Abstract Purpose. Objectives of this study were to investigate if prostate cancer stem/initiating cells (CSC) obtained from autochthonous tumors possess the molecular characteristics that allow their in vitro and in vivo recognition by cells of the innate and/or adaptive arms of the immune system, and if CSC are source of antigens for the induction of tumor-specific immune responses. Material and Methods. CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were assessed in vitro for the expression of tumor-associated antigens and susceptibility to NK and T cell killing. We also investigated NK and T cells immune surveillance against CSC by measuring the frequency of CSC-induced tumors in immunocompetent and selectively immunodeficient mice. Finally, we assessed if vaccination with dendritic cells pulsed with CSC induced a tumor-specific immune response able to delay the growth of both transplantable and autochthonous prostate tumors. Results. CSC expressed prostate cancer associated antigens, MHC I and MHC II molecules and ligands for natural killer (NK) cells. Indeed, CSC were targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with dendritic cells pulsed with apoptotic CSC induced a tumor-specific immune response that delayed tumor growth in mice challenged with prostate CSC, and caused tumor regression in TRAMP mice. Conclusions. CSC are targets of both innate and adaptive immune responses and could be exploited for the design of novel immunotherapeutic approaches against cancer. Citation Format: Elena Jachetti, Stefania Mazzoleni, Matteo Grioni, Alessia Ricupito, Chiara Brambillasca, Luca Generoso, Arianna Calcinotto, Massimo Freschi, Anna Mondino, Rossella Galli, Matteo Bellone. Prostate cancer stem/initiating cells are targets of both innate and adaptive immunity and elicit potent immune responses against autochthonous prostate tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2613. doi:10.1158/1538-7445.AM2013-2613
We recently described a lineage of proangiogenic monocytes implicated in tumor angiogenesis. These cells, identified by expression of the angiopoietin receptor (Tie2-Expressing Monocytes, TEMs), are selectively recruited to tumors and promote angiogenesis in a paracrine manner by interacting with locally derived endothelial cells (De Palma et al., Cancer Cell, 2005). Because Tie2 is specifically expressed by TEMs among the progeny of hematopoietic stem cells (HSCs), transplanting HSCs transduced by lentiviral vectors (LVs) containing the Tie2 promoter provides for selective gene expression in TEMs. Here we explored the feasibility of targeted delivery of cancer gene therapy using TEMs as selective cellular vehicles. We transduced HSCs with LVs expressing the potent anti- angiogenic factor alpha-interferon (IFN) or GFP under the control of either the Tie2 or the ubiquitously active PGK promoter, and transplanted the transduced cells into nude mice. All PGK-IFN mice died of graft failure, indicating that ubiquitous expression of IFN in the HSC progeny was severely myelotoxic. In contrast, Tie2-GFP and Tie2-IFN mice were fully reconstituted by transduced HSCs. To compare TEM-mediated delivery with systemic expression of IFN, a group of Tie2-GFP mice received an intravascular injection of PGK-IFN LV, which led to sustained IFN expression in the plasma. Eight weeks post-transplant, we injected human glioma cells intracranially and monitored tumor growth by magnetic resonance imaging. Remarkably, while all other mice displayed large brain tumors, Tie2-IFN mice were tumor free 9 days after challenge. Ten days later, tumors became detectable also in Tie2-IFN mice, although they were markedly necrotic and smaller than in Tie2-GFP mice. Serial brain sections showed that tumors of Tie2-GFP mice were viable and highly vascularized, with GFP+ TEMs surrounding typical angiogenic vessels. By contrast, tumors grown in Tie2-IFN mice had reduced vascular area and exhibited normalized/regressing blood vessels. In these tumors, we observed overexpression of IFN in a subset of tumor-infiltrating myeloid cells, likely corresponding to TEMs, suggesting that targeted expression of IFN in close proximity to neoangiogenic vessels effectively inhibited endothelial cell proliferation. Surprisingly, systemic expression of IFN not only failed to inhibit tumor angiogenesis, but also induced body wasting and severe myelotoxicity. In conclusion, targeted delivery of IFN by TEMs achieved substantial anti-tumor activity in the absence of systemic toxicity, whereas ubiquitous expression in BM-derived cells or systemic delivery were not efficacious and were highly toxic. These results provide proof of principle of a new gene therapy paradigm in which ex vivo transduction of HSCs can be used to safely deliver potent anti-cancer molecules in a tumor-targeted fashion.
Lack of robust predictive biomarkers, other than MGMT promoter methylation, makes temozolomide responsiveness in newly diagnosed glioblastoma (GBM) patients difficult to predict. However, we identified patients with long-term survival (≥35 months) within a group of newly diagnosed GBM patients treated with standard or metronomic adjuvant temozolomide schedules. We thus investigated possible molecular profiles associated with longer survival following temozolomide treatment. We investigated the association of molecular features with progression-free (PFS) and overall survival (OS). Human-derived GBM cancer stem cells (CSCs) were used to investigate in vitro molecular mechanisms associated with temozolomide responsiveness. Surgically removed recurrences allowed investigation of molecular changes occurring during therapy in vivo. Statistical analyses included one- and two-way analysis of variance, Student’s t test, Cox proportional hazards, and the Kaplan-Meier method. All statistical tests were two-sided. No association was found between survival and gene classifiers associated with different molecular GBM subtypes in the standard-treated group, while in metronomic-treated patients robust association was found between EGFR amplification/overexpression and PFS and OS (OS, EGFR-high vs low: hazard ratiodeath = 0.22, 95% confidence interval = 0.09 to 0.55, P = .001). The result for OS remained statistically significant after Bonferroni correction (Pinteraction < .0005). Long-term survival following metronomic temozolomide was independent from MGMT and EGFRvIII status and was more pronounced in EGFR-overexpressing GBM patients with PTEN loss. In vitro findings confirmed a selective dose- and time-dependent decrease in survival of temozolomide-treated EGFR+ human-derived glioblastoma CSCs, which occurred through inhibition of NF-κB transcriptional activity. In addition, reduction in EGFR-amplified cells, along with a statistically significant decrease in NF-κB/p65 expression, were observed in specimens from recurrent metronomic-treated EGFR-overexpressing GBM patients. EGFR-amplified/overexpressing glioblastomas strongly benefit from metronomic temozolomide–based therapies.
