Application of functional genomics to colorectal cancer liver metastases and blastic plasmacytoid dendritic cell neoplasms

De récents progrès techniques permettant l’acquisition de données « omiques » à l’échelle de la cellule individuelle ont marqué un réel tournant dans le domaine de la recherche en biologie. L’approche de transcriptomique en cellules uniques a notamment permis de répondre à des problématiques qui demeuraient jusqu’alors inaccessibles, en particulier en biologie du développement et cancérologie.Dans le domaine de la recherche sur le cancer, la transcriptomique en cellules uniques a fortement participé à la compréhension de la complexité de la tumeur. Pour la première fois, il est possible de quantifier la contribution des différents composants cellulaires à la progression tumorale et la résistance au traitement, ainsi que leur grande complexité. Parmi les différentes cellules du microenvironnement tumoral, les fibroblastes associés au cancer (CAF) sont une population particulièrement hétérogène et versatile, pouvant jouer à la fois un rôle antitumoral et protumoral. Malgré leur impact sur la progression de la maladie, aucun traitement efficace ne parvient à exploiter ou cibler les CAF à ce jour. Les molécules impliquées dans la communication entre les CAF et les autres acteurs cellulaires représenteraient d’intéressantes pistes thérapeutiques.Dans le cadre du projet principal de ma thèse de doctorat, nous avons appliqué l’approche de transcriptomique en cellules uniques à l’étude de l’hétérogénéité des CAF dans les métastases hépatiques de cancer colorectal. Cette étude a été menée en étroite collaboration avec les services d’anatomo-pathologie et de chirurgie de l’Institut régional du Cancer de Montpellier (ICM). Malgré le fait que les métastases constituent la principale cause de mortalité liée au cancer, leur biologie est encore loin d’être comprise. Dans cette étude, nous avons démontré l’existence d’au moins deux sous-populations de CAF, arborant des phénotypes différents. En intégrant nos données avec des jeux de données publiques, nous avons pu montrer que les CAF de cancers hépatiques (primaires et métastatiques) proviennent d’au moins trois différentes sources : les fibroblastes de l’espace porte, les cellules stellaires hépatiques et les cellules musculaires lisses vasculaires. En inférant notamment les interactions cellulaires entre les différentes populations de CAF et les cellules cancéreuses, nous avons montré que, dans les métastases hépatiques, les CAF issus des fibroblastes portaux contribuent à la réaction desmoplastique et l’angiogenèse.Recent technical progress enabling the acquisition of “omics” data at single-cell scale led to important breakthroughs in biology research. In particular, the application of single-cell transcriptomics in the recent few years allowed an unprecedented insight into several key biological questions, including cancer research and development biology.Particularly in cancer research, single-cell RNA sequencing has allowed a detailed insight in the tumour complexity. For the first time, we are able to quantify the contribution of the different cellular components to cancer progression and response to treatment. We also came to appreciate the complexity of the tumour microenvironment and the contribution of non-cancerous cells (stroma) in the tumour towards the disease progression. Among the different stromal cells, cancer-associated fibroblasts (CAFs) emerged as a heterogeneous, and versatile population with both tumour-promoting and tumour-preventing behaviours. Despite these key implications in tumour progression, no treatment to date successfully harnesses or targets CAFs in clinics. This missed opportunity points at the urgent need to deeply characterize CAFs subpopulations and elucidate how they communicate with cancer cells and the other tumour microenvironment actors. The molecules involved in this communication could represent promising targets for the development of new therapeutic strategies.The main project of my PhD thesis concerned the use of single-cell RNA sequencing to unveil CAFs heterogeneity and chart their interactions with cancer cells in colorectal cancer liver metastases (CRCLM). This study was conducted in tight collaboration with the pathology and surgery departments of Montpellier’s Cancer Institute (ICM). Despite the fact that metastases are the major cause of cancer-related death, their biology is far from understood. In this study, we demonstrated the existence of at least two main subpopulations of CAFs with distinct phenotypes. The integration of our data with public datasets further showed that in liver cancers (primary and metastatic), CAFs originate from at least three different sources: the portal fibroblasts, the hepatic stellate cells, and the vascular smooth muscle cells. Following the reconstruction of the cellular crosstalk between the CAFs and the cancer cells, we showed that in particular portal fibroblasts-derived CAFs enhance desmoplastic reaction and angiogenesis in the liver metastasis

