tumor microenvironment and tumor radiotherapy

Cancer progression is closely related to the tumor microenvironment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, signaling molecules and the extracellular matrix. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can impact the growth and evolution of cancerous cells. One of major cell components in the tumor microenvironment is myeloid-derived suppressor cells (MDSCs), which promote tumor growth and metastasis directly or indirectly by recognizing other immune cells, producing cytokines and exerting their immunosuppression functions. MDSCs have emerged as major regulators of immune responses in cancer and key targets for treating cancer. There are many limitations and side-effect in approaches of conventional cancer therapy, including radiotherapy. It has grown up to be a burgeoning field that a combination of radiotherapy and immunotherapy applied to cancer therapy. Therefore, it is fundamental to explore the immune mechanism in the process of cancer treatment. Here, we reviewed the recent progress of MDSCs in roles of the tumor microenvironment and tumor radiotherapy.     

右足底恶性黑色素瘤术后十二指肠转移内镜像个案报道

目的探讨恶性黑色素瘤术后十二指肠转移内镜像变化。方法 对1例恶性黑色素瘤术后患者临床资料进行分析,通过内镜及病理检查明确诊断。结果 恶性黑色素瘤具有高转移性,内镜像与皮肤所见黑痣相似。结论 内镜检查结合镜下病理检查,在诊断上消化道恶性黑色素瘤中起着关键作用。     

皮肤T细胞淋巴瘤的恶性炎症及肿瘤微环境的研究进展

T细胞淋巴瘤属非霍奇金亚型中的集合型淋巴瘤,是非常罕见的恶性肿瘤。研究结果显示该病预后较差。近年关于慢性炎症与肿瘤发生相关性的研究日益增多,炎症微环境和促炎因子的变化等因素可诱发肿瘤的进展,同时提示了较差的预后。目前,关于慢性炎症与实体肿瘤发生的相关性研究较多,而关于引发炎症的细胞通过异常活化推动恶性转化过程相关研究报导较少。皮肤T细胞淋巴瘤（CTCL）的特征之一是存在长期发炎的含有恶性T细胞的皮肤损伤。本文针对皮肤T细胞淋巴瘤的免疫-炎症微环境,对近两年的研究进行综述。     

结合基因表达谱探讨恶性黑色素瘤免疫微环境分型的预后预测价值

目的：探讨恶性黑色素瘤（malignant melanoma,MM）微环境分型对MM患者预后的评估价值。方法：对2010年7月 至2017年5月在南京鼓楼医院手术切除的87例原发性MM组织进行二代测序,免疫组化法检测PD-1、PD-L1、CD3+TIL、MSH2、 MSH6、PMS2和MLH1的表达。随访患者的生存时间,分析不同免疫微环境分型对患者预后的影响及其基因表达特征。结果： 根据PD-L1和TIL表达水平将87例MM患者的肿瘤微环境分为4个亚型：PD-L1+TIL+ 型或双阳型（15/87,17.24%）、PD-L1+TIL[1]型 （15/87,17.24%）、PD-L1- TIL+ 型（20/87,22.99%）、PD-L1- TIL[1]型或双阴型（37/87,42.53%）。双阳型患者的中位无病生存期显著长 于双阴型患者(P<0.05),此可能与双阴型患者存在更多CDK4、MCL1、MYC、AKT2、CCND1、FGF19等预后不良基因拷贝数扩增 相关;双阳型患者PD-1表达显著高于双阴型患者(P<0.01),可能与PD-L1、TIL分别与PD-1呈共表达和共不表达有关。结论：根 据PD-L1及TIL表达将MM 患者微环境分为4种亚型,能够区分MM患者预后,双阴型患者存在更多预后不良基因拷贝数扩增。     

Reduction of metastatic rate by immunotherapy: a comparison of the immunogenic properties of metastasizing tumor cells versus tumor cells in the primary mass.

The vasculature of a poorly immunogenic, highly metastatic transplantable fibrosarcoma (T-241) maintained in the femoral muscle of C57BL/6J mice was perfused. This permitted collection of tumor cells which had invaded into the tumor vascular channels (ie, metastasizing tumor cells). Also collected as a separate population were tumor cells from the primary tumor mass. Immunization was carried out with these cell populations in conjunction with BCG and the effect on the growth of primary tumor and metastatic rate was evaluated following rechallenge with unfractionated tumor cells. The rate of tumor growth at the primary site was not affected by any of the immunization schedules. However, immunization with venous effluent cells (metastasizing tumor cells) and BCG was two times more effective in reducing the number of pulmonary metastases than immunization using tumor cells isolated from the primary tumor mass. Passively transferred spleen cells from donors immunized with the cell populations listed above had exactly the same effect, that is, no effect on the growth of the primary tumor, but a dramatic reduction in the metastatic rate when effluent tumor cells were used to immunize cell donors. The data point to an antigenic heterogeneity with this particular transplantable tumor.