Gregory Driessens

Defining the mode of tumour growth by clonal analysis

Recent studies using the isolation of a subpopulation of tumour cells followed by their transplantation into immunodeficient mice provide evidence that certain tumours, including squamous skin tumours, contain cells with high clonogenic potential that have been referred to as cancer stem cells (CSCs). Until now, CSC properties have only been investigated by transplantation assays, and their existence in unperturbed tumour growth is unproven. Here we make use of clonal analysis of squamous skin tumours using genetic lineage tracing to unravel the mode of tumour growth in vivo in its native environment. To this end, we used a genetic labelling strategy that allows individual tumour cells to be marked and traced over time at different stages of tumour progression. Surprisingly, we found that the majority of labelled tumour cells in benign papilloma have only limited proliferative potential, whereas a fraction has the capacity to persist long term, giving rise to progeny that occupy a significant part of the tumour. As well as confirming the presence of two distinct proliferative cell compartments within the papilloma, mirroring the composition, hierarchy and fate behaviour of normal tissue, quantitative analysis of clonal fate data indicates that the more persistent population has stem-cell-like characteristics and cycles twice per day, whereas the second represents a slower cycling transient population that gives rise to terminally differentiated tumour cells. Such behaviour is shown to be consistent with double-labelling experiments and detailed clonal fate characteristics. By contrast, measurements of clone size and proliferative potential in invasive squamous cell carcinoma show a different pattern of behaviour, consistent with geometric expansion of a single CSC population with limited potential for terminal differentiation. This study presents the first experimental evidence for the existence of CSCs during unperturbed solid tumour growth.

A vascular niche and a VEGF–Nrp1 loop regulate the initiation and stemness of skin tumours

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF’s ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.

SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2–GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2–GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses, high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy—combining dendritic cells and irradiated GM-CSF secreting tumor cells—to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-γ secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery.

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an “all in vivo” strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful “all in vivo” vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

Highly Successful Therapeutic Vaccinations Combining Dendritic Cells and Tumor Cells Secreting Granulocyte Macrophage Colony-stimulating Factor

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-γ production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.

Micronuclei to detect in vivo chemotherapy damage in a p53 mutated solid tumour

Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.

Synergy between dendritic cells and GM-CSF-secreting tumor cells for the treatment of a murine renal cell carcinoma.

Dendritic cell (DC) immunotherapy for cancer certainly holds promises but definitely needs improvements, especially for enhancing tumor-specific responses able to eradicate preexisting tumors. To this end, we investigated here, for the treatment of a preimplanted murine renal cell carcinoma Renca, a new vaccination approach combining injection of DC and granulocyte macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cells. When treatment by either DC or Renca–mGM-CSF cells alone had no therapeutic effect at all, combined vaccines induced therapeutic response in 50% of the tumor-bearing mice, in a GM-CSF dose-dependent manner. Importantly, all these cured mice were protected against a rechallenge with parental Renca cells, indicating the generation of memory immune response. The combined vaccines induced elevated cytotoxic responses in all the cured mice and half of the uncured ones and a stronger systemic CD4+ T–cell-mediated interferon-γ production in the cured vaccinated mice as compared with uncured ones. In conclusion, vaccines associating DC and GM–CSF-secreting tumor cells induce high therapeutic effect in mice with preexisting renal cell carcinoma that are correlated to the induction of specific CD8+ and CD4+ T-cell responses. This original vaccination approach should be further evaluated in a clinical trial for the treatment of metastatic human renal cell carcinoma.

Abstract 4863: PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors

Tumors use tryptophan-catabolizing enzymes such as Indoleamine 2,3-dioxygenase-1 (IDO-1) to induce an immunosuppressive microenvironment. IDO-1 expression is upregulated in many cancers and described to be a resistance mechanism to immune checkpoint therapies. IDO-1 is induced in response to inflammatory stimuli such as IFN-a and promotes immune tolerance through the catabolism of tryptophan and accumulation of tryptophan catabolites including kynurenine. IDO-1 activity leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As such, IDO1 is a nexus for the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology. We have identified and characterized a new IDO-1 inhibitor. PF-06840003 is a highly selective orally bioavailable IDO-1 inhibitor. PF-06840003 reversed IDO-1-induced T-cell anergy in vitro. In vivo, PF-06840003 reduced intratumoral kynurenine levels in mice by >80% and inhibited tumor growth in multiple preclinical syngeneic models in mice, in combination with immune checkpoint inhibitors. PF-0684003 has favorable predicted human pharmacokinetic properties, including a predicted t1/2 of 16-19 hours. These studies highlight the strong potential of PF-06840003 as a clinical candidate in Immuno-Oncology. Citation Format: Joseph Tumang, Bruno Gomes, Martin Wythes, Stefano Crosignani, Patrick Bingham, Pauline Bottemanne, Helene Cannelle, Sandra Cauwenberghs, Jenny Chaplin, Deepak Dalvie, Sofie Denies, Coraline De Maeseneire, Peter Folger, Kim Frederix, Jie Guo, James Hardwick, Ken Hook, Katti Jessen, Erick Kindt, Marie-Claire Letellier, Kai-Hsin Liao, Wenlin Li, Karen Maegley, Reece Marillier, Nichol Miller, Brion Murray, Romain Pirson, Julie Preillon, Virginie Rabolli, Chad Ray, Stephanie Scales, Jay Srirangam, Jim Solowiej, Nicole Streiner, Vince Torti, Konstantinos Tsaparikos, Paolo Vicini, Gregory Driessens, Manfred Kraus. PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4863.

Abstract 1683: A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity

High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors. We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors. We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson’s disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets. These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology. Citation Format: Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Queva, Stefano Crosignani, Bruno Gomes. A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2017-1683