Metastatic colorectal cancer cells maintain the TGFβ program and use TGFBI to fuel angiogenesis.

peer reviewedColorectal cancer (CRC) cells are traditionally considered unresponsive to TGFβ due to mutations in the receptors and/or downstream signaling molecules. TGFβ influences CRC cells only indirectly via stromal cells, such as cancer-associated fibroblasts. However, CRC cell ability to directly respond to TGFβ currently remains unexplored. This represents a missed opportunity for diagnostic and therapeutic interventions. Methods: We examined whether cancer cells from primary CRC and liver metastases respond to TGFβ by inducing TGFβ-induced protein ig-h3 (TGFBI) expression, and the contribution of canonical and non-canonical TGFβ signaling pathways to this effect. We then investigated in vitro and in vivo TGFBI impact on metastasis formation and angiogenesis. Using patient serum samples and an orthotopic mouse model of CRC liver metastases we assessed the diagnostic/tumor targeting value of novel antibodies against TGFBI. Results: Metastatic CRC cells, such as circulating tumor cells, directly respond to TGFβ. These cells were characterized by the absence of TGFβ receptor mutations and the frequent presence of p53 mutations. The pro-tumorigenic program orchestrated by TGFβ in CRC cells was mediated through TGFBI, the expression of which was positively regulated by non-canonical TGFβ signaling cascades. TGFBI inhibition was sufficient to significantly reduce liver metastasis formation in vivo. Moreover, TGFBI pro-tumorigenic function was linked to its ability to stimulate angiogenesis. TGFBI levels were higher in serum samples from untreated patients with CRC than in patients who were receiving chemotherapy. A radiolabeled anti-TGFBI antibody selectively targeted metastatic lesions in vivo, underscoring its diagnostic and therapeutic potential. Conclusions: TGFβ signaling in CRC cells directly contributes to their metastatic potential and stromal cell-independence. Proteins downstream of activated TGFβ, such as TGFBI, represent novel diagnostic and therapeutic targets for more specific anti-metastatic therapies

Transcriptomic and genomic heterogeneity in blastic plasmacytoid dendritic cell neoplasms: from ontogeny to oncogenesis.

Oncogenesis and ontogeny of blastic plasmacytoid dendritic cell neoplasm (BPDCN) remain uncertain, between canonical plasmacytoid dendritic cells (pDCs) and AXL+ SIGLEC6+ DCs (AS-DCs). We compared 12 BPDCN to 164 acute leukemia by Affymetrix HG-U133 Plus 2.0 arrays: BPDCN were closer to B-cell acute lymphoblastic leukemia (ALL), with enrichment in pDC, B-cell signatures, vesicular transport, deubiquitination pathways, and AS-DC signatures, but only in some cases. Importantly, 1 T-cell ALL clustered with BPDCN, with compatible morphology, immunophenotype (cCD3+ sCD3- CD123+ cTCL1+ CD304+), and genetics. Many oncogenetic pathways are deregulated in BPDCN compared with normal pDC, such as cell-cycle kinases, and importantly, the transcription factor SOX4, involved in B ontogeny, pDC ontogeny, and cancer cell invasion. High-throughput sequencing (HaloPlex) showed myeloid mutations (TET2, 62%; ASXL1, 46%; ZRSR2, 31%) associated with lymphoid mutations (IKZF1), whereas single-nucleotide polymorphism (SNP) array (Affymetrix SNP array 6.0) revealed frequent losses (mean: 9 per patient) involving key hematological oncogenes (RB1, IKZF1/2/3, ETV6, NR3C1, CDKN2A/B, TP53) and immune response genes (IFNGR, TGFB, CLEC4C, IFNA cluster). Various markers suggest an AS-DC origin, but not in all patients, and some of these abnormalities are related to the leukemogenesis process, such as the 9p deletion, leading to decreased expression of genes encoding type I interferons. In addition, the AS-DC profile is only found in a subgroup of patients. Overall, the cellular ontogenic origin of BPDCN remains to be characterized, and these results highlight the heterogeneity of BPDCN, with a risk of a diagnostic trap