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The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases.

BackgroundMalignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets.MethodsIn the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models.ResultsOur in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts.ConclusionsThese data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1474-8) contains supplementary material, which is available to authorized users.

177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1.

BackgroundThe histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials.MethodsRadiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models.ResultsUnder hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth.ConclusionsVorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials.

BackgroundPseudomyxoma peritonei (PMP) is a rare, slow-growing abdominal cancer with no efficacious treatment options in non-resectable and recurrent cases. Otherwise, rare activating mutations in the GNAS oncogene are remarkably frequent in PMP and the mutated gene product, guanine nucleotide-binding protein α subunit (Gsα), is a potential tumor neoantigen, presenting an opportunity for targeting by a therapeutic cancer vaccine.MethodsTumor and blood samples were collected from 25 patients undergoing surgery for PMP (NCT02073500). GNAS mutation analysis was performed by next-generation targeted sequencing or digital droplet PCR. Responses to stimulation with Gsα mutated (point mutations R201H and R201C) 30 mer peptides were analyzed in peripheral blood T cells derived from patients with PMP and healthy donors. Fresh PMP tumor samples were analyzed by mass cytometry using a panel of 35 extracellular markers, and cellular subpopulations were clustered and visualized using the visual stochastic network embedding analysis tool.ResultsGNAS mutations were detected in 22/25 tumor samples (88%; R201H and R201C mutations detected in 16 and 6 cases, respectively). Strong T cell proliferation against Gsα mutated peptides was observed in 18/24 patients with PMP. Mass cytometry analysis of tumor revealed infiltration of CD3 +T cells in most samples, with variable CD4+:CD8 + ratios. A large proportion of T cells expressed immune checkpoint molecules, in particular programmed death receptor-1 and T cell immunoreceptor with Ig and ITIM, indicating that these T cells were antigen experienced.ConclusionThese findings point to the existence of a pre-existing immunity in patients with PMP towards mutated Gsα, which has been insufficient to control tumor growth, possibly because of inhibition of antitumor T cells by upregulation of immune checkpoint molecules. The results form a rationale for exploring peptide vaccination with Gsα peptides in combination with immune checkpoint inhibiton as a possible curative treatment for PMP and other GNAS mutated cancers.

Cytoreductive surgery and intraperitoneal (i.p.) chemotherapy constitute a curative treatment option in mucinous peritoneal surface malignancies of intestinal origin, but treatment outcome is highly variable and the search for novel therapies is warranted. Immunotoxins are attractive candidates for targeted therapy in the peritoneal cavity because of direct cytotoxicity, distinct mechanisms of action and tumor cell selectivity. The MOC31PE immunotoxin targets the tumor-associated adhesion protein EpCAM (Epithelial Cell Adhesion Molecule), and has been administered safely in early clinical trials. In our work, the efficacy of i.p. administration of MOC31PE alone and together with mitomycin C (MMC) was investigated in unique animal models of human mucinous peritoneal surface malignancies. In initial model validation experiments, clear differences in efficacy were demonstrated between MMC and oxaliplatin, favoring MMC in five investigated tumor models. Subsequently, MOC31PE and MMC were given as single i.p. injections alone and in combination. In the PMCA-2 model, moderate growth inhibition was obtained with both drugs, while the combination resulted in at least additive effects; whereas the PMP-2 model was highly sensitive to both drugs separately and in combination and intermediate sensitivity was found for the PMCA-3 model. Furthermore, results from ex vivo experiments on freshly obtained mucinous tumor tissue from animals and patients suggested that classic mechanisms of immunotoxin activity were involved, i.e., inhibition of protein synthesis and induction of apoptosis. The present results suggest that adding MOC31PE to MMC-based i.p. chemotherapy should be further explored for EpCAM-expressing peritoneal surface malignancies, and a phase I trial is in preparation.Mucinous peritoneal surface malignancies of intestinal origin encompass a range of clinical presentations, from the clinically benign manifestation pseudomyxoma peritonei (PMP) to aggressive mucinous peritoneal carcinomatosis from colorectal cancer. Histopathological presentation varies, with increasing cellular atypia, high epithelium to mucin ratio and signet ring cell differentiation being associated with an aggressive phenotype. The treatment is, regardless of histology, aimed at complete surgical removal of all visible tumor deposits followed by chemotherapy, typically some type of perioperative intraperitoneal (i.p.) chemotherapy, often supplemented with postoperative systemic chemotherapy. Prognosis is highly dependent on histopathological subtype, and particularly in peritoneal mucinous carcinomatosis (PMCA), the search for more efficacious chemotherapy regimens is warranted. [1][2][3] Although the benefit of cytoreductive surgery is well documented in mucinous peritoneal surface malignancies, the optimal chemotherapeutic strategy has not been proven. [2][3][4] Several drug combinations and delivery strategies are currently being pursued, mostly in early clinical trials, while preclinical assessment of drug efficacy has been impeded by lack o...

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