Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma

BACKGROUND High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment

Biopathological Significance of PIWI–piRNA Pathway Deregulation in Invasive Breast Carcinomas

International audienceSimple SummaryThe PIWI-piRNA ribonucleoproteic complexes are pivotal regulators of genome integrity, differentiation and homeostasis and their dysregulation has recently been implicated in carcinogenesis. The aim of this study was to analyze the four PIWILs gene expression in invasive breast carcinomas (IBC) at RNA level using quantitative RT-PCR and protein level using immunohistochemistry. In normal breast tissue, PIWILs 2 and 4 were solely expressed, whereas an abnormal emergence of PIWIL1 and 3 was observed in respectively 30% and 6% of IBCs. Conversely, PIWIL2 was underexpressed in 48.3% and PIWIL4 downregulated in 43.3% of IBCs. Similar patterns of PIWIL deregulation were observed in a multitumoral panel, suggesting a generic mechanism in most cancers. PIWIL2 underexpression was significantly associated with DNA methylation and strong cytotoxic immune response. Characterization of the newly recognized PIWIL-piRNA pathway in IBCs opens interesting therapeutic perspectives using piRNAs, hypomethylating drugs, checkpoints immunotherapies and anti-PIWIL 1–3 antibodies.AbstractThe PIWI proteins emerging in the development of human cancers, edify PIWI-piRNA ribonucleoproteic complexes acting as pivotal regulators of genome integrity, differentiation and homeostasis. The aim of this study is to analyze the four PIWILs gene expression in invasive breast carcinomas (IBCs): at RNA level using quantitative RT-PCR (n = 526) and protein level using immunohistochemistry (n = 150). In normal breast tissue, PIWILs 2 and 4 were solely expressed, whereas an abnormal emergence of PIWIL1 and 3 was observed in respectively 30% and 6% of IBCs. Conversely, PIWIL2 was underexpressed in 48.3% and PIWIL4 downregulated in 43.3% of IBCs. Significant positive associations were observed between PIWIL4 underexpression, HR+ status and HR+ ERBB2+ molecular subtype and PIWIL2 underexpression, PR- status, ERBB2- status and molecular subtype. Similar patterns of PIWIL deregulation were observed in a multitumoral panel, suggesting a generic mechanism in most cancers. PIWIL2-4 underexpression was mainly regulated at epigenetic or post-transcriptional levels. PIWIL2 underexpression was significantly associated with DNA methylation and strong cytotoxic immune response. PIWIL2-4 were mainly associated with genes implicated in cell proliferation. As a result of this study, characterization of the PIWIL-piRNA pathway in IBCs opens interesting therapeutic perspectives using piRNAs, hypomethylating drugs, checkpoints immunotherapies and anti-PIWIL 1–3 antibodies

The Expression of Anti-Müllerian Hormone Type II Receptor (AMHRII) in Non-Gynecological Solid Tumors Offers Potential for Broad Therapeutic Intervention in Cancer

International audienceThe anti-Müllerian hormone (AMH) belongs to the TGF-β family and plays a key role during fetal sexual development. Various reports have described the expression of AMH type II receptor (AMHRII) in human gynecological cancers including ovarian tumors. According to qRT-PCR results confirmed by specific In-Situ Hybridization (ISH) experiments, AMHRII mRNA is expressed in an extremely restricted number of normal tissues. By performing ISH on tissue microarray of solid tumor samples AMHRII mRNA was unexpectedly detected in several non-gynecological primary cancers including lung, breast, head and neck, and colorectal cancers. AMHRII protein expression, evaluated by immunohistochemistry (IHC) was detected in approximately 70% of epithelial ovarian cancers. Using the same IHC protocol on more than 900 frozen samples covering 18 different cancer types we detected AMHRII expression in more than 50% of hepato-carcinomas, colorectal, lung, and renal cancer samples. AMHRII expression was not observed in neuroendocrine lung tumor samples nor in non-Hodgkin lymphoma samples. Complementary analyses by immunofluorescence and flow cytometry confirmed the detection of AMHRII on a panel of ovarian and colorectal cancers displaying comparable expression levels with mean values of 39,000 and 50,000 AMHRII receptors per cell, respectively. Overall, our results suggest that this embryonic receptor could be a suitable target for treating AMHRII-expressing tumors with an anti-AMHRII selective agent such as murlentamab, also named 3C23K or GM102. This potential therapeutic intervention was confirmed in vivo by showing antitumor activity of murlentamab against AMHRII-expressing colorectal cancer and hepatocarcinoma Patient-Derived tumor Xenografts (PDX) models

Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo

International audienceHow mechanical stress actively impacts the physiology and pathophysiology of cells and tissues is little investigated in vivo. The colon is constantly submitted to multi-frequency spontaneous pulsatile mechanical waves, which highest frequency functions, of 2 s period, remain poorly understood. Here we find in vivo that high frequency pulsatile mechanical stresses maintain the physiological level of mice colon stem cells (SC) through the mechanosensitive Ret kinase. When permanently stimulated by a magnetic mimicking-tumor growth analogue pressure, we find that SC levels pathologically increase and undergo mechanically induced hyperproliferation and tumorigenic transformation. To mimic the high frequency pulsatile mechanical waves, we used a generator of pulsed magnetic force stimulation in colonic tissues pre-magnetized with ultra-magnetic liposomes. We observed the pulsatile stresses using last generation ultra-wave dynamical high-resolution imaging. Finally, we find that the specific pharmacological inhibition of Ret mechanical activation induces the regression of spontaneous formation of SC, of CSC markers, and of spontaneous sporadic tumorigenesis in Apc mutated mice colons. Consistently, in human colon cancer tissues, Ret activation in epithelial cells increases with tumor grade, and partially decreases in leaking invasive carcinoma. High frequency pulsatile physiological mechanical stresses thus constitute a new niche that Ret-dependently fuels mice colon physiological SC level. This process is pathologically over-activated in the presence of permanent pressure due to the growth of tumors initiated by pre-existing genetic alteration, leading to mechanotransductive selfenhanced tumor progression in vivo, and repressed by pharmacological inhibition of Ret