Dynamic changes in glioma macrophage populations after radiotherapy reveal CSF-1R inhibition as a strategy to overcome resistance.

Tumor-associated macrophages (TAMs) and microglia (MG) are potent regulators of glioma development and progression. However, the dynamic alterations of distinct TAM populations during the course of therapeutic intervention, response, and recurrence have not yet been fully explored. Here, we investigated how radiotherapy changes the relative abundance and phenotypes of brain-resident MG and peripherally recruited monocyte-derived macrophages (MDMs) in glioblastoma. We identified radiation-specific, stage-dependent MG and MDM gene expression signatures in murine gliomas and confirmed altered expression of several genes and proteins in recurrent human glioblastoma. We found that targeting these TAM populations using a colony-stimulating factor-1 receptor (CSF-1R) inhibitor combined with radiotherapy substantially enhanced survival in preclinical models. Our findings reveal the dynamics and plasticity of distinct macrophage populations in the irradiated tumor microenvironment, which has translational relevance for enhancing the efficacy of standard-of-care treatment in gliomas.

Inducing and exploiting vulnerabilities for the treatment of liver cancer.

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice.

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies.

Concomitant targeting of programmed death-1 (PD-1) and CD137 improves the efficacy of radiotherapy in a mouse model of human BRAFV600-mutant melanoma.

T cell checkpoint blockade with antibodies targeting programmed cell death (ligand)-1 (PD-1/PD-L1) and/or cytotoxic T lymphocyte-antigen 4 (CTLA-4) has improved therapy outcome in melanoma patients. However, a considerable proportion of patients does not benefit even from combined α-CTLA-4 and α-PD-1 therapy. We therefore examined to which extent T cell (co)stimulation and/or stereotactic body radiation therapy (SBRT) could further enhance the therapeutic efficacy of T cell checkpoint blockade in a genetically engineered mouse melanoma model that is driven by PTEN-deficiency, and BRAFV600 mutation, as in human, but lacks the sporadic UV-induced mutations. Tumor-bearing mice were treated with different combinations of immunomodulatory antibodies (α-CTLA-4, α-PD-1, α-CD137) or interleukin-2 (IL-2) alone or in combination with SBRT. None of our immunotherapeutic approaches (alone or in combination) had any anti-tumor efficacy, while SBRT alone delayed melanoma outgrowth. However, α-CD137 combined with α-PD-1 antibodies significantly enhanced the anti-tumor effect of SBRT, while the anti-tumor effect of SBRT was not enhanced by interleukin-2, or the combination of α-CTLA-4 and α-PD-1. We conclude that α-CD137 and α-PD-1 antibodies were most effective in enhancing SBRT-induced tumor growth delay in this mouse melanoma model, outperforming the ability of IL-2, or the combination of α-CTLA-4 and α-PD-1 to synergize with SBRT. Given the high mutational load and increased immunogenicity of human melanoma with the same genotype, our findings encourage testing α-CD137 and α-PD-1 alone or in combination with SBRT clinically, particularly in patients refractory to α-CTLA-4 and/or α-PD-1 therapy.

Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma.

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAFV600E/PTEN-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8+ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade.

Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma.

Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy.

Synchronous BRAF(V600E) and MEK inhibition leads to superior control of murine melanoma by limiting MEK inhibitor induced skin toxicity.

The BRAF inhibitor (BRAFi) treatment has led to impressive responses in BRAF(V600E) mutation-positive melanomas, but responses are not durable in many patients. As most of the BRAFi escape mechanisms involve ERK reactivation, combinations with MEK inhibitors (MEKi) are currently tested to improve BRAFi-mediated response durations. Additionally, such a combination is expected to reduce MEKi-induced skin toxicities, as these drugs are thought to have antagonistic effects on ERK activation in keratinocytes. However, preclinical in vivo data exploring the combination of BRAFi and MEKi to achieve improved tumor control in the absence of skin toxicities are limited. Using a murine Tyr::CreER(T2);Pten(LoxP/LoxP);Braf(CA/+) melanoma model, we have determined the effect of BRAFi and MEKi treatment and their combination on melanoma control and occurrence of adverse events. We found that the MEKi dosed beyond the maximum tolerable dose (MTD) led to stronger control of tumor growth than did the BRAFi, but mice had to be removed from treatment because of skin toxicity. The combination of BRAFi and MEKi reduced MEKi-associated skin toxicity. This allowed high and long-term dosing of the MEKi, resulting in long-term tumor control. In contrast to previous hypotheses, the addition of a BRAFi did not restore the MEKi-mediated downregulation of pERK1/2 in skin cells. Our data describe, for the first time, the alleviation of MEKi-mediated dose-limiting toxicity by addition of a BRAFi in a mouse melanoma model. Additional clinical Phase I studies should be implemented to explore MEKi dosing beyond the single drug MTD in combination with BRAFi.

Mimicking homeostatic proliferation in vitro generates T cells with high anti-tumor function in non-lymphopenic hosts.

