Mice Bearing Melanoma Xenografts Research Articles

Activation of human vascular endothelium in melanoma metastases induces ICAM-1 and E-selectin expression and results in increased infiltration with effector lymphocytes.

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real-time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM-1 (CD54) and E-selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3+ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF-α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8+ cells were detected in TNF-α-treated melanoma metastases compared with PBS-treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI-67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.

The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma.

Radiotherapy (RT) in patients with melanoma historically showed suboptimal results, because the disease is often radioresistant due to various mechanisms such as scavenging free radicals by thiols, pigmentary machinery, or enhanced DNA repair. However, radiotherapy has been utilized as adjuvant therapy after the complete excision of primary melanoma and lymph nodes to reduce the rate of nodal recurrences in high-risk patients. The resistance of melanoma cells to radiotherapy may also be in relation with the constitutive activation of the MAPK pathway and/or with the inactivation of p53 observed in about 90% of melanomas. In this study, we aimed to assess the potential benefit of adding RT to BRAF-mutated melanoma cells under a combined p53 reactivation and MAPK inhibition in vitro and in a preclinical animal model. We found that the combination of BRAF inhibition (vemurafenib, which completely shuts down the MAPK pathway), together with p53 reactivation (PRIMA-1Met) significantly enhanced the radiosensitivity of BRAF-mutant melanoma cells. This was accompanied by an increase in both p53 expression and activity. Of note, we found that radiation alone markedly promoted both ERK and AKT phosphorylation, thus contributing to radioresistance. The combination of vemurafenib and PRIMA-1Met caused the inactivation of both MAPK kinase and PI3K/AKT pathways. Furthermore, when combined with radiotherapy, it was able to significantly enhance melanoma cell radiosensitivity. Interestingly, in nude mice bearing melanoma xenografts, the latter triple combination had not only a synergistic effect on tumor growth inhibition, but also a potent control on tumor regrowth in all animals after finishing the triple combination therapy. RT alone had only a weak effect. In conclusion, we provide a basis for a strategy that may overcome the radioresistance of BRAF-mutated melanoma cells to radiotherapy. Whether this will translate into a rational to use radiotherapy in the curative setting in BRAF-mutated melanoma patients deserves consideration.

Salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers selectively suppress human CD20+ melanoma stem cells.

Melanoma is the deadliest type of skin cancer. CD20+ melanoma stem cells (CSCs) are pivotal for metastasis and initiation of melanoma. Therefore, selective elimination of CD20+ melanoma CSCs represents an effective treatment to eradicate melanoma. Salinomycin has emerged as an effective drug toward various CSCs. Due to its poor solubility, its therapeutic efficacy against melanoma CSCs has never been evaluated. In order to target CD20+ melanoma CSCs, we designed salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers (CD20-SA-NPs). Using a single-step nanoprecipitation method, salinomycin-loaded lipid-polymer nanoparticles (SA-NPs) were prepared, then CD20-SA-NPs were obtained through conjugation of thiolated anti-CD20 aptamers to SA-NPs via a maleimide-thiol reaction. CD20-SA-NPs displayed a small size of 96.3 nm, encapsulation efficiency higher than 60% and sustained drug release ability. The uptake of CD20-SA-NPs by CD20+ melanoma CSCs was significantly higher than that of SA-NPs and salinomycin, leading to greatly enhanced cytotoxic effects in vitro, thus the IC50 values of CD20-SA-NPs were reduced to 5.7 and 2.6 μg/mL in A375 CD+20 cells and WM266-4 CD+ cells, respectively. CD20-SA-NPs showed a selective cytotoxicity toward CD20+ melanoma CSCs, as evidenced by the best therapeutic efficacy in suppressing the formation of tumor spheres and the proportion of CD20+ cells in melanoma cell lines. In mice bearing melanoma xenografts, administration of CD20-SA-NPs (salinomycin 5 mg·kg-1·d-1, iv, for 60 d) showed a superior efficacy in inhibition of melanoma growth compared with SA-NPs and salinomycin. In conclusion, CD20 is a superior target for delivering drugs to melanoma CSCs. CD20-SA-NPs display effective delivery of salinomycin to CD20+ melanoma CSCs and represent a promising treatment for melanoma.

Abstract IA20: Targeting AMPK signaling in melanoma

Abstract Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. We have previously reported that AMPK signalling is attenuated by BRAFV600E in melanoma through phosphorylation of LKB1, a major upstream kinase of AMPK. More recently, we have also discovered that BRAF is directly phosphorylated by AMPK, leading to impaired BRAF-KSR1 association and MEK-ERK signalling (Mol Cell, 2013, 52:161). Based on this crosstalk between the AMPK and BRAF signaling pathways, we investigated the anti-tumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Co-treatment of BRAF mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared to the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells. In contrast, PLX4720 selectively targeted JARID1B negative cells, which represents the majority of cells in the tumors, and spared the JAR1D1B-positive slowing cycling population. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma. Citation Format: Bin Zheng. Targeting AMPK signaling in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA20.