CD8(+) T cells undergoing homeostatic proliferation (HP) in a lymphopenic environment acquire a central memory-like phenotype (CD44(+) CD62L(+) Ly6c(+)). Such cells are readily functional in vitro, with a strong capacity to secrete IFNγ and IL-2 and to lyse target cells upon antigen recognition. In vivo, these memory-like T cells display potent anti-tumor reactivity. When addressing whether these remarkable properties were "acquired" or dependent on sustained HP, we observed, for the first time, that memory-like T cells retained full anti-tumor functions even when removed from their lymphopenic environment and retransferred into non-lymphopenic P14/Rag2(-/-) recipients (where HP is prevented). Moreover, memory-like T cells were superior to in vitro expanded effector T cells. We next sought to determine the conditions required to reproduce such a potent phenotype in vitro, in order to obtain optimal cells for adoptive cell transfer therapy. Assessing ex vivo lymph node cultures, dendritic cells, fibroblastic reticular cells, and HP-associated cytokines, we found that stimulation of naïve T cells with anti-CD3/CD28 beads and IL-15 (IL-7 was dispensable) led to the generation of memory-like T cell with a similar phenotype. Both in vitro and in vivo memory-like T cells retained the capacity to efficiently control tumor growth in non-lymphopenic hosts upon adoptive cell transfer. A similar phenotype could be imparted to human peripheral blood leukocytes with comparable culture conditions. Our data reinforce the idea that in vitro-generated memory-like T cells could benefit adoptive cell transfer therapies.

Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma.

The development of targeted therapies and immunotherapies has markedly advanced the treatment of metastasized melanoma. While treatment with selective BRAF(V600E) inhibitors (like vemurafenib or dabrafenib) leads to high response rates but short response duration, CTLA-4 blocking therapies induce sustained responses, but only in a limited number of patients. The combination of these diametric treatment approaches may further improve survival, but pre-clinical data concerning this approach is limited. We investigated, using Tyr::CreER(T2)PTEN(F-/-)BRAF(F-V600E/+) inducible melanoma mice, whether BRAF(V600E) inhibition can synergize with anti-CTLA-4 mAb treatment, focusing on the interaction between the BRAF(V600E) inhibitor PLX4720 and the immune system. While PLX4720 treatment strongly decreased tumor growth, it did not induce cell death in BRAF(V600E)/PTEN(-/-) melanomas. More strikingly, PLX4720 treatment led to a decreased frequency of tumor-resident T cells, NK-cells, MDSCs and macrophages, which could not be restored by the addition of anti-CTLA-4 mAb. As this effect was not observed upon treatment of BRAF wild-type B16F10 tumors, we conclude that the decreased frequency of immune cells correlates to BRAF(V600E) inhibition in tumor cells and is not due to an off-target effect of PLX4720 on immune cells. Furthermore, anti-CTLA-4 mAb treatment of inducible melanoma mice treated with PLX4720 did not result in enhanced tumor control, while anti-CTLA-4 mAb treatment did improve the effect of tumor-vaccination in B16F10-inoculated mice. Our data suggest that vemurafenib may negatively affect the immune activity within the tumor. Therefore, the potential effect of targeted therapy on the tumor-microenvironment should be taken into consideration in the design of clinical trials combining targeted and immunotherapy.

Targeting BRAFV600E in an inducible murine model of melanoma.

The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.

GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors.

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis.

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-L: -cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2(-/-)γc(-/-)mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.

Reduced tumor-antigen density leads to PD-1/PD-L1-mediated impairment of partially exhausted CD8⁺ T cells.

Clinical progression of cancer patients is often observed despite the presence of tumor-reactive T cells. Co-inhibitory ligands of the B7 superfamily have been postulated to play a part in this tumor-immune escape. One of these molecules, PD-L1 (B7-H1, CD274), is widely expressed on tumor cells and has been shown to mediate T-cell inhibition. However, attempts to correlate PD-L1 tumor expression with negative prognosis have been conflicting. To better understand when PD-1/PD-L1-mediated inhibition contributes to the functional impairment of tumor-specific CD8(+) T cells, we varied the levels of antigen density and/or PD-L1 expression at the surface of tumor cells and exposed them to CD8(+) T cells at different levels of functional exhaustion. We found that the gradual reduction of cognate antigen expression by PD-L1-expressing tumor cells increased the susceptibility of partially exhausted T cells to PD-1/PD-L1-mediated inhibition in vitro as well as in vivo. In conclusion, chronically stimulated CD8(+) T cells become sensitive to PD-1/PD-L1-mediated functional inhibition upon low antigen detection; a setting which is likely involved during tumor-immune escape.

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.

Expression of the embryological morphogen Nodal in stage III/IV melanoma.