Abstract 1741: Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ)

Abstract IGF-1R is frequently up-regulated in cancers including melanoma, and is implicated in mediating resistance to chemotherapy and targeted agents via regulation of apoptosis. Here, we investigated responses to temozolomide (TMZ), a methylating agent that generates toxic O6-methylguanine (O6meG) adducts, which are removed by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6meG adducts trigger futile cycles of mismatch repair, ultimately leading to formation of replication-associated DSBs. In a first approach to test links between IGF-1R and response to TMZ, we used a panel of 10 human melanoma cell lines in which we characterized MGMT expression, activation and expression of IGF axis components, and growth inhibitory effects of TMZ. We found that TMZ GI50 values correlated with MGMT levels (r=0.79, p=0.009), and in 7 MGMT-proficient cell lines, with phospho-IGF-1R (r=0.81, p=0.038) suggesting a link between TMZ resistance and IGF-1R activation. In a second approach we used IGF-1R tyrosine kinase inhibitors (TKIs) AZ12253801 and linsitinib (OSI-906), which is currently undergoing clinical evaluation, to assess effects of blocking IGF signaling. These IGF-1R TKIs were capable of sensitizing wild-type and mutant BRAF melanoma cells to TMZ; lack of correlation with apoptosis induction suggested that other factors contributed to this effect. IGF-1R inhibition did not influence MGMT expression or activity, was not epistatic with MGMT inhibition, and TMZ-sensitized MGMT-null cells, suggesting that chemo-sensitization was MGMT-independent. We found that IGF-1R TKI sensitized melanoma cells to inhibition of poly(ADP ribose) polymerase (PARP), and influenced processing of TMZ-induced DSBs, with increased RPA focus formation within 24hr of TMZ application, and persistence of RAD51 foci at 72hr. These data suggest functional impairment of homologous recombination (HR)-mediated DSB repair. Compared with simultaneous or prior IGF-1R TKI, TMZ sensitization was greater when IGF-1R TKI followed TMZ, consistent with a model in which IGF-1R inhibitor pre-treatment reduces DSB yield. In mice bearing melanoma xenografts with a clinically relevant (V600E BRAF, wild-type p53) genotype, this sequential (TMZ→linsitinib) combination treatment was tolerable, and linsitinib achieved peak plasma levels comparable to clinical Cmax values. TMZ and linsitinib caused minor inhibition of tumor growth (gradient reduction of 13% and 25% respectively), while the combination caused supra-additive growth delay (72%) that was significantly different from control (p<0.01), TMZ (p<0.01) and linsitinib (p<0.05) groups. In summary, IGF-1R inhibition influences HR repair of TMZ-induced DSBs and sensitizes melanomas to TMZ using treatment schedules relevant to clinical practice. Citation Format: Roger N. Ramcharan, Tamara Aleksic, Shan Gao, Jordan Tanner, Nicholas Darvill, Esther Bridges, Ruth Asher, Amanda J. Watson, Geoffrey P. Margison, Emmanouela Repapi, Ji-Liang Li, Mark R. Middleton, Valentine M. Macaulay. Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2014-1741

A novel B-RAF inhibitor blocks interleukin-8 (IL-8) synthesis in human melanoma xenografts, revealing IL-8 as a potential pharmacodynamic biomarker

B-RAF mutations have been identified in the majority of melanoma and a large fraction of colorectal and papillary thyroid carcinoma. Drug discovery efforts targeting mutated B-RAF have yielded several interesting molecules, and currently, three compounds are undergoing clinical evaluation. Inhibition of B-RAF in animal models leads to a slowing of tumor growth and, in some cases, tumor reduction. Described within is a novel series of diaryl imidazoles with potent, single-digit nanomolar, anti-B-RAF activity. One compound from this series has been detailed here and has been shown to block B-RAF(V600E)-dependent extracellular signal-regulated kinase 1/2 phosphorylation in SK-MEL-28 melanoma cells as well as soft agar colony formation and proliferation. Importantly, interleukin-8 (IL-8) was identified by quantitative real-time PCR and ELISA as a product of the elevated mitogen-activated protein kinase signaling in these cells. Plasma concentrations of IL-8 in mice bearing melanoma xenografts were significantly reduced following exposure to B-RAF inhibitors. Taken together, these data suggest that IL-8 could serve as a tractable clinical biomarker.

Three-dimensional ultrasound biomicroscopy for xenograft growth analysis

We reported the use of high-frequency ultrasound biomicroscopy (UBM) in the quantitative analysis of early tumor growth in mice bearing melanoma xenografts in a noninvasive longitudinal assay. Initially, measurements of tumor width, depth and length were obtained using on-screen UBM calipers in real time and tumor volume was calculated with the standard ellipsoid formula w d l π/6. We were able to detect initiating minute tumor nodules, with the lower limit of detection at ≈ 0.01 mm 3 in volume. Successive parallel cross-sectional UBM images (33 μm step) encompassing the complete length of these tumors were also obtained and reconstructed into 3-D representations. Subsequent segmentational volumetric analysis provided a measure of tumor volume. Volume measurements using the two techniques were highly correlated when all 33 xenografts were studied (r = 0.9813, p < 0.0001) and a lower degree of correlation was measured with a subset of early small tumors (r = 0.7973, n = 16, p = 0.0004). Further analysis demonstrated that 3-D segmentational volumetric analysis yielded volume estimates that were often smaller than the caliper-and-formula calculation for most early developing xenografts. Thus, 3-D UBM imaging and segmentation is expected to be especially valuable for small tumors that were observed to grow in irregular shapes other than ellipsoids. (E-mail: acheung@sten.sunnybrook.utoronto.ca)