The characterization of factors involved in the etiology of melanoma may lead to the identification of new targets for therapy and prognostic markers. Recent data indicate that Nodal can be expressed by malignant melanoma cells and this expression seems to contribute to melanoma development and progression. The aim of this study was to investigate the potential of Nodal as a prognostic marker for stage III/IV patients and as a treatment target for melanoma immunotherapy. We analyzed, by means of immunohistochemistry, 63 patients with stage III/IV melanoma for expression of Nodal in their tumors taken during the course of their disease. Furthermore, to research potential safety issues when immunotherapeutically targeting Nodal, we assessed Nodal expression in normal human adult tissues. We found that Nodal can be expressed at all stages of melanoma progression and there is no significant increase in the frequency of Nodal expression in distant metastases. Furthermore, our data show that there is no correlation between the expression of Nodal during course of disease and survival of patients. Finally, when screening for Nodal expression in normal adult organ tissues, we found consistent expression in renal tissue. We conclude that Nodal expression cannot be used as a prognostic marker for survival of patients with stage III/IV melanoma and as this putative target is renally expressed, it is not well-suited for immunotherapeutic approaches. This study suggests that, despite strong previous suggestions, the role of Nodal in melanoma progression may be less prominent and immunotherapeutic targeting of Nodal could be potentially harmful.

Overall survival and PD-L1 expression in metastasized malignant melanoma.

BACKGROUND: Cancers are known to elude the immune system, for example, by MHC loss, FAS up-regulation, or increased secretion of TGF-beta. Recently, ligands of coinhibitory receptors like programmed cell death ligand-1 (PD-L1, B7-H1) have come to attention for their role in tumor immune escape. Various tumors have been tested for PD-L1 expression, and conflicting results were obtained regarding its correlative impact on patient survival. This study aimed to determine the prognostic relevance of PD-L1 expression for the survival of melanoma patients. METHODS: Paraffin-embedded nevi, primary melanoma, and in-transit, lymph node, and distant organ metastases from a set of 63 stages III-IV melanoma patients referred to the Netherlands Cancer Institute between 2000 and 2004 for a sentinel-node procedure or systemic therapy were studied. A large effort was invested in validating specific PD-L1 staining. In addition to immunological factors such as T-cell infiltration (CD8, CD4, and regulatory T cells), TGF-beta and MHC-I expression were assessed. RESULTS: Longitudinal analysis revealed no relevant PD-L1 expression on primary melanoma compared with metastatic disease. No significant correlations with prognosis were found regarding immunological factors, whereas known prognostic markers such as Breslow thickness and sex could be confirmed. Analyses of the overall survival of our patient cohort did not reveal a negative association with PD-L1 expression. CONCLUSIONS: Correlation of overall survival with PD-L1 expression by melanoma cells remains controversial, and future clinical studies should focus on antibody validation and time of analysis in respect to disease progression.

Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes.

The cyclin dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently deleted, silenced, or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development, showing that these genes function as tumor suppressors. Here, we describe high-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also found associations between these loci with gender, age of tumor onset, and lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with single nucleotide polymorphisms associated with chronic lymphocytic leukemia revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association studies.

Nodal as a biomarker for melanoma progression and a new therapeutic target for clinical intervention.

Nodal, an embryonic morphogen belonging to the TGF-ß superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is expressed significantly in aggressive melanoma. Surprisingly, expression of the Nodal coreceptor, Cripto-1, was detected in only a small fraction of the melanoma tumor cell population, indicating a primary role for Cripto-1-independent signaling of Nodal in melanoma. In this review, we discuss how regulatory factors present in an embryonic environment, such as Lefty, can downregulate Nodal expression and inhibit tumorigenicity and plasticity of melanoma cells. Our translational studies show that antibodies against Nodal are capable of repressing melanoma vasculogenic mimicry and of inducing apoptosis in melanoma tumors in an in vivo lung-colonization assay. Our previous work and ongoing studies suggest that Nodal may represent a novel diagnostic marker and therapeutic target in melanoma.

Homeostatic proliferation of naïve CD8+ T cells depends on CD62L/L-selectin-mediated homing to peripheral LN.

Adoptive transfer of naïve CD8(+) T cells into lymphopenic recipients results both in spontaneous proliferation and in partial activation of T cells, a phenomenon termed homeostatic proliferation (HP). HP of CD8(+) T cells is dependent on host IL-7, IL-15, and MHC-class I and has been shown to prevent T-cell tolerance, reverse T-cell anergy and support T-cell-mediated tumor control in vivo. However, the initial anatomic site of HP is still under debate. Since we observed that the earliest detectable HP occurs within LN and that T cells undergoing HP retain a CD62L(bright) phenotype, we investigated the functional role of CD62L for this process. We found that CD62L-expression on T cells is required for optimal HP and HP was impaired in lymphotoxin-alphabeta(-/-) mice, indicating the necessity for intact host secondary lymphoid organ structures. Use of the LN egression inhibitor FTY720 indicated that LN structures were pivotal to yield homeostatically proliferated T cells detected in other compartments. Consistent with these results, HP-supported control of MC57-SIY tumors depended on CD62L. Our data indicate a critical role for CD62L and LN homing for the process of HP, which has implications for adoptive immunotherapy approaches of cancer.